A Retinohypothalamic Projection in the Rat 's2

ROBERT Y . MOORE AND NICHOLAS J. LENN

Departments of Pediatrics, Medicine (Neurology) and Anatomy and
The Joseph P . Kennedy, Jr. Mental Retardation Research Center,
The University of Chicago, Chicago, Illinois 60637

ABSTRACT A direct projection from the retina to the hypothalamus was

demonstrated in the rat. Following injection of tritiated leucine or proline into
the posterior chamber of the eye, labelled protein was shown autoradiographically
in the suprachiasmatic nuclei of the medial hypothalamus, both ipsilateral and
contralateral to the injected eye. The labelling of the nucleus was heaviest in its
ventral portion but extended throughout the nucleus. No evidence for a projec-
tion to the supraoptic nucleus or any other hypothalamic nucleus was observed.
All of the known terminal nuclei of the primary and accessory optic tracts were
heavily labelled. The projection to the suprachiasmatic nucleus could not be
clearly confirmed in material prepared using the Fink-Heimer method for the
demonstration of degenerating axon terminals. Electron microscopic study of
the suprachiasmatic nucleus following orbital enucleation showed degenerating
endings making synaptic contacts with small dendrites of the suprachiasmatic
nucleus cells. These first appeared at three days after operation and were nearly
gone by seven days. Thus, the retinohypothalamic tract in the rat appears to
arise from the ganglion cells of the retina and to terminate on the smaller den-
dritic branches of the neurons of the suprachiasmatic nucleus.

It is now well-established that environ-
mental light plays an important role in
regulating hypothalamic-anterior pituitary
function in the adult mammal (cf. Harris,
'55; Wurtman, '67; Szentagothai et al., '68,
for reviews). Yet, despite the extensive in-
formation available on these neuroen-
docrine effects of light, the component of
the central retinal projection responsible
for mediating the effects has not been
identified. The simplest pathway by which
light might affect hypothalamo-hypophy-
seal function would be a direct projection
from the retina to the medial hypothala-
mus. There is substantial evidence for
such a projection in submammalian verte-
brates (Herrick, '42; Riss et al., '63; Knapp
et al., '65; Ebbesson, '68; Ebbesson and
Ramsey, '68; Bons and Assenmacher, '69)
but in mammals the literature concerning
a direct retinohypothalamic projection,
while large, is contradictory (cf. Hayhow
et al., '60; Kiernan, '67; Szentagothai et al.,
'68, pg. 59; Sousa-Pinto and Correia, '70,
for reviews). In a previous study we failed
to find convincing evidence for a retino-
hypothalamic projection in the rat (Moore,
'69) when applying the Fink-Heimer meth-
ods (Fink and Heimer, '67) at several
postoperative survival periods following
unilateral enucleation. The present study
was undertaken to re-investigate this prob-
lem using a new method for tracing cen-
tral pathways. Recent reports have demon-
strated that labelled amino acids injected
into the posterior chamber of the eye are
incorporated into protein by ganglion cells
and transported along the axons of the
optic nerve by axoplasmic flow to the ter-
minal nuclei of the optic system (Taylor
and Weiss, '65; Grafstein, '67; Sjostrand
and Karlsson, '69). In the lateral genicu-
late body the labelled protein has been
shown to be localized to nerve end-
ings both biochemically (Karlsson and
Sjostrand, '71) and by electron microscopic
autoradiography (Hendrickson, '69). These
findings suggested that an autoradio-
graphic tracing method might be useful
in demonstrating a retinohypothalamic
1The data reported here were presented in part at
the 84th meeting of the, American Association of
Anatomists (Moore et al., 71).
* Supported by USPHS grants NS-04677,NS-09313,
and HD-04583 from the National Institutes of Health.

