Biochemistry of Characterised Neurons

neurons.

VALIDITY OF SINGLE NEURON CHEMICAL ANALYSIS

CAN MICROCHEMISTRY HELP TO SOLVE THE HARD QUESTIONS OF MODERN NEUROBIOLOGY?

EZIO GIACOBINI*,     Laboratory of Neuropsychopharmacology, Dept. of Biobehavioral Sciences, University of Connecticut, Storrs, CT 06268, USA

Publisher Summary

This chapter reviews the validity of single neuron chemical analysis. To begin with, microchemical techniques and single cell analyses have found wider application within the nervous system, and single nerve cells have been more extensively studied than any other cell type. Subsequently, the analysis of minute regions or cellular components becomes crucial because of the presence of many different types of cells within small areas of the nervous system. To demonstrate the validity and the feasibility of the cellular approach one can set up a list of possible experiments. Interestingly, several single cell approaches have already been exploited with a marked degree of success and they are: 1) measurement of neurotransmitter levels in single vertebrate neurons; 2) measurement of neurotransmitter levels in the synaptic endings of identified neurons; 3) measurement of the neurotransmitter turnover and biosynthesis in single neurons in vitro; 4) measurement of the rate of axonal transport in single axons of identified neurons; 5) measurement of selective uptake mechanisms by cell body and endings of identified neurons; 6) study of the influence of behavioral and synaptic input on the regulation of specific protein synthesis; and 7) study of the problem of gene expression and protein metabolism in individual neurons.

1 AN OLD QUESTION: WHY SINGLE CELLS?

Linderstrøm-Lang and Holter were the first to initiate (1931–1932) the concept of quantitative histochemistry. They suggested that quantitative chemical analysis of biological material could be performed on tissue slices. This revolutionary concept was conceived during a time when classical non-quantitative histochemistry, mostly related to staining techniques, was a well developed science (or almost an art). Most histochemists in the thirties and forties considered this approach either a dream or the extravagance of a physical chemist unaware of the difficulties in dealing with biological material. In fact, even after Linderstrøm-Lang’s successful development of several quantitative histochemical micromethods (1939), only a very few histochemists followed the new trend. During the next two decades quantitative histochemistry was confined to a small number of laboratories, no more than a dozen throughout the world.

During this period, microchemists were looked upon (and sometimes admired) as a group of perfectionists able to handle microgram or submicrogram quantities of biological material. When was the first step from tissue slices to single cell techniques taken?

The cellular approach naturally evolved at the Carlsberg Institute in Copenhagen. The idea of dealing with single cells was particularly attractive to cell biologists and the possibility of measuring metabolic events in living unicellular organisms was fascinating to cell physiologists. Historically, the development of the original Cartesian diver by Linderstrøm-Lang (1937) and the subsequent modifications by Holter (1943) and Zeuthen (1943) to study respiration in unicellular organisms represented the first major step in this direction (Table I).

TABLE 1

INTRODUCTION OF MICROCHEMICAL METHODS IN NEUROBIOLOGY-A CHRONOLOGICAL REVIEW

The new trend spread from the Carlsberg Institute to other laboratories, mainly European and American; new approaches were tried and the first cellular assays were made with various techniques (microfluorimetry, microgasometry, microspectrophotometry, etc.) (Table I).

The application of the elegant and sensitive new tools of the Carlsberg school to cellular studies in multicellular organisms remained dormant for more than 10 years. In fact, it was not until the middle fifties that new impetus was felt in this area. Oliver Lowry, who had visited the Carlsberg Laboratory, realized the great potential of Linderstrøm-Lang’s technique in the field of cellular analysis. Although the principles and the tools were basically available, much work was needed in order to scale down the procedures to accommodate the sensitivity required for single cell analysis. This step was successfully accomplished in Lowry’s laboratory. In a series of papers from this author, the single cell barrier was broken. Enzyme activity could be measured in less than microgram of tissue, with the same accuracy as on a milligram scale. Through the brilliant work of the St. Louis school at least three orders of magnitude had been gained and single cell analysis was reached (see Lowry & Passonneau, 1972).

Simultaneously with this achievement but using a completely different approach, a Cartesian diver technique was developed for measuring enzyme activity in single mammalian neurons (Zajicek & Zeuthen, 1956; Giacobini & Zajicek, 1956; Giacobini, 1957). Shortly afterwards this technique was refined to permit analyses of subcellular components of single neurons (Giacobini, 1959a). Through the development of two of Linderstrøm-Lang’s basic ideas and techniques (quantitative histochemistry and microgasometry) single cell analysis became a reality.

The development of new techniques and approaches in the field of molecular biology during the sixties was paralleled by a period of great refinement and wider acceptance of cellular techniques (Table I).

