Trends in Neurosciences 2013 PDF

October 2013 Volume 36, Number 10 pp.
555620
Editor
Andrew M. Clark
Executive Editor, Neuroscience
Katja Brose
Editorial
555
Journal Manager
Olaf Meesters
Journal Administrators
Ria Otten
Patrick Scheffmann
Advisory Editorial Board
Silvia Arber, Basel, Switzerland
Anders Bjrklund, Lund, Sweden
J. Paul Bolam, Oxford, UK
Sarah W. Bottjer, Los Angeles, CA, USA
Barry J. Dickson, Vienna, Austria
Chris Q. Doe, Eugene, OR, USA
Craig C. Garner, Stanford, CA, USA
Yukiko Goda, Wako, Japan
Kenneth D. Harris, London, UK
Nancy Ip, Kowloon, Hong Kong
Meyer Jackson, Madison, WI, USA
Maria Karayiorgou, New York, NY, USA
Robert C. Malenka, Stanford, CA, USA
Mark P. Mattson, Baltimore, MD, USA
Freda D. Miller, Toronto, Canada
Richard G.M. Morris, Edinburgh, UK
Maiken Nedergaard, Valhalla, NY, USA
Eric Nestler, New York, NY, USA
Hitoshi Sakano, Tokyo, Japan
Greg Stuart, Canberra, Australia
Wolf Singer, Frankfurt, Germany
Marc Tessier-Lavigne, New York, NY, USA
Chris A. Walsh, Boston, MA, USA
Editorial Enquiries
Trends in Neurosciences
Cell Press
600 Technology Square
Cambridge, MA 02139, USA
Tel: +1 617 3972823
Fax: +1 617 3972810
E-mail: tins@cell.com
Looking back and looking forward
Andrew M. Clark
Science & Society
557
The Brain Prize 2013: the optogenetics

revolution
Andreas Reiner and Ehud Y. Isacoff
Opinions
561
Molecular nexopathies: a new paradigm of

neurodegenerative disease
Jason D. Warren, Jonathan D. Rohrer,

Jonathan M. Schott, Nick C. Fox,
John Hardy, and Martin N. Rossor
570
A developmental ontology for the

mammalian brain based on the prosomeric
model
Luis Puelles, Megan Harrison,

George Paxinos, and Charles Watson
579
Challenges of understanding brain function

by selective modulation of neuronal
subpopulations
Arvind Kumar, Ioannis Vlachos,

Ad Aertsen, and Clemens Boucsein
Reviews
587
Sugar for the brain: the role of glucose in

physiological and pathological brain
function
Philipp Mergenthaler, Ute Lindauer,

Gerald A. Dienel, and Andreas Meisel
598
Control of neuronal voltage-gated calcium

ion channels from RNA to protein
Diane Lipscombe, Summer E. Allen, and

Cecilia P. Toro
610
Blurring the boundaries: developmental and

activity-dependent determinants of neural
circuits
Verena Wolfram and Richard A. Baines
Cover: The mammalian brain expends tremendous energy for its physiological function. On pages 587597 of this issue,
Mergenthaler, Lindauer, Dienel, and Meisel review the tight regulation of glucose metabolism as the foundation for normal
brain function, as well as the role of disturbed glucose metabolism in the pathophysiology of diverse brain disorders.
The sweets and the cupcake-like brain on the cover signify the reliance of the brain on sugar-derived energy, and the
background images, including the angel from Albrecht Drers Apocalypsis cum figuris, motifs from Jules Vernes adventure
novels, and Daniela Nickaus explorer, illustrate the infinite intellectual and imaginative capacity of the brain. Cover design
by Malte Nickau (www.graco-berlin.de).
Editorial
Looking back and looking forward

Andrew M. Clark
Trends in Neurosciences, Cell Press, 600 Technology Square, Cambridge, MA 02139, USA
Trends in Neurosciences (TiNS) was launched over 35

years ago with the aim of publishing insightful reviews,
opinions, and commentaries covering all disciplines of the
neurosciences. TiNS started strong; among the articles
appearing in the journal that first year include those
penned by a Nobel Laureate, previous and future chairs
of the Society for Neuroscience, and numerous other leading experts. Throughout its ensuing tenure, TINS has
continued to publish timely and authoritative pieces with
the goal of synthesizing, interpreting, and drawing novel
insights into an increasingly fractured and complex literature. Although the neuroscientists toolkit has greatly
expanded over recent years, the subjects covered remain
surprisingly consistent: neuropsychiatric diseases, cellular
and molecular studies of synapse development and plasticity, and systems investigations of learning and memory,
to name but a few. The success of TINS in providing highquality coverage of such topics is reflected in its consistent
ranking among the best reviews journals in what has
grown from a handful of similar titles at its inception to
a plethora today.
Despite its long run, TiNS, until now, has had only four
editors, in chronological order: David Bousfield, Gavin
Swanson, Sian Lewis, and Rachel Jurd. Together, these
previous editors oversaw the launch and growth of the
journal, shepherded it from print to primarily electronic
distribution in the internet age, developed new means for
engaging and building a community of readers, and
commissioned countless articles that, one hopes, not only
reviewed, but also helped set trends in the field. I am
humbled to follow in their footsteps and I aim to continue
in the tradition that they started; this latest issue reflects
both the breadth of coverage and the high standard of
quality that I aim to maintain at TINS.
The neurosciences are one of the preeminent fields in
biology today, as reflected in both the amount of resources
devoted to investigations of the nervous system and the
increasing number of major prizes being awarded for
breakthroughs in this field. In this issue, in a Science
and Society article, Reiner and Isacoff highlight the work
that led to the recent receipt of one of these significant
awards, the Brain Prize 2013, which was awarded to Ernst
Bomberg, Edward Boyden, Karl Deisseroth, Peter Hegemann, Gero Miesenbock, and Georg Nagel for their development and refinement of optogenetics. Science and
Society pieces are intended to spotlight subjects of wide
interest, or topics at the intersection of the bench and the
wider world. Readers can look forward to continuing to see
more of both types in the future.
Corresponding author: Clark, A.M. (tins@cell.com).
One of the key features of TiNS opinion articles is that

they present a personal viewpoint on a particular subject,
thus advancing a novel perspective or hypothesis. In this
issue, three such opinion articles present novel perspectives on important issues in neurodegenerative diseases,
neural development, and neural coding, respectively. Warren, Rohrer, Scott, Fox, Hardy, and Rossor hypothesize
that specific neural networks are differentially susceptible
to particular pathogenic proteins and propagating protein
abnormalities, while Puelles, Harrison, Paxinos, and Watson promulgate a hierarchical classification of brain structures based upon recent findings concerning differential
gene expression during development. Finally, Kumar, Vlachos, Aersten and Boucsein note that the parallel, recurrent architecture of most real neural networks limits the
utility of selectively inactivating only particular elements
(e.g., a given cell class) one-by-one, suggesting careful
computational consideration of network structure will be
a necessary precondition of experiments seeking to unravel
network function. TiNS readers can expect to continue to
see creative, novel, and insightful opinion articles covering
other equally important topics in the future.
Brief, pointed reviews that go beyond simply reciting
the literature, to integrate and synthesize recent results
into alternative interpretations, and to suggest productive
areas for future research are the hallmark of TiNS. In this
issue, one can find three such reviews from leading groups
in molecular, cellular, and systems neuroscience. Mergenthaler, Lindauer, Dienel, and Meisel review the role
of glucose in fueling brain function and the role of breakdowns in this process in neurological disorders, while
Lipscombe, Allen, and Toro cover the mechanisms controlling the expression of neuronal voltage-gated calcium
channels (CaV). Finally, Wolfram and Baines delve into
recent findings suggesting neurotransmitter phenotype
and expression of specific ion channels in neurons are
not always hard and soft wired, respectively.
In some form or another, all of my predecessors have
expressed in this space the statement that these are exciting times to be a neuroscientist. That such a statement can
be repeated, in all earnestness, over such a long period of
time speaks to what a fascinating, dynamic, and exciting
endeavor the study of the nervous system is. Currently, the
field is faced with many challenges. According to the World
Health Organization (WHO), the socioeconomic burdens of
psychiatric and neurological diseases are only expected to
continue to increase [1]. Meanwhile, large scientific funding bodies in many countries continue to see their budgets
decline in real or inflation-adjusted terms. However, there
are also numerous possibilities and potentials for great
discoveries. The ongoing development of methods for
controlling the activity of identified neuronal subtypes
0166-2236/$ see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2013.09.002 Trends in Neurosciences, October 2013, Vol. 36, No. 10
555
Editorial
on a fast timescale offers the potential to dissect circuit
function in exquisite detail, next-generation sequencing
offers the hope to better understand the genetic basis of
neurodegenerative and neuropsychiatric diseases, and
new computational tools for mining large and complex
data sets are constantly being refined. As always, the
journal welcomes feedback on our effort to cover this
exciting ground; it would be impossible to identify and
report on all the newest trends without invaluable input
from our Advisory Editorial Board, authors, and readers.
In teaming with our colleagues at other Cell
Press journals, be they other Trends titles such as
Trends in Cognitive Sciences, Trends in Endocrinology
and Metabolism, or Trends in Pharmacological Sciences,
556
Trends in Neurosciences October 2013, Vol. 36, No. 10
or premier research journals, such as Neuron and Cell, to

provide up-to-date and authoritative coverage of the
neurosciences, TiNS hopes to be able to serve the increasingly diverse needs of this broad community. I will
be at the upcoming Society for Neuroscience conference
in San Diego, please stop by the Cell Press/Elsevier
booth to chat and pick up a free copy of TiNS, or stop
me in a poster aisle or at a symposium, to let me know
your thoughts on how the journal can best continue to
strive towards achieving this goal.
Reference
1 World Health Organization (2011) Mental Health Atlas 2011, WHO
Science & Society
The Brain Prize 2013: the optogenetics revolution

Andreas Reiner1 and Ehud Y. Isacoff1,2
1
Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California Berkeley, Berkeley,
California 94720, USA
2
Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA
The 2013 Grete Lundbeck European Brain Research Prize

was awarded to Ernst Bamberg, Edward Boyden, Karl
Deisseroth, Peter Hegemann, Gero Miesenbock, and
Georg Nagel for their invention and refinement of optogenetics. Why optogenetics? And why this sextet? To
appreciate why, we turn first to some of the core questions of neuroscience and the technical difficulties that
long obstructed their resolution.
The beauty and the agony of brain circuits

How does the brain process sensory information, drive
behavior, mediate perception, thought, and memory? Since
the late 1880s, when Santiago Ramon y Cajal used the
mysterious tendency of the Golgi stain to label only a
scattershot of neurons, and could thereby visualize the
shapes of individual cells and trace their connections,
neuroscientists have recognized that an essential part of
the explanation lies in the neuronal circuitry. Modern
morphological approaches allow the staining of entire cell
populations and visualization of the synaptic connections
therein. However, although these modern techniques, like
Cajals work originally, help in providing the physical
wiring diagram of the neural computer, they also illuminate the difficulty of the neuroscience enterprise. They
demonstrate that every part of the nervous system contains a multitude of neurons with distinct shapes, axonal
and dendritic arbors, and contact partners. The challenge
then is not only to find a way to determine who is actually
connected to whom, but how these individual synapses
transmit information, and how the many types of neurons
and connections give rise to functional networks that
perform diverse tasks.
Over the hundred years following Cajal the standard
approach to mapping this activity was to identify the
neurons active during specific sensory, motor, or cognitive
events, with the assumption that those neurons are directly involved in processing the relevant information. In these
studies, electrical signatures and cell location provided
some information about neuron type, but available
approaches could not discern the enormous variety of
cell types which was becoming evident from emerging
genetic analysis. In addition, the field lacked a method
Corresponding author: Isacoff, E.Y. (ehud@berkeley.edu).
for selectively stimulating or inhibiting activity in specific

cell types or transmission at specific synaptic connections.
The solution for discerning cell types and selectively
detecting their activity over an extended area came in the
form of genetically encoded fluorescent markers and optical reporters of action potential firing. This came from the
discovery of green fluorescent protein (GFP) as well as its
subsequent cloning and genetic engineering to generate
many color variants and to make optical reporters of cell
activity, work which was awarded the 2008 Nobel Prize in
Chemistry.
A major remaining challenge has been to develop the
means to manipulate activity in the nervous system with
the spatial, temporal, and cell-type resolution that is provided by the optical detection of activity using fluorescent
protein reporters. The 2013 Brain Prize was awarded this
year for the solution to this challenge (Figure 1).
Light as a force for exciting and inhibiting neuronal
activity
Having seen the power of light for visualization of neural
firing across a distributed network, one wonders if the
ability to focus light might also be used for pinpoint stimulation of neurons. Since 1970 efforts were made to directly
photostimulate neurons with light. The development of
light-sensitive protecting groups and their application to
photo-uncaging of ATP inspired the development of photolabile calcium chelators by Roger Tsien, Bob Zucker, and
Graham Ellis-Davis, and of caged neurotransmitters,
starting in the group of Peter Hess. These methods could
be used to stimulate specific cells that were genetically
identified by the selective expression of a fluorescent protein reporter, providing the desired cell type specificity.
However, they are best suited to stimulation of one cell at a
time, a limitation when one considers the great power both
for circuit mapping and behavioral analysis of an approach
that would enable stimulation or inhibition of multiple
cells of a genetically select type. To achieve such a goal, a
genetically encoded system that can be reversibly controlled by light seemed ideal.
Where can one turn to look for light-sensitive proteins?
The obvious place is in the vertebrate eye to the natural
worlds photoreceptor proteins, the opsins. But rhodopsin
and its cone cousins are G protein-coupled receptors that
signal to ion channels in the complex biochemical and
cellular context of the photoreceptor cell. There are several
factors which prohibit mammalian opsins from being attractive candidates. However, the photoreceptor system in
the Drosophila eye is simpler. In 2002 Gero Miesenbock
557
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TRENDS in Neurosciences
Figure 1. The award ceremony for the Brain Prize 2013. From the left: Ernst Bamberg, Edward Boyden, Karl Deisseroth, Peter Hegemann, Gero Miesenbock, Georg Nagel,
Crown Prince Frederick, and former chairman of the board of the Grete Lundbeck Foundation Nils Axelsen. Reproduced with permission from the Grete Lundbeck
Foundation.
and Boris Zemelman [1] made a critical conceptual and

experimental step by expressing a trio of Drosophila proteins (arrestin-2, rhodopsin, and its cognate Ga subunit:
dubbed chARGe) in cultured hippocampal neurons, enabling them to be excited by light. If one had to identify the
paper that launched the thousand ships of optogenetics,
this is it.
Beautiful as chARGe was, it had two shortcomings:
excitation by light was modest in strength and the approach could not be easily transferred to other systems
because of the difficulty of carrying out triple transfection.
The following year, Miesenbock combined photo-uncaging
of ATP or capsaicin with the heterologous cell-specific
expression of either the excitatory P2X or TrpV1 receptor
channels [2]. This approach also had limitations: It was
confined for use in animals that lack receptors for ATP and
capsaicin, in other words, it could not be used in vertebrates. At roughly the same time, Ed Callaways lab developed the reverse logic, expressing the Drosophila
allatostatin receptor in specific mammalian neurons and
activating it with allatostatin, which has no mammalian
receptor, to open G protein-gated inwardly rectifying K+
channels (GIRKs), and thereby inhibit action potential
firing. But allatostatin, like engineered GPCRs that are
activated only by synthetic ligands (e.g., the RASSLs described by Bruce Conklin in 1998), had no light-activated
variant, and thus lacked the rapid switching and spatial
precision that is enabled by optical control.
Was there, then, a simpler opsin to which one could
turn, allowing for exploitation of Miesenbocks conceptual
breakthrough, but with fewer components, faster speed,
and sufficient strength? The answer is yes. Opsins from
558
bacteria and single-cell algae had been studied for decades.

But they were known to be different from their relatives in
the eye and were so foreign phylogenetically that one
wondered if they could ever be imported in a functional
form into the mammalian brain.
Starting in the lab of Oesterhelt in the mid-1980s, Ernst
Bamberg and Peter Hegemann worked on two microbial
opsins from Halobacterium salinarium: bacteriorhodopsin
(bR) and halorhodopsin (hR). These opsins, although related to the seven transmembrane helix G protein-coupled
receptor opsins of the eye, are self-contained light-driven
ion pumps. In response to light, bacteriorhodopsin pumps
H+ and halorhodopsin pumps Cl across the membrane,
thereby changing the ionic balance and membrane potential. Like rhodopsin, bR and hR use retinal as a chromophore. In the late 1980s Hegemann branched out and
characterized the phototaxis in the single-cell algae Chlamydomonas reinhardtii, which appeared to depend on
another rhodopsin relative. The work culminated in two
seminal publications in 2002 and 2003 from a joint effort of
the Bamberg and Hegemann labs, both with Georg Nagel
as a first author, which described the cloning, heterologous
expression, and functional characterization in Xenopus
oocytes of these opsins, which turned out to be cationselective ion channels that were named channelrhodopsin
1 and 2 (ChR1 and ChR2) [3,4]. Importantly, Nagel et al.
demonstrated that ChR2 could be expressed to achieve
light-induced depolarization in non-neuronal mammalian
cell lines.
Here one had, it seemed, the perfect and complementary
set of genetically encoded light-driven pumps and channels. But could they be used in neurons? The reputation
Science & Society

of microbial proteins was that they are notoriously difficult
to express, and they normally operate in extreme ionic
environments very different from those of the central
nervous system. Indeed, for various reasons bR and hR
from Halobacterium salinarium and ChR1 were never
successfully transferred to neurons. But in 2005 Karl
Deisseroth and Ed Boyden, in a collaboration with Nagel
and Bamberg, and Stephan Herlitzes lab in collaboration
with Hegemann and Lynn Landmesser, successfully
expressed ChR2 in mammalian neurons and demonstrated
robust firing of action potentials in response to blue light
[5,6]. In his study, Herlitze was also able to inhibit neuronal firing using vertebrate rhodopsin, coupling it to GIRKs
or P/Q-type Ca2+ channels, and performed the first in vivo
experiments in intact spinal cord and living chicken embryo [6]. In the same year, Alexander Gottschalk, in collaboration with Nagel and Bamberg, succeeded in optically
altering the behavior of the worm Caenorhabditis elegans
[7], Hiromu Yawo demonstrated optical control of firing in
acute hippocampal slice following sterotatic injection of
ChR2-coding viruses in mouse [8], and Zhuo-Hua Pan
demonstrated functional ChR2 expression in the mouse
retina [9]. In 2007 both Deisseroth, continuing the collaboration with Nagel and Bamberg, and Boyden used a
halorhodopsin from another archaeon, Natronomonas
pharaonis, which lives in soda lakes in Egypt, to pump
Cl ions into neurons in response to light of a different
wavelength from that used to activate ChR2 and thereby to
hyperpolarize neurons and inhibit firing [10,11]. Later,
Boyden added pumps which extrude H+ to hyperpolarize
neurons.
In parallel to the development of opsins to control
neuronal firing, chemical optogenetics was developed,
thus enabling direct light-control of the signaling proteins
of the mammalian synapse. This approach was inspired by
foundational studies by Bernard Erlanger and coworkers
in the 1970s, who developed synthetic photoswitchable
tethered and diffusible ligands for muscle nicotinic receptors. The first such control of neural activity, reported in
2004, was a light-blocked K+ channel, SPARK, which could
be used to inhibit neuronal firing [12]. This was followed by
an excitatory light-activated ionotropic kainate-type glutamate receptor, LiGluR [13], an inhibitory version of
LiGluR known as HyLighter, light-activated and lightblocked neuronal nicotinic acetylcholine receptors [14],
and light-activated and light-blocked G protein-coupled
metabotropic glutamate receptors that inhibit spiking
and neurotransmitter release [15]. Although these lightgated channels and neurotransmitter receptors can be
used in the manner of the opsins to excite and inhibit
neuronal firing, and to inject calcium into neurons or glia,
their unique promise is for gating synaptic transmission
and plasticity.
Expansion of tools, explosion of neuroscience
Since 2005, when ChR2 was first used to excite neurons
in response to light, efforts in the field, led by the sextet,
culminated in the isolation of new microbial opsins and
opsin engineering to generate excitatory and inhibitory
versions with larger currents and different ion selectivity, kinetics, and excitation spectra. As envisioned, light
as a minimally invasive stimulus allowed exquisite

spatiotemporal patterning of neuronal activity, whereas
the combination with elaborate genetic methods allowed
a specificity of targeting to cell type that was never
possible before. Soon, these methods were taken from
cellular studies in vitro to behavioral studies in awake
animals.
The spread of optogenetics with microbial opsins has
been nothing short of spectacular, a product of the combined power of the method and its simplicity of use. Because retinal is bioavailable in the vertebrate nervous
system and because, unlike in visual rhodopsins, it binds
irreversibly to the microbial opsins, the user is only a
transfection, a viral infection, or genetic cross away from
an experiment. Consequently, the opsins have been
deployed across a wide range of organisms, from worm
to fly to fish to mouse to non-human primates. They have
been used to study brain waves, sleep, memory, hunger,
addiction, aggression, courtship, sensory modalities, and
motor behavior. They have also been used as models for
clinical treatment: for deep brain stimulation, on one hand,
and as a retinal prosthetic on the other.
This revolution led to hundreds of papers a veritable
deluge that even swept away entertaining qualms of
Miesenbock about the optogenetic catechism. The simplicity of the term and the power of the methodology have
conquered all. There is little question in the neuroscience
community that optogenetics deserves awards of the
magnitude of the 2013 Brain Prize. It is a great thing
that the prize has recognized the people who launched the
field both those whose basic science on opsins paved the
way, as well as those who engineered them into the
nervous system.
Acknowledgments
This work was supported by the National Institutes of Health (NIH)
Nanomedicine Development Center for the Optical Control of Biological
Function (PN2EY018241).
References
1 Zemelman, B.V. et al. (2002) Selective photostimulation of genetically
chARGed neurons. Neuron 33, 1522
2 Zemelman, B.V. et al. (2003) Photochemical gating of heterologous ion
channels: remote control over genetically designated populations of
neurons. Proc. Natl. Acad. Sci. U.S.A. 100, 13521357
3 Nagel, G. et al. (2002) Channelrhodopsin-1: a light-gated proton
channel in green algae. Science 296, 23952398
4 Nagel, G. et al. (2003) Channelrhodopsin-2, a directly light-gated
cation-selective membrane channel. Proc. Natl. Acad. Sci. U.S.A.
100, 1394013945
5 Boyden, E.S. et al. (2005) Millisecond-timescale, genetically
targeted optical control of neural activity. Nat. Neurosci. 8, 1263
1268
6 Li, X. et al. (2005) Fast noninvasive activation and inhibition
of neural and network activity by vertebrate rhodopsin and
green algae channelrhodopsin. Proc. Natl. Acad. Sci. U.S.A. 102,
1781617821
7 Nagel, G. et al. (2005) Light activation of channelrhodopsin-2 in
excitable cells of Caenorhabditis elegans triggers rapid behavioral
responses. Curr. Biol. 15, 22792284
8 Ishizuka, T. et al. (2006) Kinetic evaluation of photosensitivity in
genetically engineered neurons expressing green algae light-gated
channels. Neurosci. Res. 54, 8594
9 Bi, A. et al. (2006) Ectopic expression of a microbial-type rhodopsin
restores visual responses in mice with photoreceptor degeneration.
Neuron 50, 2333
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10 Han, X. and Boyden, E.S. (2007) Multiple-color optical activation,
silencing, and desynchronization of neural activity, with single-spike
temporal resolution. PLoS ONE 2, e299
11 Zhang, F. et al. (2007) Multimodal fast optical interrogation of neural
circuitry. Nature 446, 633639
12 Banghart, M. et al. (2004) Light-activated ion channels for remote
control of neuronal firing. Nat. Neurosci. 7, 13811386
13 Volgraf, M. et al. (2006) Allosteric control of an ionotropic glutamate
receptor with an optical switch. Nat. Chem. Biol. 2, 4752
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14 Tochitsky, I. et al. (2012) Optochemical control of genetically
engineered neuronal nicotinic acetylcholine receptors. Nat. Chem. 4,
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15 Levitz, J. et al. (2013) Optical control of metabotropic glutamate
receptors. Nat. Neurosci. 16, 507516
0166-2236/$ see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tins.2013.08.005 Trends in Neurosciences, October 2013, Vol.
36, No. 10
Opinion
Molecular nexopathies: a new

paradigm of neurodegenerative
disease
Jason D. Warren1, Jonathan D. Rohrer1, Jonathan M. Schott1, Nick C. Fox1,
John Hardy2, and Martin N. Rossor1
1
Dementia Research Centre, Department of Neurodegenerative Disease, UCL Institute of Neurology, University College London,
London, UK
2
Reta Lilla Weston Laboratories and Department of Molecular Neuroscience, UCL Institute of Neurology, University College
London, London, UK
Neural networks provide candidate substrates for the

spread of proteinopathies causing neurodegeneration,
and emerging data suggest that macroscopic signatures
of network disintegration differentiate diseases. However, how do protein abnormalities produce network
signatures? The answer may lie with molecular nexopathies: specific, coherent conjunctions of pathogenic protein and intrinsic network characteristics that define
network signatures of neurodegenerative pathologies.
Key features of the paradigm that we propose here include differential intrinsic network vulnerability to
propagating protein abnormalities, in part reflecting developmental structural and functional factors; differential
vulnerability of neural connection types (e.g., clustered
versus distributed connections) to particular pathogenic
proteins; and differential impact of molecular effects (e.g.,
toxic-gain-of-function versus loss-of-function) on gradients of network damage. The paradigm has implications
for understanding and predicting neurodegenerative disease biology.
Introduction
Neural networks are a key theme in contemporary neuroscience [13]. Operationally, a neural network can be defined as a complex system comprising nodes and links
represented by neurons and their connections [4]. Neural
networks extend over scales ranging from microscopic (neurons and synapses) to macroscopic (anatomical regions and
fibre tracts), and may be structural (defined by physical
connections; e.g., fibre tracts) or functional (defined by
physiological connections). Neuroimaging techniques, such
as functional MRI (fMRI) and diffusion tensor tractography
[4,5], coupled with methodologies such as graph theory [2,6],
have delineated intrinsic, distributed neural networks
supporting cognitive functions in the healthy brain [79].
Neural network models have been successfully applied to
common neurodegenerative syndromes [35,715], building
Corresponding author: Warren, J.D. (jason.warren@ucl.ac.uk).
Keywords: neurodegeneration; dementia; neural network; nexopathy.
0166-2236/$ see front matter
2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tins.2013.06.007
on the key insight that neurodegenerative diseases, such as

Alzheimers disease (AD) and frontotemporal lobar degeneration (FTLD), produce distinctive clinical syndromes with
regular patterns of evolution due to the spread of pathogenic
protein abnormalities via large-scale brain networks.
To date, work in neurodegenerative disease has mainly
focussed on linking clinical phenotypes to network alterations. However, it remains unclear how molecular (protein)
abnormalities translate to network damage and, thus,
clinical phenotypes; and whether pathological substrates
can be predicted reliably from macroscopic network signatures. Recent advances in genetics and histopathology
have enabled the detailed mapping of neurodegenerative
clinico-anatomical phenotypes onto specific proteinopathies, transcending broad categories such as tauopathy
or ubiquitinopathy. Histopathological patterns of protein
deposition reflect underlying molecular (biochemical or
conformational) characteristics in a range of neurodegenerative diseases, most strikingly in the FTLD spectrum
[1624]. Although the concept requires further substantiation and qualification, the diffusive intercellular or prionlike spread of pathogenic misfolded proteins holds promise
as a general mechanism for the evolution of the neurodegenerative process in a wide range of diseases [8,9,2528].
Various candidate mechanisms that might link protein
pathophysiology with intercellular miscommunication
and local circuit disruption have been identified
[3,29,30]. However, the mechanisms that translate local
effects of proteinopathies to specific patterns of large-scale
network disintegration remain largely unknown.
Here, we address this problem. We propose the term
molecular nexopathy (Latin nectere, tie) to refer to a coherent conjunction of pathogenic protein and intrinsic neural
network characteristics expressed as a macroanatomical
signature of brain network disintegration. We argue that
improved understanding of the molecular mechanisms of
network disintegration will constitute a new paradigm of
neurodegenerative disease. The essential features of the
paradigm that we propose are presented in Box 1 and
Figures 13. We now consider potential mechanisms whereby molecular dysfunction might be linked to neural circuit
disruption. We then assess the extent to which the molecular
Trends in Neurosciences, October 2013, Vol. 36, No. 10
561
Opinion
Box 1. The molecular nexopathy paradigm: key substrates and constraints