J. COMP. NEUR.,146: 1-14. 1

2 ROBERT Y. MOORE AND NICHOLAS J. LENN
projection. Autoradiography following in-
jection of labelled amino acid has been
employed in a study of avian retino-tectal
projections (Schonbach and Cuenod, '71)
and in the analysis of several projection
systems in the mammalian brain (Lasek
et al., '67; Price and Woolsey, '71). In the
present study we applied the autoradio-
graphic method to define the nuclei of ter-
mination of a retinohypothalamic pathway
in the rat and confirmed this observation
by electron microscopic study of terminal
degeneration following orbital enucleation.
MATERIALS AND METHODS
The animals used in this study were
adult albino rats of both sexes of the Holtz-
man strain (Holtzman Co., Madison, Wis.).
They were housed in clear plastic cages
placed on cage racks illuminated by fluo-
rescent fixtures. The animals were main-
tained in 12 hours of lighting (lights on
AM to 7 PM) each day. All surgical pro-
cedures were carried out under ether
anesthesia and perfusions were performed
with the animals deeply anesthetized with
pentobarbital (50 mg/kg).
Approximately 30 animals were used for
the autoradiographic study. Each received
one injection of either Lleucine-4,5"H
(19.0 Ci/mM, Amersham/Searle or 38.6
Ci/mM, New England Nuclear) or L-pro-
li11e-2,3-~H (45.7 Ci/mM, New England
Nuclear) into the posterior chamber of
one eye through a Hamilton microliter
syringe fitted with a 32 gauge needle. The
dose of tritiated amino acid was 5-10 ,Ci
in 5-10 ,l of 0.9% saline solution. The
animals were allowed to survive for 1 to
28 days before being sacrified by perfusion
with 10% formaldehyde in 0.9% saline.
Brains were removed immediately and
stored in fixative for one to seven days,
dehydrated in graded alcohols, and em-
,
bedded in paraffin. Frontal 10 sections
were cut and mounted on slides. The slides
were coated with Kodak NTB-2 emulsion
according to the procedure of Kopriwa and
Leblond ('62) and were stored in a dark-
ened refrigerator for two to four weeks.
They were then developed in Dektol and
stained with cresyl violet. Some brains
were taken from the fixative, placed over-
night in a 50% ethanol solution, and dis-
sected into the following samples; frontal
cortex, visual cortex, lateral geniculate,
superior colliculus, medial terminal nu-
cleus, ventral septal area (septal nuclei,
olfactory tubercle), medial hypothalamus
(median eminence and adjacent nuclei
caudal to optic chiasm). These samples
were dried, weighed and placed individu-
ally in Soluene (Packard Instrument CO.)
for 8-12 hours and a toluene fluor was
added before radioactivity was estimated
in a Packard Tricarb liquid scintillation
spectrometer.
To study axon degeneration using a
conventional silver impregnation method,
ten animals were subjected to unilateral
orbital enucleation. Two were sacrificed
by perfusion with 0.9% saline solution
followed by 10% formaldehyde solution
at each of five postoperative survival
periods, 24, 36, 48, 60 and 72 hours.
Brains were removed and stored in fixa-
tive for 7-14 days before being sectioned
in the frontal plane at 30 on a freezing
microtome. Every fifth section through
the optic chiasm and hypothalamus was
prepared according to the first method of
Fink and Heimer ('67). A set of adjacent
sections was stained with cresyl violet.
Electron microscopic studies were car-
ried out both on normal animals and ani-
mals subjected to bilateral orbital enuclea-
tion. After the selected survival period the
animals were anesthetized with pento-
barbital and cooled to a body temperature
of approximately 26°C. They were then
perfused via the ascending aorta with
room temperature solutions. A 30-second
washout with McEwen's ('56) solution
(pH 8.2) was followed by six minutes per-
fusion with 60 ml of 2% osmium tetroxide
in phosphate buffer, pH 7.1. Thirty min-
utes later the brains were removed and
sliced coronally. The slices were fixed for
an additional hour in the same solution
and then washed in maleate buffer (pH
5.1 OOC), treated with 2% uranyl acetate
in maleate buffer, washed, dehydrated and
embedded in Durcupan. The suprachias-
matic nucleus was identified in the light
,
microscope in 0.5 sections stained with
toluidine blue. Blocks trimmed to include
only the ventral half of this nucleus were
sectioned for examination in the electron
microscope. Fifteen blocks from nine ani-
mals were studied including some nor-
 A RETINOHYPOTHALAMIC PROJECTION IN THE RAT 3

mals and animals sacrificed at 1, 2, 3, 4, evident in the brains obtained 60 hours

5 , 6 and 7 days after enucleation. after enucleation but it was our conclu-
sion that they could not be clearly differ-
RESULTS entiated from artifact. Without the auto-
Light microscopy. In material prepared radiographic observations the Fink-Heimer
for analysis of axonal and terminal degen- material could not be interpreted as pro-
eration by the Fink-Heimer method, degen- viding any indication of a retinohypo-
erating axons were evident in the optic thalamic projection.
chiasm and tracts by 36 hours after enu- The autoradiographic material provided
cleation and abundant terminal degenera- substantial evidence for a retinohypotha-
tion appeared in the dorsal lateral genicu- lamic tract. In sections from these brains
late in cases of 48 hour survival. At no the optic chiasm and tracts showed dense
time after operation, however, was there labelling, much greater on the contralateral
clear evidence of terminal degeneration in than the ipsilateral side. The hypothalamic
any of the medial hypothalamic nuclei. nuclei themselves were free of labelling
Aberrant chiasmal fibers were found pass- save for the suprachiasmatic nuclei which
ing through nuclei adjacent to the chiasm showed numerous silver grains, both ipsi-
but these always had the appearance of lateral and contralateral to the injected
degenerating fibers of passage. Retrospec- eye, particularly in the ventral half of the
tively, once the suprachiasmatic nucleus nucleus. A diagrammatic summary of this
had been identified as a probable site of projection is shown in figure 1. The label-
termination of retinal axons (cf. fig. 1 for ling of both suprachiasmatic nuclei was
a summary of the hypothalamic projec- substantially above background. The con-
tion), i t was possible to identify structures tralateral nucleus (fig. 3) was principally
in that nucleus in the Fink-Heimer sec- labelled in the area adjacent to the in-
tions that had some likeness to degenerat- tensely labelled optic chiasm and one
ing terminals (fig. 2). These were most might infer that the apparent silver grains