In the sixties, one of the most important advances was the introduction of Lowry’s cycling technique (1963) which allowed measurements not only of enzyme activity but of substrates and intermediates in single cells in the incredible range of 10-15 moles or less. This technique was promptly applied to single neuron analysis and 15 substrates and intermediates were measured in single neurons at rest and after impulse activity (Giacobini & Grasso, 1966; Giacobini & Marchisio, 1966; Giacobini, 1968).

2 THE NEW MICROTOOLS

The new methods were not only more sensitive than the Cartesian diver, but much more rapid and versatile. Another new and important approach was the introduction of microisotopic techniques by McCaman (1968). This approach still dominates the field of microchemistry and has allowed the analysis of many neurotransmitters and their enzymes (Goldberg & McCaman, 1973; McCaman et al., 1973a) in both vertebrate and invertebrate neurons (Osborne, 1974; Giacobini, 1975).

From the already powerful arsenal of microchemical tools only one weapon was missing, i.e. the possibility of screening for new substances, intermediates, metabolites or neurotransmitters (Giacobini, 1975). This approach involved separation and identification of microquantities (picomoles or less) of aminoacids, amines or peptides in microgram samples of tissue. The microchromatographic technique, originally introduced by Neuhoff (Neuhoff et al., 1969; Neuhoff & Weise, 1970) and then extensively used and refined by Osborne (1971, 1974), allowed the screening for new mediators, not only in discrete parts of nervous tissue but in single cells. When this technique was combined with mass spectrometry (Giacobini, 1975; McAdoo, this volume) the potential for identifying and measuring levels of new molecules became apparent. The measurement of 5-HT in single Retzius leech cells with GC-MS is one example (McAdoo, this volume).

During the last 20 years single cells have become a suitable target for our chemical method. Enzyme activity, substrates, metabolites, nucleic acids, neurotransmitters, ions, etc., can be determined with the same precision and accuracy in a single cell as in samples weighing up to several grams (see Osborne, 1974).

Microchemical techniques and single cell analyses have found wider application within the nervous system, and single nerve cells have been more extensively studied than any other cell type. The presence of many different types of cells within small areas of the nervous system is the rule, consequently analysis of minute regions or cellular components becomes crucial.

For this reason neurobiology has witnessed the strongest development in, and found the widest application of micromethods than any other field of biology. This trend is still continuing and will characterize this field to an as yet undetermined extent.

3 NEW APPLICATIONS AND TRENDS OF MICROCHEMISTRY IN NEUROBIOLOGY

3.1 Studies on Developing Neurons

The analysis of small groups of neurons, nuclei or ganglia during different phases of development poses serious methodological questions. As illustrated in the diagram (Fig. 1), chick embryo sympathetic ganglia at 6-7 days of development (stage) show a dry weight of 2-3 μg (Fairman et al., 1976). By hatching this weight has increased almost 10 times. Chick ciliary ganglia weigh 10 μg d.w. at 9 days of development, and initially consist of 6000 neurons; which are then reduced by 50% by day 18 of development (Chiappinelli et al., 1976). The locus coeruleus in the chick embryo contains a few thousand cells and weighs only a few μg at the 2nd week of development. From these few examples we realize that the need for microchemical approaches might become as important for the developmental neurobiologist as for the cellular biologist.

Fig. 1 Some basic features of ganglionic growth at different stages of development, protein curve and dry weight curve of chick sympathetic ganglia.

3.2 Studies on Tissue Cultures of Neurons

A new and promising application of microtechniques is the analysis of cells from tissue cultures. In some cases, for example neuroblastoma, an almost unlimited number of cells are available for biochemical analysis. However, it is quite common that a culture at a particular stage offers only a relatively limited number of cells.

There are several potentially interesting applications of microchemistry or even single cell analysis in the expanding field of co-culture. If cells of different origin are present in the culture and viable contacts (synapses?) are established, cytochemical studies will be necessary to characterize them.

One pertinent example is dissociated rat superior cervical ganglion cells, which seem to provide cholinergic innervation to striated muscle in culture (O’Lague et al., 1976). Only one type of neuron (adrenergic) has been described in these cultures (O’Lague et al., 1976; Johnson et al., 1976). An interesting paradox has arisen with regard to whether the noradrenergic terminals are the only synaptic type found in cultures when electrophysiologically only cholinergic interactions between neurons could be demonstrated (Johnson et al., 1976). The implication of finding synapses with cytochemical characteristics of noradrenergic terminals would suggest that one type of synapse could handle both noradrenaline and acetylcholine as a neurotransmitter.