Molecular, microstructural, and functional substrates
Pathogenic proteins, including misfolded aggregates and toxic
oligomers, could promote neural network disintegration by disrupting synaptic function or maintenance [57], axonal transport or repair
[79], or via downstream trophic [58,80] or cellcell signalling
abnormalities [3,10,29,62,8185]. Disease effects would exploit intrinsic network vulnerabilities: in particular, developmental patterns
of protein expression and structural and functional interactions
across neural circuits [38,39] (Figure 1, main text). Shorter-range or
clustered dendritic and interneuronal connections appear particularly
vulnerable to some tauopathies [48,51], whereas longer-range, more
widely distributed (axonal) projections may be relatively more
vulnerable to other molecular insults (e.g., toxic oligomers derived
from amyloid precursor protein [58] or deficiency of trophic or
protective factors such as GRN [57]). The balance between molecular
net toxic gain-of-function and loss-of-function effects [57,62] might
help to determine the extent to which proteinopathies exert uniform
or graded effects across neural circuits (Figure 1, main text). Softwired alterations in synaptic function, in part reflecting pervasive
network activity patterns, might give way to subsequent (irreversible)
hard-wired structural damage and cell loss.
Disease propagation
Transcellular and, in particular, synaptically mediated diffusion of
misfolded proteins and permissive templating of protein misfolding
appear to be general principles of disease evolution across a range of
neurodegenerative proteinopathies [26,86], including prion [85], betaamyloid [3133], tau [8790], alpha-synuclein [91,92], TDP-43 [71,72],
and superoxide dismutase 1 [27]. Inoculation with beta-amyloid and
nexopathy paradigm can be reconciled with the central problems of disease evolution and phenotypic heterogeneity, and
propose experimental tests of the paradigm in future work.
How do pathogenic molecules produce specific brain
network disintegration?
Networks show variable intrinsic vulnerability to
proteinopathies
The molecular nexopathy paradigm makes no assumptions
about the instigating event that triggers the neurodegenerative process, which might be stochastic but which
results in the creation of a potentially pathogenic molecule.
However, once initiated, the topography of neurodegeneration preferentially targets network elements that are vulnerable to the instigating molecular species (Figure 1).
Emerging evidence, including inoculation experiments in
animals [3133] (Box 1), implies that a neurodegenerative
process may home to a brain region (or regions) based on
intrinsic vulnerability to the pathogenic protein. Neurodegeneration may propagate by prion-like seeding or templating of the protein abnormality (e.g., conformational
misfolding) across neural connections, in addition to physical transfer of instigating pathogenic proteins. The presence of a specific pathogenic abnormality that propagates
across a network would distinguish a neurodegenerative
molecular nexopathy from other diseases that disrupt
brain networks (for example, stroke and traumatic brain
injury): one important corollary is that compensatory or
homeostatic responses are ultimately inadequate in neurodegenerative nexopathies.
Regional neural vulnerability to a proteinopathy could
reflect anatomically restricted expression of the culprit
protein by cell populations or additional epigenetic factors
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tau triggers uptake by cells and templated conformational alteration

of normally folded protein to a potentially pathogenic, misfolded
state [26,86]. Trans-synaptic spread of tau-containing tangle pathology from entorhinal cortex neurons occurs in transgenic mice that
only express mutant MAPT in those neurons [89,90].
Macroanatomical signatures
Particular structural and functional neuroimaging profiles of network disintegration have specific molecular associations
[7,16,17,2023,68,69,74,75,9395] (Figure 2, main text). In the FTLD
spectrum, structural neuroimaging evidence suggests a scheme for
partitioning pathologies according to whether the macroanatomical
brain atrophy profile is localised or distributed, and whether atrophy
is relatively symmetric or strongly asymmetric between the cerebral
hemispheres [18,23] (Figure 2, main text). In the case of AD, early
(even presymptomatic) functional and structural alterations occur
within a specific distributed parieto temporofrontal network mediating default mode processing or stimulus-independent thought in
healthy individuals [4,11] and network disintegration tracks pathological disease staging [11,14,96]. Variant AD phenotypes, such as
posterior cortical atrophy and logopenic progressive aphasia, may
reflect differential involvement of corticocortical projection zones
that together constitute a distributed AD-vulnerable network [15,97],
although the precise pathophysiological roles of the two major
candidate pathogenic proteins (beta-amyloid and phosphorylated
tau) and the factors that modulate the profile of network damage
remain contentious [4,11,15]. Convergence of syndromes as disease
evolves may hold a solution to the problem of phenotypic heterogeneity (Figure 3, main text).
that direct the expression of the protein to particular

brain areas [3436] or determine neuronal susceptibility
to toxic events [10]. Regional differentiation of protein
expression is a fundamental feature of normal brain
development [37], establishing intrinsic specificities of
connections within neural circuits [38,39] and thereby
in turn directing the function of the circuit. Therefore,
local profiles of protein expression could confer selective
vulnerability or resistance of particular network elements
to particular neurodegenerative diseases, and would also
help drive the functional phenotypic signature of the
disease. For example, differential expression of neuroprotective factors has been linked to the relative vulnerability of particular neuronal populations in the basal ganglia
in Parkinsons disease [40], and regional expression of
genes involved in inflammatory signalling may modulate
disease onset with progranulin (GRN) mutations [41]. By
contrast, epigenetic effects during brain development
alter the regulation and expression of amyloid precursor
protein and potentially influence the later development of
AD [42]. Brain areas that are highly neuroplastic, more
specialised, or phylogenetically more recent (for example,
the language system) may be relatively more vulnerable
to proteinopathies [43], whereas primary motor and sensory cortex both show relative resistance. The process of
neurodegeneration might selectively unravel the sequence of normal ontogeny within the vulnerable network
(for example, by withdrawing essential trophic support,
repair mechanisms, or physiological signalling across
damaged synaptic connections) [29].
Regional specificity might also arise from cellular morphological factors, particularly at synaptic connections:
even if a protein is widely expressed in the brain, its effects
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(B)
Clustered no gradient
Clustered gradient
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(C) Distributed no gradient
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(B)
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Figure 1. A taxonomy of molecular nexopathy mechanisms. Here, we model

putative templates of neurodegenerative network damage at a given arbitrary
time point following introduction of a pathogenic protein. In each panel, the
stylised local neural circuit comprises nodes (N; e.g., neuronal somas) with links
(e.g., axons or dendrites) that behave as either shorter-range clustered (unfilled
lines) or longer-range distributed (filled lines) connections; the most highly
connected node behaves as a local hub (H), whereas node I is the site of an
instigating insult (wavy arrow) associated with a pathogenic protein. Grey symbols
represent unaffected or minimally affected network elements; pathogenic effects
are coded in red (filled circles representing deleted network elements and half-tone
circles representing dysfunctional network elements) and pathogenic effects are
assumed to be potentially bidirectional across network connections. The
taxonomy shown assumes two basic, interacting dichotomies arising from the
conjunction of pathogenic protein and intrinsic network characteristics: selective
targeting of shorter-range clustered neural connections [e.g., IN1N2HN3N4
N5 (A,B)] versus longer-range distributed neural connections [e.g., I-H-N5-N6,
(C,D)]; and effects that are relatively uniform [no gradient, (A,C)] or strongly graded
[gradient (B,D)] across the network. In each case, the hub H is intrinsically relatively
more vulnerable due to its high connectedness with the rest of the network [60].
Targeting of connection types could reflect subcellular compartmentalisation of
pathogenic proteins and/or local synaptic properties or other morphological
characteristics (e.g., targeting of dendritic versus axonal compartments). Gradients
of effects could be established by intrinsic polarities in network protein expression
and/or the net functional effect of a pathogenic molecular cascade (e.g., uniform
toxic gain-of-function versus graded loss-of-function effects). The model that we
propose requires propagation of disease effects across network elements, but
does not specify the precise nature of those effects: for example, propagation
could occur by direct protein transfer, protein seeding or templating in
contiguous elements, or deleterious pathophysiological signalling, all potentially
operating at different stages of disease evolution. The model predicts coherence
between culprit molecule, neural connection types predominantly targeted, and
functional (e.g., cognitive) phenotype as the neurodegenerative process scales to
the level of the whole brain (Figure 2, main text).
may propagate only in particular cell types [44] or across

specific patterns of connections [29]. Animal models have
demonstrated exquisite microanatomical and biochemical
specificity of intercellular connections in key vulnerable
structures (such as hippocampus) [45,46]. Age-related neuronal resprouting may enhance local deposition of amyloid
precursor protein in entorhinal cortex early during the
course of AD [47]. Protein expression and morphological
specificity at receptors and synapses would interact with
subcellular molecular factors. For example, tau isoforms
show distinctive subcellular distributions [48,49]. Deranged microtubular transport of abnormal tau facilitates
accumulation of aggregated tau in somatic and dendritic
rather than axonal compartments [50,51], whereas diffusible tau may further focus pathogenic effects of the protein
on local synaptic and glial connections [51]. The role of glial
elements is poorly understood, but may influence local
expression and development of network damage, reflected
Figure 2. Scaling nexopathies to large-scale brain networks. The inset cartoon

(left) shows a stylised axial view of the cerebral hemispheres in a normal brain.
Circles represent neural network elements and colours code large-scale functional
networks associated with generic clinical syndromes in previous connectivity work
[7]: the anterior temporal lobe semantic network (green); the frontoinsular
salience network implicated in behavioural variant frontotemporal dementia
(yellow); the dominant hemisphere speech production network implicated in
progressive nonfluent aphasia (magenta); the frontoparietal network associated
with corticobasal syndrome (light blue); and the temporoparietal default mode
network implicated in Alzheimers disease (dark blue). Putative shorter-range
clustered (unfilled black lines) and longer-range distributed (filled black lines)
connections between network elements are shown; connections between major
functional networks are also represented (grey lines). The middle panels show
proposed cross-sectional schemas of network breakdown (red, following Figure 1,
main text), after an instigating insult (wavy arrow) associated with a pathogenic
protein. Alongside each panel, axial and coronal MRI brain sections show
corresponding observed atrophy profiles in patients with representative,
canonical, pathologically confirmed, proteinopathies (CBD, corticobasal
degeneration associated with 4-repeat tau pathology; GRN, mutation in
progranulin gene; MAPT, mutation in microtubule-associated protein tau gene;
TDPC, TDP-43 type C pathology [19], associated with the clinical syndrome of
semantic dementia; the left hemisphere is on the left in all sections). These atrophy
profiles illustrate macroanatomical scaling of the nexopathy templates proposed
in Figure 1 (main text): (A) predominant involvement of clustered (shorter-range)
connections with uniform extension, leading to relatively focal (temporal lobe)
atrophy that is relatively symmetrically distributed between the cerebral
hemispheres; (B) predominant involvement of clustered (shorter-range)
connections with a strong gradient of network damage, leading to relatively
focal (temporal lobe), strongly asymmetric atrophy; (C) predominant involvement
of distributed (longer-range) connections with uniform extension, leading to
distributed, relatively symmetrical atrophy; and (D) predominant involvement of
distributed (longer-range) connections with a gradient of network damage, leading
to distributed, strongly asymmetric atrophy. A particular proteinopathy here
affects network connections with particular characteristics (e.g., clustered versus
distributed synaptic linkages); functional networks will be targeted according to
their specific network characteristics, but the effects of a particular nexopathy will
in general tend to spread between functional networks, while continuing to target
connections with similar properties across these networks. This would account for
empirical variability in the closeness with which proteinopathies map onto
particular functional networks (e.g., the mapping is relatively close for TDPC
pathology with the semantic network, whereas most proteinopathies involve the
salience network). The scheme makes specific predictions about the sequence of
regional involvement with particular proteinopathies (e.g., sequential involvement
of homologous contralateral temporal lobe regions with TDPC pathology) (see also
Figure 3, main text).
macroscopically in the relative extent of grey matter versus

white matter damage [52].
Proteinopathies are fitted to neural circuits
Central to the molecular nexopathy paradigm is the fit
between the pathogenic molecule and local neural circuit
(and, ultimately, large-scale network) characteristics
(Figures 1 and 2). Although data on specific local interactions of neural circuits with proteins remain limited, a
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t0
t1
t2
bvFTD
PNFA
CBS
Figure 3. Temporal evolution of disease and phenotypic heterogeneity. This

schematic illustrates the application of the molecular nexopathy concept to the
problem of phenotypic heterogeneity in neurodegenerative disease, using the
example of corticobasal degeneration. The inset cartoon (left) shows major
functional networks in a stylised normal dominant cerebral hemisphere, colour
coded as in Figure 2 (main text). The panels illustrate evolving network
involvement shortly after onset of the neurodegenerative insult (t0) and at two
arbitrary later time points (t1 and t2). The initial location of the insult (wavy arrow)
determines the clinical presentation (behavioural variant frontotemporal dementia,
bvFTD; progressive nonfluent aphasia, PNFA; or classical corticobasal syndrome,
CBS). The core corticobasal functional network (light blue in inset) is involved with
disease evolution in each case; however, variable additional involvement of other
contiguous functional networks (the salience network, speech production network
or default mode network) modulates the phenotype. Each of these phenotypes
arises from a common template of network involvement determined by the type of
neural connection predominantly involved (here, represented as longer-range
distributed intrahemispheric projections); the common nexopathy signature of
corticobasal degeneration is revealed in the temporal profile of disease evolution.
proteinopathy might spread between brain regions by

causing connected regions to develop the same intracellular protein abnormality or, less directly, by affecting the
function of those connected regions [3]. The coupling between functional and structural connectivity in neurodegenerative diseases remains poorly defined: however,
initial dysfunction could promote subsequent molecular
alterations and destruction of network elements, as shown
in lesion and tract-tracing studies in humans and nonhuman primates [5,6,53]. In more theoretical terms, the
effects of a proteinopathy on a network might be regarded
as a form of information flow, where information signifies
a change in network function (in particular, alterations in
synaptic properties) associated with the introduction of the
abnormal protein [3]. The characteristics of information
exchange across artificial neural networks have attracted
considerable interest in computational neurobiology [2,54]:
this theoretical framework might be adapted to the case of
proteinopathies. Pathogenic molecular effects often have
time constants that are much longer than those typically
associated with information flow in neural networks. However, dynamic downstream alterations in synaptic function
across circuits would occur over much shorter timescales. A
further, potentially related, factor is the role of pervasive
patterns of activity in circuit function and in predisposing
networks to the effects of neurodegenerative disease [55]:
examples include the differential and possibly usedependent susceptibility of particular motor pools to
amyotrophic lateral sclerosis (ALS) [27], or the altered
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trafficking of amyloid and tau in the isodendritic core

associated with perturbations of the sleepwake cycle in
AD [56]. Putative behavioural and activity-related factors
are likely to interact with underlying genetic and epigenetic predisposing factors, and the causal sequence in
general remains to be determined.
An important theoretical motivation for applying the
network information-processing framework to the neurodegenerative proteinopathies is the rich taxonomy of network
activity patterns that follow from relatively simple starting
assumptions when modelling the evolution of artificial neural networks [54]. In particular, it has been shown in such
artificial networks that patterns of neural activity in local
network elements can, under certain conditions, scale up to
the entire network; that the characteristics of local microcircuits strongly influence the activity pattern produced by
the network as a whole; and, furthermore, that these patterns may be highly polarised. All these are network properties predicted in the case of the neurodegenerative
proteinopathies, for which an activity pattern could be
interpreted as the cumulative effect of protein-associated
network damage integrated over time [55].
Relevant network and protein characteristics have yet
to be worked out in detail for neurodegenerative proteinopathies. However, it has been shown that brain networks
have small-world properties expressed as a high degree of
clustering among network elements and short average
path lengths between clusters [2]. These small-world properties suggest a basic dichotomy between shorter-range
neural connections within clusters (local neural circuits,
with relatively long neural path length) and relatively
sparse longer-range neural connections (with relatively
short neural path length) between clusters, in line with
highly segregated and hierarchical brain network architectures observed empirically [6] and with limited evidence
concerning the cellular pathophysiology of certain proteinopathies [48,51,57,58] (Box 1). This putative dichotomy
between clustered and distributed network connections
suggests a morphological basis for partitioning neurodegenerative diseases according to whether they produce
relatively localised versus more distributed profiles of
macroscopic brain atrophy [18,20] (Figures 1 and 2). Clustered and distributed connections could be modelled in
synthetic neural circuits and could be defined in brain
networks using anatomical methods that can measure
effective path lengths between network elements [6]; for
example, dynamic causal modelling. Alternative morphological dichotomies might also operate: for example, selective targeting of excitatory versus inhibitory projections
[44].
The degree of macroanatomical asymmetry of network
damage within and between cerebral hemispheres appears
to be a further partitioning characteristic of many neurodegenerative diseases (Figure 2), although to demonstrate
interhemispheric asymmetries, it may be necessary to
retain individual hemispheric asymmetry profiles when
pooling data in group neuroimaging studies [18,2022].
Network asymmetries could be determined, in part, by
configurational features of the host network that might
tend to polarise network activity. Such polarity has been
demonstrated in a computational model of Huntingtons
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disease [44]. Asymmetries might be predicted based on
extrapolation from network architectures in the brains of
other species. For example, the nodes of large-scale networks in the macaque brain have a highly nonuniform
distribution within the cortex and the connections between nodes are hierarchically organised [6]. Given that
the human brain shares network homologies with that of
the macaque [53], this architecture would tend to focus
the effects of neurodegenerative disease at particular
vulnerable hub regions (for example, in prefrontal cortex and posterior cingulateprecuneus [1,59]). Much
more generally, it has been shown that highly connected
network elements are intrinsically more vulnerable to
extinction following perturbing events in a variety of
hierarchical systems, ranging from ecology to economics
[60]. In the context of neurodegenerative disease, extinction might be equated to destruction of highly connected
network elements after introduction of a pathogenic
protein and susceptibility from connectedness might,
for example, predict disproportionate vulnerability of
dominant hemisphere language hubs in the progressive
aphasias [43] and medial parietal hubs binding the
default mode network in AD [1,4,15,55,59]. If functional
connections between brain regions are defined based on
the strength and direction of spontaneous activity correlations, fMRI data suggest a fundamental dichotomy
between positive connections that are dominant within
a cerebral hemisphere versus negative connections that
are dominant between hemispheres [61]: negative interhemispheric functional correlations will tend to establish
intrinsically asymmetric interhemispheric interactions
that could be exploited by neurodegenerative pathologies
(Figure 2).
It is unlikely a priori that any set of neuronal or neural
network features would confer vulnerability uniquely to a
single molecular species. However, further specificity in
the profile of network involvement (in particular, whether
strong polarity of damage is expressed across the brain)
may be driven, in part, by functional characteristics of the
pathogenic protein itself.
Directional protein dysfunction drives network
asymmetries
Across the spectrum of potentially pathogenic proteins,
there is a basic distinction between toxic-gain-of-function
(deleterious effects of protein accumulation) and loss-offunction (impaired physiological, signalling or trophic)
molecular effects [57,62]. The loss of function of a key
protein is likely to lead ultimately to the loss of function
of the affected network element and, therefore, might be
regarded in computational terms as inhibiting the affected element; the net computational effect of a toxic gain of
function is more difficult to predict. Large-scale network
asymmetries (i.e., asymmetric macroscopic atrophy profiles) might result from interaction of intrinsic connectivity
structure with a gradient of molecular effects across the
vulnerable network.
We envisage that, within an affected network, an overall
toxic gain of function will spread relatively uniformly,
whereas an overall loss-of-function effect will establish a
gradient of tissue loss due to attenuation of downstream
synaptic inputs. Such polarising network-level effects of

loss-of-function proteinopathies would be in line with a net
inhibitory action on damaged connections, because selective inhibition of network elements can generate highly
polarised network structures and self-amplifying network
activity patterns in computational models [54,61,63,64].
Proteinopathic effects would interact with (and may, in
part, be driven by) intrinsic, ontogenetic network gradients
[38,39]. Trophic effects modulate intercellular gradients in
normal morphogenesis and developmental disorders [65]
as well as in computational models [66]. Certain loss-offunction effects could become self amplifying due to additional, catastrophic mechanisms that might be specific to
particular protein alterations: an example is GRN mutations, which may inhibit neuronal repair processes leading
to accelerated collapse of network architecture [67].
Although it is unlikely that polarised protein effects
operate in pure form in the brain [57,62], for a given
disease process and disease stage, toxic gain-of-function
or loss-of-function effects may dominate at the network
level (Figure 1). Intracellularly, particular pathogenic proteins have complementary loss-of-function and toxic-gainof-function effects [62]. However, the overall primary balance of those effects across a neural network may depend
on specific molecular actions at key network elements (e.g.,
synapses) that act as the final common pathway for network damage. Additional specificity may be conferred by
biochemical characteristics and conformational signatures
of protein subtypes within broad categories, such as tau
and Tar DNA-binding protein 43 (TDP-43) [24,49]. We
currently lack such specific information for most key pathogenic proteins in the neurodegenerative spectrum [62].
There is further substantial potential for interactions
among pathogenic proteins (for example, between tau
and beta-amyloid in AD [28]). Protein-specific effects might
modulate intrinsic network connectivity properties, contributing to phenotypic variation associated with particular proteins within a common network architecture [for
example, the relatively symmetric atrophy profile associated with microtubule-associated protein tau (MAPT)
mutations versus the strongly asymmetric profile associated with TDP-43 type C (TDPC) pathology [19] within
anterior temporal lobe networks [18]].
Temporal evolution and the problem of heterogeneity
A critical feature of neurodegenerative molecular nexopathies is likely to be their pattern of evolution in time as well
as spatially within the brain. The rapidity of network
breakdown might depend on the relative proportions of
connection types affected by the pathological process, the
predominant involvement of longer-range connections corresponding to rapid spread and involvement of clustered
connections corresponding to slower spread, respectively.
This would fit with available data for certain neurodegenerative disorders. For example, patients with MAPT mutations and relatively focal anterior temporal lobe damage
have, on average, slower rates of overall brain atrophy and
survive substantially longer compared with patients with
GRN mutations associated with widespread intrahemispheric damage [68]; interhemispheric asymmetry
increases with advancing disease in association with
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GRN mutations [17]; but MAPT and GRN mutations produce similar local rates of atrophy within key structures
such as the hippocampus [69]. Taken together, such evidence suggests that disease effects are preferentially amplified if long intrahemispheric fibre tracts are implicated.
The temporal evolution of atrophy profiles associated
with a particular proteinopathy may reveal a characteristic signature of network involvement that unites apparently disparate phenotypes (Figure 3). For example,
tauopathies in the FTLD spectrum (such as corticobasal
degeneration) may present with a behavioural syndrome
due to frontal lobe involvement, with a language syndrome
due to involvement of peri-Sylvian cortices in the dominant
hemisphere, with a parietal lobe syndrome or with atypical
parkinsonism: the nexopathy paradigm predicts phenotypic convergence over time due to progressive erosion of
core frontoparietal, frontotemporal, or frontosubcortical
networks implicated in particular tauopathies [18]. There
is substantial evidence for such phenotypic convergence in
the FTLD spectrum [18,70] and related overlap syndromes
such as FTD-ALS [71]; however, precise correlations with
particular brain networks have yet to be widely established. Similarly, variant AD phenotypes have been interpreted as modulating a core temporoparietal-prefrontal
default mode network [15]. Phenotypic convergence
implies that initial stochastic insults anywhere in a vulnerable network will lead ultimately to a common signature of network breakdown, although the precise sequence
of network involvement will tend to reflect the initial locus
of pathology within the network (Figure 3).
This concept of the differential involvement of a core
vulnerable network (with increasingly complete involvement of the network over time) suggests one possible
solution to the apparent paradox of individual phenotypic
variation associated with particular proteinopathies [70].
The clinico-anatomical expression of a given proteinopathy
often varies between individuals as well as between syndromic subgroups [15,27,43]. The molecular nexopathy
paradigm requires that the neuroanatomical profile of disease evolution is not random, but adheres to a spatiotemporal template of network damage: the location of disease
onset within the vulnerable network may vary between
Box 2. Testing the paradigm: outstanding questions and future directions

Is diffusive protein spread a general mechanism of neurodegeneration?
The strength of evidence for prion-like spread, permissive templating, and direct cellcell transmission varies among proteinopathies,
and has not been established for some (e.g., fused-in-sarcoma
protein) [98]. Protein spread could occur via mechanisms other than
network pathways (e.g., extracellular diffusion [26,27]). Nonprion
diseases have not been shown to have prion-like spontaneous
infectivity and, typically, evolve more slowly [86].
Hypothesis
Diffusive protein spread is a general mechanism of network disintegration.
Key experiments
Inoculation and tracer studies in animal models [3133] and the
development of novel molecular model systems [77].
How do protein properties relate to profiles of network degeneration?
Protein properties and expression profiles remain to be related in
detail to cell morphological features, dynamics of cellular transport
mechanisms, and intercellular interactions. For various pathogenic
proteins, the final net direction of effects (loss-of-function versus
toxic gain-of-function effects) remains contentious [62]. For several
neurodegenerative diseases, the pathogenic protein species or the
pathogenic form of the protein has not been unambiguously
determined [25].
Hypothesis
Network degeneration arises predictably from interactions of pathogenic protein properties with specific network morphological and
functional characteristics.
Key experiments
Computational modelling of artificial neural networks with biologically plausible properties [44]; development of model neural circuits
in vitro [99] and in vivo [38,39]; histomorphometric and immunolabelling analyses of subcellular protein targets and putative connection vulnerabilities (e.g., dendritic versus axonal) [48,51]; correlative
phenotypic-histological studies; and cognitive science paradigms to
characterise network activity and architectures [27,30,100].
Are empirical patterns of disease evolution consistent with nexopathy models?
Certain proteinopathies have specific macroanatomical signatures of
network disintegration [4,7,11,1318,2023], but disease overlap and
heterogeneity are substantial. The problem of heterogeneity may be
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resolved, in part, by mapping longitudinal profiles of disease

evolution, using neuroimaging tools that can capture convergent
structural and/or functional changes across the brain and over time
[7,8,11]. The molecular nexopathy paradigm makes specific predictions about the sequence of regional evolution with particular
proteinopathies (Figures 2 and 3, main text). fMRI reflects synaptic
function and can assess functional connectivity of networks when
active and at rest [1,7], including potentially compensatory and
homeostatic responses [3,10,73]; in addition, diffusion tensor imaging can assess structural connectivity of white matter pathways that
bind large-scale networks [4,5].
Hypothesis
Macroanatomical spatiotemporal network signatures arise predictably from underlying molecular lesions.
Key experiments
Longitudinal quantitative tracking of regional tissue damage in
different neurodegenerative diseases in large patient cohorts (in
particular, those with defined molecular lesions) derived from multicentre collaborative platforms [101]; connectivity metrics (e.g.,
dynamic causal modelling) to assess relations between network
elements (e.g., putative shorter- versus longer-range connections)
and dynamic, physiologically motivated techniques, such as magnetoencephalography (MEG) [12]; development of new molecular
ligands (e.g., phosphorylated tau); and practical quantitative metrics
of network function and connectivity [1,2].
What are the implications for disease diagnosis, prognosis, and
treatment?
Besides predicting underlying proteinopathies, improved understanding of determinants of network disintegration would enable
the prediction and more accurate tracking of disease evolution.
Identification of specific neural network dysfunction pre-dating the
loss of structural integrity may enable modulation of culprit
molecular deficits. Specific molecular treatments could be directed
at modifying protein mechanisms that sustain network integrity [102]
or bolstering inhibitory processes to counteract excitotoxic effects
[103]. Interventions that reduce local protein concentrations might
ameliorate diffusive protein spread for many diseases.
Hypothesis
Profiles of network disintegration provide biomarkers for tracking
disease evolution and treatment response.
Key experiments
Detailed natural history studies and incorporation of network-level
metrics in clinical trials.
Opinion
individuals, but progression of the particular disease in
individuals, over time, would tend to recapitulate a characteristic pattern of network involvement. Therefore, to establish the disease template conclusively will entail detailed
natural history studies: such studies in ALS have exploited
the well-understood and highly regular organisation of the
cerebral motor pools and their connections [27,71,72]. This
example has also underlined the considerable functional
reserve inherent in many brain networks, implying that
noisy information transfer by surviving elements can support network functions until a critical stage of network
failure is reached [3,10]. Both neuroimaging and behavioural metrics will be required to capture the prodromal
phase of early network alterations as well as compensatory
or homeostatic responses [3,11,73].
The effects of a particular proteinopathy need not and
generally will not be restricted to a single vulnerable largescale network (Figures 2 and 3). Rather, nexopathy inheres
in the type of network connections affected. To the extent
that connections with particular properties are concentrated in a single functional network, the nexopathy paradigm
would predict that proteinopathies targeting those connections should principally affect that network: this may explain the existence of neurodegenerative diseases (such as
those associated with MAPT mutation and TDP-C pathology) that preferentially target the anterior temporalinferior
frontal lobe semantic network [7,18,21,69,74,75]. In general,
however, connection types will be represented in more than
one functional network (Figure 2), providing a mechanism
for the spread of proteinopathies between networks, with
further phenotypic variation and potential overlap of clinicoanatomical profiles among proteinopathies [18,70]. Functional interactions between large-scale brain networks will
also tend to obscure network specificities [76]. Ultimately,
disease spread via secondarily connected systems throughout the brain implies that network and connection specificity will be most evident earlier during the evolution of a
particular disease.
As a final important caveat on the differentiation of
nexopathies, it is unlikely that complete specificity will
apply across the entire gamut of pathogenic proteins implicated in neurodegenerative disease. Rather, we envisage a taxonomy of predictable profiles of network
disintegration: within the taxonomy, particular profiles
of nexopathy might be common to different pathogenic
proteins to the extent that those proteins share key properties that promote network damage or dysfunction. Different proteins might, for example, participate in a
common, multicomponent pathogenic cascade (perhaps
best characterised at present for AD [15,57]).
Future directions: testing the molecular nexopathy
paradigm
The molecular nexopathy paradigm requires substantiation drawing on diverse molecular, cellular, and systems
neuroscience (behavioural and neuroimaging) approaches,
including synthetic and in vitro neural circuits, transgenic
and other animal models, and dynamic macroanatomical
techniques [12]. Clinical studies will continue to have a key
role in delineating the sometimes counterintuitive phenotypes that define brain network disintegration (Figure 2);
and phenotyping should be supported by detailed correlative histological studies. If the molecular nexopathy concept can be substantiated, it would hold great potential for
understanding, tracking, and predicting the expression of
neurodegenerative proteinopathies. Indeed, the concept
need not be restricted to proteins: for example, abnormal
cellular signalling linked to carbohydrate moieties could in
principle give rise to sugar nexopathies [77]. Several
specific, testable questions follow (Box 2) that collectively
could direct future work. Beyond the principled evaluation
and monitoring of candidate therapies, if mapping network
breakdown is equivalent to mapping the expression of a
molecular lesion, then delineating such a network could be
regarded as a direct in vivo assay of the function of the
protein: a concept analogous to that proposed for the
theoretical neural nets of computational neuroscience
[78]. If neural network dysfunction were sufficiently well
specified, this could in turn help identify (or discriminate
between) candidate molecular mechanisms driving the
neurodegenerative process and suggest rational candidate
therapies.
Acknowledgements
We thank Jane Warren for proposing the term nexopathy. This work was
undertaken at University College London Hospital (UCLH)/UCL and the
National Institute for Health Research (NIHR) Queen Square Dementia
Biomedical Research Unit. The Dementia Research Centre is an
Alzheimers Research Trust Co-ordinating Centre. J.M.S. is a Higher
Education Funding Council for England Clinical Senior Lecturer. M.N.R.
is an NIHR senior investigator. N.C.F. is an Medical Research Council
(MRC) Senior Clinical Fellow. J.D.W. is supported by a Wellcome Trust
Senior Clinical Fellowship (Grant No 091673/Z/10/Z).
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569
Opinion
A developmental ontology for the

mammalian brain based on the
prosomeric model
Luis Puelles1, Megan Harrison2, George Paxinos3,4, and Charles Watson3,4,5
1
Department of Human Anatomy, University of Murcia, Murcia 30003, Spain