Fig. 1 Line drawings of the hypothalamus made from frontal sections through the
brain of a rat injected intraocularly with 10 NCi 3H-leucine. The sections were coated with
emulsion and autoradiographs were prepared. The dots on the drawings represent the loca-
tion of developed silver grains. A-D represent sections at successive rostra1 to caudal levels
of the hypothalamus. Abbreviations are as follows: A, arcuate nucleus; AC, anterior com-
missure; AH, anterior hypothalamic area; F, fornix; IC, internal capsule; INST, interstitial
nucleus of the stria terminalis; ME, median eminence; OC, optic chiasm; OT, optic tract;
PA, paraventricular nucleus of the hypothalamus; SC, suprachiasmatic nucleus; SO, supra-
optic nucleus, SOC, supraoptic commissures; VM, ventromedial nucleus.
4 ROBERT Y. MOORE AND NICHOLAS J. LENN
in the nucleus really reflected isotope in
the chiasm. This would not appear to be
the case, however, since the ipsilateral
nucleus showed a similar distribution of
label with very little in the adjacent
chiasm (fig. 4). In addition, there is no
labelling of the supraoptic nucleus immedi-
ately adjacent to the heavily labelled con-
tralateral optic tract (fig. 5). It could also
be argued that the label in the suprachias-
matic nucleus reflected isotope in fibers of
passage. The fact that both suprachias-
matic nuclei were similarly labelled would
be against such an interpretation but this
consideration prompted the electron micro-
scopic study reported below.
The autoradiographic material confirms
the observations of others on the termina-
tion of the primary and accessory optic
tracts (cf. Hayhow et al., '60, '62) with
the exception that a very profuse projec-
tion to the stratum zonale of the contra-
lateral superior colliculus was evident. A
partially quantitative estimate of the size
of these projections can be obtained from
table 1. The brains in this study were pre-
pared exactly as those for autoradiography
except that the fixed tissue was dissected
into samples which were prepared as care-
fully as possible to include only the nu-
cleus or area intended in the sample. From
these data the contralateral optic tract ap-
peared to terminate primarily in the lateral
Table 1
Inhaocular injection of 3H-proline in the rat -
distribution in visual system nuclei
DPM f mg dry weight
Brain recion tissue 5 S.E.
Right lateral geniculate 258.12 16.3
Left lateral geniculate 6414.3 2271.6
Right superior colliculus 140.12 8.7
Left superior colliculus 5878.92 359.8
Right medial terminal nucleus 39.22 3.1
Left medial terminal nucleus 295.2k 22.4
Right visual cortex 3 7 . 8 2 2.5
Left visual cortex 53.42 3.6
Right frontal cortex 38.62 5.6
Left frontal cortex 40.72 5.4
Medial hypothalamus 60.82 10.3
Ventral septal area 60.22 2.9
I Four rats were injected into the right eye with
10 pCi 3H-proline and sacrificed by perfusion with
formol-saline 15 days later. Brains were dissected,
washed, and samples were dried, weighed and dis-
solved in Soluene (Packard) before being counted.
2 The counts in the left visual cortex were sig-
nificantly greater than those in the right visual cortex
or either frontal cortex sample ( p < 0.02, two-tailed
t test).
geniculate and superior colliculus. The
ipsilateral projection to these areas, as
measured by the counts, was small. These
findings are in accord with the autoradio-
graphic observations. Similarly, both the
autoradiographic material and the count-
ing study indicated only a contralateral
projection to the terminal nuclei of the
accessory optic system. The frontal and
visual cortex samples were taken as con-
trols for the other tissue. But, in confirma-
tion of Grafstein's ('71) recent report, we
found a small and significant increase in
counts in the visual cortex contralateral
to the injected eye. This difference could
not be appreciated autoradiographically.
The medial hypothalamus sample con-
tained the median eminence and adjacent
medial hypothalamic nuclei caudal to the
optic chiasm. It did not differ significantly
from a control area, the septal area just
above the optic nerves, but both of these
were higher than the control frontal cortex
samples; suggesting that some labelled
proline or protein might have leaked into
the subarachnoid space near the entrance
of the optic nerves. The area immediately
adjacent to the optic chiasm could not be
dissected sufficiently well from the chiasm
for counting.
Electron microscopy. The neurons of
the suprachiasmatic nucleus, the glial cells
and the capillaries were normal in appear-
ance in all of the animals studied and did
not differ in their general features from
those previously described in other brain
regions in mammals. The neurons were
somewhat unusual, however, in that the
volume of cytoplasm was small, the nuclei
exhibited no deep cytoplasmic indentations
and the nucleoli were often located eccen-
trically within the nucleus.
In the normal animals and in the ani-
mals surviving one and two days after
operation, all of the presynaptic elements
and the synaptic contacts appeared en-
tirely normal and did not differ in any
significant way from the general appear-
ance of central synapses in mammals
(Palay, '58). Some axosomatic synapses
were seen and the axodendritic synapses
could be considered to occur in two groups;
one group ending on the larger proximal
dendrites and the second group on the
