The role of macromolecules in neuronal function in Aplysia

4 THE ROLE OF MACROMOLECULES IN N E U R O N A L FUNCTION IN APLYSIAt R . PgICE PETERSOrq a n d Y. PENG LOH

Departments of Anatomy and Molecular Biology, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Contents 1.

Introduction 1.1 R N A in Aplysia neurons 1.2 R N A and stimulation

179 180 181

2.

Protein 2.1 Protein specificity in neurons 2.2 Neuronal activity and protein synthesis in "simple systems"

185 187 189

3.

Conclusions References

200 201

I" This work was funded primarily by NIH grant NS08759.

177

4 THE ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN APLYSIA R. PRICE PETJZRSON and Y. PENG LOH Departments of Anatomy and ikfolecdur Biology, University of Pennsyhmia, Philadelphia, Pennsylvania 19174

1. Iutroduction

The work to be described in this review will revolve around the idea that the macromolecules RNA and protein play a role in plastic and specific electrophysiological behavior of neurons. As the reader will see, this notion is based more on faith than evidence. We will point out the evolution of this faith and describe various experiments which both support and disavow it. Most of the data will be drawn from work on identified neurons of Aplysiu since this is the animal we’ve chosen to work with. We have also included some work on macromolecules in Aply.siu neurons which may not pertain directly to the problem, but is included to compliment other discussions. Until the 1950s the main article of faith was that proteins and RNA in neurons were utilized metabolically in neural activity. That is, somehow they were necessary for firing activity and the more active a neuron became, the greater its consumption of these macromolecules. This notion, although no longer the most popular, is still seen occasionally (Bocharova et al., 1972). Over the past 15 years this idea has generally given way to the ideas of molecular genetics that RNA and protein serve more to regulate and define a cell’s functional potentialities rather than serve as metabolic substrates. The inability to clearly prove or disprove that macromolecules have a role in neuronal function has frequently been attributed to imperfections of the biological systems being studied and the search for a simple system lacking these imperfections has led, among others, to the use of Aplysia (in practice this trades one set of imperfections for another). This is a large marine mollusc (Gastropoda, the ‘sea hare’) species which exists in most of the oceans of the world, but which is reliably available only from California (Pacific BioMarine Supply Co., Venice, California). Information is available on the neuroanatomy (Bullock and Horridge, 1965), general biology (Eales, 1921), identification and properties of abdominal ganglion neurons (Frazier et al., 1967) and microchemical methods for single neuron chemistry (Peterson, 1972) in Aplysia. In addition a bibliography of essentially everything published on Aplysiu (1955-1971) has been compiled by Dr. Felix Strumwasser, California Institute of Technology, Pasadena, California, and is probably available from him. The most recent review related to this area is that of Castellucci et al. (19 72). 179

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R. PRICEPETERSONAND Y. PENGLOH

1. I R N A in Aplysia Neurons In the late 1960s four separate laboratories began working on nucleic acids in single neurons of Aplysia, all of them concentrating on the giant neuron R2 (the terminology ol Frazier et al., 1967, although inelegant, is useful). This large (0.5-0.8 mm diameter) neuron is heavily pigmented, has no dendrites and has a multibranching axon whose main branch connects primarily with other ganglia. R2 has a homologous giant neuron in the left pleural ganglion (LPG). The neuron can be synaptically driven by stimulating any of the five nerves of the ganglion, but the neuron apparently has no synapses upon its perykaryon. Lasek and Downer (1971) measured the D N A content of the neuron R2 and discovered it to be highly polyploid, although not polytene. They found that during development this neuron doubles its original diploid genome sixteen times to achieve a DNA content of 0.131/zg which is 131,000 times that of an Aplysia sperm. Subsequent work (personal communication) indicated only a very small satellite on CsC1 gradients which was not tissue specific and no evidence for differential gene amplification. The melting curve of Aplysia neuronal DNA is quite smooth (Tin ---- 82°C in SSC) and suggests a low G + C content (Peterson, unpublished). This vast amount of DNA per neuron is presumably related to the large size of the cell and is also reflected in RNA content per cell. In adult Aplysia giant neurons the D N A content forms two classes, 0.067/~g and 0.131/zg--likewise the RNA values fall into two modes of 1.6/~g and 3.2/~g (Peterson, 1972). This RNA value works out to about 5 ~ (w/v) which is comparable to other reported values for vertebrates (Edstrom, 1956) and is about 100 times higher per unit surface area than reported elsewhere (Edstrom and Pigon, 1958). When incubated in artificial sea water neuron R2 will readily incorporate either uridine or cytidine into RNA. With an extracellular level of 1-5 x 10 -5 M uridine, R2 incorporates 5-10 × 10-lm moles into RNA/hr at 18°C, for a minimum turnover rate of approximately 0.005 H/hr. Labeled uridine is initially incorporated into the nucleus where it first appears in heterodisperse high molecular weight (MW) RNA and in peak at 38S. Aside from tRNA, appreciable amounts of label do not appear in the cytoplasm until 80-90 rain of incubation. Evidence has previously been presented (Peterson, 1970) that the 38S nuclear RNA represents a precursor which is processed through 31S and 21S species to form the final 18S and 27S rRNAs. This incorporation is 90 700inhibited by actinomycin-D (5/~g/ml), although the differential inhibition produced by lower doses in other systems was not observed. As in other systems, transfer of labeled methyl groups from methionine occurs predominantly into ribosomal and 4S RNA. In comparing gel electrophoretic patterns of labeled RNA from R2 with that of other neurons, differences were noted in two areas; one was the regular occurrence of a 14S peak in R2 cytoplasmic RNA (absent in other neurons) and the other was the variety of rates of labeling of various RNA species in different neurons. It is worthwhile to note that in incubation systems as this, it is likely that marked metabolic changes are occurring with time. Previous reports emphasized the neurophysiological durability of the Aplysia preparation, with normal microelectrode recording possible for up to 6 weeks in vitro (Strumwasser and Bahr, 1966). Recently Schwartz et al. (1971) reported that uptake of amino acid dropped by 90 ~o within 15 hr after removal from the animal. We have confirmed this finding and also find that nucleotide uptake drops dramatically after several hours in vitro. Thus electrical activity is not a particularly good indicator of metabolic state.