Fremantle Hospital, Fremantle 6160, Australia
3
Neuroscience Research Australia, Sydney 2031, Australia
4
University of New South Wales, Sydney 2052, Australia
5
Faculty of Health Sciences, Curtin University, Perth 6845, Australia
2
In the past, attempts to create a hierarchical classification of brain structures (an ontology) have been limited
by the lack of adequate data on developmental processes. Recent studies on gene expression during brain
development have demonstrated the true morphologic
interrelations of different parts of the brain. A developmental ontology takes into account the progressive
rostrocaudal and dorsoventral differentiation of the neural tube, and the radial migration of derivatives from
progenitor areas, using fate mapping and other experimental techniques. In this review, we used the prosomeric model of brain development to build a hierarchical
classification of brain structures based chiefly on gene
expression. Because genomic control of neural morphogenesis is remarkably conservative, this ontology should
prove essentially valid for all vertebrates, aiding terminological unification.
What is ontology?
The concept of ontology (see Glossary) was borrowed from
the realm of philosophy by information scientists, who now
use it as a way to represent an existing domain of knowledge in the form of a hierarchical taxonomy [1]. The
availability of a brain ontology is vital for the field of
neuroinformatics. There have been several attempts to
create a brain ontology, the most notable of which are
NeuroNames [24], the Biomedical Information Research
Network (BIRN) [5], and the Brain Architecture Management System (BAMS) [6,7]. However, these ontologies are
largely based on traditional topographic classification of
parts of the adult brain, whereas the discovery of gene
targeting in mice [8] has revealed details of gene expression, lineage mapping, and causal inductive mechanisms
during development, leading to a new form of hierarchical
classification based on ontogeny.
Conventional ontologies have the weight of tradition,
but they do not include fundamental ontogenetic data, such
Corresponding author: Watson, C. (c.watson@curtin.edu.au).
Keywords: ontology; gene expression; neuromeres; forebrain; midbrain; hindbrain.
0166-2236/$ see front matter .
Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved. http://
dx.doi.org/10.1016/j.tins.2013.06.004
570
as neuromeric developmental units, genoarchitectonic evidence for natural boundaries between brain parts, or their
inner subdivisions (as opposed to many arbitrary classical
divisions unrelated to causal mechanisms). Therefore, they
have little power to encapsulate emergent understanding
of brain development and structural evolution. By contrast, a developmental ontology connects adult neuroanatomy with the tradition of comparative embryology; it
contemplates developmental structural units shared
Glossary
Diencephalon: the caudal subdivision of the forebrain that joins the midbrain to
the secondary prosencephalon; it contains three major alar domains (pretectum, thalamus, and prethalamus), as well as the corresponding tegmental
regions.
Evo-devo: an approach to the analysis of brain structure based on the merging
of concepts drawn from evolution and embryonic development.
Hodology: the study of connections within the central nervous system (odos
is Greek for a road).
Neuromeres: transverse unitary subdivisions of the neural tube that share a
common dorsoventral structure (floor, basal, alar, and roof plates), but each
have differential molecular identities and fates; they comprise the secondary
prosencephalon, diencephalon (prosomeres), the midbrain (mesomeres), and
the hindbrain (rhombomeres).
Ontogeny: Greek for the genesis of being: the process of development.
Ontology: a formal conceptualization of the structure of a knowledge base,
usually in the form of a hierarchical classification.
Pallium: major subdivision of the telencephalon, usually visualized as covering
and surrounding the subpallium; in mammals, it gives rise to the cerebral
cortex and several claustroamygdaloid pallial nuclei.
Prosencephalon: Greek for forebrain: the part of the brain that appears at the
rostral end of the neural tube.
Secondary prosencephalon: the rostral major subdivision of the developing
forebrain that separates from the diencephalon caudally (early in development,
both are encompassed within the primary prosencephalon); the secondary
prosencephalon includes the telencephalon, the eye, and the hypothalamus.
Subpallium: a major subdivision of the telencephalon usually visualized
topographically as lying under the pallium, at the brain baseit generates the
so-called basal ganglia, including the striatum, pallidum, diagonal-basal area,
and preoptic area.
Tagma: a meaningful higher-level unit of biological structure, comprising
segments that share a general character (e.g., the Drosophila thorax tagma as
opposed to the abdominal tagma).
Telencephalon: a dorsal subdivision of the secondary prosencephalon that
forms the pallium and subpallium.
Topography: a system for describing and representing position relative to
external references.
Topology: a system for describing the relative position of the components of a
structure irrespective of external references and any nondisruptive deformations; topology attends exclusively to the invariant neighborhood relations
between the components.
Opinion
(A)
NP
(B)
M
SpC
(C)
M
D
PPH
SP
PH
PMH
MH
SpC
(D)
p2
CSP
p1
m1
is
p3
m2
r2
RSP
r1
r3
r9
r4 r5 r6 r7 r8
r10 r11
SpC
(E)
MTt
PlsTt
Pt
Th
Tel
POTel
PtTg MTg
ThTg
PTh
PlsTg r1
PThTg
r2
PedHy
r3
PPHy
r4
is
SpC
r11
r9 r10
r5 r6 r7 r8
Alarbasal
boundary
Alarbasal
boundary
Figure 1. A series of diagrams of lateral views of the developing mouse brain. (A) The neural primordium (NP), which is a hollow tube with no subdivisions. In (B), the
rostral (left) part of the neural tube shows the appearance of the forebrain (F), midbrain (M), and hindbrain vesicles (H), with the developing spinal cord (SpC) on the right. In
(C), the forebrain vesicle has two divisions, the secondary prosencephalon (SP) and the diencephalon (D), and the hindbrain is divided into four regions: the prepontine
hindbrain (PPH), the pontine hindbrain (PH), the pontomedullary hindbrain (PMH), and the medullary hindbrain (MH). In (D) from the top, more subdivisions appear in the
forebrain [caudal secondary prosencephalon (CSP or hp1); rostral secondary prosencephalon (RSP or hp2); and prosomeres 13 of the diencephalon (p1, p2, and p3)],
midbrain [mesomere 1 and 2 (m1 and m2)], and hindbrain [isthmus (is) and rhombomeres 111 (r1 to r11)]. In (E), some parts of the forebrain have become further
differentiated: the caudal prosencephalon has formed the main part of the telencephalon; the rostral secondary prosencephalon has formed the preoptic telencephalon
(POTel), the terminal hypothalamus (THy), and the peduncular hypothalamus (PedHy)); and prosomeres 13 have formed the pretectum (Pt), thalamus (Th), and
prethalamus (PTh), respectively. In this diagram, the diencephalon and midbrain are further subdivided by the alarbasal boundary, which bounds distinct tegmental
regions [prethalamic tegmentum (PThTg); thalamic tegmentum (ThTg); pretectal tegmentum (PtTg); midbrain tegmentum (MTg); and preisthmic tegmentum (PIsTg)]. The
dorsal part of the midbrain is divided into the main midbrain tectum (MTt) and smaller preisthmic tectum (PIsTt). Created by L. Puelles for the Allen Brain Institute (http://
developingmouse.brain-map.org).
among vertebrates, as revealed by fate-mapping studies,

and is consistent with evolutionarily conserved gene patterns. Because of this, a developmental ontology has the
capacity to stimulate insights into causation. Our proposal
of a developmental ontology for the adult mouse brain is a
simplified version of the extended version designed by L.
Puelles for the Allen Developing Mouse Brain Atlas (http://
developingmouse.brain-map.org). The new ontology is consistent with the most recent version of the prosomeric
model [9], a known paradigm for structural and molecular
analysis of vertebrate brains (Figure 1). Note that this

review centers on the ontology and does not aim to explore
the prosomeric model itself.
Comparing traditional and developmental ontologies
All adult brain ontologies start with the recognition of
three basic elements: forebrain, midbrain, and hindbrain.
Even at this level, the developmental ontology is distinctive, in that it includes the isthmus within the hindbrain,
rather than in the midbrain, as found in traditional
571
Opinion
(A)
(B)
Roofplate
Pallium
Thalamus
Pretectum
Diencephalon
Telencephalon
p1
p2
Striatum
nd
Cervical
exure
ain
r4
br
Terminal
hypothalamus
Eye
Hi
ain
Anterior
commissure
Cerebellum
r1
r2
r3
br
Preopc
area
Midbrain
Diencephalon
nd
Peduncular
hypothalamus
Telencephalon
Cerebellum
is
Hi
Dg
Septal
roofplate
Cephalic
exure
STh
Dg
Midbrain
m1 m2
Substana
nigra
zli
Prethalamus
p3
Pallidum
Roofplate
Hypothalamus
Cervical
exure
r11
Spinal
cord
Spinal
cord
Neurohypophysis
Eye
Neurohypophysis
Figure 2. The segmental organization of the developing brain. (A) A diagram of a lateral view of developing mouse brain at a stage later than the last element shown in
Figure 1 (main text). The telencephalon is now divided into the pallium and subpallial regions [striatum, pallidum, diagonal domain (Dg), and preoptic area]. The septal
roofplate (gray shading) extends from the telencephalic roof to the developing anterior commissure (ac). Within the terminal hypothalamus, the eye vesicle, the
neurohypophysis, and the mamillary bodies (M) are differentiating. Within the peduncular hypothalamus, the subthalamic nucleus (STh) is developing. The red line
represents the alarbasal boundary, also in the midbrain and diencephalon. In the diencephalon, this molecular boundary is for a short distance pulled to the diencephalic
roof as the zona limitans (ZLi), which largely separates p2 and p3 at alar plate levels. The gray area above the cephalic flexure represents the most rostral area of Sonic
hedgehog (Shh) expression in the floor plate. The diagram shows that the developing substantia nigra extends rostrally from the midbrain into the diencephalon. Other
abbreviations are as in Figure 1. (B) This is another version of (A), to show the alarbasal boundary from the spinal cord to the rostral hypothalamus. The basal plate is in
green and the alar plate is in pink.
ontologies. The importance of transgenic fate mapping to

the understanding of such delimitation has been emphasized previously [1012].
There are many novel features at the next hierarchical
level of the developmental ontology. In the forebrain, three
components are recognized: the telencephalon, the hypothalamus (including the eye vesicle), and the diencephalon
(Figure 1). The telencephalon and hypothalamus constitute the secondary prosencephalon [12] (Figure 1C), which
can be separated from the diencephalon proper on the basis
of gene expression patterns [1315]. This is further supported by experimental evidence showing that the holoprosencephalic malformation syndrome selectively affects
the telencephalon and the hypothalamus [16], and the
Otx2 / Emx1+/ double mutant mouse selectively loses
the diencephalon, but retains the hypothalamustelencephalon complex [17,18]. In traditional ontologies, the
hypothalamus was incorporated within the diencephalon,
because it was incorrectly assumed to be the equivalent of
the basal and floor plates of the thalamus [19]. Recent fate
maps and molecular findings [14,15] indicate that the
hypothalamus is topologically rostral to the thalamus
and other parts of the diencephalon, and that the telencephalic and eye vesicles represent bilateral alar evaginations. The neural tube ends rostrally at the terminal wall
(between the mamillary body and the anterior commissure) with a distinct bow-like specialized median domain,
named the acroterminal area, across the tuberal (basal)
and chiasmatic (alar) hypothalamus and the preoptic area
[16]. Although it does not evaginate, the preoptic area
belongs to the telencephalic subpallium on the basis of
shared molecular profiles [20]. This classification is further
supported by the finding that the preoptic area contributes
tangentially migrating neurons to the telencephalic pallium, similar to the other parts of the subpallium, but not to
the hypothalamus [2124].
572
In the hindbrain, the developmental ontology recognizes

12 segments (the isthmus and 11 rhombomeres)
(Figure 1D,E), principally on the basis of fibroblast growth
factor 8 (Fgf8) [10,25] and homeobox (Hox) gene expression
[26], whereas the traditional ontology divides the hindbrain into pons and medulla oblongata, classic gross divisions that are not consistent with the rhombomeric
subdivisions [12]. In the developmental ontology, the cerebellum is separated from the basilar pons, and properly
classified as a dorsal outgrowth of the isthmus and the first
rhombomere, a result of several fate-mapping studies [27
29] (Figure 1D,E).
At the third level of the developmental ontologic hierarchy, the telencephalon is divided into pallium and subpallium, as it is in traditional ontologies (Figure 2A).
However, as noted above, the new subpallium includes
the preoptic area [20], parallel to three other distinct
radial subpallial domains, identified in mediolateral order
as diagonal domain (old substantia innominata, covered
superficially by the diagonal band formation), pallidum,
and striatum (Figure 2A). Each of these domains extends
radially from the ependyma to the pial surface and has
diverse stratified derivatives. The striatum contacts the
pallium across the palliosubpallial boundary [30]. The
four subpallial domains are elongated along the septoamygdaloid axis, and they form at their ends composites
known as the subpallial amygdala and the subpallial
septum [30] (Figure 3). The hypothalamus is divided into
terminal (rostral) and peduncular (caudal) neuromeric
portions, continuous dorsally with the preoptic area
(unevaginated telencephalon) and the evaginated telencephalic hemisphere, respectively [16] (hypothalamic prosomeres hp2 and hp1; Figure 2A). At this third level, the
diencephalon is divided caudorostrally into three diencephalic prosomeres: the pretectum (prosomere 1), the thalamus (prosomere 2), and the prethalamus (prosomere 3),
Opinion
(A)
(B)
Striat
Pall
Striat
Sept
Pall
Parasept
Diag
Preopt
Preopt
Diag
Central
Amygd
Figure 3. A diagram to show the main subdivisions of the subpallium examined in a coronal plane. Panel (A) represents the four main histogenetic domains of the
subpallium: striatum (Striat), pallidum (Pall), diagonal domain (Diag), and preoptic area (Preopt). These extend radially from the ventricle to the pial surface. Panel (B) shows
the secondary subdivisions of these domains along the septoamygdaloid axis. Each subpallial histogenetic domain shows diversely differentiated septal (Sept), paraseptal
(Parasept), central, and amygdaloid regions (Amygd). This concept recapitulates and expands the idea of an extended subpallial amygdala.
including corresponding tegmental components [14,16,

3133] (Figure 2A). Conventional ontologies assign the
diencephalic tegmental territory to either midbrain or
posterior hypothalamus. The diencephalic tegmentum
contains rostral parts of the ventral tegmental area and
substantia nigra, which are in fact isthmo-meso-diencephalic plurisegmental entities [11,16,3436].
Subsequent hierarchical levels of subdivision of the
forebrain, midbrain, and hindbrain are based on the
well-known dorsoventral regionalization of the neural tube
into roof plate, alar plate, basal plate, and floor plate,
introduced by His [37]. These longitudinal zones can
now be more precisely defined by their differential molecular profiles. The longitudinal extent of the floor plate,
ending at the mamillary floor, is marked by genes such as
Sonic hedgehog (Shh), netrin 1 (Ntn1), and LIM homeobox
transcription factor 1-beta (Lmx1b), although these markers are not unique to this zone [15]. The basal plate of the
forebrain, midbrain, and hindbrain is defined by the presence of either NK2 homeobox 1 (Nkx2.1) or 6 (Nkx6.1) [38].
At early developmental stages, expression of paired box
(Pax) genes (Pax6 in the forebrain; Pax 3 and Pax7 in the
midbrain and hindbrain) broadly characterizes the alar
plate [39,40]. Genes of the Zic family are expressed at the
dorsal part of the alar plate, next to the roof plate [41].
Consistent with a specific set of gene expression patterns
[e.g., Nkx2.2, patched 1 (Ptc1), or gamma glutamylcysteine
synthetase 1 (Gsh1)], the developmental ontology includes
a distinct liminal band at the ventral rim of the alar plate.
The liminal region is defined as the junction of basal and
alar plates.
This broad-brush picture of the developmental ontology
is based upon the redefinition of brain organization suggested by the prosomeric model, which attempts to connect
developmental discoveries with adult neuroanatomy
across the whole brain. This picture needs more detailed

explanation in three areas: (i) the issue of the now obsolete
columnar forebrain axis of Herrick [42]; (ii) the definition of
neuromeres; and (iii) the organization of the pallial and
subpallial telencephalic territories.
Herrick and the columnar forebrain axis
Twentieth-century ideas on forebrain subdivisions were
dominated by the columnar model of the forebrain put
forward by Herrick [42]. Contradicting the earlier analysis
of His [43], Herrick proposed that the brainstem axis
simply extended rostrally as a straight line into the forebrain, so that the thalamushypothalamus complex (then
called the diencephalon) stood in direct axial continuity
with the telencephalon rostrally and the midbrain caudally. This concept was not based on developmental findings,
and it disregarded the ostensible cephalic flexure, present
in all vertebrates. It was based instead on an ill-fated ad
hoc attempt to extrapolate brainstem functional columns
into the forebrain. The columnar hypothalamus was held
to be exclusively basal (and connected directly with the
midbrain tegmentum), matched with an exclusively alar
thalamus. The columnar concept of forebrain organization
has not stood the test of developmental gene expression
patterns and related causal mechanisms, whereas these
recent discoveries have strongly endorsed the earlier axial
conception of His, in which the telencephalon is dorsal to
the hypothalamus, and the diencephalon proper lies caudal
to it [16,4447] (Figure 2B).
This fundamental error has had a major impact on the
interpretation of the mutual topographic relations of the
telencephalon, eyes, hypothalamus, and diencephalon by
many authors [19,48]. In the updated revised model of the
forebrain that underpins our developmental ontology,
the term diencephalon accordingly includes only the
573
Opinion
prethalamus, thalamus, and pretectum, each with a piece
of basal tegmentum, and excludes the hypothalamus
(Figure 2B).
Within the developmental ontology, the hypothalamus
is divided first into a rostral part (terminal hypothalamus)
and a caudal part (peduncular hypothalamus) [16]
(Figure 2A). The intrahypothalamic boundary separating
these parts runs parallel to the subsequent course of the
fornix tract; the boundary lies just rostral to the fornix and
the neighboring medial forebrain bundle and lateral forebrain bundle (cerebral peduncle) (Figure 2A). This boundary also separates the preoptic area from the diagonal
region in the evaginated telencephalon, and also divides
the mamillary and retromamillary areas one from another
[16] (Figure 2A). The evaginated eyes and surrounding
supraoptic and suprachiasmaticanterior-hypothalamic
areas are all alar derivatives of the terminal hypothalamus, whereas the tuberal region, the attached neurohypophysis, and the mamillary bodies are corresponding
basal derivatives (see [16]). The peduncular hypothalamus
(marked by its role as bed of the medial and lateral
forebrain bundles and the fornix) contains the main part
of the deep paraventricular nucleus and the radially migrated entopeduncular complex as its principal alar derivatives, and includes retrotuberal and retromamillary
formations in its basal subregion. The basal subregion
contains the subthalamic nucleus (a migrated retromamillary derivative), which was previously not considered to be
a part of the hypothalamus [16,49] (Figure 2A).
Neuromeres
Periodic transverse outpouchings in the neural tube wall
were first recognized over a century ago ([12,50,51]). Orr
[51] called them neuromeres (prosomeres in the prosencephalon, mesomeres in the midbrain, and rhombomeres
in the hindbrain), thus viewing them as neural segments
within the general plan of head segmentation (Figure 1D).
The neuromere concept fell into disuse with the rise in
popularity of Herricks columnar paradigm, basically because brain segments did not offer at that time a recognizable functional significance. However, interest in
neuromeres has returned in the molecular era because
distinct molecular profiles characterize these developmental units and their fate-mapped derivatives, thus dispensing with the old myth that neuromeres were transient
early phenomena. The molecular segmental scenario provides an opportunity for causal explanations of brain
structure, an endeavor that completely collapsed with
the old columnar theory. Moreover, physiologists have
identified various examples of brainstem functional circuitry that relate to neuromeric compartments [52,53]. In a
similar way, functionally distinct prethalamic, thalamic,
pretectal, and midbrain circuits are now recognized to be
neuromerically organized, after their interpretation as
longitudinal zones was abandoned. Therefore, any developmental ontology is obliged to take account of these
transverse components of the neuraxis.
The boundary between diencephalon and midbrain is
marked by the caudal boundary of the forebrain expression
of Pax6 [54]. The boundary between midbrain and hindbrain is sharply defined by the interface of the expression
574
territories of orthodenticle homeobox 2 (Otx2) and gastrulation brain homeobox 2 (Gbx2) [55,56]. The midbrain
proper can be subdivided into rostral and caudal mesomeres (m1 and m2; Figure 1) [11]. The existence of a thin m2
component of the midbrain, separating the isthmus from
the inferior colliculus, was previously recognized by early
embryologists [57]. Gene-mapping studies support the
existence of m2, defined by coexpression of Otx2 and
Pax2 [58]. Its alar subregion has been called the preisthmic domain (which includes the nucleus sagulum,
the cuneiform gray, and subbrachial nucleus) and its basal
structures include the retrorubral A8 catecholaminergic
cells and the rostral-most part of the dorsal raphe nucleus
[11,12,58].
The developing hindbrain is overtly segmented in its
central part (r2r6). Its rostral (isthmus, r1) and caudal
(r7r11) parts are not overtly segmented, but are differentially coded molecularly into cryptic neuromeric compartments [12,26,5961] (Figure 1D,E). The rostral hindbrain
is influenced by the isthmic organizer [6264] and can be
divided into the isthmus and rhombomere 1, which contribute to the formation of the cerebellum in the same
general way as the hypothalamus builds the telencephalon
[6466]. The isthmus itself is defined by the early expression of Fgf8 [25]. The remainder of the hindbrain is marked
by the diversified expression of Hox genes and ephrins [66
71].
Rhombomeres r2r6 are usually recognized as overt
bulges separated by constrictions in the embryonic hindbrain [51,66,67,71]. However, the boundaries of cryptorhombomeres r7r11 were distinguished in the chick on the
basis of fate mapping and differential Hox gene expression,
and the same subdivisions exist in the mouse [72]. In fact,
the series of rhombomeres seems to be conserved among all
vertebrates [73,74].
These segments in the hindbrain are molecularly distinct developmental units [26,67,75,76]. For example, we
can now recognize 11 neuromeric parts of the trigeminal
sensory column across r1r11. The vestibular column can
be similarly subdivided segmentally across r1r9. Raphe
nuclei have been recently analysed in the rhombomeric
context, identifying some 45 separable cell groups across
the whole hindbrain [77]. Differential histogenetic behavior due to regional changes in the molecular identity of both
progenitors and derived neurons correlates with the development of differentiated transverse blocks of hindbrain
structure [26,60,66,71]. Because of shared dorsoventral
patterning, many rhombomeres contain similarly placed
nuclei and, because early observable boundaries largely
become invisible as development proceeds, such serial
nuclei form plurineuromeric sensory columns, but the
unique molecular identities and consequent differential
hodological or functional properties of individual segments
often persist [52]. Fate- mapping has shown that the
neuromeric developmental units are still separated by
cryptic boundaries as the hindbrain matures [60,66,71]
and, therefore, represent necessary conceptual levels for
structural classification in any modern ontology.
Three major tangential migrations cause significant
alterations to the anatomy of the hindbrain region. Pontine
neurons migrate from the caudal rhombic lip (r6r7) to the
Opinion
ventral surface of r3 and r4, where they form the pontine
nuclei [71,72,7781]. More caudal rhombic lip derivatives
(r8r11) migrate via an extramural tangential stream into
the lateral reticular nucleus and external cuneate nucleus,
and via an intramural stream into the inferior olive [60,81
83]. The mammalian facial motor nucleus migrates from
the medial part of r4 to reach its familiar superficial
position in r6 [18,84]. Recently, the interpeduncular nucleus has been shown to form by tangential migratory convergence of diverse alar and basal cell populations at the
isthmic and r1 midline [85,86].
The new developmentally based hindbrain ontology
purposefully avoids using the classic subdivision of pons
and medulla. The term pons is particularly misleading as
a regional descriptor. The pons of the human brain mistakenly refers to a region that apparently extends from the
midbrain to the caudal border of the sixth rhombomere (the
caudal end of the facial nucleus). In fact, the basilar
pontine formation arises from the rhombic lip in r6 and
r7, and migrates to the ventral margin of r3 and r4
[29,72,81,87]. In the mouse, the crossed pontocerebellar
fibers of the middle cerebellar peduncle grow across r2 into
the r1 entrance into the cerebellum, thus encircling the
trigeminal root; this gives the impression that r2 also
forms part of the pons. Therefore, the term pons as a
regional descriptor of a zone ventral to the cerebellum
reaching from the midbrain to the medulla must be abandoned, although the r3r4 region can be called pontine
hindbrain. The word pons sensu stricto can be properly
applied to the nuclei and crossing fibers of the basilar
pontine formation. Note that there is a substantial prepontine hindbrain, often misidentified as a caudal midbrain domain, which includes isthmus and rhombomeres 1
and 2. The trapezoid body and neighboring trapezoid and
superior olivary and periolivary nuclei of the auditory
pathway, as well as the nucleus abducens, are strictly
retropontine, being associated with r5. The pyramidal
decussation lies in r11.
Pallium and subpallium
The telencephalic hemisphere is not an axial vesicle, because there are two of them. It is formed by an idiosyncratic
patterning mechanism that generates pallial and subpallial regions that are already at neural plate stages [8891].
The traditional columnar misconception that the longitudinal axis of the brain ends in the telencephalon has
resulted in the widespread assumption that the embryonic
pallium lies dorsal to the embryonic subpallium; in reality,
the pallium is topologically caudal to the subpallium,
as shown conclusively by neural plate fate maps
[16,89,90,92]. The rostral end of the brain axial dimension
is identified by landmarks in the roof, the floor, and the
alarbasal boundary. The rostral end of the roof plate has
been fate mapped to the locus of the anterior commissure
[16,89]; the floor plate ends at the mamillary pouch [16];
the alarbasal boundary is continuous from left to right
just under the suprachiasmatic nucleus [16]. These three
points are equally rostral-most and can be traced molecularly from early neural plate stages onwards. The median
wall interconnecting them builds the terminal wall of the
neural tube. The telencephalon sits alongside the dorsal
alar subregion of this wall, with the preoptic area extending into the bed of the anterior commissure. Differential
gene expression defines the telencephalic palliosubpallial
boundary [9294] (Figure 2A). The presence of specific
regional molecular profiles has profound histogenetic
and functional consequences, with the subpallium becoming the almost exclusive source of inhibitory (GABAergic)
telencephalic neurons [95], and the pallium becoming the
main source of excitatory (glutamatergic) neurons [96,97].
The GABAergic subpallial neurons that migrate tangentially into the developing pallium express distal-less homeobox (Dlx), LIM homeobox 6 (Lhx6), and various other
genes [95].
The amniote pallium can be divided on anatomical and
molecular grounds into septal, medial, dorsal, lateral,
ventral, and amygdaloid zones [97]. In mammals, the
medial pallium forms hippocampal and parahippocampal
structures; the dorsal pallium forms the neocortical mantle, and the lateral and ventral pallium are held to form the
olfactory cortical structures with attached pallial claustroamygdaloid nuclei [97,98]. The intimate relation of the
mammalian claustrum with the insular cortex is still not
well understood. Non-mammalian vertebrates show the
same pallial domains, but mammals are the only animals
that have a fully developed neocortex [9,99].
Further developmental ontologic subdivisions of each of
the pallial and subpallial zones can be recognized [100].
The medial pallium differentiates into the diverse components of the hippocampal formation: dentate gyrus, CA
fields, subiculum, presubiculum, parasubiculum, entorhinal cortex, and retrosplenial cortex [101]. The dorsal pallium includes the typical central group of neocortical areas
(orbitofrontal, parietal, occipital, and temporal) and the
mesocortical areas at its periphery (cingulate, perirhinal,
periorbital, and insular cortex) [102]. The lateral pallium
and ventral pallium each contribute to the olfactory areas,
the insular claustrum, and the amygdala [98].
The subpallium is adjacent to the ventral pallium along
the palliosubpallial boundary. It is organized bilaterally
as several bands of differentially specified radial histogenetic territories, stretching along the septoamygdaloid axis
(Figure 3). Each subpallial band has septal, paraseptal,
central, and amygdaloid differentiated portions, wherein
the major derivatives are the central ones (striatum, pallidum, diagonal-basal domain, and preoptic area;
Figure 3). The best-known paraseptal element is the striatal nucleus accumbens, which is complemented by analogous paraseptal parts within the pallidal, diagonal, and
preoptic zones. The concept of the extended amygdala [103]
antedated the realization that some amygdaloid nuclei
share properties with subpallial sectors beyond the apparent limits of the traditional amygdala, extending supraand infracapsularly all the way to the septum. The present
ontology is consistent with this view, with its medial
(diagonal) and lateral (pallidal) portions of the stria terminalis complex and central amygdaloid striatal elements
[30]. The other major components of the diagonal-basal
domain are the substantia innominata, the basal magnocellular nucleus, and the nuclei of the diagonal band.
Ontological representation of these regions is complicated
by the fact that some classic anatomic entities (such as the
575
Opinion
Box 1. The future of brain ontology
Is the new ontology flexible enough to incorporate future
discoveries in gene expression during development?
How will future changes to the ontology be moderated and
approved for inclusion?
Who will host the current ontology and its future derivatives?
What are the barriers to extending the ontology to include all
vertebrate classes?
In cases where a neuron group migrates from its place of origin to
another brain region, should it be classified according to the
region of origin, the final location, or both?
septum and the amygdala) comprise developmentally distinct sectors deriving from the pallium, striatum, pallidum,
and diagonal area, respectively and also encompassing
tangentially migrated cells derived from the alar hypothalamus and the preoptic area [22,30,96,97].
Concluding remarks
A new brain ontology based on modern developmental
criteria and models provides a more accurate and complete
view of the natural morphologic interrelations of different
parts of the brain. Neuronal populations have been classified on the basis of progressive rostrocaudal and dorsoventral differentiation of the neural tube. Radially migrated
(stratified) derivatives can be traced from progenitor areas
based on fate mapping, gene mapping, and other experimental evidence. Overall, this demonstrates a consistent
relation between embryonic patterns and adult structure,
which is expressed in the form of a developmental ontology.
Because genomic control of neural morphogenesis is remarkably conservative, this ontology should prove to be
essentially valid for all vertebrates, aiding terminological
unification. The new ontology will undoubtedly be contested, and many details are still subject to revision that
will be based on further discoveries (Box 1).
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Opinion
Challenges of understanding brain

function by selective modulation of
neuronal subpopulations
Arvind Kumar, Ioannis Vlachos, Ad Aertsen, and Clemens Boucsein
Bernstein Center Freiburg (BCF), and Neurobiology and Biophysics, Faculty of Biology, University of Freiburg, Freiburg, Germany
Neuronal networks confront researchers with an overwhelming complexity of interactions between their
elements. A common approach to understanding neuronal processing is to reduce complexity by defining
subunits and infer their functional role by selectively
modulating them. However, this seemingly straightforward approach may lead to confusing results if the
network exhibits parallel pathways leading to recurrent
connectivity. We demonstrate limits of the selective
modulation approach and argue that, even though
highly successful in some instances, the approach fails
in networks with complex connectivity. We argue to
refine experimental techniques by carefully considering
the structural features of the neuronal networks involved. Such methods could dramatically increase
the effectiveness of selective modulation and may lead
to a mechanistic understanding of principles underlying brain function.
Introduction
Structural features of networks on multiple scales
The mammalian brain is often referred to as the most
complex biological system, not only because of the large
number of cells (1011) but mainly because of the multitude
of interactions between them. Even if all connections (1015)
were known, a system with such complexity could not be
successfully treated at all levels of detail simultaneously.
Instead, it is advisable to focus on an appropriate spatial
scale and to reduce the lower levels to putative functional
units, approximating their intrinsic fine structure with the
help of fewer variables, if not ignoring them entirely.
At the macroscopic scale (1 cm), the brain can be described as a network of different areas or regions. Together
with this anatomical modularity, neurophysiological evidence indicates that these different regions in the brain
are to some extent specialized to process specific sensory,
motor, or cognitive information, and that they are interconnected in a non-trivial fashion [1,2]. At mesoscopic scales
(1 cm), brain regions are themselves often anatomically
segregated and can be subdivided into layers, subfields,
or nuclei. Examples are the hippocampal formation, the
interconnected cortical layers or columns, and the networks
Corresponding authors: Kumar, A. (arvind.kumar@biologie.uni-freiburg.de);
Boucsein, C. (clemens.boucsein@biologie.uni-freiburg.de).
2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tins.2013.06.005
in the limbic system such as the basal ganglia and amygdala. Thus, at macro and mesoscopic levels the brain is best
considered as a network of networks (NoN). Finally, at
microscopic scales (1 mm), brain tissue can be defined
as a network of neurons, which can be broadly categorized
as excitatory and inhibitory. At this spatial resolution, brain
tissue conforms most appropriately to the term network
because the interconnected nodes are naturally defined
(individual neurons), and their connectivity can, to some
extent, be measured experimentally. Below this scale, specialized, spatially extended cells can add additional degrees
of freedom through compartmentalization and non-linear
dendritic integration [3,4] which might affect computations
performed at higher levels. However, although different
spatial scales of network organization render the system
complex, the presence of specific network motifs (Figure 1)
raises the hope that different spatial scales can be described
using common principles.
Such conceptual reduction of complexity is commonly
referred to as model-building. Their mathematical formulation makes them accessible for theoretical studies, opening the opportunity to study their behavior beyond the
limitations that often constrain experiments on real
brains. Commonly used models typically fall into one of
three classes: feedforward models, ring models, and models
with recurrent connectivity (Figure 1). The simplest model
is the feedforward network, which allows an intuitive
understanding of its working principles merely from visual
inspection of its graphical description (Figure 2A). In such
a feedforward network it is straightforward to characterize
the separate contributions of all populations by controlled
modulation of their activity.
Several networks in the sensory and motor periphery
may conform (at least approximately) to such simple architecture regarding their intra- and inter-network connectivity hence they can be treated as effectively feedforward.
Examples include cortico-striatal interactions as well as
many networks in the sensory and motor periphery, and
the hippocampus with its largely feedforward, trisynaptic
pathway. In fact, at short time-scales (such that the neuronal activity cannot travel over the full loop), even the
entorhinal cortex-hippocampal and the thalamo-cortical
loop (thalamus and layer IV) can be studied as an effectively
feedforward network. Finally, particular random recurrent
networks can also be reduced to an effective feedforward
structure [5].
579
Opinion
(A)
successful approach to networks that show a slightly more

complex connectivity structure beyond simple feedforward circuitry.
(D)
Feedforward
e.g., rena to LGN
Inhibitory recurrent
network e.g., striatum,
central amygdala
(E)
(B)
Apparent feedforward
e.g., entorhinal cortex,
and hippocampus
No exc. recurrence
e.g., CA1
(F)
(C)
Recurrent exc. inh.