distal portions of the dendrites including
 A RETINOHYPOTHALAMIC PROJECTION IN THE RAT
spines. Some synaptic endings contained
larger (approximately 1000 A diameter)
synaptic vesicles but this could not be cor-
related with their site of synaptic contact.
In the animals surviving three to five
days after enucleation there was a small
but readily identifiable incidence of modi-
fied synaptic endings. These endings, in
all cases, were characterized by a con-
striction of their size involving either an
unusually smooth or an unusually crenated
outline (figs. 6, 7). They were increased
in density both because of increased pack-
ing of their synaptic vesicles and an in-
crease in the density of their matrix. In
addition, the synaptic vesicles and mito-
chondria had lost their integrity to a var-
iable degree. The mitochondria showed
increased density and loss of internal struc-
ture. Some of the degenerating endings
contained large dense cored vesicles. There
were no examples of degeneration showing
neurofilaments in the endings. The syn-
aptic contacts formed by the degenerating
endings and the post-synaptic structures
contacted were not modified in any way.
In virtually all cases the post-synaptic
members were small dendrites (figs. 6, 7).
The large majority of nerve endings in the
suprachiasmatic nucleus, however, were
entirely unmodified. On occasion an ap-
parently degenerating ending not making
a synaptic contact was observed sur-
rounded by astrocyte cytoplasm, but in no
case where the plane of section was favor-
able did this appear to represent other
than the frequent surrounding of nerve
endings by a process of astrocyte cyto-
plasm. In particular, there was no evidence
of phagocytosis of the degenerating end-
ings by astrocytes. At this stage many
myelinated axons also were modified in
that their axoplasm was increased in
density and often appeared separated from
the surrounding myelin. The myelin was
well preserved.
In the animals surviving six and seven
days only a few examples of nerve endings
exhibiting degenerative changes were ob-
served. The changes in the myelinated
axons were similar to those noted with
the shorter survival period.
It is not evident from this material that
the population of endings undergoing de-
generative changes following enucleation
5
is defined by the presence of a particular
type of synaptic vesicle. Some contained
large, dense cored vesicles but this was not
the rule. It was clear, though, that a small
proportion of the synaptic endings forming
axodendritic synapses with the more distal
portions of dendrites in the nucleus will
undergo degeneration following removal
of the eye. These observations provide con-
firmation at the ultrastructural level for
the autoradiographic findings indicating a
direct projection from the retina to the
suprachiasmatic nucleus.
DISCUSSION
The light and electron microscopic ob-
servations of the present study provide
substantial experimental evidence for the
existence of a retinohypothalamic projec-
tion in the rat. These observations indicate
that this projection originates in the gan-
glion cells of the retina and traverses the
optic nerve and chiasm to terminate bilat-
erally in the suprachiasmatic nuclei of the
hypothalamus. The projection is heaviest
to the ventral portion of these nuclei but
the entire nucleus appears to receive fibers
of retinal origin. There was no evidence for
a projection to any other region of the
hypothalamus. Two techniques were em-
ployed to obtain these observations. The
first utilized an autoradiographic method
for tracing the visual pathways. This
method is based upon the now well-estab-
lished finding that tritiated amino acids
injected into the posterior chamber of the
eye will be incorporated into proteins in
retinal ganglion cells and then transported
along the axons of these cells to their
termination (Taylor and Weiss, '65; Graf-
stein, '67, '69; Sjostrand and Karlsson, '69;
Karlsson and Sjostrand, '71). Previous
autoradiographic studies have shown the
labelled protein in the lateral geniculate
neuropil in the rabbit (Sjostrand and
Karlsson, '69) and, with electron micro-
scopic autoradiography (Hendrickson, '69),
the radioactivity was shown to be associ-
ated with synaptic endings in the monkey
lateral geniculate. Most of the brains used
for autoradiography and for scintillation
counting in the present study were from
animals surviving seven days or longer
after injection of the labelled amino acid
and, thus, demonstrated protein trans-
6 ROBERT Y. MOORE AND NICHOLAS J. LENN
ported in the slow as well as the rapid
phases of axoplasmic transport (Grafstein,
'69; Karlsson and Sjostrand, '71). The dis-
tribution of labelled material shown in the
counting study indicated that protein was
transported into all of the principal regions
of termination of the retinal projection and
this was confirmed by the autoradiographic
studies. There was no indication in any
of the autoradiographic material of trans-
location of the label beyond the boundaries
of optic tract or nuclei such as the lateral
geniculate or medial terminal nucleus.
This implies that the label demonstrated
in the suprachiasmatic nucleus repre-
sented an area of projection rather than
an artifact of technique. The strongest con-
firmation of this comes from the second
part of our study, the electron microscopic