One of the critical questions related to this kind of experiment is the number of cholinergic neurons present in the original culture. In a study performed on single cells (before and after denervation) the number of cholinergic neurons present in normal L7 lumbar ganglia of the cat was estimated to be about 13% (Buckley et al., 1967b). In the rat superior cervical ganglion the percentage of cholinergic neurons may be less than in the cat (Yamauchi, 1973). Single cell analysis would make it possible to evaluate precisely the percentage of cells present in the culture which have different enzymatic characteristics, i.e. the number of cholinergic and adrenergic neurons.

We should be aware of the fact that if sympathetic neurons cultured in vitro are able to originate both lines of cholinergic and adrenergic cells, this might simply be an expression of their genetic potential under particular conditions in vitro. Accordingly, this phenomenon may not reflect (a) the in vivo situation or (b) be related to the normal functional activity of the ganglion.

4 PRESENCE OF SEVERAL NEUROTRANSMITTERS IN THE SAME NEURON

In certain invertebrate neurons the presence of several putative neurotransmitters, such as acetylcholine, serotonin, octopamine, etc. (see Saavedra’s and McAdoo’s chapters in this book) has been demonstrated (Hanley et al., 1974; Brownstein et al., 1974). It will be important to discover whether the very low levels of certain neurotransmitters found in these cells reflect (a) a genetic noise level not entirely repressed, or (b) a true function of the molecule in neurotransmission processes.

If (b) is the case then another experiment would be necessary: namely to find evidence for the simultaneous release of several neurotransmitters from the endings of the same neuron. Such a finding would of course contradict the so-called Dale principle (see Burnstock, 1976).

Release experiments at this level are technically very difficult to perform but perhaps not impossible (Gerschenfeld, 1973; Gerschenfeld et al., 1976).

Another related problem which might be investigated with microchemical techniques is whether some nerve cells synthesize and release more than one neurotransmitter.

The discussion of Burnstock (1976) illustrates extensively the viewpoint that in some species during development or during hormone dependent cycles, many nerve cells might synthesize, store and release more than one transmitter. Actually this problem has already been attacked by neurochemists in invertebrate ganglia (see Evan’s and McAdoo’s chapters in this volume).

5 THE FUTURE: NEW APPLICATIONS OF MICROCHEMISTRY IN NEUROBIOLOGY

Factors and mechanisms involved in the regulation of neurotransmission have so far been studied mostly at the level of the whole brain, in discrete regions of the CNS and in ganglia.

There is no doubt that single cell studies would be very useful. To demonstrate the validity and the feasibility of the cellular approach one could set up a list of possible experiments. When we tried to set up the following guidelines we were actually surprised to find out that several single cell approaches had already been exploited:

5.1 Measurement of Neurotransmitter Levels in Single Vertebrate Neurons

This has already been successfully performed in invertebrate giant cells as illustrated by several examples in this volume.

5.2 Measurement of Neurotransmitter Levels in the Synaptic Endings of Identified Neurons

This step would provide more information with regard to the different composition of cell body and synapsis and the dynamic aspects of neurotransmission biochemistiry in the different parts of the neuron. See Dowdall’s chapter in this volume.

5.3 Measurement of the Neurotransmitter Turnover and Biosynthesis in Single Neurons In Vitro

This approach has already been exploited by Koike et al. (1972), Cedar et al. (1972), Cedar and Schwartz (1972) and Goldman and Schwartz (1974) by injecting ³H choline, ³H adenine and ³H-5-HTP intracellularly in order to examine directly the synthesis of ACh, 5-HT and c-AMP in identified nerve cell bodies of the Aplysia. Osborne’s chapter in this volume discusses this approach.

5.4 Measurement of the Rate of Axonal Transport in Single Axons of Identified Neurons

This approach has been used in conjunction with the experiments reported in c) above.

5.5 Measurement of Selective Uptake Mechanisms by Cell Body and Endings of Identified Neurons

This approach has already been initiated by Pentreath and Cottrell (1972, 1973) and Osborne et al. (1975).

5.6 The measurement of the parameters suggested at points 5.1 to 5.5 above, should be performed under special experimental conditions including denervation, reinnervation, electrical stimulation and drugs.

Experiments in these directions have already been attempted by Osborne and Cottrell (1972), Osborne (1972b), Juorio and Killick (1972) and Osborne and Pentreath (1976). See the chapter by Berry and Pentreath in this volume.

5.7 Study of the Influence of Behavioral and Synaptic Input on the Regulation of Specific Protein Synthesis

This should be performed in a single and identified neuron. This direction has already been pursued by Gainer and Barker (1974) on isolated abdominal ganglia from Aplysia. See Berry’s chapter in this