network e.g., CA3,
neocorcal layers
Fully recurrent
Figure 1. Organization of neuronal networks at different spatial scales. (AC)

Neuronal network at microscopic scales (1 mm). There are three main motifs at
this scale: (A) a recurrent network composed solely of inhibitory neurons; for
example, the striatum or the central amygdala. The neurons are maintained in their
spiking state by excitation from other networks. (B) A recurrent network composed
of both excitatory and inhibitory neurons but with little or no recurrent connectivity
among excitatory neurons; for example, the CA1 subfield of the hippocampus. (C)
A recurrent network with both excitatory and inhibitory neurons and mutual
connectivity between the two populations; for example, the individual layers of the
neocortex and the CA3 subfield of the hippocampus. (DF) Beyond the scale of
1 mm, networks in the brain are in fact networks of networks (NoN). These NoNs
can be classified into three different categories as follows. (D) Feedforward
connection between two networks is the simplest NoN motif. Such motifs are most
common in the sensory and motor periphery. (E) Next, NoNs such as the
entorhinal and hippocampus loop are effectively feedforward, although there may
be multiple projections from one field to other fields (e.g., entorhinal cortex
connects to dentate gyrus, CA3, and CA1). (F) Finally, in fully recurrent NoNs the
constituent neuronal networks (nodes) form both feedforward and feedback
connections. Such network motifs are commonly observed both at anatomical
(e.g., the inter-layer connectivity in the neocortex) and functional levels (e.g., the
network of various subnetworks involved in visual information processing).
Abbreviations: exc., excitatory; exc. inh., excitatory and inhibitory; LGN, lateral
geniculate nucleus.
With recent improvements in brain stimulation technology it has become possible to modulate neuronal networks and/or groups of neurons with unprecedented
specificity. Because selective stimulation of neuronal networks has been very informative regarding the functional
interactions in early sensory and peripheral motor circuits (essentially feedforward structures), it seems only
natural to use these tools in the same approach to other
brain areas as well, and to all levels of structural detail,
even down to the single cell level. However, several considerations concerning the dynamics of network activity
reveal fundamental problems in extending this seemingly
580
Limitations of the selective activity modulation

approach
Beyond the first stages of sensory processing or the
penultimate stages of motor processing, most networks
in the brain cannot be approximated by a feedforward
structure. Higher brain areas exhibit more recurrency for
which it is non-trivial to reveal the specific activity
patterns that implement a presumed function and to
identify the elements involved. To demonstrate the problems that may occur in recurrent networks, we converted our feedforward network into a recurrent
network by adding one more connection from the third
to the second population (Figure 2A,B). In this model it is
necessary to modulate the activity of P2 and P3 both
separately and simultaneously to understand their functional roles in the network.
In the case of more interacting neuronal populations or
neurons (both referred to as nodes in the sequel), modulating the activity of single nodes within the network yields
only limited information about network function [6,7]
when activity in one node inhibits a second and excites a
third, and these in turn are coupled and feed back onto the
first, even brief consideration of the network behavior
reveals its complexity. Thus, for a network of five interacting nodes, to assess the functional contributions of its
elements and their possible interactions it would be necessary to modulate the activity of one, two, three, and four
nodes simultaneously, and to record the corresponding
network activity in each case. In general, for a network
of N nodes, we would need to selectively modulate all single
nodes, and all combinations of two to (N 1) nodes, separately to identify the relevant subset(s) of nodes (cf. [8]).
For a network of N interacting elements, the number of all
possible subsets of nodes is given by Bells number [9]:
BN
N
1
X
N 1!
Bk
k!N 1 k!
k0
[1]
Unfortunately, BN grows faster than exponentially with

N. For a system consisting of six populations (the minimal
population size to study thalamo-cortical interactions),
B6 = 203, and for a system with ten populations,
B10 = 115 975. Thus, as the number of interacting nodes
in a network grows, the number of node combinations that
need to be modulated rapidly becomes prohibitively large.
For example, even considering only five layers of a
neocortical column (those containing the majority of all
cell bodies), to comprehend the contribution of each layer to
a particular function we would need B5 = 52 different
combinations of single and multi-layer stimulations. Obviously, to treat neocortical networks that way is an oversimplification, and ignores that a large fraction of the
inputs originate from the surround [10]. Moreover, interlayer connectivity [11] implies that activity dynamics and
layer-specific responses are influenced by the activity of
various neuron types and networks in different layers
[12,13]. Thus, modulation of a specific neuron type in a
Opinion
(A)
(C)
P1
1
P2
2
P3
3
P1
1
P2
2
P3
3
(B)
P1
1
P2
2
P5
5
P3
3
P4
4
Figure 2. Only simple networks of interacting neuronal populations can be studied using selective modulation of individual populations. (A) In a feedforward network it is
straightforward to characterize and understand the contribution of each population onto the next. (B) Any deviation from the strict feedforward motif requires control of the
activity of more than one population. In this three-population network we need to stimulate, in addition to single populations, pairs of populations to understand network
function. (C) In a recurrent network of networks (NoN) (Figure 1D) with N populations, we need BN (Equation 1) different combinations of simultaneous stimulation of
multiple populations to understand the impact of one population upon the others. Unfortunately, the number BN grows with N faster than exponentially. In the toy example
with five populations, this requires 52 different stimulation patterns involving 15 populations. Abbreviation: l15, firing rates of respective neuron populations P1P5.
given layer [14] is not sufficient to understand the dynamics [12] and stimulus response [13] of a cortical column.
Indirectly, this issue was already encountered when Lee
et al. [15] attempted to extract the contribution of specific
pyramidal neurons to the fMRIBOLD (blood oxygen leveldependent) signal, a problem complicated even more by
recurrent inter- and intra-layer connections [16].
This combinatorial problem becomes even more intractable when nodes interact in a non-linear fashion (as is
most often the case in brain networks) or when couplings
are dynamic and activity-dependent. In both cases, one
would need to linearize the system and perform the experiments and associated analyses around a specific operating
point for each of which Bells number applies and,
hence, to understand the full system, Bells number would
2
3
4
Figure 3. Scheme of the dynamical space of a five-population network of networks

(NoN). The population activity represents the average activity of neurons in the
given neuronal population. In a dynamical system description, the function of the
NoN, in an abstract sense, can be described as the transition from one state to
another (i.e., control of the network activity dynamics). Here the states are
schematically indicated by colored circles; arrows mark the transitions between
states. Indeed, often more than one transition from any given state is possible.
Controllability provides the necessary criteria to determine whether a system is
controllable; in other words, whether it can be steered from one dynamical state
to another in finite time. Abbreviation: l15, firing rates of different neuron
populations.
need to be multiplied by the number of operating points

chosen.
Finally, we note that most experiments thus far rely on
steady-state responses upon stimulation of one or more
nodes. However, when dealing with interactions among
dynamical systems it is important to consider both transient and steady-state responses to identify the functional
subset of the network because steady-state responses alone
can be misleading [17].
Taken together, these considerations demonstrate that,
even if a thorough understanding of the functional network
mechanisms is not mandatory (e.g., in medical applications), the sheer number of possibilities to affect network
behavior by selective modulation of its elements renders a
successful outcome of such experiments rather unlikely.
Common techniques for selective activity modulation
Assuming that the above-mentioned combinatorial issues
with selective modulation could be resolved (see below for
new perspectives), another important issue is the availability of experimental methods for selectively modulating
the activity of neuronal networks. Deciding which methods
to apply for studying a given network partly depends on the
research goals: if we wish to understand thoroughly the
working principles underlying both, the function and the
functioning of the network, we need to identify the functional role of each element and characterize their interactions. However, for the technical task of controlling of
prosthetic devices by brain activity, local field potentials
(LFPs) can be utilized [18], even though the mechanisms
underlying their generation are still under debate [19] and
the LFP is unlikely to affect brain activity (but how LFP
might influence spiking see [20,21]).
The earliest successful selective modulation experiments probably date back to early Greco-Roman times
when Galen (AD 129199) discovered that mental impairments of gladiators were associated with specific head
injuries. Since those early, involuntary brain lesion studies, experimenters have sought to refine loss-of-function
approaches and, indeed, lesions of brain structures by
targeted ablations at different levels of detail have provided important insights into the functional organization of
581
Opinion
Populaon acvity (sp/s)
10
8
(B)
12
10
8
6
4
2
0
500 550
100
600
650
Network ID
Populaon acvity (sp/s)
(A) 12
700
Key:
Out-degree
k-shell-out
0.0
0.2
50
4
2
0
0
0
500
1000
1500
2000
2500
Out-degree
3000
0.4
0.6
0.8
1.0
Figure 4. Estimation of embeddedness in numerical simulations of spiking neuronal networks. (A, inset) Network response (peri-stimulus time histogram, PSTH) for
identical stimulation of 30 different subpopulations of 250 neurons each in an example network. (A) We estimated the sum of the PSTHs for 100 different networks (see [42]
for details) upon stimulation of different subpopulations (250 neurons). Two networks with small-world properties are highlighted (dark blue, orange dots). The random
networks which do not show much out-degree variance are indicated by triangles. The network response increases with the out-degree, but the same out-degree can also
give different responses. (B) Average correlation coefficient (r, sorted) between the sum of PSTHs as a function of out-degree and k-shell-out indices. Both these measures
predict the network response; however, neither alone describes it completely. (Figure adapted from [42]). Abbreviation: sp/s, spikes per second.
the CNS [22]. First, pieces of brain tissue were physically

removed and, later, silenced by injection of toxic chemicals
or local tissue cooling (Table 1). A major refinement in
selective modulation came with electrical stimulation.
Since 1870 [23] it is used both to identify the function of
brain areas and as a therapy to intervene with aberrant
activity dynamics associated with brain disorders. Moreover, electrical stimulation also allows activation rather
than silencing of nerve tissue. However, the effects of
electrical brain stimulation (including behavioral effects)
are generally not well understood, partly because they may
involve a multitude of pathways [24].
All these classical approaches affect the activity of
more or less the entire cell population in a locally confined
volume of brain tissue, most likely (through axonal stimulation) together with cells downstream and upstream. This
implies that they operate on multiple levels simultaneously, involving a hierarchy of networks. With recent developments in optogenetic methods these caveats have been
largely eliminated, leading to an unprecedented richness of
tools for selective activation/inactivation. Transgenic or
viral transfection with light-gated ion channels now allow
a specific group of neurons to be stimulated while recording
neuronal activity without stimulus artifacts [25,26]. Thus,
it seems only natural to combine the formerly successful
modulation approaches with these new capabilities. In the
light of the aforementioned theoretical considerations,
however, we should appreciate that the simple modulation
approach will rarely (only in structures reducible to feedforward networks) lead to reliable guidelines for clinical
intervention, let alone to a thorough understanding of the
neuronal mechanisms involved.
What is a network, and how can its function be
investigated?
Defining networks
Even assuming we could deal with the combinatorial
explosion associated with selective modulation, another
582
fundamental issue needs to be resolved: how to define

the network which should be stimulated or whose function needs to be understood? Classical methods used to
modulate brain activity activate/inactivate cells in a spatially localized fashion. Hence, it seems intuitive to consider anatomically proximate neurons as a functional
unit. Indeed, neighboring neurons often appear to be
responsive to similar stimulus features, or are co-active
during similar tasks as expressed in the concept of
functional maps. However, experimental data show that
neurons involved in cognitive and motor functions are
distributed throughout the brain [27]. Likewise, primary
sensory areas can be modulated by other stimulus modalities [2831], and spatial grouping of neurons with
similar stimulus preferences does not seem to be a general feature of cortical networks [32]. The applicability of
proximity-based grouping breaks down entirely if cellspecific markers are employed, as in many optogenetic
approaches. Here, only a subpopulation of cells within a
given volume is modulated based on the expression of
particular biomarkers. At the level of such subpopulations, feedforward circuits have been identified [33], but
the respective cells are also involved in other circuits
within the same tissue volume and, thus, modulating
their activity may have confounding effects.
In addition, it can be problematic to demarcate functionally specialized neuronal subpopulations from the outset. Various classification schemes based on neuron
location, morphology, gene expression and spike patterns
have been suggested [3437]. Undoubtedly, many of these
features will relate to the functional role of the respective
populations: activating excitatory cells has very different
effects on the network than activating inhibitory neurons.
Also, applying different criteria may lead to overlapping
boundaries between putative neuron types, and differentiation is still limited for key neuronal populations.
For instance, the subdivision of neocortical pyramidal
cells based on their firing pattern seems to be of limited
Opinion
Table 1. Selective activity modulation approaches

Stimulation method
Electrical microstimulation
Temporal resolution
Good
Spatial resolution
Poor, because axons from distal
regions can also be stimulated
Depends on the diffusion of the
chemical
Specificity
Poor. To some extent it is possible to select
between stimulating somata and axons [62]
Good
Chemical injection, e.g.,

neurotransmitter agonists
and antagonists
Magnetic stimulation
(e.g., transcranial magnetic
stimulation, TMS)
Cryogenic
(cooling of brain tissue)
Optogenetic stimulation
Poor
Good

regions can also be stimulated
Poor
Good

regions can also be affected
Moderate. Depends on the penetration
of light and expression pattern of the
optogenetic tools. Potential for
improvement by two-photon
techniques
Poor
Good. However, thus far

all transfected neurons
are stimulated
simultaneously [63]
Good. Even specific neurons and their

components can be targeted
Experimental methods for selective modulation of the activity of particular parts of the brain have been developed for various spatial and temporal scales.
usefulness in specific network types: propagation of spiking activity in feedforward networks is not influenced by
the type of neuron model, and feedforward networks with
integrate-and-fire neurons [38,39] behave similarly to
those with detailed neuron models [40]. Likewise, in a
study of the response of different types of neuron models
(firing-rate model, conductance-based leaky-integrateand-fire neurons) to higher-order input activity correlations, the input statistics proved much more important
than the neuron type [41].
At the microscopic scale, it is instructive to investigate
how a given neuron influences local and downstream network activity, given its other properties (morphology, gene
expression, firing pattern, neurotransmitter type) [42].
Recent experiments suggest that neurons indistinguishable by existing classification schemes might form functional subpopulations [43]. Activity-dependent labeling
showed that a comparatively small group of neocortical
pyramidal cells might provide the major background drive
in their respective networks [44]. Similarly, long-range
projection targets of cortical output cells are predicted
by their intracortical connectivity [45], suggesting that it
might be informative to classify them by their afferents
and efferents. The density of outgoing projections, together
with their overall activity, could be combined into a single
variable, quantifying how strongly a cell (cell group) influences the network activity. This, together with the type of
neurotransmitter and, to some extent, the synaptic properties, may be used to define the embeddedness of a
neuron (Box 1) [42].
Selectively modulating individual neurons and recording the network response allows the effective embeddedness of a neuron in vivo to be estimated experimentally. In
addition, selectively visualizing the presynaptic sources
[46,47] or postsynaptic targets [48] of a neuron may provide useful insights into its structural embeddedness.
The structural embeddedness of a neuron in its local
microcircuit may also be estimated by juxtacellular recording in vivo and labeling afterwards [49]. Next to these
indirect methods, new approaches for measuring neuronal
embeddedness need to be established. Indeed, optogenetic
tools rank among the most promising candidates in this
respect.
Controllability as a concept for selective modulation in

complex networks
When considering the brain as a dynamical system, firing
rates of neurons or average population rates of participating
networks are often used as dynamic variables. Traditional
approaches such as eigenvalue or Schur decomposition,
transforming a network into a new system with only selfinteractions or feedforward interactions, respectively, are
extremely successful in helping to understand physical
systems. However, it has thus far not been possible to
map these concepts to experimental studies of neuronal
networks [5].
Although it could be held that the ultimate goal of
neuroscience is to understand the functional role of each
neuronal network and neuron type in the brain, there are
fundamental problems with this. We discuss here a new
Box 1. Embeddedness
The concept of embeddedness was initially coined for socioeconomic networks to understand the effect of social relations on
economic decision-making [64]. Within the context of neuroscience,
embeddedness measures the impact of the spiking activity of a
neuron on the spiking activity of the surrounding network. It can be
experimentally estimated by stimulating a neuron and counting the
total number of extra spikes in the network [65] (Figure 4).
When a neuron is activated, it sends spikes to its postsynaptic
neurons; some of which will produce spikes and send those to their
postsynaptic neurons, and so on. Thus, to estimate the impact of
activating a neuron we need to know all possible direct and indirect
(involving multiple synapses) paths from the stimulated neuron to
other neurons in the network. That is, the sum of the powers of the
connectivity matrix (SNAN) can be used to estimate the number of
extra spikes induced by stimulating a neuron or a group of neurons
[66]. When the embeddedness is estimated using the connectivity
matrix, we refer to it as structural embeddedness.
Although the connectivity matrix is an important determinant, so
far it has not been possible to associate a particular graph property
alone with embeddedness because synaptic and cellular properties,
ongoing activity, neuromodulators, etc. also shape the impact of a
neuron on the dynamics of the network. The experimental estimate
of extra spikes implicitly includes all these other factors as well.
When the effective connectivity matrix Aeff (measured from the
neuronal activity) is known, the effective embeddedness could be
estimated using the powers of Aeff. Finally, graph-theoretical
measures such as out-degree, k-shell index, eigenvalue centrality
etc. are also related to the embeddedness (Figure 4). For details see
[42].
583
Opinion
Box 2. Controllability
Consider a network of neurons or networks:
l Il Al BU j
[I]
where l is the column vector of firing rates of the N neurons

(neuronal populations in an NoN), A is the connectivity matrix (size
N N), representing the connectivity among the neurons or the
neuronal populations, respectively, I is the identity matrix describing
the fact that the activity in a network would change in the absence of
inputs from other nodes and external sources, U (size R 1) represents the R external inputs, B is the connectivity of the neuronal
populations with the external inputs (size N R), and j is the noise in
each of the neuronal populations.
All activity states of an N-node network can be described in an Ndimensional space (Figure 3). Controllability determines whether a
linear system (Equation I) allows a transition from one dynamical
state to another in finite time [50]. The N NR controllability matrix
(C) is defined as:
C B AB A2 B . . . AN1 B
[II]
For a fully controllable system, the matrix C should have a full rank, in
other words, Rank(C) = N. This ensures that all dynamical states of
the system are accessible.
Because the exact values of the matrices A and B are not known
for most neuronal systems, C cannot be evaluated. However, the
notion of controllability can be extended to the concept of structural
controllability [51]. To estimate the structural controllability matrix
CS, all non-zero entries in the matrices A and B are replaced with 1 s,
reducing the connectivity matrices to binary adjacency matrices
[67]. If Rank(CS) = N, the system is controllable [51] and the
controllability of a network can be determined, even if the exact
values of the connection strengths are not known.
Furthermore, the concepts of driver nodes [52] and power
dominant set [53] allow us to calculate the minimal set of nodes
that need to be stimulated to control the network dynamics and
suffices to visit all activity states of the network.
Driver nodes: the set of the minimum number of nodes to achieve
full control over the network dynamics. To identify the driver nodes
we need to find the maximum matching in the network, which
refers to the maximum set of links that do not share starting or end
nodes [52]. A node is matched if a link in the maximum matching set
points towards it; otherwise, it is unmatched. The unmatched nodes
are the driver nodes that need to be directly stimulated to control the
network dynamics. For instance, in Figure 2C the first node P1 is
unmatched.
Power dominant set (PDS): when the nodes have self-couplings
(Figure 2C, dotted arrows), every node could become a driver node
[53]. For such systems the control nodes are the minimum number of
nodes from which the rest of the nodes are only one link away [53]. In
Figure 2C, the nodes P1, P3, P5 or P1, P2, P4 constitute the PDS.
paradigm based on the concept of controllability (Box 2),

which might be a promising candidate to assist in understanding the function and dynamics of neuronal networks.
Interestingly, in this paradigm we could potentially avoid
the combinatorial explosion. Based on the dynamical description of neuronal network activity (Equation I in Box
2), the function of the brain could be considered as the
ability to find desired trajectories of the network activity
state (Box 2) because different behavioral states can be
mapped onto these activity states. Following this interpretation, a more pertinent question in understanding brain
function would be to know how the overall system dynamics could be controlled, rather than to determine how one
particular neuron population affects the activity of the
others.
However, even in this more utilitarian view, the problem still arises concerning which specific nodes should be
584
stimulated, and how to steer the network to a desired

state, even when the connectivity matrix A is known. A
trivial, but biologically non-implementable, solution to
control the network dynamics is to stimulate all nodes
externally. By contrast, a systematic search for the set
of nodes to stimulate for controlling the system leads to the
afore-described combinatorial explosion.
Engineers have successfully applied the concept of controllability to predict whether a given system can be
steered from one dynamical state to another in finite time
[50] (Box 2). For a dynamical system, controllability
depends on the matrices A and B. Fortunately, in the
absence of exact values of A and B, the notion of structural
controllability (Box 2) [51] can be effectively used to determine the controllability of a system, and it is possible to
identify a minimum set of nodes, the stimulation of which
is sufficient to control the full network state [52]. Thus,
equipped with the knowledge of A, we can bypass the brute
force approach and identify the driver nodes whose stimulation can control the dynamical repertoire of the neuronal network.
The estimation of the driver nodes explicitly assumes
that the nodes/neurons do not have self-connections, which
is true for most neurons. However, at the system dynamics
level the self-connections capture the intrinsic dynamics of
the nodes in other words, node dynamics in the absence of
influences from other nodes and neuron refractoriness.
Ignoring the intrinsic dynamics implicitly means that
the activity state of the node does not change when there
are no influences from other nodes (i.e., l = 0 or infinite
time constant) [53]. Typically, the activity of biological
neurons/networks decays to zero in the absence of external
inputs. Thus, the absence of self-connections can only be
justified in restricted cases, for example, when the dynamics of the constituent networks of a NoN exhibit attractor
dynamics and can exhibit asynchronous-irregular self-sustained activity [54]. Furthermore, it has been shown that
the power dominant set (PDS) (Box 2) is the set of driver
nodes when intrinsic dynamics and self-connections are
included in the dynamics [53]. In this context it is important to note that hubs, which are typical choices for external brain stimulation, usually are not part of the driver
nodes and PDS [52].
Extending the framework of controllability to neuronal
networks, we argue that the concepts of driver nodes [52]
and the PDS [53] have emerged as interesting alternatives
for choosing the nodes to be stimulated. Once the appropriate set of driver nodes is known, the controllability
Gramian [55] can be used to determine the input pattern
needed to drive the system to a desired state. Because the
number of neurons in a set of driver nodes or PDS is
typically substantially less than the number of nodes in
the network, the total number of stimulations required will
always be significantly less than Bells number. However,
the framework of controllability still requires simultaneous manipulations of multiple nodes. Therefore, we will
certainly need technology that is capable of varying the
activity of more than few nodes independently. In addition
to the notion of controllability, other graph-theoretical
measures [56,57] may also be used to make educated
choices of the stimulation sites.
Opinion
Concluding remarks
The considerations outlined above identified two major
pitfalls when applying selective activity modulation to
understanding brain function: (i) the need for simultaneous control of the activity of multiple neuronal populations leads to a severe combinatorial explosion; (ii) using
gene expression, firing patterns, morphological or spatial
location criteria alone to define functional groups of neurons is highly ambiguous. Interestingly, in other scientific
disciplines it has been realized that the seemingly intuitive
approach of selective activity modulation of parts of a
complex system to study its function can be misleading.
For instance, systems biologists now agree that genes
themselves act as a complicated network of dependencies,
and that single-gene/single-function mapping is the exception rather than the rule [58]. We argue that the dynamic
and nonlinear nature of interactions within large neuronal
networks in the mammalian brain makes it unlikely that
single-neuron-type/single-function relationships hold for
these systems, either. Recent cross-modal studies even
suggest that the separation between areas concerning
their involvement in different functions may be less clear
than previously thought [2831].
Instead, brain function is more likely to be the result of
coordinated activity distributed over multiple brain areas.
Assuming that brain function can be understood as an
interaction of subnetworks of different types of neurons
ignores this insight, as well as the rich repertoire of activity
dynamics that can be displayed by neuron subtypes
depending on the activity of the embedding network
[59]. Finally, the approach of defining cell populations
based on currently available markers can only be a starting
point and needs to be complemented by more sophisticated
criteria. Embeddedness, graph-theoretical measures, and
criteria derived from linear and nonlinear control theory
[60] may be promising candidates in this respect.
Future directions
We have shown that knowing the network connectivity of
neurons and neuronal populations can help in choosing the
most appropriate network node(s) for activity modulation
to help understand the function and dynamics of networks
in the brain. Instead of needing to go painstakingly
through all possible combinations, such approaches (e.g.,
based on controllability) could potentially evade some of
the inherent limitations of selective activity modulation
approaches. Indeed, recent developments of optogenetic
tools provide interesting avenues in this direction. Hence,
future research on the development of experimental tools
should be driven by the following goals:
(i) To define tools that can be used to control the activity
of multiple neuronal populations simultaneously.
(ii) To identify biomarkers for classifying neurons
according to their recent spiking activity [43,61].
(iii) To extract the functional and anatomical connectivity
of different types of neurons and neuronal populations, and to use these to measure their embeddedness and the controllability of the network [42].
(iv) To design selective activity modulation experiments
based on the network-related features extracted with
the above-mentioned approaches.
In parallel, it is also important to develop new computational models, where selective activity modulation-based
experimental approaches can be tested under fully controlled conditions. As a starting point, we provide an online
system (Neural System Prediction and Identification Challenge, nuSPIC) for extracting the function of relatively
small networks of spiking neurons designed to perform a
specific task. Its web interface provides several tools for
performing a variety of experiments, including selective
activity modulation. We believe that nuSPIC provides
useful models for calibrating the efficacy of experimental
approaches and to help interpret their results. Once we
have tools to control selectively the activity of multiple
neuronal populations simultaneously, and the knowledge
of their mutual connectivity, we will be in a position to
develop a deeper understanding of their contribution to
brain function.
Acknowledgments
We thank Nikos Logothetis, Moshe Abeles, Stefan Rotter, and Yexica
Aponte for helpful discussions and comments on the initial version of the
manuscript. Partial funding by German Federal Ministry of Education
and Research (BMBF) grant 01GQ0420 to BCCN Freiburg, 01GQ0830 to
the Bernstein Focus Neurotechnology (BFNT) Freiburg/Tuebingen, and
the BrainLinks-BrainTools Cluster of Excellence funded by the German
Research Foundation (DFG) grant EXC 1086, is gratefully acknowledged.
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Introduction. Maarten van Steen
Review
Sugar for the brain: the role of glucose

in physiological and pathological brain
function
Philipp Mergenthaler1, Ute Lindauer2, Gerald A. Dienel3, and Andreas Meisel1
1
Department of Experimental Neurology and Department of Neurology, Center for Stroke Research, NeuroCure Cluster of
Excellence, Charite University Medicine Berlin, Berlin, Germany
2
Experimental Neurosurgery, Department of Neurosurgery, TUM-Neuroimaging Center, Technical University Munich, Munich
Cluster for Systems Neurology (SyNergy), Munich, Germany
3
Department of Neurology, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock,
AR, USA
The mammalian brain depends upon glucose as its main

source of energy, and tight regulation of glucose metabolism is critical for brain physiology. Consistent with its
critical role for physiological brain function, disruption of
normal glucose metabolism as well as its interdependence with cell death pathways forms the pathophysiological basis for many brain disorders. Here, we review
recent advances in understanding how glucose metabolism sustains basic brain physiology. We synthesize
these findings to form a comprehensive picture of the
cooperation required between different systems and cell
types, and the specific breakdowns in this cooperation
that lead to disease.
Nobody realizes that some people expend tremendous energy merely to be normal. Albert Camus,
Notebooks 19421951.
Glucose metabolism: fueling the brain