examination of degenerating terminals in
the suprachiasmatic nucleus following
orbital enucleation. No degenerating ter-
minals were evident until three days after
operation and by six to seven days few re-
mained. It is unlikely that the changes
noted in these terminals were artifact
(e.g., of the type described by Cohen and
Pappas, '69). They were not present in
normal specimens, did not appear until
three days after operation in the enu-
cleated brains, and had a consistent rela-
tionship with a particular postsynaptic
element. Many of the degenerating end-
ings were in contact with small dendritic
elements exhibiting membrane specializa-
tions characteristic of a synaptic contact.
This would appear to establish that the
degenerating endings not only were of
retinal origin but that they made synaptic
contacts on suprachiasmatic nucleus neu-
rons. No other hypothalamic nuclei were
examined electron microscopically because
neither the autoradiographic material nor
the Fink-Heimer material indicated a pro-
jection to any other area of the hypo-
thalamus.
The prior evidence for a retinohypothal-
amic tract in mammals has been obtained
in a variety of species and using both nor-
mal brains and experimental material. A
very balanced review of this evidence has
been presented by Nauta and Haymaker
('69). The strongest proponents of the pro-
jection have been Frey ('37, '47, '50, '51),
Knoche ('56, '57) and Bliimcke ('58). In
addition, Polyak ('57) observed in Marchi
material from the rat that a few fibers
from the chiasm appeared to enter the
hypothalamus but could not be traced
further. His description of the visual path-
ways in the cat and monkey do not note a
similar group of fibers. In contrast to these
studies presenting evidence for a direct
retinohypothalamic projection, the major-
ity of workers carrying out experimental
studies have failed to fmd experimental
evidence for this projection in mammals
including Bodian ('37); Hayhow et al.
('60); Giolli ('63); Tigges ('66); Hayhow
('66); Campos-Ortega and Glees ('67);
Kiernan ('67); Szentagothai et al. ('68);
Garey and Powell ('68); Moore ('69);
Campbell and Hayhow ('71); Kostovic
('71). A recent exception to this was the
work of Sousa-Pinto ('70) and Sousa-Pinto
and Correia ('70) who found evidence in
Fink-Heimer material for a widespread pro-
jection into the preoptic, periventricular
and the medial tuberal region (ventro-
medial and arcuate nuclei) after enuclea-
tion in the rat. This projection is reminis-
cent of that described by Knoche ('56,
'57) and Bliimcke ('58) as the '<retino-
hypothalamische Bahn." The photographs
of experimental material ( Sousa-Pinto and
Castro-Correia, '70) show some structures
that are clearly normal axons exhibiting
pseudodegeneration. In addition, it is diffi-
cult to interpret some of the dark profiles
identified electron microscopically in the
arcuate nucleus by Sousa-Pinto ('70) as
degenerating endings. Some of these might
well be oligodendroglial processes or the
dark profiles described by Cohen and
Pappas ('69). As noted above we found
evidence only for a projection to the
suprachiasmatic nucleus which, while
bilateral, appeared very small. We did not
obtain evidence for a more widespread
distribution of retinal afferents in either
the Fink-Heimer material or the labelled
material. If there was a significant projec-
tion to the tuberal hypothalamus, it should
have been identifiable in the autoradio-
graphic material since the projection to the
suprachiasmatic nucleus was readily evi-
dent. This is particularly true if the pro-
jection was as widespread as suggested by
Sousa-Pinto and Correia ('70) and in-
volved as large a proportion of the ter-
 A RETINOHYPOTHALAMIC PROJECTION IN THE RAT
minals of the arcuate nucleus as indicated
by Sousa-Pinto ('70). Similarly, one would
have expected the medial hypothalamic
samples prepared for scintillation counting
to have been clearly above background if
a significant number of retinal axons pro-
jected to this area. It is apparent that this
technique is extremely sensitive to small
differences over background as the differ-
ence between the visual cortex samples
(cf. table 1 ) was small but highly signifi-
cant. Our autoradiographic observations
are at variance with those of OSteen and
Vaughn ('68) who injected labelled
5-hydroxytryptophan into the eye. This dif-
ference undoubtedly reflects the different
substances injected, one, a precursor for
protein synthesis and the other a biogenic
amine precursor. Lastly, our observations
are in accord with a number of scattered
findings available in the literature. Both
Roussy and Mosinger ('35) and Rieke
('50) noted small axons leaving the
chiasm in normal material to enter the
suprachiasmatic nucleus. Pate ('37) ob-
served what is described as transneuronal
atrophy in the cells of the suprachiasmatic
nucleus of the cat contralateral to an
enucleation. Campbell ('69), using the
Fink-Heimer method, observed apparent
degenerating terminals in the hedgehog
suprachiasmatic nuclei bilaterally after a
unilateral enucleation. Recently, we have
found a retinal projection in the hamster
using the autoradiographic method (Eich-
ler and Moore, '72) that is identical to the
one described here in the rat. In summary,
we would agree with Ebbesson ('70) that
the retinohypothalamic tract should be