ROLE OFMACROMOLECULES

IN

NEURONAL FUNCTION IN Aplysia

181

1.2 RNA and Stimulation There have recently been several studies (Peterson and Kernell, 1970; Kernell and Peterson, 1970; Peterson and Erulkar, 1972; Berry, 1969; Berry and Cohen, 1972; Wilson and Berry, 1972) attempting to define what the relation is, if any, between stimulation and RNA synthesis in neuron R2. Those focusing on stimulation and protein synthesis are summarized in another section of this chapter. The general format of these experiments has been to isolate the abdominal ganglion in saline containing labeled nucleoside, monitor the activity of R2 and stimulate the neuron to fire. This has been done with a variety of stimulation times and methods. R2 is then dissected out and the RNA extracted and counted for radioactivity. Generally uptake into the stimulated neuron is then compared with an unstimulated neuron or group of neurons. The points of agreement of these reports are that stimulation does enhance uptake of labeled uridine into RNA (when uridine concentration is above 1.5 tzu) and that synaptic stimulation (EPSPs) is effective whereas direct electrical stimulation is not. The latter point is a rather clear-cut finding and is useful in interpreting a great many earlier conflicting results in vertebrates--that is, generally when no effects were reported, stimulation was antidromic and when increases were reported, orthodromic stimuli had been used. Since the effective stimulus appears to be orthodromic synaptic input rather than antidromic or direct electrical stimulation, the question arises as to what aspect of the synaptic stimulus is the trigger for RNA synthesis? Three possibilities exist: (a) the ionic shifts associated with the EPSP, (b) the synaptic transmitter and (c) 'trophic' factors. Since direct electrical activation and action potentials, which produce ionic shifts similar to the EPSP, were without effect, the first alternative seems unlikely. Since we don't know what 'trophic" factors are, experimental approaches to test for their function seem fruitless at this time. This is not the case with transmitters, although the excitatory transmitter to R2 is unknown. However, it is known that ACh is the excitatory transmitter in the superior cervical sympathetic ganglion and Gisiger (1971) has exploited this possibility. He first showed that ortho- and not antidromic stimulation has the same stimulatory effect on labeled nucleoside incorporation into RNA as in Aplysia. He then was able to demonstrate that the effects of stimulation was abolished by d-Tubocurarine and Mecamine. ACh applied in the absence of stimulation produced an increased incorporation into RNA similar to stimulation and this ACh effect was not blocked by either tetrodotoxin or KC1 depolarization. These experiments argue strongly that the transmitter itself is adequate to explain the increase in labeled nucleoside incorporation. Once stimulation is begun, the increased rate of incorporation seems to be constant for many hours (Berry, 1969). The degree of increase is correlated not only with frequency of stimulation (Gisiger, 1971 ; Peterson and Erulkar, 1972) but also with the duration of stimulation at a constant frequency (Peterson and Erulkar, 1972). The only exception to this is an apparent decrease in rate of incorporation during the initial 10-15 min (Gisiger, 1971, and Peterson and Erulkar, 1972). This is apparent, rather than real, because it is probably due to a decrease in rate of phosphorylation of labeled uridine precursor, rather than a decrease in rate of transcription (Orrego, 1967). That synaptic activation enhances uptake of labeled precursors into RNA is not in itself a particularly significant finding. The relevant question is whether expression of the genome of the post-synaptic neuron is, to any degree, regulated by incoming synaptic bombardment.

182

R. PRICE PETERSON AND xtr. PENG LOH

This would be reflected in RNA by a qualitative change in the species of RNA transcribed; it might or might not be accompanied by a quantitative change in the gross amount of RNA synthesized. However, in the case where a quantitative change did occur, this would be an argument for regulation. In any measurement of synthetic rate by radioisotope, accurate knowledge of the specific activity of the precursor is at least as important as that of the product. Thus, in order to be confident that the enhanced incorporation into RNA reflects enhanced synthesis, accurate determination of the precursor UTP pool specific activity is necessary. Berry and co-workers have been much more careful in this regard than other workers in the area. However, no one has yet been able to determine the UTP specific activity due to the very small amount of UTP in a single neuron. Initially Berry (1969) tried to account for possible changes in pool radioactivity by expressing his results as cpm in aeid-precipitable material divided by cpm in the acid-soluble fraction. Although this tends to normalize for different sized tissue samples, it is several steps removed from pool specific activity. In a subsequent paper (Berry and Cohen, 1972) the methodology was refined so that radioactivity in UTP in the stimulated and control neuron was measured and they were found to be identical. However, the inability to measure the amount of UTP prevented again a measure of UTP specific activity--thus the enhanced uptake into RNA might have reflected pool shrinkage or increased RNA synthesis. Most recently, in an effort to understand this effect of stimulation, Wilson and Berry (1972) repeated the experiment with decreasing concentrations of precursor in the medium which should have had no effect on the results. However, below 1.5 FM no enhancement of incorporation into RNA was observed. The significance of this finding remains unclear. Two other indirect arguments have been proposed to support the notion that this increase in label represents increased synthesis. The first is that if the increase were due solely to pool effects, then one would expect the increased label to appear equally in all classes of RNA. Both Peterson and Kernell (1970) in Aplysia and Gisiger and Gaide-Huguenin 0969) in sympathetic ganglia have presented evidence based on gel electrophoresis of the labeled RNA, that this does not happen and in fact that the increase is in predominantly high molecular weight RNA. However, this argument is considerably weakened by the fact that different classes of RNA also have different turnover rates and in some cases there are precursor-product RNAs. Therefore increased labeling of only certain classes of RNA could still reflect merely increases pool specific activity and labeling of the more rapidly turning over RNA species. The second argument is based on recent experiments (Peterson and Erulkar, 1972) where R2 was synaptically stimulated in the absence of labeled precursor for l0 rain, rested for 20 min and then incubated (without stimulation) for a further l0 rain in labeled uridine. It was reasoned that if there were pool changes associated with stimulation, these would not effect the incorporation results when the labeled precursor was added 20 rain after the cessation of stimulation. In these experiments the stimulated R2s incorporated significantly more labeled uridine (67 ~ ) than the unstimulated controls, a result suggesting that there is a true increase in rate of synthesis independent of what the pool may be doing during stimulation. To return to a point made earlier, it should be borne in mind that the aim of this work is to determine whether or not synaptic input regulated, to any degree, the expression of the post-synaptic genome. I have discussed efforts to demonstrate this by showing increased rate of transcription and the problems encountered with precursor pools. Alternatively,

ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN

Aplysia

183

if one could show qualitative changes in transcription, these would be independent of overall rates of synthesis and avoid the pool pitfalls. Three types of experiments have been done, aimed at demonstrating changes in types of RNA transcribed--RNA from stimulated and unstimulated neurons has been compared by gel electrophoresis, by its degree of methylation and by its ability to hybridize to D N A in the presence of a large excess of non-neuronal Aplysia RNA. When labeled RNA from a stimulated and an unstimulated R2 neuron is extracted and electrophoresed on gels, the patterns are similar in the lower molecular weight regions. However, the stimulated neuron RNA generally is increased in the middle to higher molecular weight regions in a broad heterodisperse band (Peterson and Kernell, 1970). Gisiger (1971) has obtained similar results with sympathetic neurons--a marked increase in the higher molecular weight region of 30-40S and above. A second approach, suggested by Prof. J.-E. Edstrom, was based on the fact that methyl groups are transferred from methyl labeled methionine primarily to rRNA and tRNA with very little going into other classes of RNA. Therefore if, when comparing total RNA from stimulated neurons incubated in all-methyl methionine, the degree of methylation were the same, it would argue against qualitative transcriptional changes. On the other hand, increased methylation would signify stimulation induced increases in rRNA and/or tRNA whereas decreased methylation would indicate increases in other types of RNA, perhaps mRNA. In these experiments ~4C uridine was also present to monitor changes in total RNA synthesis. Additionally the total RNA per neuron was measured so that results could be expressed as specific activities. Table 1 shows the results of these experiments. The point to be made is that whereas uridine incorporation into the stimulated neuron RNA was up 150 ~o (line 3) the methyl incorporation was up only 64 ~o (line 4). Therefore the pattern of transcription is changed during stimulation, the increased transcription occurring primarily in unmethylated species. This conclusion must be tempered by the possibility that rather than representing modified transcription, this simply indicates the production of undermethylated RNA. The third type of experiment to test for qualitative changes in transcription with synaptic stimulation was competitive hybridization. In these experiments we are directly measuring the degree of difference in the RNA between the stimulated and unstimulated neuron by comparing the ability of these RNAs to hybridize to DNA template in the presence of a large excess of common competitor RNA (Aplysia gonad RNA). (The strategy of competing unstimulated RNA vs. stimulated RNA is prevented by the small amount of RNA in a single neuron). We would expect that the gonadal RNA contains many nucleotide sequences common to both neuronal RNAs. Thus if the uncommon neuronal sequences hybridize to the same degree we would conclude that no qualitative changes in transcription had occurred with stimulation. However, if the RNA from the stimulated neuron hybridizes to a greater or lesser degree, then we would conclude that synaptic stimulation had modified genetic expression. The previously described experiments led us to expect that if changes occurred, they would be small---a point which is also suggested by the lack of demonstrable effect of stimulation on protein synthesis (see below). Unlabeled DNA and RNA were prepared and purified from Aplysia gonad. Labeled RNA was prepared from 1 hr synaptically stimulated R2 neurons and unstimulated LPG controls. Hybridization was performed in a liquid system at 60°C for 20 hr with a ratio of labeled RNA to DNA of 4.0. These experiments are described in detail in Peterson and v.N. 2/2~]a

184

R. PRICEPETERSONANDY. PENGLO~ TABLE

1. Control

Stimulated

1.40 4- 0.05 (8) 3.2 ± 0.1 (4)

1.59 4- 0.08 (13)

2. Moles 3H methyl-methionine ( × 10-'5) incorporated into protein/cell

89 4- 17 (13)

415 4- 57 (13)

3. Moles t4C uridine (× 10 -~5) incorporated into RNA/~g RNA

13 4- 0.9 (11) P < 0.001

334-5 (I1)

4. Moles 3H methyl ( × 10- ~5) incorporated into RNA/tLg RNA

3.8 4- 1.2 (lO) P < 0.02

5.9 4- 1.1 (10)

1. RNA (/~g/cell)

4- = S.E.M. ( )=n 60 rain incubation plus stimulation (37/min) in 4.5 x 10-5. ZH methyl-T-methionine and 8.0 x 10-s M ~4C uridine. Erulkar (1972) as well as the control experiments for background binding, saturation, time course and hybridization of heterologous RNA. Assuming that D N A - R N A hybridization kinetics are similar to those of D N A - D N A , the C o T value o f 200 used was high enough to allow hybridization of the highly and moderately redundant species and about one-half of the single copy sequences. The R N A from nine pairs of stimulated and unstimulated neurons was hybridized in the presence of increasing amounts of competitor RNA. This generated eighteen competition curves (each point on each curve was the mean of triplicate samples). Figure 1 shows the averages of these two sets of nine curves. The dashed line is the mean competition curve o f the R N A from the unstimulated control neurons, the solid line that from the stimulated. Little difference is seen in the two curves until the 25/zg level is reached, at which there is a significant 5 ~ difference. At the plateau levels of 50-100/~g there is a 4 ~ difference which is also significant (p < 0.01). This means that the gonadal R N A was common to 96 ~ of the control L P G labeled R N A but c o m m o n to only 92 ~ of the stimulated R2 RNA. F o r comparison a control series was run comparing L P G R N A with R N A from R2 neurons which had been penetrated by the recording microelectrode but not stimulated. In this case both curves had plateau values (within 0. I Yo of each other.) F r o m this data we can say 8 ~o of the R N A in a stimulated giant neuron is different from gonadal as opposed to 4 ~o in an unstimulated neuron. Does it follow that stimulation induced the transcription of new genetic information ? N o t necessarily. In the unstimulated neuron 4Yo of the labeled R N A is "neuron specific" (i.e., not contained in the gonad). The transcription which is induced by stimulation m a y be either more of this same "neuron specific" R N A o r it may be new transcripts--this experiment does not differentiate between the two possibilities.

ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN

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FIG. I. This shows the summary of nine pairs of competition curves. This shows the degree of competition obtained when the less homologous yeast RNA is used as competitor. Of the upper curves, the dashed line is the curve for RNA from the unstimulated LPG and the solid line that for RNA from the stimulated R2. In summary these three experiments, eleetrophoresis, methylation and hybridization, leave little doubt that synaptic input does regulate to some degree, genetic expression. The extent to which this is carried through to translation will be seen in the next section. 2. Protein

Amongst the many classes of measurable protein changes which may be associated with neuronal activity, three types have commonly been looked for: (a) Quantitative changes and rate of synthesis. (b) Qualitative changes. (c) Changes in the quantity and activity of enzymes. Yet another approach is to study the effects of inhibitors of macromolecular synthesis on neuronal activity. One of the earliest examples of the former approach was put forward by Hyden in 1943 (Hyden, 1943). Using a cytospectrophotometric method, he found that motor anterior horn cells from guinea pigs, which had been exhausted by running, showed a diminution of cytoplasmic protein content compared to resting control animals. The decrease, he suggested, was associated with the function. In another set of experiments where spinal ganglion cells of cats and rabbits were subjected to intense electrical irritation, immense decrease of cytoplasmic protein was also reported. These observations were summarized by Hyden as follows: "the function of the intensity"

186

R. PRICE PETERSON AND Y. PENG LOH

activated " . . . cell appears to be correlated with an immense destruction of proteins and a recuperative p r o c e s s . . , the intense protein-rebuilding process, which may extend to the major part of the cell body substance, is carried on in close connection with the function of the cell" (Hyden, 1943). This idea of a "metabolic" role of proteins in nerve cell activity soon ebbed away. Gradually, there was conceptual change from the metabolically used protein to a regulatory role of proteins, as revealed by Hyden's subsequent work (Hyden and Lange, 1970). The focus since the 1960s has been on the correlation of complex behavior such as learning with the synthesis of proteins--perhaps of a specific nature unique to the task. For example, Hyden studied the effect of transfer of handedness in rats on protein synthesis in hippocampal nerve cells both quantitatively and qualitatively by electrophoresis of the soluble, protein fraction (Hyden and Lange, 1970). His results which showed both qualitative and quantitative changes in proteins have however been subjected to criticism (Rose, 1970). Subsequently many workers have done similar experiments varying the animal species, task, and assay techniques. A review of such experiments is given elsewhere (Glassman, 1969). Experiments of this nature have yielded both negative and positive results, which make it difficult to understand the biochemical processes of learning. In order to seek more consistent results, so-called "simple" systems have been sought. One of these, a preparation consisting of a cockroach leg attached to the metathoracic ganglion, was subjected to shock avoidance learning. The effect of this on the rate of incorporation of aH leucine into protein as well as changes in activity of acetylcholinesterase in the ganglion were measured (Kerkut et al., 1970, 1971, 1972). Cockroach leg preparations trained to keep the metathoracic leg out of saline showed significantly greater incorporation of labeled leucine into the metathoracic ganglion, over the control animals. Fractionation of the proteins in the ganglion showed that three distinct bands increased in incorporation during learning. Moreover, it was found that drugs inhibiting protein synthesis also inhibit learning, thus suggesting the participation of protein in learning. An investigation into the acetylcholinesterase (ACHE) activity showed good correlation between the decrease in AChE activity with degree of learning. The activity of AChE was subsequently claimed to be composed of two isoenzymes. The major isoenzyme with Km of 5.88 × l0 -s M in untrained ganglia showed a decrease in activity after training, due to a change in K,, to 1.33 × l0 -4 M. These workers have suggested that the AChE enzyme undergoes some reversible conformational change with learning. However, later work from the same laboratory failed to confirm these results, finding that with electrophoretically fractionated enzyme both peaks showed the same Km and this was unchanged by training (C. Lambertsen, personal communication). The mechanisms of learning and retention appear to be more complex than a one protein, one enzyme hypothesis and may involve a series of biochemical events. While Kerkut's results are certainly of value, this multicellular system is still complex. To approach an even simpler level of organization, investigators have turned to neurochemical studies at the single cell level. Molluscan nervous systems such as those of Aplysia californica and Otala lactea (land snail) fulfilled many of the criteria for an "ideal" preparation of neurochemists in this field. With the abdominal ganglion of Aplysia californica one can isolate and identify approximately 30 of the 2000 neurons. Many of the identifiable cells have been characterized by their morphology, neurophysiology, and neurosecretory function (Frazier et aL, 1967). Similarly with the snail brain, a number of single neurons have been identified and characterized (Gainer, 1972a). .

.

.

ROLEOF MACROMOLECULESIN NEURONALFUNCTIONIN Aplysia

187

2.1 Protein Specificity in Neurons

Before correlative studies can be made, it is essential first of all to ask the question: Is there neuronal protein specificity parallel to neuronal differentiation within the ganglion ? Several groups (Wilson, 1971; Gainer, 1971, 1972a; Loh, 1972) have attempted to answer this question by looking at labeled proteins from different identified neurons. In the case o f the abdominal ganglion of Aplysia, the ganglion is first incubated in 3H leucine and then the individual identified neurons are dissected and homogenized, solubilizing approximately 90 ~o of total protein. The soluble proteins are then fractionated on SDS polyacrylamide gels according to their molecular weight. Figure 2 shows a suggested functional m a p of some of the identifiable neurons of the

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Endogenously bursting neuron (Frazier et. al., 1967; Chen et. al., 1971). R15 also shows parabolic bursting and a circadian rhythm (Strumwasser, 1965; Lickey, 1967; Jaeklet, personal communication).

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t

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Possibly neuroseeretory neuron on morphological evidence, (Frazier et. al., 1967).

Bag cells, neurosecretory cells containing egg laying protein, (Kupfermann, 1967; Strumwasser 1969). FIG. 2. Diagram of the dorsal surface of the abdominal ganglion of Aplysia indicating the most common position of some identified cells and their characteristics (nomenclature according to Frazier et aL, 1967). Similar characte,'istics in other Aplysia neurons have been less well studied.

188

R. PRICE PETERSON AND Y. PENG LOH

abdominal ganglion of Aplysia. The molecular weight distribution of labeled proteins as shown on SDS acrylamide gels by Wilson is shown in Fig. 3. It is evident that each of the neurons studied differ in their protein composition as shown by this method. This technique, however, is unable to show any functional significance of the dominant peaks of the neurons.

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~G. 3. SDS-polyacrylamide gel-labeling patterns from extracts of single R2, R15 cell bodies obtained after ganglion incubation in leucine all-containing medium for 4hr. 8.5 × 106 cpm = 1/zg L-leucine. A gel slice is l t m m in length. A portion of the gels, in front of the tracking dye, was extruded and counted as a single piece; its length is indicated by the corresponding number of gel slices. Protein molecular weight calibration, as determined by migration relative to the brompbenol blue tracking dye, is indicated. 45 epm background was subtracted. (From Wilson, 1971.)