The mammalian brain depends on glucose as its main
source of energy. In the adult brain, neurons have the
highest energy demand [1], requiring continuous delivery
of glucose from blood. In humans, the brain accounts for
approximately 2% of the body weight, but consumes approximately 20% of glucose-derived energy, making it the
main consumer of glucose (approximately 5.6 mg glucose
per 100 g human brain tissue per minute [2]). Glucose
metabolism provides the fuel for physiological brain function through the generation of ATP, the foundation for
neuronal and non-neuronal cellular maintenance, as well
as the generation of neurotransmitters. Therefore, tight
Corresponding author: Mergenthaler, P. (philipp.mergenthaler@charite.de).
Keywords: glucose metabolism; metabolic coupling; apoptosis; brain-body axis;
metabolic brain disease.
Glossary
Autophagy: an intracellular recycling pathway that can be activated under
conditions of metabolic stress to inhibit cell death. It involves the lysosomal
degradation of cytoplasmic proteins or entire organelles for catabolic
regeneration of nutrient pools [61].
Bloodbrain barrier (BBB): the permeability barrier arising from tight junctions
between brain endothelial cells, restricting diffusion from blood to brain. Entry
into the brain is limited to molecules that can diffuse across membranes (e.g.,
oxygen and other gases, or lipid-permeable compounds) or have transporter
molecules (e.g., glucose transporters). Neuroactive compounds (e.g., glutamate or adrenalin) in the blood are restricted from entering into the brain.
Functional activation: a response by the brain to a specific stimulus (e.g.,
sensory stimulation) that increases cellular activity and metabolism above the
resting and/or baseline value before onset of the stimulus. Brain activation
has the same meaning but is a more general term that includes increased
activity during abnormal or disease states.
Glutamateglutamine cycle: the release of the neurotransmitter glutamate
from excitatory neurons, its sodium-dependent uptake by astrocytes, its
conversion to glutamine by glutamine synthetase in astrocytes, the release
of glutamine and uptake into neurons followed by the conversion to glutamate
by glutaminase and its repackaging into synaptic vesicles.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH): a glycolytic enzyme that
reduces NAD+ to NADH and converts D-glyceraldehyde-3-phosphate to 1,3bisphospho-D-glycerate, an intermediary metabolite in the generation of pyruvate.
Glycolysis: a cytoplasmic pathway for metabolism of one molecule of glucose
to produce two molecules of pyruvate, with phosphorylation of 2 ADP to form 2
ATP and reduction of 2 NAD+ to 2 NADH. Cytoplasmic oxidation of NADH can
be achieved by conversion of pyruvate to lactate by the LDH reaction or via the
MAS (see Figure 2A in main text). The MAS is required to generate pyruvate for
oxidation in the TCA cycle, whereas LDH removes this substrate from the cell.
Net production of lactate in the presence of adequate levels and delivery of
oxygen is sometimes termed aerobic glycolysis, contrasting the massive
production of lactate under hypoxia or anoxia (anaerobic glycolysis).
Hexokinase (HK): the enzyme catalyzing the first step in glucose metabolism: the
irreversible conversion of glucose to Glc-6-P in an ATP-dependent reaction. The
brain has different HK isoforms that have specific functions. HKI is the major
isoform in brain for the glycolytic pathway; it has a broad substrate specificity and
is feedback-inhibited by Glc-6-P. HKII is a minor, hypoxia-regulated isoform in the
brain that controls neuronal survival depending on the metabolic state. HKIV
(glucokinase, GK) is a minor isoform of hexokinase in the brain that has an
important role in glucose-sensing neurons; it is specific for glucose and is not
inhibited by Glc-6-P.
Ketogenic diet: a diet that has a high fat and low carbohydrate content so that
plasma levels of ketone bodies (acetoacetate and b-hydroxybutyrate) increase
and serve as alternative oxidative fuel.
Metabolic coupling: a synergistic interaction between different cells or cell
types in which compounds produced in one cell are used by another cell.
Neurovascular unit: groups of neurons, astrocytes, endothelial cells, vascular
smooth muscle cells, and pericytes that are involved in local signaling
activities, metabolic interactions, and regulation of blood flow.
Tricarboxylic acid (TCA) cycle: a mitochondrial pathway for oxidation of pyruvate
to produce 3 CO2 and generate FADH2 and NADH that are oxidized via the electron
transport chain with conversion of oxygen to water and formation of approximately 32 ATP per glucose molecule. This ATP yield is less than the theoretical
maximum due to proton leakage across the mitochondrial membrane.
587
Review
(E)
Cell
fate
(D)
Oligodendrocytes
(C)
Hypothalamus
Neurons
Glc
AgRP
Lac
Astrocytes
POMC
GLUT1
Endothelial
cells (BBB)
Liver
Gastrointesnal
tract
Ne
Pericytes
(B)
uro
e
s i g ndoc
n a rin
ls
e
(A)
Pancreas
Glucose
Aerent and
eerent vagus
Figure 1. The role of glucose in brain function. Glucose (Glc) is the main source of energy for the mammalian brain. (A) Specialized centers in the brain, including proopiomelanocortin (POMC) and agouti-related peptide (AgRP) neurons in the hypothalamus, sense central and peripheral glucose levels and regulate glucose metabolism
through the vagal nerve as well as neuroendocrine signals. (B) Glucose supply to the brain is regulated by neurovascular coupling and may be modulated by metabolismdependent and -independent mechanisms. Glucose enters the brain from the blood by crossing the bloodbrain barrier (BBB) through glucose transporter 1 (GLUT1), and
(C) glucose and other metabolites (e.g. lactate, Lac) are rapidly distributed through a highly coupled metabolic network of brain cells. (D) Glucose provides the energy for
neurotransmission, and (E) several glucose-metabolizing enzymes control cellular survival. Disturbed glucose metabolism on any of these levels can be the foundation for
the development of a large variety of disorders of the brain (see section on Disease mechanisms).
regulation of glucose metabolism is critical for brain physiology and disturbed glucose metabolism in the brain
underlies several diseases affecting both the brain itself
as well as the entire organism.
Here, we provide a comprehensive overview of the functional implications and recent advances in understanding
the fundamental role of glucose metabolism in physiological and pathological brain function. Although brain energy
metabolism has been investigated for decades, certain
aspects remain controversial, in particular in the field of
energy substrate consumption and utilization. It is beyond
the scope of this review to resolve these controversies;
rather, it is our aim to highlight conflicting concepts and
results to stimulate discussion in key areas. To this end, we
review the bioenergetics of neurotransmission, the cellular
composition of a metabolic network, the regulation of
cerebral blood flow (CBF), how peripheral glucose metabolism and energy homeostasis are sensed and controlled by
the central nervous system (CNS), and the tight regulation
of cellular survival through glucose-metabolizing enzymes.
Glucose is required to provide the precursors for neurotransmitter synthesis and the ATP to fuel their actions, as
well as the energy demands of the brain that are not
related to signaling. Cellular compartmentation of glucose
transport and metabolism is intimately related to local
regulation of blood flow, and glucose-sensing neurons govern the brainbody nutrient axis. Glucose metabolism is
connected to cell death pathways by glucose-metabolizing
588
enzymes. Thus, disruption of pathways of glucose delivery

and metabolism leads to debilitating brain diseases. We
highlight the multifaceted role and complex regulation of
glucose metabolism in the CNS as well as the physiological
and pathophysiological consequences of balanced and disturbed glucose metabolism (Figure 1).
Glucose metabolism: the bioenergetic basis for
neurotransmission
The largest proportion of energy in the brain is consumed for
neuronal computation and information processing [3]; for
example, the generation of action potentials and postsynaptic potentials generated after synaptic events (Figure 1D),
and the maintenance of ion gradients and neuronal resting
potential [1,4]. Additionally, glucose metabolism provides
the energy and precursors for the biosynthesis of neurotransmitters (for a comprehensive overview, see [5]). Importantly,
astrocytic glycogen seems to be directly relevant for learning
[6]. Furthermore, the glycolytic end-product lactate appears
to have a role in long-term memory formation [7], although
the exact mechanism has not yet been established. Lactate
injections [7] alter the intracellular redox state and pH due to
co-transport of H+ with lactate, and lactate receptors may
also have a role in linking brain energy metabolism and
neurotransmission [8,9]. However, oxidative metabolism in
both neurons and astrocytes appears to contribute to sustained learning effects after training, and glycogen can
supply carbon for synthesis of glutamate during learning [6].
Review
(A)
(B)
Glc
GLUT
Glc
GLUT1
BBB
Cytosol
Glycolysis
Glyc
(astrocytes)
MCT1
Glc
Glc
Pentose-phosphate-pathway
Glc-6-P
Glc
Lac release
from brain
GLUT3
NADP+
GLUT1
PPP
Glc
NADPH
Fru-6-P
Glyc
Glc-6-P
NAD+
Glc-6-P
NALS
Gal-3-P
Pyr
Lac
MAS
NADH
MAS
Acetyl CoA
TCA
Glu
NADH
NAD+
Release
from cell
LDH
Lac
TCA
& ETC
Neuron
Gln
ANLS
Lac
MCT2
Pyr
Glc
Extracellular
uid
Glu-Gln
cycle
Lac
Pyr
MCT1, 4
Glu
Gln
TCA
Astrocyte
Oxidave metabolism
Figure 2. Generation of energy in the brain and three models for the fate of lactate derived from glucose metabolism in the brain. (A) Major pathways of glucose
metabolism. Hexokinase uses ATP to phosphorylate glucose (Glc) to glucose-6-phosphate (Glc-6-P) in the first irreversible step of the glycolytic pathway. Glc-6-P regulates
hexokinase activity by feedback inhibition [19], and it is a branch-point metabolite that has alternative metabolic fates. Glc-6-P can continue down the glycolytic pathway to
generate pyruvate that can then be used in mitochondria by oxidative metabolism via the tricarboxylic acid (TCA) cycle. It can also enter the pentose phosphate shunt
pathway (PPP) to generate NADPH for management of oxidative stress and precursors for nucleic acid biosynthesis and, in astrocytes, Glc-6-P is a precursor for glycogen.
Most of the glucose carbon derived from the PPP re-enters the glycolytic pathway downstream of Glc-6-P. The glycolytic pathway produces a net of 2 ATP per molecule of
glucose and oxidation of pyruvate via acetyl coenzyme A (acetyl CoA) in the TCA cycle produces approximately 30 ATP for a total of approximately 32 ATP. Formation of
pyruvate from glucose requires regeneration of NAD+ from NADH produced by the glyceraldehyde-3-phosphate dehydrogenase reaction by the malate-aspartate shuttle
(MAS). NADH cannot cross the mitochondrial membrane, and the MAS transfers cytoplasmic NADH to the mitochondria, where it is oxidized via the electron transport
chain (ETC). When glycolytic flux exceeds that of the MAS or the TCA cycle rate, or during hypoxic or anoxic conditions, NAD+ is regenerated by the lactate dehydrogenase
(LDH) reaction that converts pyruvate to lactate. Because intracellular accumulation of lactate would cause reversal of the LDH reaction, lactate must be released from the
cell by monocarboxylic acid transporters (MCT). Exit of lactate eliminates pyruvate as an oxidizable substrate for that cell and limits the ATP yield of glycolysis per molecule
glucose to two. (B) Three models for the fate of lactate generated in the brain from blood-borne glucose or astrocytic glycogen. The astrocyte-to-neuron lactate shuttle
(ANLS) was proposed on the basis of glutamate-evoked increases in glucose utilization and lactate release by cultured astrocytes (reviewed in [29]). In brief, the model
states that Na+-dependent uptake of the neurotransmitter glutamate from the synaptic cleft by astrocytes generates a demand for 2 ATP in astrocytes, one to extrude Na+
and one to convert glutamate into glutamine in the glutamateglutamine (GluGln) cycle. The model states that this ATP is generated by the glycolytic pathway and is
associated with release of lactate from astrocytes and its uptake by nearby neurons, where it is oxidized. Thereby, astrocyteneuron metabolic coupling is linked with the
glutamateglutamine cycle and excitatory neurotransmission. Thus, during brain activation, glycolytic upregulation is stated to occur in astrocytes, with astrocyte-derived
lactate providing the major fuel for neurons. The neuron-to-astrocyte lactate shuttle (NALS) is based on the kinetics of glucose uptake into brain cells in response to
increased metabolic demand and different model assumptions compared with the ANLS [27]. Here, glucose is predicted to be predominantly taken up into neurons due to
their high energy demand and the higher transport rate of the neuronal glucose transporter 3, GLUT3, compared with the astrocytic glucose transporter 1, GLUT1 [16].
Lactate is posited to be generated by neurons and taken up by astrocytes. The lactate release model [5] is based on the observed mismatch between total glucose utilization
and oxidative metabolism and measured lactate release from brain during brain activation in vivo. If lactate were produced and locally oxidized, total and oxidative
metabolism would be similar in magnitude. However, the increase in oxidative metabolism varies with experimental condition and pathways stimulated, and it is much less
than that of total glucose utilization [5]. Astrocytes have a faster and greater capacity for lactate uptake from extracellular fluid, and for lactate dispersal among gap junctioncoupled astrocytes compared with neuronal lactate uptake and shuttling of lactate to neurons [17]. Astrocytic endfeet surround the vasculature, and can discharge lactate to
perivascular fluid for efflux from brain.
It has been suggested that action potentials have been

rendered highly efficient through evolution [10] and, thus,
most of the energy consumed in the brain is used on synaptic
activity [3,10,11]. The human cortex alone requires approximately 31023 ATP/s/m3 [1], and the energy expenditure to
release one synaptic vesicle is calculated to be approximately 1.64105 molecules ATP [3]. Consequently, a model of
energy use in the brain suggests that a considerably larger
amount of energy is spent in the gray matter compared with
the white matter [12]. In essence, the brain increases its
utilization of glucose upon activation [13].
Glucose uptake in the brain: how are neurons and
astrocytes fed?
Dependence of the brain on glucose as its obligatory fuel
derives mainly from the bloodbrain barrier (BBB; see
Glossary), and its selective permeability for glucose in the
adult brain. Glucose cannot be replaced as an energy source,
but it can be supplemented, as during strenuous physical

activity when blood lactate levels are elevated [14] or during
prolonged starvation [15] when blood levels of ketone bodies
are elevated and BBB monocarboxylic acid transporter
(MCT) levels are upregulated. Because entry of neuroactive
compounds (e.g., glutamate, aspartate, glycine, or D-serine)
into the brain is restricted by the BBB, these compounds
must be synthesized from glucose within the brain. The BBB
and its transport properties sharply contrast with muscle
and liver that do not have tight junctions between their
vascular endothelial cells and have different transporter
levels for various compounds, enabling these organs to
metabolize glucose, monocarboxylic acids, fatty acids, amino
acids, and ketone bodies.
The large blood-to-brain concentration gradient drives
the facilitative transport of glucose across the endothelial
membranes via glucose transporter 1 (GLUT1) into extracellular fluid (Figures 1B and 2). The steady-state brain
589
Review
tissue glucose concentration is approximately 20% of that
in arterial plasma. GLUT1 further mediates glucose uptake from extracellular fluid into astrocytes, oligodendroglia, and microglia, whereas GLUT3, which has a higher
transport rate than GLUT1, facilitates neuronal glucose
uptake (Figures 1C and 2B) [16]. Glucose transport capacity exceeds demand over a wide range, and the higher
transport rate of GLUT3 ensures that neurons have sufficient glucose supplies under varying glucose levels and
different activity states [5]. Although astrocytes are generally believed to be involved in the uptake and distribution of brain metabolites [3,17,18], modeling predicts that
most glucose diffuses from endothelial cells through the
gaps between the surrounding astrocytic endfeet, and
throughout the extracellular fluid to more distant brain
cells, facilitating rapid GLUT3-mediated uptake into neurons [16]. However, some glucose may also be taken up into
astrocytic endfeet, followed by its diffusion down its concentration gradients to other gap junction-coupled astrocytes, with release to extracellular fluid at sites more
distant from the capillary [3,17,18].
Local rates of glucose utilization are driven by functional activities (Figure 1D) that consume ATP and generate
ADP, which is an obligatory co-substrate for energy-producing reactions. Intracellular glucose is phosphorylated
by hexokinase I (HKI) to form glucose-6-phosphate (Glc-6P), thereby trapping the molecule in the cell and, thus,
creating a sink that draws more glucose into the cell
(Figure 2A). The intracellular glucose pool size is maintained as the net balance between rates of its influx, efflux,
and metabolism. The Km (half-saturation constant) of HKI
for glucose is low [19] and, therefore, HKI can operate at
maximal velocity as long as the intracellular glucose
exceeds approximately 0.81 mmol/L. Glc-6-P governs
HKI activity by feedback inhibition, such that the in vivo
activity of HKI in resting, awake brain is only approximately 5% of its maximal capacity measured in vitro. Thus,
dis-inhibition of HKI by consumption of Glc-6-P can stimulate HKI flux by up to 20-fold, a capacity that greatly
exceeds the four-to sixfold rise in the cerebral metabolic
rate of glucose (CMRglc) during seizures and ischemia
[20,21]. Glc-6-P is not only metabolized via the glycolytic
pathway to generate ATP, but is also the substrate for the
pentose phosphate shunt pathway (PPP) that generates
NADPH to manage oxidative stress and to synthesize
nucleic acid precursors (Figure 2A). Phosphofructokinase
is considered to be the major regulator of the glycolytic
pathway due to its allosteric regulation by many metabolites (e.g., inhibition by ATP, citrate, H+, and activation by
ADP, AMP, fructose-6-P, fructose-1,6,-P2, fructose-2,6,-P2,
and ribose-1,5,-P2) that act in concert to integrate the
fluxes of the glycolytic and tricarboxylic acid (TCA) cycle
pathways. Glucose metabolism is also the source for biosynthesis of other compounds required by the brain, including complex carbohydrates that are components of
glycoproteins and glycolipids, amino acids, one-carbon
donors for methylation reactions, and the supply of neurotransmitter precursors [5,22]. To summarize, CMRglc is
controlled in each cell by the rate of ADP production (i.e.,
ATP demand) and regulation of rate-controlling enzymes
by metabolites.
590
In astrocytes, Glc-6-P is the precursor for glycogen, a

polymer comprising glucose residues. Glycogen is the only
energy reserve in the brain (Figure 2A,B). In normal brain,
glycogen turnover occurs at normal glucose levels, consistent with its role as an important local energy buffer for
astrocytes, and it is mobilized by functional activation or
energy deficits [23,24]. Modeling predicts that glycogenolysis reduces astrocytic glucose utilization by maintaining
levels of Glc-6-P sufficient to sustain high feedback inhibition of HKI, thereby sparing glucose for neurons [25].
During severe hypoglycemia or aglycemia, low rates of
glycogenolysis equivalent to only a few percent of normal
glucose utilization rates are sufficient to prolong neuronal
functions [5,22].
To maintain glycolytic flux, NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) must be
oxidized. Regeneration of NAD+ can occur by two mechanisms: the malateaspartate shuttle (MAS) or the lactate
dehydrogenase (LDH) reaction. The MAS is required for
generation of pyruvate as an oxidative fuel because LDH
activity is associated with lactate release (Figure 2A, see
figure legend for details).
In resting awake brain, most of the glucose is completely
oxidized to CO2 and water, and nearly a stoichiometric
amount of O2 (i.e., 6 O2 per glucose) is consumed. Due to
biosynthetic reactions and slight efflux of lactate from the
brain, the oxygen:glucose consumption ratio is generally
approximately 5.55.8. Under different conditions ranging
from deep anesthesia to the conscious state, the rate of
neuronal glucose oxidation is approximately proportional
to glutamatergic neurotransmission [26], indicating that
the rate of the major energy-producing pathway in neurons
(the TCA cycle, Figure 2) is directly related to the energy
demands associated with the flux through the glutamate
glutamine cycle (Figure 2B) [5].
During brain activation, glycolysis is usually preferentially upregulated compared with oxygen consumption [5],
and the oxygen:glucose utilization ratio falls. Because this
phenomenon occurs in normal, normoxic subjects that have
excess oxygen delivery to the brain, it is sometimes called
aerobic glycolysis to distinguish it from the large rise in
glycolysis during hypoxia or anoxia. Stimulation of glycolysis generates increased amounts of lactate that cannot
only be released from brain (Figure 2), but can also have
various important functions, including serving as supplementary oxidative fuel for astrocytes and neurons, modulating redox signaling of metabolic state, regulating blood
flow [5], or functioning as a mediator of metabolic information [9].
Metabolic interactions among astrocytes and neurons,
and lactate shuttling
Both neurons [16,27,28] and astrocytes [18,29] have been
described as the main consumers of glucose. The cellular
contributions to overall glucose utilization has been a
controversial issue for decades because current technology
does not have adequate spatiotemporal resolution to quantify metabolic activity in single cells in vivo. Two conflicting
concepts describe the predominant cellular fate of glucose
during brain activation and propose different directions
and magnitudes of shuttling of lactate among neurons and
Review
astrocytes. A third model is derived from demonstration of
substantial lactate release from brain, irrespective of the
originating cell type (Figure 2B) [5,17].
The astrocyte-to-neuron lactate shuttle (ANLS;
Figure 2B) claims that glutamatergic neurotransmission
stimulates astrocytic lactate production that serves as an
important neuronal fuel during activation [29]. However,
this notion remains controversial because glutamate does
not stimulate glycolysis in most astrocyte preparations, the
cellular origin of lactate in vivo is unknown, substantial
lactate oxidation by neurons has not been demonstrated
during brain activation, and studies supporting this model
[29] have been challenged [5,22]. Furthermore, the neurotransmitter glutamate itself may directly support energetics of perisynaptic astrocytes, because the glial glutamate
aspartate transporter (GLAST) forms a macromolecular
complex linking glutamate uptake with its oxidation [30]
which can provide ATP to meet the astrocytic energy
demands. Glutamate oxidation at the site of its uptake
eliminates the need for glycolysis to generate ATP and the
ANLS [31]. The notions that glycogen-derived lactate is
necessary as the bioenergetic basis for neuronal memory
consolidation [7,32] and that glucose-derived lactate from
oligodendrocytes is required to support axons [33,34] demand direct experimental proof of the magnitude and
contribution of lactate shuttling compared with other energy sources.
The neuron-to-astrocyte lactate shuttle (NALS,
Figure 2B) is based on different assumptions than the
ANLS and accounts for the kinetics of glucose transporters
in neurons and astrocytes. The NALS model predicts
predominant neuronal glucose uptake during activation,
with transfer of lactate to astrocytes such that the direction
of metabolite flux can be context-dependent [16,27]. Astrocytes have key roles in lactate uptake from extracellular
fluid and lactate dispersal to other astrocytes via gap
junctional communication; these processes occur at rates
that are two- to fourfold faster than lactate uptake by
neurons or astrocytic transfer of lactate to neurons [17].
Thus, astrocytes are poised to take up lactate from interstitial fluid and release lactate from their endfeet to perivascular fluid for discharge to lymphatic drainage systems
and venous blood [5,17]. The total glucose utilization substantially exceeds oxidative metabolism of glucose, with
release of sizeable quantities of lactate released from
activated brain (Figure 2B) [5,22].
The use of lactate as a supplemental fuel varies with its
availability and physiological state of the subject. In sedentary subjects, the brain lactate level exceeds that in
blood, facilitating the efflux of lactate from activated brain
regions to blood. By contrast, strenuous physical activity
increases glycolysis in muscles and increases blood lactate
levels, reversing the direction of the lactate gradient from
blood to brain and flooding the entire brain with lactate.
Under these conditions, lactate is oxidized in the brain in
amounts that increase with blood lactate level. However,
lactate oxidation in brain during exercise accompanies
increased CMRglc and release of brain-derived lactate to
blood [35], suggesting separate routes for lactate efflux and
influx. Thus, increased blood lactate level represents a
glucose-sparing physiological state in which use of a
supplemental oxidative fuel helps maintain availability

of glucose for the glycolytic and pentose phosphate shunt
pathways that provide critical functions for the brain.
Glucose metabolism and the regulation of CBF
Under resting conditions, local CBF is highest in brain
regions with the highest local glucose metabolism. All
brain regions are metabolically active at all times, but
there is a large heterogeneity among various brain structures. During functional activation, the increase in local
CBF usually parallels the increase in CMRglc, whereas the
increase in oxygen metabolism is lower [36]. However,
there is at least one example where, under peripheral
somatosensory stimulation, local CBF in the ipsilateral
cortex can decrease despite increased CMRglc [37].
This close correlation of CBF and CMRglc (and, to a
lesser extent, the cerebral metabolic rate of oxygen,
CMRO2) demands highly dynamic and fine-tuned mechanisms to adapt local glucose and oxygen delivery and
carbon dioxide removal via the blood to the actual demand
of active brain regions. The traditional metabolic hypothesis of neurovascular coupling [38], mediated by vasoactive
metabolic products such as lactate, CO2/H+, or adenosine,
has recently been replaced by the currently favored neuronal hypothesis. It suggests that neuronal energy demand is communicated to the vasculature (either
directly or indirectly by astrocytes) within the neurovascular unit in an anticipatory, feed-forward manner by
vasoactive neurotransmitters or products of synaptic signaling, and that vasodilation occurs independently of glucose metabolism-induced signaling (Figure 1B; reviewed in
[39]). The proposed feed-forward regulation is a reliable
basis for fast adaptation of the regional blood flow to the
actual local level of neuronal activity, avoiding risky drops
in glucose and oxygen concentrations, which might occur
during exclusive metabolic regulation. However, recent
studies suggest that changes in the lactate:pyruvate ratio
and, therefore, the cytosolic NADH:NAD+ ratio [40] or
increased lactate production [41,42] are at least partially
responsible for vasodilation during neuronal activation.
Thus, neurovascular coupling regulated by feed-forward
signaling may be supplemented or modulated by metabolism-dependent mechanisms [43].
Experimental studies show that direct glucose-sensing
mechanisms are unlikely to be involved in the activityinduced regulation of CBF. Neither hyperglycemia nor
mild-to-moderate hypoglycemia significantly changes the
blood flow responses to functional activation [44,45]. In
addition, during acute hypoglycemia, resting CBF only
increases significantly when blood and brain glucose are
dramatically reduced (for a detailed review, see [46]).
The consequences of impaired adaptation of CBF to
CMRglc are under active investigation. Artificial reduction
of the CBF response during functional activation had no
impact on evoked neuronal activity in an acute experimental setting [47]. However, it is assumed that chronic global
hypoperfusion of the brain may be not only a consequence,
but also an early cause of neurodegeneration in vascular
dementia and Alzheimers disease (AD) [48] (see below).
Thus, fine-tuned CBF-CMRglc-CMRO2 regulation is indispensable for healthy brain.
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Brainbody axis: central control over peripheral glucose

metabolism
Given that the brain relies on exogenous nutrient supplies,
it is not surprising that it can increase these supplies,
especially glucose, by regulating systemic homeostasis
and food intake [49,50] (Figure 1A). Specialized neuronal
networks in the hypothalamic arcuate nucleus and in the
hindbrain sense, integrate, and regulate energy homeostasis and glucose levels, and signal to the periphery through a
dedicated neuronal network [4951]. Indeed, central glucose sensing and peripheral regulation of glucose metabolism are tightly linked [52]. In addition to their peripheral
action, hormones [4951], including insulin [53] and glucagon-like peptide-1 (GLP-1) [54,55], mediate peripheral glucose uptake through neuronal signaling cascades.
Furthermore, brain insulin receptors [56] and other metabolic receptors and transporters, such as glucose transporters [57,58], mediate metabolic signaling in the brain.
In the hypothalamus, pro-opiomelanocortin (POMC)
[59], melanin-concentrating hormone (MCH) [60], and neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons
sense peripheral glucose levels and regulate energy metabolism in an antagonistic fashion [49]. Defective neuronal maintenance in these cells has severe consequences for
(A)
peripheral metabolism. Defective autophagy [61] in POMC

neurons can lead to lifelong metabolic defects, such as
peripheral glucose intolerance and obesity [62,63]. However, disrupted autophagy in glucose-sensing AgRP neurons
promotes leanness and reduced food intake [64]. Interestingly, glucokinase (GK) is expressed in select neuronal
populations in hypothalamic glucose-sensing formations
[65], and a protein complex containing GK and Bcl-2
antagonist of cell death (BAD) might regulate peripheral
[66], as well as central glucose sensing.
Besides hormones and nutrients, both afferent and
efferent metabolic signals link hindbrain nuclei and the
gastrointestinal tract through the vagal nerve [51]. Thus, a
complex interplay between the brain, in particular the
hypothalamus, and peripheral systems control glucose
supply to the brain [49,51], peripheral nutrient uptake
[49,51] and utilization [67], as well as feeding [6769].
Notably, the regulation of energy homeostasis through
the brain is not limited to glucose metabolism, but also
includes most other major energy-producing systems with
close links between these systems [49,51,64,70]. The mechanisms of the brainbody interaction in the regulation of
glucose metabolism have recently been reviewed in greater
detail [49,50].
(B)
HKII
x
O2
Glc
PEA15
Glc
GLUT
TIGAR
VDAC
OMM
Cytosol
IMM
Glc
Cell
survival
HKII
Glc-6-P
PEA15
PPP
NADPH
(C)
HKII
Fru-6-P
TIGAR
PEA15 O2
Glc
TIGAR
Gal-3-P
GAPDH
OMM
Pyr
IMM
Lac
TCA
VDAC
Cell
death
Figure 3. The connection between glucose metabolism and cell death. (A) Glucose metabolism and cell death regulation intersect at several levels. Glucose-metabolizing
enzymes, including hexokinase II (HKII), glucokinase (GK), the fructose-2,6-bisphosphatase Tp53-induced glycolysis and apoptosis regulator (TIGAR), glyceraldehyde-3phosphate dehydrogenase (GAPDH), and others, are involved in the regulation of cell death through different mechanisms. Phosphoprotein-enriched in astrocytes (PEA15)
might function as a molecular linker between HKII and TIGAR under certain conditions. Flux through the pentose phosphate pathway (PPP) generates NADPH, which is
important for neuronal redox environment and inhibits cell death. (B,C) The expression of HKII in neurons is upregulated under hypoxic conditions. Together with PEA15, it
functions as a molecular switch to regulate neuronal viability depending on the metabolic state [72]. HKII and PEA15 interact and bind to mitochondria through the outermitochondrial membrane voltage-dependent anion channel (VDAC). During hypoxia, HKII protects cells from cell death, whereas during glucose deprivation, where HKII
detaches from mitochondria and the interaction with PEA15 is destabilized, HKII promotes cell death [72]. HKII also interacts with TIGAR under hypoxic conditions [77].
Similar to PEA15, which increases the capacity of HKII to protect neurons, TIGAR increases the glycolytic activity of HKII. However, the exact mechanistic link is presently
unknown. Abbreviations: Gal-3-P, glyceraldehyde-3-phosphate; Glc, glucose; Glc-6-P, glucose-6-phosphate; GLUT, glucose transporter; Fru-6-P, fructose-6-phosphate; Lac,
lactate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; Pyr, pyruvate; TCA, tricarboxylic acid cycle; OMM, outer mitochondrial membrane; IMM, inner
mitochondrial membrane. HKII was rendered in PyMOL using structure 2nzt (RCSB Protein Data Bank).
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Glucose metabolism and the regulation of cell death
Glucose metabolism is evolutionarily linked to the regulation of cell death [71] (Figures 1E and 3A), and this link is
tightly controlled in a similar fashion in many cell types,
arguing for a universal role of coregulated metabolic and
apoptotic pathways. Neurons and cancer cells are among
the cell types that rely almost exclusively on glucose
metabolism for energy generation, and recent evidence
suggests that these cells use similar mechanisms to adapt
to substrate deprivation and promote survival [72,73].
Hexokinase II (HKII), a hypoxia-regulated HK isoform
in the brain, has been demonstrated to control neuronal
survival depending on the metabolic state [72] (Figures 1E
and 3). HKII restricts or inhibits apoptosis in a variety of
different cell types depending on whether it is bound to
mitochondria [72,74] and on the availability of glucose [72].
Furthermore, the capacity of HKII to phosphorylate glucose is involved in sensing the metabolic state of the cell
(Figure 3). In addition, HKII elicits its antiapoptotic function through a molecular interaction with phosphoprotein
enriched in astrocytes/phosphoprotein enriched in diabetes (PEA15/PED) [72]. HKII protects against neuronal cell
death after hypoxia [72] and in the presence of oxidative
stress [75]. However, HKII increases neuronal cell death
under glucose deprivation, thereby functioning as a molecular switch that regulates neuronal survival depending on
the metabolic state. Importantly, the capacity of HKII to
phosphorylate glucose and its interaction with PEA15 both
mediate this effect [72] (Figure 3B,C).
Intriguingly, controlled regulation of glucose metabolism protects both neurons and cancer cells from apoptosis
through related mechanisms [72,73], highlighting the universal importance of sensing the metabolic state of a cell. In
both cell types, HKII protects from cell death under hypoxic conditions [72], and increased activity of the PPP
provides the reducing environment for inhibiting cytochrome c-mediated apoptosis, thereby preventing cellular
damage through oxidative stress [73]. Furthermore, PPP
inhibition after selective experimental activation of glycolysis and the concomitant deprivation of NADPH triggers
apoptosis in neurons [76]. However, it has not been established whether the fructose-2,6-bisphosphatase Tp53-induced glycolysis and apoptosis regulator (TIGAR), which
promotes flux through the PPP and interacts with HKII
under hypoxic conditions [77], promotes preferential PPP
flux in neurons and thereby cooperates with HKII to
prevent cell death. Nevertheless, increased HKII activity
upon interaction with TIGAR [77] is reminiscent of the
increased capacity of HKII to inhibit neuronal apoptosis
when it is bound to PEA15 [72] (Figure 3B,C).
Finally, other enzymes of the glycolytic cascade, including GAPDH, have also been demonstrated to inhibit cell
death under certain conditions [78]. However, GAPDH has
also been suggested to mediate neuronal apoptosis after
DNA damage [79]. Thus, glycolytic enzymes can regulate
neuronal cell death in a context-dependent fashion, exerting both pro- and antiapoptotic effects.
Control of apoptosis by glucose-metabolizing enzymes
appears not to be a one-way street. As an example, the Bcl2 family member BAD interacts with GK (also known as
hexokinase IV) in liver and pancreatic b cells to modulate
apoptosis in response to changes in glucose levels [66].