considered a regular component of the
accessory optic system in vertebrates. At
the present time it would be appropriate
to conclude that the tract innervates the
suprachiasmatic nucleus in mammals, but
the possibility of a more widespread pro-
jection, either as a more general feature
of the projection or as a specialized aspect
in selected species, remains open for
further investigation.
The principal reason for attempting to
demonstrate a retino-hypothalamic tract
has been to provide a correlation for avail-
able data and a basis for further investiga-
tion in neuroendocrine regulation. Lesions
in the area of the suprachiasmatic nucleus
7
abolished the constant estrous response to
light in the female rat, whereas bilateral
destruction of the primary optic tracts had
no effect on this response to light (Critch-
low, '63). Similarly, Butler and Donovan
('71) reported that rats with knife cuts
isolating the hypothalamus exhibit per-
sistent vaginal estrus if the suprachiasma-
tic nucleus is included in the isolated zone
but with smaller isolations excluding the
suprachiasmatic region persistent diestrus
occurred. And, in a series of investiga-
tions, Halasz ('69) has elegantly demon-
strated the importance of the suprachias-
matic region in neuroendocrine regulation.
Recently, we have found that lesions
transecting either the primary optic tracts
or the primary optic tracts and the acces-
sory optic tracts do not affect the diurnal
rhythm in adrenal corticosterone content
in the rat whereas small lesions in the
suprachiasmatic nuclei, sparing the optic
chiasm, abolish the rhythm (Moore, '72).
These data give further support to the con-
cept that a retinohypothalamic tract pro-
jecting to the suprachiasmatic region is
essential for the maintenance of certain
neuroendocrine responses to light.
ACKNOWLEDGMENTS
The authors are grateful to Mrs. Francis
Karapas, Mrs. Aina Svanbergs and Miss
Nancy Bubula for their devoted and skilled
technic a1 assistance.
LITERATURE CITED
Bliimcke, S. 1958 Zur Frage einer Nerven-
f aserverhindung zwischen Retina und Hypo-
thalamus. I. Anatomische und experimentelle
Untersuchungen an Meerschweinchen und
Katzen. 2. Zellforsch., 48: 261-282.
Bodian, D. 1937 An experimental study of the
optic tracts and retinal projection of the Vir-
ginia opossum. J. Comp. Neur., 66: 113-114.
Bons, N., and I. Assenmacher 1969 Presence
de fibres retiniennes degenerees dans la region
hypothalamique supra-optique du Canard apres
section d u n nerf optique. C. R. Acad. Sci.
(Paris), 269: 1535-1538.
Butler, J. E. M., and B. T. Donovan 1971 The
effects of surgical isolation of the hypothala-
mus on reproductive function in the female
rat. J. Endocrin., 49: 293-304.
Campbell, C. B. G. 1969 The visual system of
insectivores and primates. Ann. N. Y. Acad.
Sci., 167: 388-403.
Campbell, C. B. G . , and W. R. Hayhow 1971
Primary optic pathways in the Echidna, Tachy-
glossus aculeatus: A n experimental degenera-
tion study. J. Comp. Neur., 143: 119-136.
8 ROBERT Y. MOORE AND NICHOLAS J. LENN
Campos-Ortega, J., and P. Glees 1967 The sub-
cortical distribution of optic fibers i n Saimiri
Sciureus (squirrel monkey). J. Comp. Neur.,
131: 131-142.
Cohen, E. B., and G. D. Pappas 1969 Dark
profiles i n the apparently normal nervous sys-
tem: A problem i n the electron microscopic
identification of early anterograde axonal de-
generation. J. Comp. Neur., 136: 375-396.
Critchlow, V. 1963 The role of light i n the
neuroendocrine system. In: Advances i n Neuro-
endocrinology. V. Nalbandov, ed. Univ. of
Illinois Press, Urbana, pp. 377-401.
Ebbesson, S. 0.E. 1968 Retina projections i n
two teleost fishes (Opsanus tau and Gymno-
thorax funebris). A n experimental study with
silver impregnation methods. Brain Behav.
E d . , I: 134-154.
1970 On the organization of the cen-
tral visual pathways in vertebrates. Brain
Behav. Evol., 3: 178-194.
Ebbesson, S. 0. E., and J. S. Ramsey 1968 The
optic tracts of two species of sharks (Galeocerdo
cuvier and Ginglymostoma cirratum). Brain
Res., 8: 36-53.
Eichler, V. B., and R. Y. Moore 1972 The pri-
mary and accessory optic systems of the golden
hamster. In preparation.
Frey, E. 1937 Vergleichend-anatomischeUnter-
suchungen iiber die basale optische Wurzel, die
Commissura transversa Gudden und iiber eine
Verbindung der Netzhaut mit dem vegetativen
Gebiet im Hypothalamus durch eine “dorsale
hypothalamische Wurzel” des Nervus opticus
bei Amnioten. Schweiz. Arch. Neur. Psychiat.,
39: 255-290.
1947 Degenerationsstudien iiber das
optische Gebiet im Hypothalamus des Meer-
schwinchens. Acta Anat., 4: 123-136.
1950 Neue anatomische und experi-
mentelle Ergebuisse iiber das optische Gebiet
im Hypothalamus. Schweiz. Arch. Neur.
Psychiat., 66: 67-86.
1951 Uber die Hypothalamische Opti-
cuswurzel des Hundes. Bull. Schweiz. Akad.
Med. 7: 115-127.
Fink, R. P., and L. Heinzer 1967 Two methods
for selective silver impregnation of degenerat-
ing axons and their synaptic endings in the
central nervous system. Brain Res., 4: 369-374.
Garey, L. J., and T. S. P. Powell 1968 The
projection of the retina i n the cat. J. Anat.
(Lond.), 102: 189-222.
Giolli, R. A. 1963 An experimental study of
the accessory optic system in the cynomologous
monkey. J. Comp. Neur., 121: 89-108.
Grafstein, B. 1967 Transport of protein by
goldfish optic nerve fibers. Science, 157:
196-198.