ROLE OF MACROMOLECULESIN NEURONAL FUNCTION IN Aplysia 189 We have made a somewhat more extensive study of the molecular weight distribution of protein from identified Aplysia neurons on SDS acrylamide gels. Some of the results are shown in Fig. 4 (A-D). Figure 4A is an example of the reproducibility of this method. Each of the neurons chosen differ in the following properties: R2 is a cholinergic neuron (McCaman and Dewhurst, 1970; Giller and Schwartz,' 1971), R15 exhibits spontaneous activity (Alving, 1968; Chen et al., 1971) and has a circadian rhythm (Strumwasser, 1965; Lickey, 1969; Jacklet, personal communication), L7 is a motoneuron associated with the gill withdrawal reflex and L6 is an endogenously firing neuron (Chen et al., 1971). The unique protein patterns of each neuron is evident. Thus, there appears to be protein specificity among individual neurons possessing characteristic physiology. Another example of protein specificity comes from the data of enzyme studies. Only the cholinergic neurons, L10, L11, and R2 in the abdominal ganglion of Aplysia possess high choline acetyltransferase activity (MeCaman and Dewhurst, 1970; Giller and Schwartz, 1971). The distribution of the aromatic amino acid decarboxylase activity showed a low level in many neurons except for R15 where it is high (Weinreich et al., 1972). Generally, the activity of enzymes would bear a quantitative relationship with the enzyme protein. However, it is possible that the high activities in certain neurons may merely reflect the low Km values of the enzyme species in a particular neuron. In the snail nervous system (Otala lactea), Gainer (1972a) has shown neuronal proteins, specific to identified, electrophysiologicaUy characterized neurons (see Fig. 5). Studies of the nature just described emphasize protein specificity parallel to neuronal differentiation in "simple" nervous systems in molluscs.

2.2 Neuronal Activity and Protein Synthesis in "Simple Systems" The specific proteins inherent to each functionally distinct neuron (see above) suggests that proteins may specify the neuronal characteristics observed in individual neurons. Knowing the steady-state protein pattern of the neuron, one can now begin to induce or modify the neuronal activity and look for any measurable effects on protein synthesis. The correlation of electrophysiological activity and behavior with proteins synthesis can be divided into three classes: Class I Correlation of protein with a simple behavior which could be induced experimentally. Class II

Correlation of a protein with a stable differentiated neurophysiological or pharmacological property.

Class III Correlation of a protein with a transient property such as synaptie coupling. 2.2.1 Correlation of a protein with a simple behavior which can be induced experimentally Kupfermann (1967) reported that the application of a sea water extract of isolated bag cells of the abdominal ganglion of the Aplysia induces egg laying in the animal. Later, Strumwasser et al. (1969) found that egg laying is induced only during the mating season, that bag cells extracts from both sexually mature and immature animals can induce egg laying and there is a stereotyped behavior pattern accompanying egg laying.

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FIG. 4. SDS electrophoretic patterns of extracts of somas of R2, L7, RI5 and L6 neurons (A-D respectively). The whole ganglion was incubated for 3 hr in a buffered (I0 mM Tris) artificial sea water medium (pH 7.8), containing 13/~M L-all leucine. Single soma isolation was then carried out and homogenized in a SDS-mercaptoethanol Tris buffer. The extract was electrophoresed on a 1.5 mm diameter, 15~ polyacrylamide gel with a 2.5 % stacking gel, Tris-glycine system for 4½hr using a constant voltage of 50 V for 2 hr and 70 V for a further 2½ hr. An internal marker, cytoehrorne C, was co-electrophoresed with each gel. The gels containing the separated proteins were stained in coomassie blue, destained and sliced into 1 mm slices and 0.5 mm slices for R2 only. Each slice was then counted in Toluene PPOPOPOP scintillationfluid containing 10 % NCS and 2 ~o 4N NH4 OH (Wilson, 1971). Background = 7. Counting was carded out to no more than 10~ error. In Fig. 4(A), the radioactivity (cpm minus background) was plotted on the ordinate against the relative mobility (relative to cytochrome C) on a log scale on the lower abscissa. Note the reproducibility of this method. In Fig. 4 (B-D), the radioactivity slice was plotted on the ordinate and the slice number on the lower abscissa. The upper abscissa of Fig. 4 (A-D) shows the molecular weight (M.W.).

Toevs a n d B l a c k e n b u r y (1969) concluded f r o m their studies that the active egg laying agent is the same as the bag cells specific protein. This p r o t e i n has a m o l e c u l a r weight o f a p p r o x i m a t e l y 6000, is sensitive to pronase, heat a n d trypsin t r e a t m e n t b u t n o t to R N a s e or D N a s e (Toevs, 1969). Recently, Arch (1972) studied the effect o f electrical a n d high K ÷ s t i m u l a t i o n o n bag cell p r o t e i n release. The a b d o m i n a l g a n g l i o n was removed from the

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Aplysia and incubated in medium containing aH leucine for 12 hr. At the end of the incubation period the ganglion was rinsed and then perfused with drops of the artificial medium. 1 ml fractions were collected and 200 tzl aliquots from each fraction were dried on filter discs, acid precipitated, washed and counted. Figure 6 shows the histograms of these counts as a function of time showing the enhanced release of labeled material after stimulation in high K + medium or 30 see electrical stimulation applied to the right connective nerve. Control experiments showed that release occurred only from the bag cells. With inter-

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perfusate fractions 1-3 collectedat the outset of control perfusion in a high-potassium stimulation experiment. The second distribution was derived from fractions 8-10 which immediately preceded high-potassium stimulation. Fractions 11-13 were the first three collected after the onset of high-potassium perfusion, and fractions 18-20 were collected at the end of high-potassium perfusion. The bottornmost distribution was derived from an aqueous supernatant of an homogenate of the bag cells. Sample preparation from this supernatant was the same as that described for perfusate samples. Due to variation in the length of each gel, the distance of tracking dye migration, and the fractionation procedure comparison of molecular weights from gel to gel is approximate. In this figure the distributions have been aligned according to the location of the 12,000 M.W. marks. (From Arch, 1972).

