Synaptic Differentiation of Single Descending Fibers Studied by Triple Intracellular Recording in the Frog Spinal Cord

Alexander E. Dityatev and H. Peter Clamann

Department of Physiology, University of Bern, CH-3012 Bern, Switzerland

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Dityatev, Alexander E. and H. Peter Clamann. Synaptic differentiation of single descending fibers studied by triple intracellular recording in the frog spinal cord. J. Neurophysiol. 79: 763-768, 1998. Evoked excitatory postsynaptic potentials (EPSPs) were simultaneously intracellularly recorded in two lumbar motoneurons located in spinal segments 8-10 in response to intraaxonal stimulation of a descending fiber. Their mean amplitudes, paired-pulse facilitation, and short- and long-term posttetanic potentiation were compared to reveal possible functional differences among synapses formed by one axon on different postsynaptic targets. The mean amplitudes of EPSPs recorded in two motoneurons were significantly different in most experiments. This amplitude difference was related to the location of motoneurons in that it was twofold larger in motoneurons separated by >1 mm than in motoneurons located within 200 µm of one another and also that the amplitude of EPSPs recorded in motoneurons located in the tenth segment was regularly smaller than the amplitude recorded in the ninth segment. The estimation of binomial model parameters suggests that the difference in mean EPSP amplitude was due mostly to differences in the maximal number of quanta prepared for release (binomial parameter N) and in mean release probability rather than to differences in quantal size. The ability of connections formed by a single axon on different motoneurons to undergo use-dependent synaptic modulations was different on scales of milliseconds, seconds, and tens of minutes as revealed by the measurements of effects of paired-pulse and tetanic stimulation. The difference in magnitude of short-term posttetanic potentiation in connections formed by a single descending axon was significantly correlated with the difference in mean probability of release in these connections. Thus our data revealed a functional nonuniformity of synapses formed by individual descending fibers on widely separated motoneurons, most likely innervating different muscles. This process can be one of the mechanisms by which a fine descending control of recruitment of motoneuronal populations is achieved.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

The synaptic connections formed by a single neuron on several different postsynaptic targets can differ widely in their synaptic efficacy. This result has been shown in a number of systems and species including interneuron-interneuron synapses in the locust central nervous system (CNS) (Laurent and Sivaramakrishnan 1992), neuromuscular junctions in the lobster and crayfish (Cooper et al. 1995; Katz et al. 1993), reticulospinal synapses in the lamprey (Brodin et al. 1994), and Ia-afferent connections on motoneurons of the cat (Clamann et al. 1985; Honig et al. 1983; Koerber and Mendell 1991; Lüscher and Vardar 1989; Lüscher et al. 1989). In the latter, spike-triggered averaging or intraaxonal stimulation of Ia-afferents was used to compare efficacy of synapses [measured as the mean amplitude of excitatory postsynaptic potentials (EPSPs)] formed by a single fiber onto several postsynaptic targets or by several axons onto a single motoneuron. Among these researchers, however, only Koerber and Mendell (1991) studied differences in the use-dependent modulation of synaptic transmission in synapses formed by individual axons in the CNS of vertebrates, due probably to the difficulty in finding and identifying ensembles of connected neurons. The data obtained for Ia-afferent connections showed that EPSP amplitude modulation during high-frequency stimulation depends mostly on the identity of the postsynaptic motoneuron. In contrast, it was shown that short-term use-dependent modulation in reticulospinal connections of the lamprey depended on properties of both pre- and postsynaptic partners (Brodin et al. 1994). The question of differentiation of presynaptic terminals has not been studied in the frog spinal cord, which is a convenient model to search for pairs of connected neurons. Here, both reticulomotoneuronal and Ia-motoneuronal individual connections can be studied (Babalian and Shapovalov 1984; Grantyn et al. 1984).

This issue acquires functional importance in studies of the modulatory control of CNS function, for example, in the motor system. It is well known that muscle force is varied by modulating the drive onto a whole motoneuron pool, whereupon a population of motoneurons are recruited into activity in order of their sizes. The mechanism for this recruitment order is not known. The original suggestions involved differences in the sensitivity to synaptic input of the postsynaptic structure, the motoneuron, (reviewed in Burke 1981; Henneman and Mendell 1981). Later suggestions included differences in presynaptic structures including weighted or exclusive inputs to motoneuron subpopulations (Burke 1981; Lüscher and Clamann 1992). In terms of a binomial model of synaptic efficacy, presynaptic factors are the probability of vesicle release (P) and the maximal number of vesicles prepared to be released (N). Postsynaptic factors include the number or density of receptors, the distance of synaptic boutons from the cell body, and the geometry of the dendrites and cell body. Several lines of evidence suggest a significant role of the presynaptic factors in synaptic differentiation. First, when the differences in the levels of paired-pulse facilitation and tetanic modulation in synapses formed by a single fiber were examined, the synapses producing EPSPs of smaller initial amplitude showed stronger facilitation (Koerber and Mendell 1991). Second, when stimulation of a fiber produced facilitation in one synapse and depression in another, the depression could be abolished by reduction of extracellular calcium concentration, after which both synapses exhibited homosynaptic facilitation (Katz et al. 1993). Third, quantal analysis revealed differences in the releasable pool and the probability of release in inhibitory synapses formed by a single interneuron in the locust CNS (Laurent and Sivaramakrishnan 1992). Fourth, synapses having higher efficacy were shown to have a larger number of presynaptic dense bars and a larger presynaptic calcium influx (Cooper et al. 1995; Govind et al. 1994; Katz et al. 1993).