- 1969 Communication between soma
and synapse. In: Advances i n Biochemicat
Psychopharmacology. E. Costa and P. Green-
gard, eds. Raven Press, New York, 1 : 11-25.
1971 Transneuronal transfer of radio-
activity in the central nervous system. Science,
172: 177-179.
Harris, G. W. 1955 Neural Control of the
Pituitary. Arnold, London, pp. 60-102.
Hayhow, W. R. 1966 The accessory optic sys-
tem i n the marsupial phalanger, Trichosurus
vulpeca. J. Comp. Neur., 126: 653-672.
Hayhow, W. R., A. Sefton and C. Webb 1962
Primary optic centers of the rat in relation to
the terminal distribution of the crossed and
uncrossed optic nerve fibers. J. Comp. Neur.,
1 1 8 : 295-322.
Hayhow, W. R., C. Webb and A. Jervie 1960
The accessory optic fiber system in the rat.
J. Comp. Neur., 115: 187-215.
Hendrickson, A. 1969 Electron microscopic
autoradiography: Identification of origin of
synaptic terminals in normal tissue. Science,
165: 194-196.
Herrick, C. J. 1942 Optic and postoptic systems
i n the brain of Amblystoma tigrinum. J. Comp.
Neur., 77: 191-353.
Karlsson, J.-O., and J. Sjostrand 1971 Synthe-
sis, migration and turnover of protein i n retinal
ganglion cells. J. Neurochem., 18: 749-767.
Kiernan, J. A. 1967 On the probable absence
of retino-hypothalamic connections in five
mammals and a n amphibian. J. Comp. Neur.,
~ ~ - - ___
131: 405-408.
Knapp, H. D., F. Scalia and W. Riss 1965 The
optic tracts of Rana pipiens. Acta Neurol.
Scand., 41: 325-355.
Knoche, H. 1956 Morphologisch-experimentelle
Untersuchungen uber eine Faserverhindung
der Retina mit den vegetativen Zentren des
Zwischenhirns und mit der Hypophyse. Z. Zell-
forsch., 45: 201-264.
1957 Die retino-hypothalamische Bahn
von Mensch, Hund und Kaninchen. Z Miker.-
anta. Forsch., 63: 461486.
1960 Ursprung, Verlauf und Endigung
der Retino-hypothalamische Bahn. Z. Zell-
forsch., 51: 658-704.
Kopriwa, B. M., and C. P. Leblond 1962 Im-
provements i n the coating technique of auto-
radiography. J. Histochem., 10: 269-285.
Kostovic, I. 1971 The termination of acces-
sory optic fibers in the rat. Brain Res., 31:
202-206.
Lasek, R.,B. J. Joseph and D. G. Whitlock 1968
Evaluation of a n autoradiographic neuroana-
tomical tracing method. Brain Res., 8: 319-336.
McEwen, L. M. 1956 The effect on the iso-
lated rabbit heart of vaginal stimulation and
its modification by cocaine, hexamethonium
and ouabain. J. Physiol. (Lond.), 131: 678-689.
Moore, R. Y. 1969 Visual pathways controlling
neuroendocrine function. In: Progress i n En-
docrinology. Excerpta Medica, Amsterdam, pp.
490-494.
1972 Visual pathways mediating neu-
roendocrine regulation, In: Proceedings of a
Workshop on the Pineal Gland. D. C. Klein, ed.
Raven Press, New York. In pxess.
Moore, R. Y., F. Karapas and N. J. Lenn 1971
A retinohypothalamic projection in the rat.
Anat. Rec., 169: 382-383.
Nauta, W. J. H., and W. Haymaker 1969
Retino-hypothalamic connections. In: The
Hypothalamus. W. Haymaker, E. Anderson and
W. J. H. Nauta, eds. Charles C Thomas, Spring-
field, Illinois, pp. 187-189.
 A RETINOHYPOTHALAMIC PROJECTION IN THE RAT
OSteen, W. K., and G. M. Vaughn 1968 Radio-
activity in the optic pathway and hypothalamus
of the r a t after intraocular injection of
tritiated 5-hydrosytryptophan. Brain Res., 8:
209-212.
Palay, S. L. 1958 The morphology of synapses
in the central nervous system. Exptl. Cell Res.,
Suppl., 5: 275-293.
Pate, J. R. 1937 Trans-neural atrophy of the
nucleus ovoidens following eye removal in cats.
Anat. Rec., 67: 39.
Polyak, S . 1957 The Vertebrate Visual Sys-
tem, Univ. Chicago Press, Chicago.
Price, J. L., and T. A. Woolsey 1971 Auto-
radiographic demonstration of pathways in the
mammalian CNS. Anat. Rec., 169: 406.
Rieke, W. 0. 1958 Optico-hypothalamic path-
ways in the rat. Anat. Rec., 130: 363-364.
Riss, W.,H. D. Knapp and F. Scalia 1963 Optic
pathways in Cryptobranchus allegheniensis, as
revealed by the Nauta technique. J. Comp.
Neur., 121: 31-43.
Roussy, G., and M. Mosinger 1935 L‘hypo-
thalamus chez l’homme et chez le Chien. Rev.
Neurol. (Paris), 63: 1-35.
Schonbach, J., and M. Cuenod 1971 Axo-
plasmic migration of protein. A light micro-
scopic autoradiographic study in the avian
9
retino-tectal pathway. Exp. Brain Res., 12:
275-282.
SjBstrand, J., and J.-0. Karlsson 1969 Axo-
plasmic transport in the optic nerve and tract
of the rabbit: A biochemical and radioauto-
graphic study. J. Neurochem., 16: 833-844.
Sousa-Pinto, A. 1970 Electron microscopic ob-
servations on the possible retinohypothalamic
projection in the rat. Exp. Brain Res., 11:
528-538.
Sousa-Pinto, A., and J. Castro-Correia 1970
Light microscopic observations on the possible
retinohypothalamic projection in the rat. Exp.
Brain Res., 11: 515-527,
Szentagothai, J., B. Flerko, B. Mess and B. Halasz
1968 Hypothalamic Control of the Anterior
Pituitary, Akademiai Kiado, Budapest.
Taylor, A. C., and P. Weiss 1965 Demonstra-
tion of axonal flow by the movement of tritium
labelled protein in mature optic nerve fibers.
Proc. Nat. Acad. Sci. U. S. A., 54: 1521-1527.
Tigges, J. 1966 Ein experimenteller Beitrag
zum subkortikalen optischen System von Tupaia
glis. Folia Primat., 4: 102-123.
Wurtman, R. J. 1967 Effects of light and
visual stimuli on endocrine function. In: Neuro-
endocrinology. Volume 2. L. Martini and W. F.
Ganong, eds. Academic Press, New York, pp.
19-59.
 PLATE 1
EXPLANATION OF FIGURES