mittent application o f high K + a gradual decline in the amount released was observed. This, however, was thought to be a result of depletion of the store o f the protein rather than deterioration of the tissue, since stimulation after 3½ hr of rest showed recovery and significant release again. The release process was also found to be Ca + + dependent. Qualitative study on SDS polyacrylamide gel of the perfusate showed the presence of a 75,000 M.W. (molecular weight) and 25,000 M.W. product and an increase of a less than 10,000 M.W. protein. A homogenate o f the bag cells also showed a predominance of the 10,000 M.W. protein suggesting that it is this protein that is released with stimulation. A final test of the biological activity of the 10,000 M.W. product released was performed by injecting the

ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN

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perfusate into the haemicoel of a test animal. Within about 45 min, egg laying was induced. The work demonstrated how a neurophysiologieal event caused either by high K + or electrical stimulation can give rise to a defined behavior, mediated through the release of a specific protein from the neurosecretory bag cells. At the single cell level, the isolated snail brain has also proven to be useful preparation for such studies. As previously mentioned, Gainer (1972a),having shown, from SDS electrophoretic separation, that there are patterns of protein synthesis, specific to single, identifiable neurons in the land snail Otala lactea, proceeded to study the effect of experimentally induced diapause (active or dormant periods) on the electrophysiology and protein synthesis in these neurons. In particular, cell 11 was most interesting. A difference in electrical activity was evident whether the animal was in an active or dormant period (see Fig. 7). It was found that the synthesis of a 5000 M.W. protein was inhibited during the dormant period (see Fig. 7). None of the other cells studied showed differences in protein pattern synthesis with diapause. Evidence was also given that this 5000 M.W. protein may be neurosecretory material. Thus, here is another example of how a "simple" behavior such as diapause may be correlated with electrophysiological characteristics and protein metabolism of a single neuron. 2.2.2 Correlation of a protein with a stable differentiated neurophysiological or pharmacological property Cell R15 shows a pacemaking and bursting activity which seems to be both Na + and CIdependent while L3 has a pacemaking activity dependent only on Na +. Wilson (quoted by Strumwasser, 1972), making use of the neurophysiological properties of these two cells, tried to find protein species which may be associated with these characteristics of the neurons. It was found that both L3 and R15 had major peaks resolved on SDS polyacrylamide gels at 12,000 M.W. and another 6000-9000 M.W. He was able to show that under conditions which interfered with pacemaking activity in R15 (e.g. Na + or C1- free medium) the 60009000 M.W. protein was not synthesized. Thus, it appears that there is some correlation between endogenous pacemaking activity of neuron and protein synthesis, although a direct ionic effect on translation, independent of electrical activity, must be considered. The converse of this approach was tried by Schwartz, Castellucci, and Kandel (1971) who inhibited protein synthesis and monitored neurophysiological activity. Anisomycin, at low concentration, was found to be a very effective inhibitor of protein synthesis in Aplysia and prolonged incubation in this inhibitor had no effect on the resting membrane potential of the neurons. The R15 cell was tested for spontaneous activity after 1, 2, 3, 4 and 22 hr of incubation in anisomyein. Figure 8 shows that after 22 hr of inhibited protein synthesis the firing pattern has been considerably modified, although the endogenous activity was not eliminated. It has been shown that deterioration of the bursting rhythm also happens in controls and this may be a result of depressed protein synthesis which occurs with prolonged incubation in sea water, in the absence of inhibitors. If, as Wilson suggests, there is a correlation between 6000-9000 M.W. protein and bursting, the results of Schwartz et al. (1971) would imply either that they are not causally related, or there is a large pool of this protein. Postsynaptic potentials (PSPs) produced by the activities of endogenously active interneurons may be recorded from R15 and L7. In R15 the characteristic input are diphasic, non-cholinergic PSPs which persisted in the presence of anisomycin for 30 hr. Similarly the inhibitory postsynaptic burst recorded in L7 was present after 30 hr of inhibition of

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FIG. 7. Patterns of electrical activity of cell 11. A, B, and D show three modes of spontaneous (pacemaker) activity which are typically recorded from cell 11 in active animals: A, strong burst pattern; B, beating pacemaker pattern; and D, weak burst; C, recording from cell 11 from an estivated (dormant) snail. Note the absence of spontaneous electrical activity, but that a full sized action potential could be evoked by the passage of intracellular depolarizing current. Calibration pulse preceding evoked spike: 20 mV, 100 msec. E, spike evoked by a short pulse of current in the same cell as shown in C after complete isolation of the cell body from the ganglion. Calibration pulse preceding spike: 20 mV, 50 msec. Upper beam: zero potential line and current monitor; lower beam; membrane potential (From Gainer, 1972a). Electrophoretic SDS microgel protein patterns from the extracts of the cell body of cell 11 isolated from active (filled circles, solid line) and dormant (open circles, broken lines) snails after 20 hr incubation of the ganglion in media containing L-3H leucine. Each labeling pattern in the figure is an average of three independent microgel patterns obtained from separate, identified neurons (i.e., each point represents the average disint./min minus background/gel slice of three separate gels at the specified relative mobilities). See legend to Fig. 5 for an interpretation of the coordinates of the graph. (From Gainer, 1972a.) p r o t e i n synthesis (see Fig. 9). This a g a i n w o u l d suggest t h a t i m m e d i a t e p r o t e i n synthesis is unnecessary f o r this t y p e o f stable neural activity. O n the one h a n d , W i l s o n f o u n d t h a t the suppression o f R 1 5 ' s e n d o g e n o u s activity was c o r r e l a t e d with the absence o f synthesis o f a 6000-9000 M . W . protein, while o n the o t h e r h a n d , Schwartz et al. f o u n d t h a t the bursting c o n t i n u e d in the absence o f p r o t e i n synthesis. These results need n o t necessarily c o n t r a d i c t each other, b u t m a y be i n t e r p r e t e d as the l a c k o f d e p e n d e n c e o f immediate p r o t e i n synthesis f o r s p o n t a n e o u s activity. The results o f

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Schwartz et al. also suggest that generation of PSPs from interneuron inputs is not dependent on immediate protein synthesis. L10 has 14 identifiable follower cells and this interneuron can mediate excitation, inhibition and conjoint excitation and inhibition to three different classes of follower cells. There is pharmacological evidence that these actions are effected by the release of one transmitter only, acetylcholine (Kandel et al., 1967; Watchel and Kandel, 1967, 1971; Blackenship, Watchel and Kandel, 1971). Subsequent studies showed that L10 contains a much higher level of choline acetyltransferase activity over other cells (McCaman and Dewhurst, 1970; Giller and Schwartz, 1971). Thus we have an example of where a known protein, choline acetyltransferase, may be correlated with a functional, pharmacological property of the neuron. 2.2.3 Correlation o f a protein with a transient property Although de novo protein synthesis does not appear to be necessary for basic neurophysiological activity of neurons, such as PSP generation, perhaps it is for a more complex