However, it is presently unknown whether GK has a role in
regulating neuronal viability depending on the metabolic
state in select glucose-sensing neuronal populations (see
above). Furthermore, BclXL, an antiapoptotic Bcl-2 family
member, has been suggested to increase the metabolic
efficiency of neurons by decreasing a proton leak within
the F1FO ATPase and across the inner mitochondrial
membrane [80,81].
Disturbed metabolism is closely linked to cell death
pathways and autophagy [61]. Indeed, GAPDH [78], as
well as many players in the apoptotic cascades, regulate
autophagy [61]. However, whether these mechanisms
involve (dysfunctional) glucose sensing through glycolytic enzymes or if they are controlled by coregulated
apoptotic and/or autophagic pathways (e.g., by the
Bcl-2 family [82]), remains to be established. Thus,
disturbed signaling through these pathways is thought
to be the pathophysiological basis for a variety of
diseases (Box 1).
Disease mechanisms
Neurons are largely intolerant of an inadequate energy
supply and, thus, the high energy demand of the brain
predisposes it to a variety of diseases if energy supplies are
disrupted. Various CNS pathologies are the consequence,
and sometimes also the cause, of disturbed central or
peripheral glucose energy metabolism, which can be affected at almost every level of the cellular or biochemical
metabolic cascades (Figure 1). Changes in the glucose
metabolism of affected patients can efficiently be measured
by positron emission tomography (PET) imaging in a large
variety of clinical scenarios (Box 2).
Neuroglycopenia is a neurodevelopmental syndrome
characterized by mental retardation or developmental
delay, anomalous coordination and muscle tone, as well
as thalamocortical hypometabolism [83]. This syndrome is
further characterized by infantile drug-resistant seizures,
developmental retardation, and microcephaly in many
cases [84]. It can be caused by persistent hypoglycemia
during development or by deficiency in the major glucose
Box 1. Glucose metabolism, cell death, and

neurodegeneration
Glucose metabolism and the regulation of cell death are tightly
coupled [66,71,72] (Figure 3, main text).
Autophagy can be activated upon metabolic stress (e.g., starvation) of cells, as well as upon other stressors, including hypoxia
and inflammation, to promote cellular survival under these
conditions [61]. Autophagy, in turn, can be regulated by cell
death and metabolic pathways [61], including key regulators of
glucose metabolism [78].
Defective autophagy, oxidative stress, and bioenergetic stress
have been linked to the development of neurodegenerative
diseases [61,96,106].
Disrupted axonal nutrient supply and a defective metabolic
network are associated with neurodegeneration in the CNS and
peripheral nervous system [33,34].
Future research will elucidate the role of defective glucose
metabolism and the extent of the involvement of members of
the glycolytic cascade [72,7678] in the pathophysiology of
neurodegenerative diseases.
593
Review
Box 2. Glucose metabolism and functional brain imaging

Under physiological conditions, cerebral glucose metabolism is
tightly correlated with neuronal activity [13]. Therefore, imaging of
local CMRglc can be used to visualize areas of increased neuronal
activity. The most frequently used methods of brain metabolic
imaging are the detection of radiolabeled glucose by PET in vivo or
for diagnostic imaging (Figure I), and by ex vivo autoradiography
[107]. The glucose analogs [18F]fluoro-2-deoxyglucose or 2-deoxy[14C]glucose are injected intravenously, transported into the brain,
and phosphorylated by HK to 2-deoxyglucose-6-phosphate (2-DG-6P). Fluorescent 2-DG derivatives can be used for fluorescence imaging
in animal models [108], but quantitative determination of CMRglc with
the fluorescent analogs requires evaluation of the kinetics for
competition with glucose for transport and phosphorylation [5].
Labeled 2-DG-6-P is trapped in the tissue and then detected. In an
experimental setting, autoradiography of previously sliced tissue
provides a higher spatial resolution than does external imaging. To
achieve further information on the metabolic pathways of glucose,
nuclear magnetic resonance spectroscopy after [13C]glucose infusion
can be applied as a powerful tool to measure a large number of
metabolic fluxes of glucose noninvasively [109]. Due to low temporal
resolution, these methods do not enable measuring fast dynamic
changes in glucose metabolism during neuronal activation.
-66
-62
-58
-54
-50
-46
-42
-38
-34
-30
-26
-22
-18
-14
-10
-6
-2
+2
+6
+10
+14
+18
+22
+26
+30
+34
+38
+42
+46
+50
Hypometabolism
Hypermetabolism
18
Figure I. Example of a diagnostic [ F]fluoro-2-deoxyglucose PET-imaging

study. As illustrated in this 23-year-old female patient after a 2-month course of
severe anti-NMDA-receptor encephalitis, these patients typically show
widespread frontotemporal cortical hypermetabolism as well as bioccipital
and cerebellar cortical hypometabolism [103]. For visualization, hyper- and
hypometabolism are color coded across the entire brain as depicted in the
lookup table. Images are from superior (top left) to inferior (bottom right). For
details on the voxel-based statistical analysis to demonstrate hyper- and
hypometabolism and the corresponding cohort study see [103]. Images
courtesy of R. Buchert.
transporter of the BBB, GLUT1 (Figure 1B). Indeed, more

than 10% of early-onset absence epilepsy [85] and up to 1%
of the common idiopathic generalized epilepsies [86] have
been ascribed to GLUT1 deficiency caused by mutations in
594
the solute carrier family 2 (SLC2A1) gene. Early diagnosis

of the GLUT1 deficiency syndrome is important because
adherence to a ketogenic diet [15] is an effective treatment
for most patients [83]; in general, a ketogenic diet efficiently suppresses epileptic seizures in childhood drug-resistant
epilepsy [87]. The anticonvulsant effect of restricted dietary carbohydrate intake further underscores the relevance of glucose-derived energy for neuronal excitability
[15]. Furthermore, inhibition of glycolysis with the glucose
analog 2-deoxyglucose in experimental seizures is an effective treatment and illustrates a role for glycolysis-derived NADH in the metabolic regulation of genes involved
in epilepsy [88]. Interestingly, BAD appears to be involved
in the regulation of neuronal energy substrate utilization
independent of its apoptotic function [89]. However, it
remains unclear whether GK [66], which has a restricted
expression pattern in the brain, or another HK isoform has
a role in facilitating glucose sensing by BAD in this context.
A thromboembolic occlusion of a brain-supplying artery
leads to an acute disruption in the blood supply to a specific
brain territory, causing cerebral ischemia (Figure 1B). Within minutes, glucose depletion and associated compromised
bioenergetic pathways cause extensive neuronal death in
the core of the infarction and, over time, in the surrounding
tissue [90,91]. Cellular models suggest that increased levels
of the mitochondrial-bound glycolytic enzyme HKII protect
neurons from cell death in ischemia [72] (Figure 1E).
Spreading depression is a self-propagating wave of
neuronal depolarization in the cortex, which is associated
with a variety of neurovascular diseases, including stroke,
subarachnoid hemorrhage, traumatic brain injury [92],
and migraine [93]. Spreading depolarizations (SD) disturb
cortical glucose metabolism [94], although interestingly it
has been demonstrated that hyperglycemia increases the
cortical resistance against SD [95].
Although neurodegenerative diseases are not classically
thought to be caused by disturbed metabolism, bioenergetic
defects are emerging as important pathophysiological mechanisms (Box 1) [3] in several disorders. One of the earliest
signs of AD is a reduction in cerebral glucose metabolism,
and both human studies and animal models suggest that
disturbed glucose metabolism is associated with AD progression [96]. In a mouse model of AD, GLUT1 expression
was reduced both at the BBB as well as in astrocytes, which
was paralleled by impaired glucose transport and reduced
cerebral lactate release during neuronal activation [97]
(Figure 1B). Dysregulated glucose metabolism in metabolic
disorders such as obesity or type 2 diabetes mellitus has
been linked to AD progression and cognitive impairment
[96]. However, a large clinical trial could not demonstrate a
beneficial effect of aggressive glucose lowering on cognitive
outcome in patients with diabetes [98].
In patients with Parkinsons disease (PD), widespread
cortical hypometabolism is accompanied by glucose hypermetabolism in the external pallidum and possibly other
subcortical structures [99]. In one model of PD, HKII,
which regulates neuronal viability depending on the metabolic state [72] (Figure 1E), has been suggested to inhibit
degeneration of dopaminergic neurons [100].
Disturbed metabolism in myelin-producing cells is associated with axonal degeneration. In the brain, defective
Review
lactate transporter levels in oligodendrocytes are linked to
axonopathy [34] (Figure 1C) and, in the peripheral nervous
system, disrupted oxidative phosphorylation in Schwann
cells is related to severe neuropathy [33]. However, demyelination without extensive axonal loss in an animal model
of multiple sclerosis [101] hints to a complex underlying
mechanism.
Autoantibodies against the NR1 subunit of the
N-methyl-D-aspartate receptor (NMDAR) may inhibit
glutamatergic transmission (Figure 1D) by blocking,
crosslinking, and initiating the internalization of the
receptor. Patients with anti-NMDAR encephalitis have
distinct symptoms that occur over weeks and months.
The symptoms start with fever, psychosis, and seizures,
and progress to abnormal movements, respiratory failure, dysautonomia, and coma [102]. As a consequence of
impaired NMDAR function, and correlating with disease
activity, a characteristic pattern of [18F]fluoro-2-deoxyglucose (FDG)-PET abnormalities (Box 2, Figure I)
includes increased frontotemporal and decreased occipitoparietal glucose metabolism. Despite these severe and
long-lasting symptoms and changes in metabolism, most
patients do not have pathological findings in diagnostic
magnetic resonance imaging (MRI) studies. Furthermore, normalization in cerebral glucose metabolism
accompanies recovery [103].
Finally, disrupted central glucose sensing, insulin
signaling, and defective hypothalamic circuits have been
implicated in the pathophysiological mechanism of type 2
diabetes mellitus and obesity [49,59,6264,67] (Figure 1A).
At the same time, dysregulated glucose metabolism in
diabetes mellitus can injure the brain through both hypoand hyperglycemia [104]. Furthermore, cachexia, a severe
complication after cerebral ischemia, has been ascribed in
Box 3. Outstanding questions

Does modeling accurately predict actual energy use by the brain?
Can metabolic substrates be interchanged? Are there functional
consequences of using glucose and other metabolites? Future
studies need to address the consequences of oxidative versus
glycolytic metabolism for different brain functions.
What metabolic substrates support neurons and other cell types
under different functional states of the brain? What is the
relevance of shuttling of metabolic substrates between different
cell types? Does the direction of metabolic shuttling between cells
depend on the (physiological or experimental) context?
How does metabolic coupling among brain cells and shuttling of
metabolites sustain brain activity?
Are there additional anatomical or cellular networks in the brain
that control peripheral glucose metabolism? Do cell death pathways contribute to central glucose sensing? How does disturbed
peripheral metabolism influence central glucose sensing and
regulation? How does disrupted central glucose sensing cause
systemic metabolic disorders?
What is the functional connection in the regulation of cell death
pathways through different members of the glycolytic cascade?
What is the functional impact of this connection for different cells
of the brain (e.g., neurons, astrocytes, oligodendroglia, etc.)? How
does dysbalanced metabolism and subsequent dysregulation of
cell death pathways contribute to neurodegenerative diseases or
other acute or chronic disorders of the brain?
How can knowledge about cerebral glucose metabolism be
exploited for refined therapies of neurodegenerative disorders
or other diseases of the brain?
part to the dysregulation of the hypothalamuspituitary

adrenal axis and perturbed efferent signaling [105]. Given
the role of hypothalamic structures in glucose and nutrient
sensing (see above and [49,51]), disturbed central glucose
sensing and impeded central regulation of peripheral
metabolism (see above) may contribute to the development
of cachexia after CNS damage.
Concluding remarks
Glucose metabolism is closely integrated with brain physiology and function. Although recent studies have shed
light on the complex regulation of biochemical, cellular,
and systemic pathways, many features of the exact regulation remain controversial or elusive (Box 3). The advent
of novel and refined biochemical or genetic tools, screening
methods, imaging technologies, and systems analyses will
enable the study of cellular, subcellular, and even biochemical mechanisms in the cell or in vivo with unprecedented
temporal and spatial resolution. In addition to studying
individual biochemical or cellular pathways and their
control over intracellular signaling cascades (e.g., programmed cell death), peripheral homeostasis or brain
activity, future challenges lie in integrating the parts of
the puzzle to form a conclusive picture of the cooperation
between different systems and cell types. Ultimately, a
thorough understanding of these mechanisms will lead to
better insight into the pathophysiology of multiple diverse
disorders of the brain and enable the development of novel
treatment strategies.
Acknowledgments
We are grateful to the members of our labs for their contribution to our
underlying research. We would like to thank Ralph Buchert, Department
of Nuclear Medicine, Charite, for kindly providing and analyzing the PET
images. This work was supported by the Seventh Framework Programme
of the European Union (FP7/2008-2013) under Grant Agreements 201024
and 202213 (European Stroke Network), the Deutsche Forschungsgemeinschaft (NeuroCure Cluster of Excellence, Exc 257; SyNergy, Munich
Cluster for Systems Neurology, Exc 1010), the Bundesministerium fur
Bildung und Forschung (Center for Stroke Research Berlin, 01 EO 08 01),
a Canadian Stroke NetworkEuropean Stroke Network Transatlantic
Collaboration grant, and the National Institutes of Health (DK081936).
Disclaimer statement
P.M. has received research funding from Sanofi. A.M. has received
research funding and speaker honoraria from Bayer Vital GmbH, Sanofi,
and Wyeth Pharma GmbH.
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958972
597
Review
Control of neuronal voltage-gated

calcium ion channels from RNA to
protein
Diane Lipscombe, Summer E. Allen, and Cecilia P. Toro*
Department of Neuroscience, Brown University, Providence, RI 02912, USA
Voltage-gated calcium ion (CaV) channels convert neuronal activity into rapid intracellular calcium signals to
trigger a myriad of cellular responses. Their involvement
in major neurological and psychiatric diseases, and importance as therapeutic targets, has propelled interest in
subcellular-specific mechanisms that align CaV channel
activity to specific tasks. Here, we highlight recent studies that delineate mechanisms controlling the expression of CaV channels at the level of RNA and protein. We
discuss the roles of RNA editing and alternative premRNA splicing in generating CaV channel isoforms with
activities specific to the demands of individual cells; the
roles of ubiquitination and accessory proteins in regulating CaV channel expression; and the specific binding
partners that contribute to both pre- and postsynaptic
CaV channel function.
Ten CaV genes, thousands of different CaV mRNAs, and
many more functionally different proteins
. . .different Ca currents show so many differences in
fundamental properties that we find it easier to
assume that there are more than one type. [1]
CaV channels are present at every critical step of information transfer in the nervous system, from signal detection
to perception, and from neuronal development to programmed apoptosis. Strategically located at points of sensory detection and on both sides of the synapse, CaV
channels have a defining role in integrating signals and
influencing synaptic strength. All but one of the ten mammalian genes that encode the pore-forming a1 subunit of CaV
channels are expressed in the nervous system (Box 1). Each
CaV gene has a distinct expression profile reflecting its
functional specialization to support specific cellular tasks.
For more than 50 years, the functional diversity of CaV
currents among different cell types has been attributed to
the expression of multiple channel types [1]. Indeed, individual neurons can express multiple CaV genes, consistent
with the varied subcellular distribution of calcium channel
Corresponding author: Lipscombe, D. (Diane_Lipscombe@brown.edu).
Keywords: voltage-gated calcium channels; alternative splicing; splicing factors;
synaptic transmission; synaptic proteins; ubiquitination.
*
Current address: Oregon Hearing Research Center, Oregon Health & Science
University, Portland, OR 97239, USA.
598
subtypes controlling a range of neuronal functions. The

value of highly selective toxins and drugs used to tease out
the relative contributions of CaV channels within single
cells is hard to overstate. Yet this is not always straightforward: for example, until recently, the absence of pharmacological tools to distinguish CaV1.2 and CaV1.3
channels [2], which collectively give rise to most dihydropyridine-sensitive Ca currents in neurons, has stalled
progress in defining their individual contributions. Even
when specific toxins are available, the challenges associated with isolating the functional contributions of closely
related CaV channels are magnified several-fold when
considering the thousands of channel isoforms potentially
generated from single genes by alternative pre-mRNA
splicing and RNA editing.
All ten mammalian calcium channel, voltage-dependent,
alpha 1A through 1I, and 1S genes (CACNA1A through
CACNA1I and CACNA1S) contain multiple sites of alternative splicing; each gene has the potential to generate thousands of unique splice isoforms controlled by the expression
and relative activities of cell specific splicing factors (Box 1)
(Figures 1 and 2). Typically, these sites are located in coding
regions that tolerate sequence variation and that are not
essential for structural integrity: N termini, cytoplasmic
loops III and IIIII, extracellular linkers that connect
transmembrane a-helices S3 and S4 in domains III and
IV, and the C termini (Figures 2A,B and 3). Consequently,
these hypervariable regions are hotspots for cell specific
posttranslational modification, second messenger action
on, and protein binding to, CaV channels (Figures 3 and
4). The tethering of appropriate CaV channels to presynaptic
active zones or to postsynaptic specializations depends on
specific nanodomain-level protein interactions (Figures 5
and 6), as discussed below for the most recently discovered of
these interactions. A common feature across these studies is
the ability to resolve molecular-level detail of specific RNAs
and proteins within subpopulations of neurons and functionally distinct subcellular compartments. Novel techniques have been developed and applied to identify the
following: splicing factor binding sites on CaV channel
pre-mRNAs; CaV channel RNAs in specific populations of
neurons; and CaV proteins at active zones. These methodologies marry disciplines to yield a more comprehensive,
multidimensional view of CaV channels and their regulation
from RNA to protein. Here, we highlight these and other
recent studies elucidating cell specific control of CaV RNA
Review
Box 1. By any other name

CaV channels are highly specialized plasma membrane proteins that
convert small changes in the membrane potential into rapid, localized
increases in intracellular calcium. Only a limited number of ion
channels, including several ligand-gated ion channels, allow the
passage of calcium. However, CaV channels are exquisitely selective
for calcium over other cations as long as extracellular calcium
concentrations exceed approximately 10 mM. In the absence of
extracellular calcium, CaV channels lose their high selectively to
calcium and readily conduct a range of cations.
Most CaV channels have a dual function: they support ionic current
that changes the membrane potential, and they permit the flow of
calcium across the plasma membrane that serves as an intracellular
second messenger. In mammals, ten genes encode the ten major a1
subunits of CaV channels (CACNA1A through CACNA1I, and CACNA1S). They have unique expression profiles, cellular function, and
pharmacology, and are associated with various diseases. The
numerous aliases used to identify and distinguish the different CaV
channel genes, proteins, and currents are shown for seven different
species (Table I).
The pore-forming a1 subunits of CaV channels fall into three classes
based on sequence homology: CaV1 (1.1, 1.2, 1.3, and 1.4), CaV2 (2.1,
2.2, and 2.3) and CaV3 (3.1, 3.2, and 3.3). The classes are functionally
distinguished by a combination of pharmacological tools and
biophysical properties, although pharmacological differences across

CaV1 (dihydropyridine sensitive), CaV2 (v-agatoxin-IVA inhibits CaV2.1;
v-conotoxin GVIA inhibits CaV2.2), and CaV3 channels more reliably
distinguish among CaV subtypes. Pharmacological, genetic, and
functional studies have helped delineate the different cellular roles
controlled by CaV channels. Presynaptic calcium entry, and subsequent transmitter release, are mediated by CaV2 channels (mainly
CaV2.1 and CaV2.2) throughout the nervous system, by CaV1.3
channels in IHCs [73] and by CaV1.4 channels in retina [74,75].
Dendritic postsynaptic CaV1 (mainly CaV1.2) channels regulate calcium
entry that controls gene expression [76,77]. Dendritic CaV2.3 channels
[32] are implicated in acquisition of dendritic phenotype [78] and
oscillatory burst discharge in the reticular thalamus [79]. Postsynaptic
CaV1.3 channels underlie pacemaking in certain cells [80], are
important for brain stem neuron development [63], and are implicated
in calcium-dependent death of dopaminergic neurons [2]. The roles of
presynaptic CaV2 channels in synaptic transmission and short-term
plasticity [58] and the role of postsynaptic CaV1 channels in activitydependent gene expression in neurons have been reviewed elsewhere
[81]. A functional, correctly targeted CaV channel depends on its
association with other calcium channel subunits, including CaVb,
CaVa2d, and other proteins; some of these aspects of CaV channel
regulation are detailed in other recent reviews [82].
Table I. Voltage-gated calcium channel nomenclature

Protein name
CaV1.1
CaV1.2
CaV1.3
CaV1.4
CaV2.1
CaV2.2
CaV2.3
CaV3.1
CaV3.2
CaV3.3
Old
a1S
a1C
a1D
a1F
a1A
a1B
a1E
a1G
a1H
a1I
Current type
L
P/Q
N
R
T
Gene name
Human
CACNA1S
CACNA1C
CACNA1D
CACNA1F
CACNA1A
CACNA1B
CACNA1E
CACNA1G
CACNA1H
CACNA1I
Mouse, Rat
Cacna1s
Cacna1c
Cacna1d
Cacna1f
Cacna1a
Cacna1b
Cacna1e
Cacna1g
Cacna1h
Cacna1i
processing, posttranslational CaV protein modifications,

and CaV proteinprotein interactions. Collectively, this
work demonstrates exciting progress made in defining the
molecular mechanisms underlying the expression of CaV
channel isoforms in specific neurons, and in linking their
expression to critical cell functions.
Cell specific alternative pre-mRNA splicing and RNA
editing regulate CaV channel function
. . .the more you look, the more you see. (Robert M.
Pirsig, 1974)
Alternative pre-mRNA splicing is particularly prevalent in the mammalian brain, and is essential for normal
neuronal development, axon targeting, neuronal excitability, and neural circuit formation. This form of pre-mRNA
processing occurs in the nucleus of the cell, and is controlled by the concerted actions of a set of cell specific, RNAbinding proteins called splicing factors. Trans-acting splicing factors bind to consensus cis-sequence motifs in premRNAs to influence the action of the spliceosome, promoting or repressing the inclusion of alternatively spliced
exons, and promoting or repressing the use of alternative
splice acceptor or donor sites at intronexon boundaries
Zebrafish
cacna1sa,b
cacna1c
cacna1da,b
cacna1f
cacna1aa,b
cacna1ba,b
cacna1g
cacna1i
Pufferfish
1S-a,b,c
1C
1D-a,b,c,d
1F-a,b,c
1A-a,b
1B
1E-a,b
1G-a,b
1H-a,b
1I
Drosophila
Ca-a1D
Caenorhabditis elegans
egl-19
cac
unc-2
Ca-a1T
cca-1
(Figure 1). Nova, rbFox, and polypyrimidine tract binding

(PTB) families of splicing factors direct inclusion or skipping of alternatively spliced exons in several neural gene
pre-mRNAs, including CACNA1 genes (Box 1) (Figure 2).
These splicing factor protein families have the capacity to
either repress (silence) or enhance exon inclusion, in different target pre-mRNAs or within the same pre-mRNA,
depending on the position of their consensus binding
motifs, either upstream (50 ) or downstream (30 ) relative
to the target exon [37] (Figures 1 and 2C). Genome-wide
maps of splicing factor-binding sites reveal certain common features in the mechanism of action of certain splicing
factors and bring us closer to defining a working splicing
code [8]. These splicing factor genomic maps have advanced understanding of cell specific regulation of channel
structure and function. In addition, several studies have
shown that alternative splicing can be controlled by epigenetic factors [9], signaling factors including cellular protein
kinases and phosphatases [10], and by cell specific alternative splicing of splicing factors themselves [5,6].
Splicing factors that target CACNA1 pre-mRNAs
CACNA1 pre-mRNAs are targets of splicing factors from
the Nova, rbFox, PTB, and Muscleblind-like 2 (Mbnl2)
599
Review
pre-mRNA
mRNA
Repressor
(A)
Enhancer
+
(B)
(C)
Enhancer
Repressors
(D)
+
Figure 1. Splicing factors repress and enhance alternative exon inclusion during pre-mRNA processing. Splicing factors bind to consensus nucleotide sequences in introns
or target exons of pre-mRNAs to enhance or repress (silence) recognition of the exon by the spliceosome during pre-mRNA processing. Alternatively spliced exons
(colored), constitutive exons (gray), and introns (black connecting lines) are illustrated. The position of splicing factor binding, relative to the target exon, is often predictive
of splicing factor action. For example, members of the Nova, rbFox, and polypyrimidine tract binding (PTB) splicing factor protein families tend to repress or silence exon
inclusion when they bind their respective nucleotide sequence-binding motifs upstream (A) or within (D) the target exon, and enhance exon inclusion when they bind their
respective binding motifs downstream of the target exon (B). Alternatively spliced cassette exons are excluded or included during pre-mRNA splicing (A,B). Mutually
exclusive alternative splicing involves two or more exons, only one of which is selected during pre-mRNA processing (C,D). Mutually exclusive exons often start or end with
an incomplete codon. In this case, there is a shift in the reading frame in mRNAs that either lack both mutually exclusive exons or contain both, and early protein
termination (not shown). Often, exon selection is influenced by the concerted action of several splicing factors (D). The expression levels and activities of individual splicing
factors depend on many cellular factors, including those involved in development, neuronal activity, and defining neuronal subtype.
RNA-binding protein families [47] (Figure 2D). Moreover,

multiple rbFox splice isoforms act on the same CACNA1
pre-mRNA exon, but with different consequences [11].
Importantly, developmental changes in the patterns of
CaV splice isoform expression can parallel the changes
in expression levels of key splicing factors. For example,
as PTB expression levels fall in the developing brain, levels
of neuronal PTB (nPTB) increase [12,13]. PTB controls the
choice between a pair of mutually exclusive exons in CACNA1C (CaV1.2), e8 and e8a, via binding to motifs in CACNA1C pre-mRNA upstream of e8a [14] (Figure 2C). As PTB
levels decrease during development, the ratio of e8a- to e8containing CaV2.2 mRNAs increases because PTB no longer represses e8a. In this case, nPTB does not substitute
for PTB as a repressor of e8a [14]. E8a and e8 encode
alternate forms of transmembrane S6 in domain I of
CaV1.2 in a region that is critical for normal channel
function (Figure 2B). E8a and e8 are of special interest
because certain mutations in either exon cause the severe
multi-organ hereditary disorder, Timothy Syndrome [15].
The severity of neurological symptoms in individuals with
Type I and Type II Timothy Syndrome depends on which
exon carries the mutation, presumably reflecting the different expression patterns of e8 and e8a in the brain [16].
CACNA1B (CaV2.2) pre-mRNAs are targets of Nova2,
and several Nova2-binding sites align with previously identified alternatively spliced exons. For example, highthroughput sequencing combined with cross-linking immunoprecipitation methods (HiTS-CLIP) showed that Nova2binding sites are located within the intron immediately
600
upstream, and overlapping, a short six-nucleotide exon

e31a in CACNA1B pre-mRNAs [17] (Figure 2C). The positioning of RNA-binding motifs for Nova2 upstream of e31a
predicts Nova2 repression of e31a. The HiTS-CLIP data are
consistent with earlier reports that e31a-containing CaV2.2
mRNAs are found at very low levels in the rodent brain
where Nova2 is expressed. By contrast, e31a-containing
CaV2.2 mRNAs dominate in peripheral ganglia, which do
not express Nova2 [18]. The same splicing factor can act
differently on multiple alternatively spliced exons within
the same pre-mRNA. This is the case for Nova2; in contrast
to its silencing action on e31a, it binds motifs downstream of
e24a to augment e24a expression in brain (Figure 2C,D)
[18]. Intriguingly, there are clusters of Nova2-binding sites
in other regions of CACNA1B that do not map to known
alternatively spliced exons, and the functional significance
of these is not yet known.
RbFox and Nova splicing factor protein families have
overlapping networks of synaptic protein targets, consistent
with the collective action of multiple splicing factors on
several alternative CACNA1 exons [17] (Figures 1 and
2C,D). Loss of rbFox2 results in reduced expression of
Nova1, perhaps reflecting the regulation of Nova1 premRNA splicing by rbFox2 [17]. Several important studies
have shown profound consequences, including gross neurodevelopmental abnormalities, of disrupting or eliminating
splicing factors that regulate alternative splicing of multiple
targets [5,7,19]. However, because the splicing of hundreds
of neuronal pre-mRNAs, including those that encode other
splicing factors, are disrupted in these studies, the relative
Review
(A) Transmembrane-spanning (S1S6), domains (IIV), and intracellular regions of CaV channels
S1
S1
S6
S1
S6
II
S6
S1
III
III
S6
IV
IIIII
IIIIV
N terminus
C terminus
(B) Locaon of alternave exons in CaV channels

33
8/8a
Key:
CaV1.2
CaV1.3
CaV1.4
CaV2.1
CaV2.2
CaV2.3
CaV3.1
CaV3.2
CaV3.3
21/22
8a/8b
31a
31a 32
24a
12
out
14
16
in
17
9*
9/9a
9
2/2a
18a
11
9b
10*
14
10a10/10a
17
25c
20a
46
(C) Splicing factors target exons in CaV pre-mRNAs
Channel
25
31
Nova
24
CaV2.1
Cacna1a
24a
25
31
31a
CaV2.2
32
Cacna1b
E12 corcal
neurons
PTB
CaV1.2
pre-mRNA
7
Adult corcal
neurons
CaV1.2
8a
31 32 33
8
PTB
8a
35
rbFox
9*
10
Cacna1c
CaV1.3
31 32 +
34
10
rbFox
CaV1.2
pre-mRNA
9*
45a
45 45
33
34
Exon
Locaon
Splicing
factor
Refs
24a
IIIS3-IIIS4
Nova
[18]
31a
IVS3-IVS4
Nova
[18]
24a
IIIS3-IIIS4
Nova
[4]
31a
IVS3-IVS4
Nova
[18]
18a
II-III
rbFox
[91]
8a
IS6
PTB
[14]
9*
I-II
rbFox
[90]
33
IVS3-IVS4
rbFox
[90]
8b
IS6
rbFox
[5]
11
I-II
rbFox
[5]
29
IVS3-IVS4
rbFox
[5]
Gene
32
Nova
CaV2.2
pre-mRNA
31a
43
44
(D) Known exons regulated by splicing factors
Brain
24a
42a
47
37
24
34
35
37a/b
35a
40b
42
26
26
1a/b/c
CaV2.1
pre-mRNA
31a
31a/b
31/32
25a
Cacna1d
35
CaV1.1
Cacna1s
Figure 2. Alternative splicing is extensive in voltage-gated calcium ion (CaV) channels and is regulated by the action of cell-specific splicing factors. (A) Major domains and
regions located on a schematic of the a1 subunit of a CaV channel. There are four structurally homologous domains: I, II, III, and IV. Each domain comprises six
transmembrane spanning a-helices (S1S6). The intracellular regions that link the domains are labeled III, IIIII, and IIIIV. The N terminus and C terminus are intracellular.
This naming system is used to identify the major regions of CaV channels. For example, the third transmembrane spanning a-helix in domain IV is referred to as IVS3. (B)
Location of alternative exons mapped on to a schematic of the a1 subunit of a CaV channel. The 24 transmembrane-spanning domains are shown (gray) as well as
extracellular and intracellular regions. The approximate locations of alternative exons are shown (circles) and color coded to indicate specific CaV channel subtypes. Exon
numbers are indicated and the numbering system used follows mouse calcium channel, voltage-dependent, alpha 1A-1I, 1S (Cacna1a-Cacna1i, Cacna1s) gene numbering.
Alternate first exons are shown at the start of the N termini, and mutually exclusive alternative exons are designated as X/Y. The following references were used to compile
information for CaV1.2 [14]; CaV1.3 [83]; CaV1.4 [84]; CaV2.1 [85]; CaV2.2 [83], accession # FJ609386; CaV2.3 [83,86]; CaV3.1 [87]; CaV3.2 [88]; CaV3.3 [89]. (C) Splicing factor
target exons in CaV pre-mRNAs. Two splicing patterns are shown for three splicing factor RNA-binding protein families that have similar mechanisms of action: Nova, rbFox
and polypyrimidine tract binding (PTB). The first example (top) illustrates the action of Nova on alternatively spliced exon cassettes, e24a and e31a, in CaV2.1 and CaV2.2
pre-mRNAs. Nova enhances inclusion of e24a during pre-mRNA splicing of CaV2.1 and CaV2.2 by binding to elements in the respective introns downstream of the target
exons (intronic splicing enhancer, ISE), whereas Nova represses (or silences) inclusion of e31a during pre-mRNA splicing by binding element upstream and within or
overlapping the target exon (intronic splicing silencer, ISS; exonic splicing silencer, ESS). Nova is expressed in brain where CaV2.1 and CaV2.2 mRNAs containing e24a and
lacking e31a dominate [18]. In the second example (lower), PTB represses inclusion of mutually exclusive e8a during splicing of CaV1.2 pre-mRNA. PTB is expressed in
embryonic brain and, therefore, CaV1.2 mRNAs lacking e8a and containing e8 dominate early during development. In adult cortical neurons, PTB levels are reduced and it
no longer represses e8a. By contrast, rbFox is upregulated during development and represses inclusion of e9* and enhances inclusion of e33 [14,90]. In adult cortical
neurons, CaV1.2 mRNA containing e8a, lacking e9*, and containing e33 dominate [14]. (D) Alternatively spliced exons of CaV channel genes are shown together with splicing
factors known to regulate their expression. Cassette exons are either included or excluded. E8a/e8 of CaV1.2, and e8b/e8a of CaV1.3 are mutually exclusive (A,B). Therefore,
repression of e8a during pre-mRNA splicing of CaV1.2 promotes inclusion of e8, and repression of e8b during pre-mRNA splicing of CaV1.3 promotes inclusion of e8a.
CaV1.2 e8a is strongly repressed by PTB but more weakly repressed by the neuronal homolog, nPTB, despite their similar RNA-binding motifs [14]. Based upon
[4,5,14,18,90,91].
601
Review
e17a I K727
e17 E K727
Synprint S L718
CaV2.2 683
CaV2.1 690
Start
End
Inseron
Phosphorylaon
PKC P S774
1 del. S R756
IIS6 IIIII linker
CaMKII P S784 (2.2)