2 Photomicrograph of the suprachiasmatic nucleus contralateral to a

unilateral enucleation, 60 hour survival period. Fink-Heimer, method
1. X 1280.
3 Autoradiograph of a section through the suprachiasmatic nucleus
contralateral to an intraocular injection of 10 pCi of 3H-leucine,
seven days prior to sacrsce. The section is turned so that the ven-
tral surface of the brain would be to the right. The optic chiasm
below the suprachiasmatic nucleus is designated OC. Cresyl violet
stain. X 700.

10
A RETINOHYPOTHALAMIC PROJECTION IN THE RAT PLATE 1
Robert Y. Moore and Nicholas J. Lenn

11
 PLATE 2
EXPLANATION OF FIGURES

4 Autoradiograph of a section through the suprachiasmatic nucleus

ipsilateral to that shown in figure 3. The orientation of the section
is identical to that in figure 3. The optic chiasm is designated OC.
Cresyl violet stain. x 700.
5 Autoradiograph of a section showing the optic tract ( O T ) and the
supraoptic nucleus contralateral to the injected eye from the same
brain shown in figures 3 and 4. Cresyl violet stain. x 700.

12
A RETINOHYPOTHALAMIC PROJECTION IN THE RAT PLATE 2
Robert Y. Moore and Nicholas J. Lenn

13
A RETINOHYPOTHALAMIC PROJECTION IN THE RAT PLATE 3
Robert Y. Moore and Nicholas J. Lenn

EXPLANATION O F FIGURES

6 Electron micrograph of the suprachiasmatic nucleus from an animal three days after
enucleation. The arrow points to a degenerating bouton forming a synaptic contact
with a distal dendritic branch. X 35,000.
7 Electron micrograph of the suprachiasmatic nucleus from a n animal surviving five days
after enucleation. Two degenerating boutons are shown, one with a clear synaptic
contact (arrow). X 40,000.

14