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Fro. 9. A: spontaneous occurrence of a characteristic diphasic synaptic potential at a noncholinergie synapse in cell RI 5 in the presence and in the absence of anisomyein. Part 1 : the control was incubated in the absence of inhibitor for 21 hr. Part 2: three successivesynaptie potentials occurring spontaneously from a cellincubated for 30 hr in the presence of 18 ~Manisomycin. Intervals between the PSPs were 92 and 133 see. B: spontaneous spike and synaptic potentials in L7 in the presence and in the absence of anisomycin. Part 1 : the control ganglion was incubated in artificial sea water for less than 1 hr. Part 2: the experimental ganglion was incubated in the presence of 18 tzM anisomycin for 28 hr. (From Schwartz et aL, 1971.) phenomenon such as short-term changes in synaptic efficacy or modification of neuronal activity by electrical stimulation. Since the early twentieth century there has been an interest in the effect of electrical stimulation on protein synthesis in nerve cells. The giant neuron R2 in the abdominal ganglion of Aplysia provides an ideal preparation for this study, by virtue of its size and its quiescent electrophysiological eharacter. Since it has been shown that excitatory, synaptic activity produced enhancement of R N A synthesis in R2 (Berry, 1969; Peterson and Kernell, 1970) it was interesting to find out if the newly synthesized R N A was translated. F o r these reasons, Wilson and Berry (~972) studied the effect of excitatory synaptic stimulation on protein synthesis in R2. The left pleural giant cell (LPG) which is homologous to R2 (Hughes and Taue, 1963) provided a good intra-animal control. Protein patterns resolved on SDS polyacrylamide gels were similar for both R2 and LPG. An unstimulated R2 from

ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN Aplysia

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another animal was generally used as a second control. Two stimulation procedures were used by Wilson and Berry (1972). Type I

Type H

Stimulation via a suction electrode on the left connective. The stimulus was delivered once per second for 4 min every 15 min and consisted of either single pulses or bursts of 2-3 pulses at 25 per see and the voltage was varied to evoke one spike per stimulus in R2. Stimulation of the left connective or brachial nerve with a single shock or burst of 2--4 shocks 60-72 times per min. The number of stimuli per burst and their voltage were adjusted to give a single spike from R2 per burst. One nerve was stimulated until the R2 response was exhausted, then the other was stimulated then back to the first, etc. Stimulation continued for 1 hr.

Experiments were carried out to measure (i) the total incorporation of aH leucine into protein and (ii) quantitative or qualitative changes in protein profiles in stimulated R2 and unstimulated LPG. After Type I stimulation 4-5 hr followed by 1 hr in aH leucine no significant change of total incorporation over the control was found in R2. While this method may not be sensitive enough, any small quantitative changes or new protein species should be detectable on SDS polyacrylamide gels. Therefore another series of experiments were done where the ganglion was given either Type I or Type II stimulation. Type I stimulation was generally continued for 9 hr while the ganglion was immersed in a medium containing 3H leucine. The ganglion was subjected to stimulation for one hour in a medium without the aH leucine, but was subsequently incubated in labeled medium for another hour, after which single cell isolation and gel electrophoresis were carried out. Neither type of stimulation showed either quantitation or qualitative effects on the pattern of protein synthesized. While RNA precursors may be a limiting factor, another experiment was done where unlabeled uridine was added to the medium during Type I stimulation for 4-5 hr. The ganglion was then incubated in 3H leucine containing medium for 1 hr before single cell analysis. Again, the results were no different than before. Wilson and Berry (1972) then concluded that excitatory, synaptic activity has no effect on the total incorporation or pattern of protein synthesized in the giant neuron R2. The effect of inhibition of protein synthesis on the electrical properties of R2 was investigated by Schwartz et al. Antidromically conducted action potentials and orthodromieally mediated synaptic potentials were elicited by stimulating the fight connective nerve and the siphon or genital-pericardial nerve respectively. The ganglion was incubated for 25 hr in the presence of anisomycin before testing, and it was found that normal antidromic spikes and excitatory postsynaptic potentials can be evoked by stimulation (see Fig. I0). Schwartz and his co-workers also tested the effect of anisomycin on the ability of neurons to the increase in synaptic efficacy, known as posttetanic potentiation. This means of electrical modification may be an example of synaptic plasticity of a simple form. There is evidence that stimulation of the right connective gives rise to a cholinergic monosynaptic EPSP in R15 (Fraizer et al., 1967; Gerchenfeld, Ascher and Taut, 1967) which is potentiated after a tetanous pulse (5 per sec) (Schwartz et aL, 1971). Incubation for 31 hr in anisomycin, which most probably blocked synthesis of choline acetyltransferase, did not deprive the cell of the ability to show potentiation (see Fig. 11).

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FIG. 10. AntidromicaUy conducted action potential and orthodrornically mediated synaptic potentials in the absence of protein synthesis. Antidromic spikes were evoked in R2 by stimulating the fight connective repetitively, and postsynaptie potentials were evoked by stimulating the siphon and the genital-pericardial nerves. Several traces are superimposed in all cases. The ganglion had been incubated for 25 hr in the presence of anisomycin. (From Schwartz, et aL, 1971.) Next, neuronal correlates of another type of neurophysiological modification was considered--habituation and dishabituation. Stimulation of~the siphon nerve produces a complex EPSP of both mono- and polysynaptic components in the gill motoneuron L7. Repeated stimulation decreases this EPSP (habituation) which recovers after a rest period or stimulation via another pathway (dishabituation). Figure 12 shows a gradual decrease of PSP in L7 with repeated stimulation of the siphon nerve (habituation), the recovery after 15 min, a further rehabituation, dishabituation induced by stimulating the left connective, and then recovery again after a 15 min rest period. The lower plot (Fig. 12) shows the lack o f effect of inhibition of protein synthesis on the synaptic efficacy, associated with habituation and dishabituation. Up to 6 hr of incubation in anisomycin has been tested for loss of the ability, of the cell, to respond to simple synaptie, modification of neuronal behavior, but no effect was found. Somewhat less work, using this approach, has been carried out on other in vitro preparations. Toschi and Giacobini (1965) looked at the effect of puromycin on the generation of impulse activity of crayfish stretch-receptor neuron. The experiments were carried out on one of the slowly adapting cells while the contralateral cell from the same animal acted as the control. The results showed that with puromycin there was no impairment of impulse