2 del. S R793 (2.1)
S
E
I
P
e16 S I667
CaV2.2 583
CaV2.1 590
e17 S Y705
e16 E N704
1 del. S L754
Acidic
Basic
Hydrophobic
Polar
Dierent
2 del. S A738
Channel amino acid #
Synprint S A722
Key:
e18a I A755
CaV2.2 781
CaV2.1 790
CaMKII P S896,S898
PKC P S898
CaV2.2 871
CaV2.1 884
1 del. E P947
2 del. E P947
Synprint E H963
CaV2.2 963
CaV2.1 968
2 del. E P1001
Synprint E R984
CaV2.2 1048
CaV2.1 1062
1 del. E L1139
CaV2.2 1114
CaV2.1 1162
IIIII linker IIIS1

Figure 3. The IIIII intracellular loop regions of voltage-gated calcium (CaV) 2.1 and CaV2.2 have divergent sequences. Amino acid alignments for CaV2.1 (rat sequence:
NM_012918) and CaV2.2 (rat sequence: AF055477) are shown for approximately 700 amino acids encoding the IIIII intracellular loops as well as sequence in IIS6 and IIIS1
(Clustal Omega software [92]). The approximate locations of boundaries between transmembrane and intracellular domains are indicated. The chemical nature of each
amino acid is coded as follows: acidic (green) D, E; basic (purple) K, R, H; hydrophobic (orange) A, F, I, L, M, P, V, W; and polar (blue) C, G, N, Q, S, T, Y. The black solid bars
below the alignments indicate regions of differences in amino acid type between CaV2.1 and CaV2.2. White circles indicate amino acids of interest, the amino acid numbers
are shown and they are coded as follows: (S) alternative exon, deletion (del.), or synprint area start; (E) alternative exon del., or synprint area end; (I) alternative exon
insertion; (P) phosphorylation. References: Alternative e16, e17, and e17a of CaV2.1 [85]; CaV2.2 e18a [93,94]; CaV2.2 synprint [95]; CaV2.1 synprint [96]; PKC and CaMKII
phosphorylation [97]; CaV2.1 D1 and D2 [98]; CaV2.
contribution of splicing events within a single gene family,

such as CACNA1, cannot be distinguished.
Individual alternatively spliced exons have measurable
cellular and behavioral consequences
Many individual splice sites in CACNA1 genes are evolutionarily conserved, and it is often assumed that each adds
functional value and fitness to the cells in which it is
expressed. Another possibility is that the functional impact
of any individual splicing change may not be discernible at
the cellular or behavioral level. Alternative pre-mRNA
splicing of genes may have evolved because essential proteins operate over a broader dynamic range than would be
possible from the activity of a single protein. This feature of
alternative splicing could be critical during development,
particularly in neurons that adapt to, and continue to
function in the face of, dramatic changes in morphology
and activity [20]. Yet, there are several notable examples
showing that exon choice in an influential gene can indeed
modulate behavior. For example, reproductive dominance
in honeybees is controlled by alternative splicing of exon 5
in a gene homologous to the gemini transcription factor of
Drosophila [21], and cell specific expression of a transient
receptor potential cation channel, subfamily V, member 1
(TRPV1) splice isoform confers unique infrared sensing
capabilities to vampire bats [22].
602
Recently, the cellular and behavioral consequences of an

alternatively spliced exon in the mouse Cacna1b gene were
demonstrated. Enhanced expression of the nociceptorspecific exon 37a splice isoform of CaV2.2 increased cellular
sensitivity to inhibition by activated m-opioid receptors and
behavioral sensitivity to spinal morphine-induced analgesia
[23] (Figure 4). Cell specific expression of e37a in wild type
mice may help explain why CaV2.2 channels in nociceptors
are particularly sensitive to inhibition by drugs, transmitters, and hormones that act through G protein-coupled
receptors. Additional alternatively spliced exons in Cacna1b
and in the closely related Cacna1a (CaV2.1) and Cacna1e
(CaV2.3) genes modify amino acid sequences in the cytoplasmic domains of CaV channels. The actions of G proteins
and second messengers on other ion channels may also be
influenced by cell specific alternative splicing of ion channel
pre-mRNAs in other parts of the nervous system.
Cell specific inclusion of exons in Cacna1 genes can also
impact disease states. Already mentioned are the different
symptoms in Type I and Type II Timothy Syndrome determined by which exon, e8a or e8, in CACNA1C carries the
mutation [16]. Other examples include the different consequences of Cacna1a (CaV2.1) mutations associated with
familial hemiplegic migraine on the short (D47) and long
(+47) splice isoforms of CaV2.1 [24], and an epilepsycausing mutation in mouse Cacna1h (CaV3.2) that results
Review
Key:
RIM
CaV2
CaV2
src TK
e37b
e37a
GPCR
i/o
e37a
i/o
Only G
G + src
Only src
Figure 4. Alternative splicing influences G protein-coupled receptor (GPCR)

inhibition of voltage-gated calcium (CaV) 2.2 channels. Illustration showing a Gi/o
protein-coupled receptor (green) inhibiting presynaptic CaV2.2 channels (blue) by
different signaling mechanisms. The GPCR is activated by neurotransmitter
released from a neighboring neuron. Following activation, the Gbg dimer
dissociates from Gi/o, binds to and directly inhibits both CaV2.2 channel splice
isoforms [e37a (orange) and e37b (pink)]. A second G protein-dependent pathway,
that requires src tyrosine kinase (TK) activation but that is independent of Gbg,
inhibits e37aCaV2.2. This schematic is based on data published in [23,99]. A
prediction that follows from these studies for e37aCaV2.2 channels, which are
more distant from GPCRs, is that they will not be inhibited by membrane-delimited
Gbg, but may still be inhibited by src TK. This is because the latter mechanism
involves a diffusible second messenger.
in different channel phenotypes depending on whether

alternative exon 25 is expressed [25].
RNA editing of CACNA1D mRNA
Additional cell specific alterations in amino acid sequences
can arise from RNA editing, introducing potentially even
finer specialization of CaV activities. In RNA editing, posttranscriptional deamination of adenosine to inosine (read
as guanosine during translation) is catalyzed in RNA by
the adenosine deaminase acting on RNA (ADAR) family of
enzymes. Evidence for different RNA-edited versions of
CaV channel mRNAs was first reported for the Drosophila
CaV2 homolog cacophony (Dmca1A) (Box 1, Table I) [26].
Recently, central nervous system (CNS)-specific editing of
mammalian CaV1.3 RNAs by the ADAR protein ADR2 has
been carefully documented and shown to generate four
distinct sequences within the C terminus IQ domain:
IQDY, MQDY, IRDY, and MRDY [27]. By contrast, only
one IQ sequence, IQDY, is found in CaV1.3 channels
expressed outside of the CNS [27]. The IQ domain forms
part of a critical calmodulin-binding site in CaV channels
that mediates calcium-dependent inhibition. The latter is
an important feedback control on subsequent calcium entry; cytoplasmic calcium inhibits the further gating of the
ion channel through which it flowed. Editing of the IQ site
(e.g., to MQ) reduces calcium-dependent inactivation and,
as a consequence, is thought to participate in shaping the
rhythmicity of action potential firing in neurons of the
suprachiasmatic nucleus [27].
Cell specific RNA editing could exert similar control
over CaV channels in other populations of neurons, and
Figure 5. The density of voltage-gated calcium (CaV) 2 channels at presynaptic

nerve terminals is controlled by RIM and the CaVa2d-1 subunit. The figure shows
that CaV2 channel trafficking to, or retention at, presynaptic nerve terminals
depends on the association of CaV2 proteins with both RIM and CaVa2d-1
[30,38,45,46,49]. The details of where RIM and CaVa2d-1 interact with CaV2, in
which intracellular compartment, are not known but the figure is intended to
capture the finding that overexpression of CaV2 channels does not lead to an
increase in functional presynaptic CaV2 channels (left) unless CaVa2d-1 is also
overexpressed (right) [45,46]. These data suggest that CaVa2d-1 limits the insertion
into, or the stabilization of, CaV2 channels at the active zones of the presynaptic
nerve terminal [45].
could alter amino acid sequences of other critical domains

that regulate specific functions. Because of the potential
importance of CaV1.3 channels in promoting calciumdependent cell death in dopaminergic neurons in the substantia nigra pars compacta and the proposed connection
between CaV1.3 activity and Parkinsons disease [28], it
will be interesting to know which edited versions of CaV1.3
RNAs dominate in these neurons. Mapping the cell specific
expression patterns of the ADAR2 protein and mapping
ADAR2-binding sites to specific RNAs would provide
valuable information in this regard. However, as is
the case for splicing factors, ADAR2 activity depends
on several factors, including phosphorylation-dependent
prolyl-isomerase Pin1 that controls its nuclear localization
[29], the WW domain containing E3 ubiquitin protein ligase
2 (WWP2) that promotes its proteasomal degradation [29],
as well as other second messengers.
Mechanisms that control numbers of CaV channels at
the cell surface
CaV channel activity depends not only on the pattern of
expression of functionally different splice and RNA edited
isoforms, but also on the overall expression level of CaV
channel proteins in specific subcellular compartments.
Counting CaV2 channels at active zones of different synapses by quantitative molecular and ultrastructural analyses recently demonstrated a tight correlation between
presynaptic CaV2.1 and CaV2.2 channel number and vesicle release probability [30,31], whereas the third member
of the CaV2 gene family, CaV2.3, was shown (with the
exception of the interpeduncular nucleus) to be mostly
restricted to postsynaptic structures, particularly dendritic shaft plasma membrane [32]. Thus, the overall activities
of presynaptic CaV2.1 and CaV2.2 channels have a major
influence on the efficacy of transmission at mammalian
synapses. We know a great deal about G protein-coupled
receptor-mediated control of CaV2 channels, particularly
CaV2.2. These well-studied signaling pathways terminate
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Review
Vesicle
Synapsin
CaV1.3
Ub
Ribbon
CaN
MAP1A
CaM
SNAP-25
CaV
CaV2.2
NSF
VAMP
Syntaxin
Presynapc
CSP
V-ATPase
SV2
Presynapc
PP2A
Vesicle
Vesicle
Munc18
RIM1 CaSK
Dynamin
AP-2
Ribeye
Endophilin
Dynacn
CRMP
RIM2
14-3-3 Clathrin
CaBP4
Whirlin
Ub
Harmonin
CaV
CaV2
Laminin
HA
eIF3
eIF
A
PP2
P
AKA 0
5
1
/
9
7
Ub
P2B
MA
CaV1.2
Postsynapc
PKA
CaV1.3
Sha
nk
3
Erb
De
nsi
in
n CaM
KII
Figure 6. Presynaptic and postsynaptic voltage-gated calcium ion (CaV) channels are associated with several membrane-anchoring and signaling proteins that define their
function. Illustrated are a subset of proteins shown to bind presynaptic CaV2.2, postsynaptic CaV1.2, and pre- and postsynaptic CaV1.3 channels. Not all proteins reported to
bind to these CaV channels are shown, but those illustrated have been confirmed by more than one proteomics screen and/or by a functional assay. In addition, some G
proteins, chaperone, and cytoskeletal proteins are not included. The proteins shown are approximately grouped by function, but beyond that there is no significance to
location in the synapse or relative to each other. Data were collected using the following references: CaV2.2: cysteine string protein (CSP) [100]; collapsin response mediator
protein 2 (CRMP) [101]; laminin [102]; microtubule-associated protein 1A (MAP1A) [103]; ubiquitin (Ub) [35,36]; other proteins were identified by [54,55,104]. CaV1.2: protein
phosphatase 2A (PP2A) [105]; protein kinase A (PKA) [106]; A kinase (PRKA) anchor protein 79/150 (AKAP79/150) [107109], HA [110], Ub [34,111], eukaryotic translation
initiation factor 3 (eIF3) [112]; MAP2B [108,113]; and dynamin [112]. CaV1.3 presynapse: whirlin [114]; regulating synaptic membrane exocytosis 2 (RIM2) [48]; harmonin
[42]; Ub [42], ribeye [60,115]; and calcium-binding protein 4 (CaBP4) [116,117]. CaV1.3 postsynapse: Shank [118]; erbin [81]; densin [64]; calcium/calmodulin-dependent
protein kinase II (CaMKII) [64]; and eIF3 [112].
on CaV2.2 channels and account for the short-term presynaptic effects of many neurotransmitters, hormones, and
drugs that modulate synaptic transmission. By contrast,
the cellular mechanisms that control the overall number of
CaV channels at active zones, which likely contribute to
long-term changes in synaptic plasticity, were only recently elucidated [31].
Not surprisingly, cellular mechanisms analogous to
those critical in setting the overall expression levels of
postsynaptic receptors, including the ubiquitin proteasome
system (UPS) [33], also control surface expression of presynaptic CaV2.1 and CaV2.2 channels [3436]. In addition,
proteinCaV2 channel interactions described recently appear critical in establishing the number of CaV2 channels
specifically at presynaptic terminals [30,37,38] (Figure 5).
Such molecular interactions are dynamic and subject to
modulation, thereby regulating the density and number of
presynaptic CaV2 channels during synaptic plasticity
[30,31].
Ubiquitin regulates CaV1 and CaV2 channels
Ubiquitination of several neuronal proteins is a critical
posttranslational mechanism to modify the trafficking,
endocytosis, and activity of synaptic receptors and ion
604
channels to adjust synaptic strength [34,39]. Ubiquitin

covalently attaches to intracellular lysines of target proteins and, depending on the type of conjugate (i.e., monoubiquitination or polyubiquitination), can promote
internalization, modify protein function, or target protein
for degradation via the UPS [40]. Activity-dependent ubiquitination of postsynaptic AMPA receptors is described
and known to regulate synaptic plasticity. Until recently,
CaV channels were rarely considered important targets of
ubiquitin despite functional evidence that ubiquitin-dependent changes in synaptic efficacy involved presynaptic
components [41]. Four reports now show that CaV1.3,
CaV1.2, and CaV2.2 channels are all targets of posttranslational modification by ubiquitin [3436,42]. These
reports implicate the UPS in controlling activity-dependent pre- and postsynaptic calcium levels. Ubiquitination
of CaVa1 subunits is influenced by their association with
CaVb subunits, known to regulate plasma membrane targeting of CaV, and by cell specific alternative splicing. CaVb
subunit binding to CaV1.2 and CaV2.2 reduces CaVa1
ubiquitination and protects channels from proteasomal
degradation [34,36]. Similarly, CaV2.2 channel splice isoforms with restricted expression (e37a containing) have
reduced ubiquitination compared with other broadly
Review
expressed isoforms (e37b containing) and are associated
with increased channel current densities [35]. Thus, the
influence of the UPS on CaV2.2 channels is cell specific,
reflecting the particular expression profile of certain alternative splice forms. Cav2.2 was recently identified as a
target of ubiquitin based on a large-scale proteomics analysis of diGly-modified lysine residues of proteins expressed
in a human colorectal carcinoma cell line [43]. Modified
lysines in the region of Cav2.2 encoded by e37a and e37b
were not identified in this screen, suggesting that ubiquitination at this site is cell specific. Future large-scale
proteomics analyses that enrich for neuronal ion channels,
and other membrane proteins, hold promise for mapping
major sites of posttranslational modifications of critical
synaptic proteins.
CaVa2d and RIM regulate CaV2 expression
Two protein families have recently grabbed the limelight
as being essential for trafficking or tethering CaV2 channels to nerve terminals: CaVa2d and regulating synaptic
membrane exocytosis (RIM) (Figures 5 and 6). CaVa2d-1,
first shown to interact with CaV channels approximately 25
years ago, is a glycosylphosphatidylinositol (GPI)-anchored extracellular protein [44]. This auxiliary subunit
of CaV2 channels promotes membrane trafficking, speeds
channel activation and inactivation, and binds the analgesics gabapentin and pregabalin. CaVa2d-1 has now been
shown to act as a rate-limiting factor controlling the number of CaV2.1 channels at presynaptic terminals [45]. Evidence for a limiting factor in CaV2 channel trafficking to
presynaptic nerve terminals emerged from studies in cultured hippocampal neurons. Whereas somatic Ca currents
were reliably enhanced in cultured neurons transiently
expressing exogenous CaV2.1 or CaV2.2 channels, synaptic
transmission was not [45,46]. However, coexpressing
CaVa2d-1 along with CaV2.1 or CaV2.2 channels in these
neurons augmented channel function, facilitated CaV2
coupling to exocytosis, and enhanced synaptic transmission [45] (Figure 5).
RIM proteins are also critical CaV2 channel partners at
nerve terminals. The reported actions of RIM proteins on
presynaptic CaV channels are diverse: they augment
CaV2.1 currents in presynaptic calyx of Held terminals
[37], decrease voltage-dependent inactivation (VDI) of
CaV2.2 channels, interfere with the inhibitory actions of
m-opioid receptors on CaV2.2 channels expressed in HEK293 cells [47], and slow CaV1.3 channel inactivation in
inner hair cells [48]. However, their importance in tethering CaV2 channels to active zones is central to explaining
their in vivo function [38,49]. Meticulous high-resolution
analyses of the molecular and morphological architecture
of glutaminergic synapses show that RIM protein numbers
scale proportionately with presynaptic CaV2.1 channel
numbers at active zone areas [30]. Moreover, the numbers
of these complexes scale with, and predict, vesicle release
probability, consistent with critical function [30] (Figure 6).
Controlling subcellular targeting of CaV channels
Synapse-specific location of CaV2 channels
CaV2.1 and CaV2.2 channels couple differentially to neurotransmitter vesicle release machinery according to synapse
type. In hippocampal interneurons, CaV2.1 channels mediate the release of neurotransmitter from parvalbumin (PV)expressing fast-spiking basket cells, whereas CaV2.2 channels mediate the release of neurotransmitter from cholecystokinin (CCK)-expressing basket cells [50,51]. The physical
distance between presynaptic channels and calcium sensors
of exocytosis are predicted to differ between CaV2.1 and
CaV2.2. These predictions as based on the differential ability
of BAPTA and EGTA, which are fast and slow calcium
chelators, respectively, to inhibit synaptic events mediated
by CaV2.1 and CaV2.2 channels [50,51]. CaV2.1 channels at
PV nerve terminals are predicted to be within nanometers of
the calcium sensor that leads to synchronous GABA release,
whereas CaV2.2 channels at CCK terminals are predicted to
be within micrometers of the Ca sensor and are thought to
underlie the highly asynchronous GABA release of CCK
neurons [5052]. However, the precise molecular identity of
CaV2.2 and CaV2.1 splice isoforms at these synapses is not
yet determined. Therefore, it is possible that different splice
isoforms of CaV2, which are known to influence binding to
release machinery, contribute to synapse-specific differences in calcium-dependent exocytosis.
Alternative splicing of auxiliary CaV subunits, as well as
other interacting proteins, is also likely to contribute to the
functional diversity among different synapses. For example, the four CACNA2D1-4 genes that encode mammalian
CaVa2d14 subunits are each subject to alternative premRNA processing. Similarly, the regulating synaptic
membrane exocytosis 1 and 2 (Rims1 and Rims2) genes
generate five principal RIM proteins by the use of independent promoters (RIM1a, RIM1b, RIM2a, RIM2b, and
RIM2g) and both Rims genes contain sites of alternative
splicing [49]. This provides substantial capacity for synapse-specific differences in Ca-dependent transmitter release. Finally, asynchronous transmission can occur as a
consequence of prolonged CaV2 channel openings, a phenomenon that is significantly augmented in CaV2.1 channels associated with CaVb2a subunits [53]. CaVb subunits
strongly influence the rate of CaV2 channel inactivation,
and CaVb2a subunits in particular slow channel inactivation significantly compared with CaVb1, CaVb3, and CaVb4
subunits (e.g., [53]).
Proteins in nanodomains with synaptic CaV2 channels
Although lacking resolution at the level of specific synapses, a proteomics screen combined with affinity purification
and high-resolution quantitative mass spectroscopy has
identified over 200 proteins that interact closely with CaV2
proteins in rodent brain [54]. This valuable data set
includes a subset of proteins isolated from brain synaptosomes that were previously identified to interact with
CaV2.2 channels [55], as well as cytoskeletal and chaperone proteins and a complex of novel proteins involved in
both exocytosis and endocytosis (Figure 6). Functional
analyses, validating the significance of several of these
proteinprotein interactions, suggest the interactions
are critical to the subcellular specialization of CaV2 channels. For example, the collapsin response mediator protein
2 (CRMP2)CaV2.2 interaction controls CaV2.2 channel
current densities in sensory neurons [56,57]. Disrupting
this interaction using a cell permeable blocking peptide
605
Review
reduced CaV2.2 trafficking to presynaptic terminals, spontaneous excitatory postsynaptic currents in the spinal
dorsal horn, and inflammatory and neuropathic pain
[56]. The absence of gross behavioral or motor effects in
these mice supports a unique role for CRMP2CaV2.2 in
maladaptive responses in sensory neurons to noxious stimuli. Other protein interactions that, similar to CRMP2,
are mediated via C termini of CaV2 channels may be
important in stabilizing the presynaptic molecular architecture (Figure 6).
Perhaps the best-studied region of CaV2 channels is the
synprint site located in the IIIII intracellular linkers of
CaV2.1 and CaV2.2 (Figure 3). The synprint region binds
synaptic soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins syntaxin 1A
and SNAP-25 and synaptotagmin (Figure 6). This tripartite interaction (CaV2SNAREsynaptotagmin), which is
so critical to the release process, is reviewed in detail
elsewhere [58]. Less studied is the interaction between
endocytotic proteins and presynaptic CaV2 channels, most
notably adaptor-related protein complex 2 (AP-2; adaptor
protein for clathrin-mediated endocytosis) [54]. AP-2 was
shown to associate with CaV2.2 and CaV2.1, but not
CaV1.2, via the synprint region to control endocytosis,
but not exocytosis, at calyx of Held nerve terminals [59].
The IIIII linker sequence of CaV channels is highly variable among CACNA1 genes (Figure 3) and contains several
sites of alternative pre-mRNA splicing (Figure 3). Therefore, alternative splicing of CaV2.2 and CaV2.1 pre-mRNAs
generates IIIII splice isoforms that have different capacities to interact with SNAREs [58] and potentially with AP2.
Proteins that bind and modulate pre- and postsynaptic
CaV1.3
CaV1.3 channels are found both pre- and postsynaptically in
cochlear inner hair cells (IHCs). Presynaptic CaV1.3 channels regulate transmitter release, interact with ribeye (the
ribbon synapse protein that promotes channel clustering),
and negatively regulate the size of the ribbon body [60,61]
(Figure 6). Harmonin also associates with presynaptic
CaV1.3 channels at the onset of hearing in 2-week-old mice.
This interaction promotes CaV1.3 ubiquitination and leads
to a decrease in channel surface expression. In the deaf
circler mouse, dfcr, a mutant allele of the harmonin gene
disrupts the harmonin interaction with CaV1.3, leading to
abnormally high CaV1.3 currents in IHCs [42]. Postsynaptic
CaV1.3 channels also have a critical role in the auditory
brainstem, where they are required for the normal development of the inhibitory medial nucleus of the trapezoid body
(MNTB) to the lateral superior olive (LSO) projection
[62,63]. There is a substantial reduction in the number of
MNTBLSO axons over the first 2 weeks of life, concomitant
with strengthening of the remaining synapses. Thus, postsynaptic CaV1.3 channels in the LSO neurons are thought to
be vital for the development and maturation of inhibitory
MNTBLSO projections [62]. Postsynaptic CaV1.3 channels
isolated from brain are also found in a complex with densin
and calcium/calmodulin-dependent protein kinase II (CaMKII), where they localize to dendritic spines [64]. Densin
promotes calcium-dependent facilitation of CaV1.3 channels
606
[64]. Alternative pre-mRNA splicing of CACNA1D premRNA must generate functionally distinct presynaptic
and postsynaptic CaV1.3 channels. Defining the major splicing patterns of CaV1.3 isoforms, their differential expression
patterns, and functional properties should provide valuable
insight into structural domains essential for presynaptic
and postsynaptic function.
Concluding remarks and challenges
The number of molecularly distinct CaV proteins that can
be generated from CACNA1 genes is stunning. Cell specific
gene expression, alternative pre-mRNA splicing, RNA
editing, posttranslational modifications including ubiquitination, miRNA targeting, and subcellular specific proteinprotein interactions are all used according to the
demands of the cell.
At the level of RNA, we still lack cell specific information
about which mRNA is expressed and when. Several cell
type-specific transcriptome data sets have been generated
from the combined application of methods, including highthroughput mRNA sequencing, pooling of genetically homogenous cells, and enriching for polysomal RNAs [13,65].
These methods are cataloguing CACNA1 transcripts according to neuronal subtype and developmental stage, although
substantial variability in the composition of certain transcripts has been observed across different studies and stochastic variations in transcriptomes are evident even in
genetically similar cell types [13]. Genetically targeted
and affinity-based miRNA profiling (miRAP) has also generated the first comparative analysis of cell specific miRNAs
in glutamatergic and GABAergic neurons of neocortex and
cerebellum [66], giving insights into miRNA-dependent
control of mRNA translation and stability in different classes of neuron. In addition, recent genome-wide mapping of
splicing factor-binding sites by CLIP-seq, as already discussed, locates sites of splicing factor binding relative to
target exons in pre-mRNAs that, combined with other information, could eventually lead to a splicing code [8]. These,
and other sequence-based data sets not mentioned here, are
publicly accessible (http://genome.ucsc.edu/) and recently
integrated with the encyclopedia of DNA elements project
(ENCODE, http://www.genome.gov/10005107).
At the protein level, major challenges remain but exciting technological advances are being made that combine
high-resolution imaging with proteomic analyses of single
synapses [67]. It should soon be possible to visualize and
distinguish among CaV isoforms with the spatial resolution
needed to place them at individual active zones and synapses. Similarly, it may be possible to define unique patterns of posttranslational modifications, and characterize
unique proteinCaV interactions that occur in highly localized, specialized subcellular regions of neurons. Such information should reveal the molecular mechanisms that
underlie functional specialization at the level of individual
synapses, and may suggest new therapeutic strategies to
target specific regions or neural circuits within the brain.
At the gene level, several hereditary diseases and disorders originate from point mutations or trinucleotide
expansions in CACNA1 genes, but a major, as yet unexplained finding implicates CACNA1C (CaV1.2) in determining an individuals susceptibility to a range of
Review
psychiatric illnesses. In the most recent and largest genome-wide association study of its kind, four risk loci were
identified with significant and overlapping links to autism
spectrum disorder, attention-deficit/hyperactivity disorder, bipolar disorder, major depressive disorder, and
schizophrenia [68]. Two of these risk single nucleotide
polymorphisms (SNPs) are located in calcium channel
genes, CACNA1C (CaV1.2) and CACNB2 (CaVb2) [69
72]. The widespread expression of CaV1.2 channels in
the body, including muscle cells of the cardiovascular
system, endocrine cells, and smooth muscle, adds to the
intrigue. The cellular mechanisms described here that
individualize CaV channel function according to cell type
and alternative pre-mRNA splicing, in particular, could be
selectively disrupted in individuals exhibiting psychiatric
illness without involvement of non-CNS systems.
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609
Review
Blurring the boundaries:

developmental and activity-dependent
determinants of neural circuits
Verena Wolfram and Richard A. Baines
Faculty of Life Sciences, University of Manchester, Manchester, UK
The human brain comprises approximately 100 billion

neurons that express a diverse, and often subtypespecific, set of neurotransmitters and voltage-gated
ion channels. Given this enormous complexity, a fundamental question is how is this achieved? The acquisition
of neurotransmitter phenotype was viewed as being set
by developmental programs hard wired into the genome. By contrast, the expression of neuron-specific ion
channels was considered to be highly dynamic (i.e., soft
wired) and shaped largely by activity-dependent mechanisms. Recent evidence blurs this distinction by showing that neurotransmitter phenotype can be altered by
activity and that neuron type-specific ion channel expression can be set, and perhaps limited by, developmental programs. Better understanding of these early
regulatory mechanisms may offer new avenues to avert
the behavioral changes that are characteristic of many
mental illnesses.
Neurons express diverse signaling properties
Neural circuits in organisms as diverse as worms, flies, and
humans exhibit remarkably similar design and developmental principles [13]. Circuit function depends on the
concerted action of distinct classes of sensory neuron,
regulatory interneuron and motor neuron. The function
of each neuronal subtype is defined by its position, axon
trajectory, synaptic connectivity, neurotransmitter expression, and electrophysiological properties. An important but
unanswered question is how do neurons acquire subtypespecific properties? The answer undoubtedly depends on
the relative contributions of both developmental programs
(e.g., the type-specific transcription factor expression) and
activity-dependent mechanisms.
Although the identification of the developmental and
activity-dependent mechanisms that shape axon trajectory
and neurotransmitter phenotypes has progressed [46],
the same is not true for the regulation of expression of
ionic currents in early embryonic neurons. Progress has
been hampered by the lack of suitable model systems in
which genetics and electrophysiology can be combined at
the level of identifiable neurons. Recent developments in
the fruit fly, Drosophila melanogaster, and zebrafish,
Corresponding author: Baines, R.A. (Richard.Baines@manchester.ac.uk).
2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tins.2013.06.006
610
Danio rerio, which allow such recordings, have started

to yield important first clues. In this review, we consider
these recent findings that directly relate to the question of
how to build a neural circuit. We present an updated view
of the potential contribution of differing regulatory mechanisms operative during development that determine active signaling properties of embryonic neurons.
Neuronal specification
Neuronal specification occurs early during embryogenesis
and, for simplicity, can be considered to comprise three
broad processes: proliferation, migration, and differentiation. Although all of these processes are crucial for the
proper formation of neural circuits, we concentrate on
neuronal differentiation in this review. It is during neuronal differentiation that neurons first acquire their specific,
and often unique, properties; these include axon projection,
dendrite arborization, neurotransmitter specification, and
ion channel expression. Until recently, the three former
properties were considered to be highly stereotypic and
were described as being hard wired. In support of this,
many developmental transcription factors have been identified as important determinants that specify axon pathfinding and neurotransmitter specification. By contrast,
much less is known with regard to the developmental
determinants that orchestrate ion channel gene expression. Indeed, this aspect of neuronal function has been
shown to be dynamic and under extensive control of extrinsic neuronal activity [7] and activity-dependent homeostatic mechanisms [8]. Implicit in these studies is the
concept that, unlike specification of neurotransmitters,
the emergence of electrical properties in embryonic neurons is dependent on network activity and, as such, could
be considered to be soft wired. However, the distinction
between hard- and soft-wired properties is blurred by
recent evidence that shows that neurotransmitter phenotype is influenced by activity and that ion channel expression can be set by subtype-specific intrinsic developmental
mechanisms.
Neurotransmitter specification
In comparison to ion channels, our understanding of the
regulatory mechanisms that specify neurotransmitter expression is more comprehensive. The choice of neurotransmitter not only determines the functional modality of any
given neuron, but is also critical for the functionality of the
Review
circuit to which each neuron contributes. As such, the
specification of neurotransmitter phenotype is a key step
for each and every neuron during embryonic development.
Several studies have shown that neurotransmitters are set
by neuron subtype-specific transcriptional programs and,
as such, could be considered to be a hard wired characteristic. However, as we describe here, recent experiments
overturn this view by showing that the choice of neurotransmitter can be respecified by neural activity [5].
The setting of neurotransmitter phenotype through cell
type-specific developmental programs could be considered
consistent with both the robustness and stability of neurotransmitter expression throughout the life of a neuron.
There are several considerations that strengthen such a
hypothesis. First, many neuron types generally express only
one classical neurotransmitter [most likely acetylcholine
(ACh), glutamate, GABA, serotonin, noradrenaline (nAdr),
or dopamine (Da)] and often neurons with different classical
transmitters develop from distinct pools of neuronal precursors [9]. Second, the restricted number of neurotransmitters and, in many cases [e.g., ACh, Da, nAdr, or 5hydroxytryptamine (5-HT)], the requirement of gene cassettes to produce that neurotransmitter is indicative of tight
transcriptional regulation. Third, many circuits, such as
sensory input circuitry or the motor network output, must
maintain reliable transmission, which would be ensured by
an early and stable encoding of neurotransmitter phenotype. Indeed, several studies (described below) describe
genetic programs, active during development, that specify
neurotransmitter expression.
Transcription factor specification of neurotransmitter
phenotype
In different phyla, neurotransmission at the peripheral
neuromuscular junction (NMJ) is mediated by different
neurotransmitters. For example, in the nematode Caenorhabditis elegans, NMJs comprise a mixture of excitatory
cholinergic and inhibitory GABAergic synapses [1013].
The excitatory NMJs of dipteran insects (i.e., Drosophila)
and chordates (i.e., vertebrate) utilize glutamate and ACh,
respectively [1416]. The expectation that neurotransmitter expression is tightly regulated at NMJs has been partly
met by the identification of transcription factors of the LIM
domain and homeodomain (HD domain) families, which
are differentially expressed in motor neurons, where they
orchestrate the developmental decisions of which neurotransmitter to express [1719].
Neurotransmitter specification is arguably best understood within the eight classes of C. elegans motor neuron
(AS, DA, DB, DD, VA, VB, VC, and VD). For example,
expression of the UNC-3 transcription factor is sufficient
to specify a cholinergic phenotype in type A and B motor
neurons (VA, VB, DA, DB, and AS) (Figure 1A) [17], whereas
the HD transcription factor UNC-30 is required for the
GABAergic phenotype of D motor neurons (VD, DD)
[20,21]. In addition, AST-1, an E-twenty six (ETS) domain
transcription factor, is sufficient to coordinate expression of
genes required to synthesize Da [22]. Such observations are
consistent with a simple, perhaps even a one factorone
transmitter code. Acquisition of an appropriate neurotransmitter phenotype often requires coordinated expression of
several genes, including enzymes that are essential for the

synthesis of transmitters, vesicular transporters, and, in
some cases, autoreceptors. Coregulation of such gene cassettes by transcription factors is facilitated in one of two
ways: either members of gene cassettes are organized within
a single transcriptional unit or operon [23,24] or, when
dispersed across the genome, are coordinately regulated
by means of common cis-regulatory elements [25].
It seems unlikely, even in the relatively simple central
nervous system (CNS) of C. elegans, that a one factorone
transmitter code is sufficient for all neurotransmitter
choices. Indeed, although UNC-3 specifies a cholinergic
phenotype in A and B motor neurons [17], all cholinergic
neurons are not specified by UNC-3; neither are all UNC-3
neurons cholinergic. For example, UNC-3 is not required
for the cholinergic phenotype of the AIY interneuron,
which is, instead, governed by the interplay of TTX-3
and CEH-10 [26]. UNC-3 expression is also observed in
the noncholinergic ASI chemosensory neuron that releases
neuropeptide-like proteins, such as N-acetylneuraminate
pyruvate lyase (NPL1) [27,28]. These latter observations
support the existence of more complicated and contextspecific transcription codes.
Developmental studies in Drosophila motor neurons
have made important contributions to understanding
the mechanisms of neuronal differentiation. Conserved
transcription factors, such as Even-skipped (Eve), Islet,
Lim3, and Hb9, have been shown to have pivotal roles in
neuronal subtype specification [2932]. These transcription factors are differentially expressed between motor
neurons and subsets of interneurons, supporting a concept
of combinatorial activity [30,31,33]. Interestingly, it is in
interneurons where the potential to specify neurotransmitter phenotypes has been shown. For example, Islet is
required for both serotonergic and dopaminergic interneuron phenotypes and, moreover, ectopic expression is sufficient to initiate expression of the Da-synthesizing enzyme
tyrosine hydroxylase in some, but not all, neurons. Importantly, ectopic expression must occur early during neuronal development to alter transmitter phenotype,
suggestive of the presence of a critical period [33].
In vertebrates, mature NMJs are cholinergic and most, if
not all, motor neurons express Islet-1, Islet-2, Lim3 (Lhx3),
and Hb9 (MNR2/MNX1), at some stage during their development. Expression of Islet-1, as well as MNR2 and Lhx3,
has been associated with a cholinergic phenotype. Thus,
ectopic expression of MNR2 in interneurons, normally
expressed in paired box 6 (PAX6+) motor neuron progenitors,
is sufficient to activate a motor neuron-like developmental
program including the expression of choline acetyltransferase (ChAT), the rate-limiting enzyme in the synthesis of ACh
[18]. However, it is clear that MNR2 (Hb9) alone is insufficient to determine a cholinergic phenotype because it is also
expressed in noncholinergic neurons, such as mouse ventral
spinal glutamatergic interneurons [34]. Transmitter choice
can also be achieved by the active suppression of alternative
transmitter phenotypes. Islet-1 participates in an early fate
decision between zebrafish primary motor neurons and
interneurons by repressing the interneuronal GABAergic
phenotype (Figure 1A) and, hence, the motor neuron
phenotype is established [19].
611
Review
(A)
Caenorhabdis elegans
UNC-3
Zebrash
Islet
ACh
Chicken
GABA
Drosophila
Islet
Mouse
5-HT
(B)
GABA
DBX1
_ _ _
+ + +
Low
acvity
High
acvity
+
+ +
_ _
_
_
_ _
_ _ _
ChAT
MNR2
+ +
+
+ +
+ + +
_ _
_ _ _
+ + +
Figure 1. Specification of neurotransmitter phenotype. (A) Neurotransmitter phenotypes are genetically specified early during development by expression of
developmental transcription factors. Transcription factors act by activating or repressing the transcription of proteins that are important for the synthesis and transport
of neurotransmitters. Often, transcription factors are part of combinatorial codes and act together in a complex. Examples of transcription factors are given for neurons of
representative species across different phyla. Arrows indicate that the transcription factors are required for expression of the respective neurotransmitter, whereas T-bars
indicate a repressive effect. (B) Activity-dependent switching of neurotransmitter phenotypes. Studies in Xenopus demonstrate that enhanced (shown on the right) as well
as reduced (shown on the left) neuronal activity is sufficient to induce a respecification of the neurotransmitter phenotype in neurons of the spinal cord to maintain an
appropriate excitationinhibition balance. Decreased activity favors an increase in neurons expressing excitatory neurotransmitters [acetylcholine (ACh) and glutamate,
orange circles], whereas an increase in activity leads to increased numbers of GABA-expressing neurons (blue circles) [48]. Abbreviations: 5-HT, 5-hydroxytryptamine;
ChAT, choline acetyltransferase; DBX1, developing brain homeobox 1; UNC-3, uncoordinated-3.
Studies of vertebrate interneurons have also provided

substantial clues to the complexity of neurotransmitter
specification. Interneurons are either inhibitory, expressing GABA or glycine, or excitatory, mostly expressing
glutamate. In mouse dorsal horn neurons, two transcription factors, T cell leukemia, homeobox 3 (TLX3) and
ladybird homeobox 1 (LBX1), determine whether glutamate or GABA is expressed. Whereas TLX3 promotes the
glutamatergic phenotype and suppresses the GABAergic
phenotype [35], LBX1 promotes the GABAergic phenotype
and suppresses the glutamatergic phenotype [36]. By manipulation of either TLX3 or LBX1, a neuron can be pushed
toward excitatory or inhibitory neurotransmission, respectively. In neurons where both TLX3 and LBX1 are coexpressed, TLX3 antagonizes the function of LBX1, thus
612
ensuring that, ultimately, only one of these two transmitters, with diametrically opposite effects, is specified [36].
The above example demonstrates nicely how two transcription factors compete to specify neurotransmitter phenotypes. However, often multiple transcription factors
work together to regulate the same neurotransmitter phenotype in different neuronal subsets and, as such, form a
combinatorial code. Evidence for combinatorial activity
includes the observations that Islet-1 represses the interneuron-specific GABAergic phenotype in zebrafish motor
neurons [19]. However, when Islet-1 is misexpressed in
GABAergic interneurons, not all go on to acquire a motor
neuron identity. Indeed, only interneurons that express
the transcription factor Lhx3 seemingly have this potential. Lhx3 is also expressed in motor neurons, suggesting
Review
(A)
(B)
Acvity
(C)
Figure 2. Specification of ion channel expression in embryonic neurons. To acquire the unique electrical properties that will underpin the contribution of a neuron to a
circuit, there are two more probable scenarios. (A) The neuron expresses a common default set of ion channels, making it almost indistinguishable from other neurons (as
indicated by the consistency in color). Once part of a network (C), activity-dependent mechanisms shape the final cocktail of ion channels expressed. (B) Individual neuron
subtypes express distinct sets of ion channels before the formation of networks, regulated by differential developmental programs (indicated by the different colors of the
surrounding neurons). These differences in ion channel repertoire convey distinct functionality to each neuron that might be postulated to reduce the time required to
produce functional networks. Similarly, once part of a functional network (C), activity-dependent mechanisms act to fine-tune those electrical properties.
that it is the coexpression of Islet-1 and Lhx3 that facilitates the manifestation of a motor neuron identity [37] and
suppression of a GABAergic phenotype [19]. Similarly, in
mouse, the GABAergic phenotype in excitatory interneurons is repressed by developing brain homeobox 1 (DBX1)
[38]. Intriguingly, both DBX1 and Islet-1 are also
expressed in GABAergic interneurons [3941], indicating
that their ability to repress the GABAergic phenotype is
context dependent.
Similar to that observed in C. elegans [22,26], consensus-binding sequences have also been described for selected vertebrate transcriptional regulators. For example, the
mouse ETS domain transcription factor Pet-1 binds a cisregulatory element that directs expression of serotonin
pathway genes, including tryptophan hydroxylase 2
(Tph2), the rate-limiting enzyme in the synthesis of serotonin, and solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (Slc6a4), the serotonin
transporter [42]. Moreover, Pet-1 is required throughout
development and in to adult life to establish and maintain
the serotonergic phenotype. Similarly, the homeodomain
transcription factor paired-like homeodomain 3 (Pitx3),
together with its interactor Nurr1, regulates genes required for Da synthesis, again through binding to specific
promoter elements [43]. These types of observation are
consistent with these transcription factors acting as terminal selectors of neurotransmitter phenotypes [44].
Activity-dependent respecification of neurotransmitter
phenotype
When considering motor neurons and sensory neurons, a
permanent and stable neurotransmitter phenotype might
impart stability for function, which would be consistent
with the role of these types of neuron. By contrast, within
more complex central interneuron networks, neurotransmitter plasticity could offer a mechanism to maintain the
important balance between neuronal excitation and inhibition (E/I). An appropriate E/I balance has been hypothesized to be critical for neuronal development, and
disturbance has been linked to an increased probability
for neurological disorders, such as seizure, autism, and

schizophrenia [45]. However, it recently became apparent
that both central interneurons and motor neurons can
undergo activity-dependent respecification of neurotransmitter phenotype [46,47]. These experiments were carried
out in Xenopus embryos where different classes of spinal
cord neuron show distinct patterns of spontaneous Ca2+
spike activity [46]. Experimentally decreasing this activity, by expressing the Kir2.1 K+ channel, resulted in increased expression of excitatory neurotransmitters,
glutamate and ACh, over the inhibitory transmitters,
GABA and glycine. By contrast, increasing Ca2+ spike
activity, by overexpressing a voltage-gated Na+ channel,
was sufficient to induce a compensatory increase in inhibitory transmitter expression [46]. Such activity-dependent neurotransmitter respecification is achieved
through a step in which excitatory and inhibitory neurotransmitters are coexpressed [46], but whether it always
results in a complete replacement of transmitters remains
to be evaluated (Figure 1B).
Activity can also regulate the occurrence of the neuromodulator serotonin, with increasing activity resulting
in a reduction of serotonergic neurons [48]. Where examined, neurotransmitter respecification by activity is
transduced via an activity-dependent regulation of transcription factors, such as Tlx3 and Lmx1b [47,48]. As
discussed above, in chick and mouse spinal cord, Tlx3
acts as molecular switch that favors glutamatergic over
GABAergic neurotransmission. Ectopic expression of
Tlx3 is sufficient to increase glutamatergic neurons at
the expense of GABAergic cells, whereas loss-of-function
mutants show the opposite effect [36]. Neurotransmitter
switching, which might be important to maintain an
appropriate E/I balance [46,47], is confined to a brief
critical period before synapse formation. The details of
how electrical activity acts to respecify neurotransmitter
phenotype remain to be determined. However, it is clear
that activity can alter transmitter phenotype at a time
when genetically determined programs were widely believed to predominate.
613
Review
Box 1. Development of electrical properties of Drosophila embryonic central neurons

The first appearance of each current, recorded in motor neurons, and
its continuity throughout development are shown in Figure I.
Important behavioral outputs, such as first muscle movements to
hatching, are also indicated. Time is counted from initial egg laying in
hours.
The first currents to express are the voltage-activated delayed rectifier
K+ conductance and ligand-gated ACh currents. These are followed by
voltage-activated Ca2+ and Na+ currents. A-type K+ currents, including

Shaker and Shal, coincide with the appearance of the first action
potentials, which are preceded in these neurons by excitatory
postsynaptic potentials (EPSCs). Ionic currents similarly develop in a
time-dependent manner in Xenopus neurons; however, here, voltagegated Ca2+ currents precede other currents [53]. Similar to Drosophila,
the A-type K+ current shows a late onset in Xenopus [52].
ICa
ACh response
INa
IA
CNS
Acon potenals
IK
EPSCs
12
13
14
15
16
17
Onset of coordinated
movement
First
Movements
18
19
20
Hours AEL
21
Hatching
Figure I. Time line for development of motor neuron electrical properties. Abbreviations: ACh, acetylcholine; AEL, after egg laying; CNS, central nervous system.
Adapted from [51].
Ion channel expression

Just as neurotransmitters underpin neuronal communication within a CNS, so a well-tuned set of voltage-gated ion
channels is essential for the ability of the receiving neuron
to integrate synaptic information and to instigate an appropriate neuronal output. In contrast to neurotransmitters, the identification of transcription factors capable of
regulating ion channel gene expression is more limited.
Questions also remain as to whether the same transcription factors coregulate both aspects of neuronal differentiation. As we describe below, early indications suggest that
this is indeed so, raising the possibility of coregulation of
multiple aspects of neuronal signaling through common
developmental mechanisms.
Ion channel expression, plasticity, and homeostasis
Electrophysiological analysis of mature neurons has generated a paradox. This is because, although it is possible to
distinguish, and even identify, neuron subtypes by their
characteristic expression of ion channels, the same
electrophysiological behavior can, in silico, be induced
by multiple disparate sets of underlying ion channels
[49,50]. Therefore, a key question is the extent to which
ion channel gene expression is regulated by developmental, as opposed to activity-dependent, mechanisms. The
former might be expected to result in fixed expression
levels of ion channel genes, whereas the latter might
achieve appropriate circuit outputs through more varied
expression patterns.
It could be envisaged that the ion channel repertoire of
embryonic neuron subtypes is indistinguishable from one
another, representing a default, developmentally determined, state (Figure 2). The stereotypic and sequential
expression of specific ion channels by developing neurons is
614
consistent with this view [5153]. By contrast, numerous

studies have highlighted the importance of activity-dependent homeostatic regulation of ion channel expression in
both developing and mature neurons [5456]. Where observed, homeostatic regulation of neuronal electrical properties is considered important for network stability by
maintaining a constant neuronal output (i.e., action potential firing) in response to changes in network synaptic
activity, rapid turnover of ion channels, or unpredictable
network perturbations [57]. A key substrate for homeostatic plasticity is the repertoire of voltage-dependent conductances expressed by neurons, in particular voltage-gated
Na+ conductances [5862]. Because of the presumed importance that activity has in establishing, refining, and
maintaining the ion channel repertoire of a given neuron,
the role of developmental transcriptional programs has
only very recently been appreciated. Now a new picture
emerges in which developmental factors specify neuron
subtype-specific ion channel expression profiles before circuit formation. Of course, these properties are likely open
to subsequent activity-dependent modification once network activity is established.
Ion channel specification by developmental factors
Computational modeling has suggested that a fixed neuronal output can be achieved by an almost random combination of many ion channels, creating an almost indefinite
parameter space [49]. Electrophysiological analysis of biological neurons shows that the actual parameter space is
more restricted. Elegant work from the Marder laboratory
and colleagues has shown that the expression of ion channels, in a mature neuron, does not vary randomly but
rather in a coordinated fashion, with pairs or sets of
channels exhibiting either transcriptional correlation or
Review
(A)
IKv
dMN
incl. * RP2 and ** aCC
*
**
vMN
incl. RP1, RP3, RP4 and RP5
WT dMN
AC
200 pA/pF
PC
WT vMN
10 ms
Dorsal muscles
Ventral muscles
40 mV
60 mV
90 mV
(B)
MiP
CaP
MiP (dMN)
500 pA
10 ms
CaP (vMN)
20 mV
80 mV
40 mV
Figure 3. Embryonic motor neurons show distinct electrophysiological properties before active synapse formation. (A) Diagram of the ventral nerve cord in a late-stage
Drosophila embryo. Dorsal motor neurons (dMN, in magenta) express the transcription factor Even-skipped and project to dorsal muscles. Ventral motor neurons (vMN, in
green) express the transcription factor Islet and project to ventral muscles. Voltage clamp experiments show differences in outward K+ currents between the two motor
neuron populations that are independent of synaptic activity. Magnitude of currents are normalized to cell capacitance. (B) Diagram of the zebrafish spinal cord depicting
two distinct primary motor neurons, the dorsal projecting, Islet-1 expressing MiP (magenta) and the ventral projecting, Islet-2 expressing CaP (green). Voltage clamp
recordings show that K+ currents are larger in ventral motor neurons compared with dorsal motor neurons. Adapted and modified from [67] (A) and [66] (B).
anticorrelation [63,64]. This may be indicative that, similar to neurotransmitters, ion channels are also regulated in
a gene battery-like manner. Transcription linkage of ion
channels might also explain why neurons of the same type
or origin can be distinguished by their electrophysiological
properties (i.e., by the ion channels they express). In
addition, it points toward single neurons expressing a
restricted ion channel repertoire that may be acquired
through early developmental mechanisms.
Where it has been investigated, ion channel expression, similar to the expression of neurotransmitters, is
detected very early during embryonic development and
before synapse formation [51,53] (Box 1). It is tempting to
speculate that this is because both are coregulated by the
same developmental mechanisms. This early expression
is independent of external factors, such as synaptic activity, and is seen specifically for ion channels required for
basic neuronal excitation, such as voltage-gated Na+,
Ca2+, and K+ channels [52]. However, the majority of
these studies were confined to mostly unidentified neurons and one cannot exclude the possibility that neurons
express a default set of ion channels in a time-dependent
manner. What has been missing is a comparative analysis of the acquisition of electrical conductances within
individual neurons of particular subtypes (e.g., motor

neurons). One of the first model organisms where such
a comparative analysis was possible is Drosophila due, in
greater part, to the stereotypic position of identifiable
embryonic motor neurons and their accessibility to
electrophysiology.
Within the past few years, it has been established in
both Drosophila and zebrafish that distinct motor neurons
exhibit different electrical properties before synapse formation (Figure 3) [6567]. These initial studies provided
the first clear indications that the expression of at least
some ion channels is regulated by early developmental
mechanisms and that these are specific to particular
neuronal subtypes. In accordance with this hypothesis,
ion channels have been identified as potential targets of
differentially expressed transcription factors in worms,
flies, and vertebrates [17,65,68]. Specific examples include
Ca2+ and K+ voltage-activated channels in C. elegans that
contain a motif (COE motif), which is recognized by the
UNC-3 transcription factor, a determinant of the cholinergic phenotype of type A and B motor neurons [17].
However, no electrophysiological data are available for
these motor neurons. By contrast, the terminal selector
gene TTX3 is required for the normal expression of
615
Review
Box 2. Slowpoke and Shaker K+ currents contribute to
action potential firing
Slowpoke
Slowpoke is the Drosophila homolog of vertebrate Ca2+-gated K+
channels (BKs) [77,78]. BKs are activated by membrane depolarization and simultaneous increase in intracellular Ca2+ and contribute
to the repolarizing phase of the action potential and the afterhyperpolarization [79]. Depending on cell context, BKs can both
dampen or increase action potential firing [65,80].
Shaker
Shaker is the Drosophila homolog of vertebrate Kv1.1, a voltagedependent K+ channel, carrying A-type current (fast activating and
inactivating) [8183]. Shaker and its homologs are activated by
membrane depolarization. They are involved in repolarization of the
action potential and, as such, can modulate firing frequency [84] and
spike propagation [85]. Often, the presence of the A-type channel
and its localization leads to a reduction or dampening of action
potential firing [86].
outward K+ currents in AIY interneurons [69], although in

an earlier study, no specific voltage-gated ion channel
target was identified [26]. In Drosophila dorsally projecting motor neurons, Eve has been shown to regulate the
expression of Slowpoke, a Ca2+ and voltage-gated K+
channel of the BK family [65]. In addition, Islet-1 knockout
was shown to influence the transcription of several ion
channel genes in zebrafish motor neurons, as evidenced by
microarray studies [68].
Perhaps the most conclusive studies to date for the early
developmental regulation of ion channel expression come
from Drosophila, where a direct link between the differential expression of transcription factors, ion channels, and
electrophysiological properties has been recently demonstrated. In the ventral nerve cord of the Drosophila larva,
motor neurons can be readily identified by cell body position, axonal projection, and muscle targets [7072]. Differences in axonal targeting, to either dorsal or ventral body
wall muscles, have been linked to subtype-specific expression of developmentally important transcription factors, in
particular expression of Eve being required for dorsal axon
targeting and Islet for ventral axon targeting [29,73].
Whereas Eve is expressed in motor neurons innervating
dorsal muscles, so-called dorsal motor neurons [29], Islet,
Lim3, and Hb9 are expressed in motor neurons that target
ventral muscles, termed ventral motor neurons [30,32,33].
Recently, it has been shown that dorsal and ventral motor
neurons differ in their electrophysiological properties
(Figure 3A) [65,67]. Specifically, dorsal motor neurons
exhibit larger outward K+ currents (Figure 3) and fire
fewer action potentials (Figure 4) [67]. Two transcription
factors, Eve and Islet, have been linked to subtype-specific
ion channel gene expression. In dorsal motor neurons [29]
Eve is sufficient to downregulate, but not abolish, the
expression of slowpoke [65]. By contrast, in ventral motor
neurons, Islet is both necessary and sufficient to repress
completely the Shaker K+ current, an A-type K+ current
analogous to vertebrate Kv1.1 [67] (Box 2). Whereas Slowpoke is expressed in both motor neuron types, albeit to
varying amounts, Shaker is absent from ventral motor
neurons. This is indicative that not only type, but also
the relative level, of ion channels are regulated by
616
transcription factors during early development. Such

mechanisms would facilitate the acquisition of subtypespecific ion channel repertoires on which plasticity and
homeostasis can subsequently act during later postembryonic stages.
Concluding remarks
Coordinated gene expression, coupled with activity-dependent refinement, underpins the formation of functional
neural circuits. The examples described above illustrate
the many interactions so far documented between developmental mechanisms (i.e., transcription factors) and neuronal activity in the specification of neurotransmitter
phenotype and ion channel expression in developing neurons. Although these are early days, several themes are
beginning to emerge that will hopefully be explored over
coming years.
The first is that the canonical view of properties such as
specification of neurotransmitters being developmentally
hard wired whereas expression of ion channels is set,
largely, as part of activity-dependent feedback (i.e., soft
wired) is blurred by recent experiments. The demonstration that neuron subtype-specific electrical properties are
set by developmental mechanisms is particularly important because it is consistent with the viewpoint that neural
circuit function and, therefore, behavior, are encoded to
some degree within the genome. Although not a new
concept [74], these first glimpses of developmental determination of ion channel gene expression offer substantial
evidence to support this view. Much might be gained from
re-evaluating whether some neurological disorders arise
from incorrect developmental specification of ion channel
gene expression during early neurogenesis. By contrast,
activity-dependent respecification of neurotransmitter
content overturns the long-held view that this neuronal
property is developmentally fixed. Interesting questions
include the precise timing during which the expression of
both neurotransmitter-associated and ion channel genes
are available to modification during neurogenesis and
whether change to one might instigate obligatory change
to the other.
A second important theme to emerge is that multiple
aspects of neuronal differentiation are regulated by common factors. Perhaps one of the best examples is from
Drosophila, where the transcription factor Islet is seemingly able to regulate axon guidance, neurotransmitter
phenotype, and expression of electrical properties. Might
we consider Islet to be a terminal selector and will all of its
target genes be identifiable by the presence of conserved
cis-regulatory motifs similar to what has been observed for
some C. elegans transcription factors (e.g., ttx-3 and unc-3)
[17,22,26]? Related questions include whether there are
additional terminal selectors that orchestrate separate or
overlapping cassettes of gene targets. Although recent
studies in C. elegans support this view [75,76], whether
this will also hold true for other organisms remains to be
determined. Taken to the extreme, this might mean that
we will be able to predict key neuronal properties based on
the profile of such transcription factor expression.
Although these, and other, important questions remain, the prospect is bright. The recent combination of
Review
(A)
(B)
vMN
dMN
+ Islet
Islet
Sh
- Islet
Sh
Figure 4. Islet is deterministic for motor neuron subtype electrical properties in Drosophila. (A) A dorsal (aCC) motor neuron labeled by DiI applied to its neuromuscular
junction (NMJ) with its target muscle (muscle 1). The dorsal motor neuron expresses the Shaker K+ channel, which conducts a fast activating-inactivating potassium current
akin to the A-type current (Box 2, main text). The traces show typical recordings of action potential firing obtained by current injection at the soma (10 pA for 500 ms) via
whole-cell current clamp. (B) A ventral motor neuron labeled by DiI applied to its NMJ with its target muscle (muscle 6). The ventral motor neurons do not express the
Shaker K+ channel due to transcriptional repression mediated by Islet. In the absence of Shaker, the neuron fires comparatively more action potentials during 500 ms of
10 pA current injection [compare to (A)]. When Islet is ectopically expressed in dorsal motor neurons, their endogenous Shaker K+ current is diminished. By contrast, loss of
function of Islet in ventral motor neurons results in a pronounced Shaker K+ current [67]. This demonstrates that, at least in Drosophila, Islet forms part of an developmental
decision-making process that is critical to specifying subtype-specific electrical properties in developing motor neurons before neural circuit formation.
electrophysiology and molecular genetics, in worms, flies,

and fish (to name just a few), offers the prospect of making
progress to answer these and other related questions to
determine the relative contribution of developmental
versus activity-dependent mechanisms for the formation
of neural circuits.
Acknowledgments
We thank Matthias Landgraf, Stefan Pulver, and Gino Poulin for their
comments. We also thank Carlo Giachello for his help in constructing the
figures. We are also grateful to Rosa Moreno and Angeles Ribera for
permission to reproduce their work in this review. The experiments
described from the Baines group were supported by the Wellcome Trust.
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October 2013 Volume 36, Number 10 pp.

Looking back and looking forward

Science & Society

The Brain Prize 2013: the optogenetics

Andreas Reiner and Ehud Y. Isacoff

Molecular nexopathies: a new paradigm of

Jason D. Warren, Jonathan D. Rohrer,

A developmental ontology for the

Luis Puelles, Megan Harrison,

Challenges of understanding brain function

Arvind Kumar, Ioannis Vlachos,

Sugar for the brain: the role of glucose in

Philipp Mergenthaler, Ute Lindauer,

Control of neuronal voltage-gated calcium

Diane Lipscombe, Summer E. Allen, and

Blurring the boundaries: developmental and

Verena Wolfram and Richard A. Baines

Looking back and looking forward

Trends in Neurosciences (TiNS) was launched over 35

Corresponding author: Clark, A.M. (tins@cell.com).

One of the key features of TiNS opinion articles is that

Trends in Neurosciences October 2013, Vol. 36, No. 10

or premier research journals, such as Neuron and Cell, to

Science & Society

The Brain Prize 2013: the optogenetics revolution

The 2013 Grete Lundbeck European Brain Research Prize

The beauty and the agony of brain circuits

for selectively stimulating or inhibiting activity in specific

Science & Society

Trends in Neurosciences October 2013, Vol. 36, No. 10

and Boris Zemelman [1] made a critical conceptual and

bacteria and single-cell algae had been studied for decades.

Science & Society

Trends in Neurosciences October 2013, Vol. 36, No. 10

as a minimally invasive stimulus allowed exquisite

Science & Society

Trends in Neurosciences October 2013, Vol. 36, No. 10

Molecular nexopathies: a new

Neural networks provide candidate substrates for the

on the key insight that neurodegenerative diseases, such as

Trends in Neurosciences October 2013, Vol. 36, No. 10

Box 1. The molecular nexopathy paradigm: key substrates and constraints

tau triggers uptake by cells and templated conformational alteration

that direct the expression of the protein to particular

Trends in Neurosciences October 2013, Vol. 36, No. 10

(C) Distributed no gradient

Figure 1. A taxonomy of molecular nexopathy mechanisms. Here, we model

may propagate only in particular cell types [44] or across

Figure 2. Scaling nexopathies to large-scale brain networks. The inset cartoon

macroscopically in the relative extent of grey matter versus

Trends in Neurosciences October 2013, Vol. 36, No. 10

Figure 3. Temporal evolution of disease and phenotypic heterogeneity. This

proteinopathy might spread between brain regions by

trafficking of amyloid and tau in the isodendritic core

Trends in Neurosciences October 2013, Vol. 36, No. 10

synaptic inputs. Such polarising network-level effects of

Trends in Neurosciences October 2013, Vol. 36, No. 10

Box 2. Testing the paradigm: outstanding questions and future directions

resolved, in part, by mapping longitudinal profiles of disease

Trends in Neurosciences October 2013, Vol. 36, No. 10

Trends in Neurosciences October 2013, Vol. 36, No. 10

44 Wickens, J.R. et al. (1995) Effects of local connectivity on striatal

Trends in Neurosciences October 2013, Vol. 36, No. 10