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FIG. 11. A: posttetanic potentiation of a presumably cholinergic EPSP in the absence of protein synthesis. Intracellular records were from cell R15, which was hyperpolarized to prevent firing. The right connective was stimulated every 10 sec to evoke control elementary monosynaptic EPSP. After a brief tetanization (5 per see, 30 see), the test EPSP was again evoked at the control rate. Posttetanic potentiation was produced every hour. Examples are given of the EPSP and its subsequent potentiation from the same R15 before (top line) and during incubation in the presence of anisomycin for 2 and 8 hr. B: posttetanic potentiation in cell RI5 in the presence (C)) and in the absence ( 0 ) of anisomycin. The mean value of about ten EPSP amplitudes prior to the tetanus was taken as 100 K. Ganglia were batched in artificial sea water for 1 hr and then incubated in the presence (experimental) or in the absence (control) of anisomycin for an additional period of 1-3 hr (part 2), 3-8 hr (part 3), 7-21 hr (part 4). Several ganglia were tested in artificial sea water within 2 hr of removal from the animal (part 1). The mean percentage values of amplitudes of the initial EPSPs (-)-SEM) at various time intervals after tetanization are illustrated. The amplitude of the initial EPSP (mv) and the number of ganglia tested (n) were: part 1 : control, 13.4 (n = 10); part 2: control, 5.3 (n = 5); experimental, 14.0 (n = 8); part 3: control, 5.2 (n = 6); experimental, 9.7 (n = 13); part 4: control, 10.7 (n = 2); experimental, 9.2 (n = 5). The initial amplitude of the EPSP roughly correlated with the percentage of facilitation observed: the smaller the EPSP the larger the facilitation. This might partially explain the scatter of the data. (From Schwartz et aL, 1971 .)

g e n e r a t i o n in t h e e x p e r i m e n t a l a n i m a l s , f o r p e r i o d s u p to 12 hr. T h u s , there seems t o be n o d e p e n d e n c e o n i m m e d i a t e p r o t e i n synthesis, f o r t h e e v o k i n g o f g e n e r a t o r p o t e n t i a l s in a n e u r o n b y m u s c u l a r stretch. T h e c o n c l u s i o n d r a w n f r o m this r e v i e w is t h a t e l e c t r o p h y s i o l o g i c a l a n d s h o r t - t e r m s y n a p t i c m o d i f i c a t i o n s o f n e u r o n a l a c t i v i t y d o n o t r e q u i r e the synthesis o f n e w p r o t e i n . I t w o u l d a p p e a r , t h e r e f o r e , t h a t e i t h e r p r o t e i n s a r e n o t r e q u i r e d at all o r t h a t t h e p r o t e i n s r e q u i r e d f o r t h e n e u r o n a l f u n c t i o n s tested, t u r n e d o v e r at a r e l a t i v e l y slow rate. H o w e v e r , P.N. 212~r

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this data does not rule out that there are other m o r e c o m p l e x functions o f neurons w h i c h m a y require d e n o v o synthesis.

3. Conclusions F r o m the literature reviewed it is clear that in m o l l u s c a n neurons, as in other neurons, m a c r o m o l e c u l e s do specify s o m e neuronal functions such as w h a t transmitters a cell will use and egg-laying h o r m o n e s . There are two other types o f neuronal f u n c t i o n where the relation between m a c r o m o l e e u l e s and function is not so clear.

ROLE OF MACROMOLECULES IN NEURONAL FUNCTION IN

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Correlation of macromolecules with a stable differentiated electrophysiological property. Class l I l Correlation of macromolecules with a transient property such as synaptic coupling or stimulation.

Class H

The experiments of Wilson (quoted by Strumwasser, 1972) with R15 permit the interpretation (among others) that Class II type correlation of a spontaneous electrophysiological property with protein synthesis does exist. That protein synthesis is required to give rise to the endogenous neurophysiological activity is suggested in the observation of Schwartz and his co-workers (1971) where the activity decreases with time as does protein synthesis in their control experiments. The persistence, although with deterioration, of the electrophysiological activity for 22 hr in anisomycin may be explained by a slow turnover of protein. Conversely if the neurophysiological activity is inhibited by the removal of Na + and CIthere is perhaps a feedback mechanism which inhibits further synthesis of the 60009000 M.W. protein. Class III represents correlation of macromolecular synthesis with changes in synaptic coupling of parameters of stimulation. Posttetanic potentiation, habituation and dishabituation show no dependence on protein synthesis. Although experiments have been cited demonstrating modification in transcription of RNA with stimulation, experiments designed to show translation of this RNA have failed to do so. There is no evidence for either qualitative or quantitative changes in translation. If there existed a causal relationship between protein and synaptic activity, modification of one should produce changes in the other. This does not occur. An argument that has been proposed to explain this lack of effect has been to suggest that the proteins which specify functions have very large pools and are therefore not amenable to change or depletion in experiments lasting 1 or 2 days. We are left then with a demonstrable effect on RNA (albeit in the absence of "plastic response") but with no apparent function for this RNA. Since this increased RNA remains for some hours in the neuron, it is probably not being used metabolically. If it is not being used for either metabolism or translation, it might be used to directly alter synaptic coupling, although how this would be accomplished is a bit hard to visualize. Finally, because of the time necessary for synaptic stimulation to induce transcriptional changes and for this new RNA to get to where it might function, any synaptic modification which occurs in less than 30 min is almost certainly unrelated to synthetic changes in macromolecules. In summary, it would seem that the time has come to begin exploring other mechanisms than synthesis of macromolecules for control of neuronal function, because, with a few exceptions, the evidence appears to argue against this notion.

References ALVING,B. O. (1968) Spontaneous activity in isolated somata of Aplysia pacemaker neurons. J. Gen. Physiol. 51, 29-48. ARCH,S. (1972) Peptide secretion from the isolated parietovisceral ganglion of Aplysia californica. J. Gen. PhysioL 59, 47-59. BERRY,R. W. (1969) Ribonucleic acid metabolism of a single neuron: correlation with electrical activity. Science, 166, 1021. BERRY,R. W. and COHEN,M. J. (1972) Synaptie stimulation of RNA metabolism in the giant neuron Aplysia californica. 2. Neurobiol. 3, 209-223.

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