In the present work we have examined the roles of pre- and postsynaptic factors in synaptic differentiation in the connections formed by a single reticulo- or propriospinal fiber with lumbar motoneurons of the frog by a comparison of the following: 1) the coefficient of variation of EPSP amplitude, 2) parameters of a binomial model, and 3) use-dependent modulation. We show that synaptic efficacy, measured as mean EPSP amplitude, depends more strongly on the identity of the postsynaptic target than on the presynaptic axon if the presynaptic structures have similar origins (i.e., both arise from the reticular formation or from propriospinal neurons). However, this postsynaptically determined regulation of the synaptic strength is achieved largely via changes in presynaptic parameters. We took the further step in the study of differentiating among presynaptic terminals by making simultaneous triple intracellular recordings. In this way the effect of one afferent on two motoneurons could be studied simultaneously. Changes in efficacy occurring in the course of, or because of, the experiment are thus not a factor. This detail seems to be especially important for a comparison of long-term modulation of synaptic transmission in the synapses formed by one fiber on two different postsynaptic targets. If two motoneurons are tested consecutively, the strong tetanic stimulation applied to the axon during a test of the first cell could modulate the transmission from the axon to the second cell and thus affect the results of the succeeding test of the second axon-motoneuron pair. Our data extend previous findings by showing that long-term modulation of synaptic transmission can be different in connections formed on two motoneurons by the same axon.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

The experiments were carried out on frogs, Rana esculenta. Animals were cooled in ice for 30 min and killed by decapitation. After dorsal laminectomy, the spinal cord and the whole brain stem were removed and placed in a chamber superfused with Ringer solution, the composition of which has been described in Babalian and Shapovalov (1984). Single axons and motoneurons were impaled and recorded at 15-17°C with glass microelectrodes filled with 3 M KCl and having input resistances of 40-50 MOmega and 25-30 MOmega , respectively. The microelectrodes were covered by aluminum foil to minimize stimulus artifact. The axons were impaled in the anterior white matter of the spinal cord 2- to 3-mm rostral to the closest motoneurons in the 8th to 9th segments. To find a triplet consisting of one axon connected to two motoneurons, two motoneurons were first impaled and then an axon was sought. In one-third of the cases, if the axon transmitted to one of the motoneurons it also transmitted to the other. In a number of experiments, only double recordings were performed. In these cases after acquisition of data from one pair, the motoneuron or axon was left and a new partner was found near the previous one. To identify the origin of the penetrated axon, a strategy similar to that employed by Babalian and Shapovalov (1984) was used. After a pair or triplet of connected elements was found, the reticular formation of the brain stem was stimulated at the level of the 10th cranial nerve with a beveled metal bipolar electrode. The impaled axons were identified as reticulospinal if stimulation of the reticular formation <50 V evoked a spike with a latency of 0.5-2 ms in the axon. Otherwise, it was assumed to arise from a propriospinal neuron. In a separate study, 50 V provided supramaximal stimulation of supraspinal fibers and the reticulospinal axons impaled in the reticular formation evoked EPSPs with the same parameters as EPSPs evoked by axons impaled in the spinal cord and physiologically identified to be reticulospinal (A. E. Dityatev, N. M. Chmykhova, A. L. Babalian, G. U. Dityateva, H. P. Clamann, unpublished data). The identified axon was then intracellularly stimulated through the microelectrode with 0.5-ms current pulses of 1-10 nA at 3-s intervals, with trains of 20 stimuli applied at a frequency of 50 Hz every 10 s, or with theta-burst stimulation (bursts of 5 stimuli, 10-ms interstimulus interval, delivered at 5 Hz during 1 min). All EPSPs analyzed in this work were presumably monosynaptic according to their short and stable latency, an absence of failures during tetanic stimulation, and the presence of numerous close appositions between varicosities of descending axons and lumbar motoneurons as revealed in another series of our experiments where successful labeling of pairs was achieved (Birinyi et al. 1996; Dityatev et al. 1995).

The coefficient of variation (CV) of EPSP amplitudes was calculated as the ratio S/X, where X is the mean amplitude and S2 is the variance of the amplitudes. The mean probability of release P was estimated as the ratio between X and the difference between maximal EPSP amplitude and SD of baseline noise. Or, expressed mathematically P = X/(EPSPmax - SDbaseline noise). This formula takes into account the contribution of noise in the measured maximal amplitude and is valid not only for the simple binomial model but also for release processes with nonuniform release probabilities or nonuniform contributions of quanta released. In the former case, it provides the value of mean release probability if the individual release probabilities are not too small. The other parameters of transmitter release were estimated on the basis of the binomial model by the method of moments: quantal size Q S2/[X(1 - P)] and maximal quantal content N = X2(1 - P)/[S2P] (Voronin et al. 1992). The method provided reasonably good estimates of binomial parameters as was found in our computer simulations. For instance, when 100 data sets of 200 amplitudes were simulated using binomial model with P = 0.6, N = 10, Q = 100, and standard deviation of noise equal to 40, the estimated parameters were P = 0.63 ± 0.03, N = 8.5 ± 1.3, and Q = 115 ± 13. The mean values are given in the text with ±SD (default) or ±SE.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

In 14 experiments we found 23 triplets consisting of one descending axon connected to two lumbar motoneurons separated by 1-2 mm. In 21 cases, intracellular stimulation of the axon evoked short-latency, fast EPSPs in both motoneurons; the latency was 2.5 ± 0.6 (SD) ms and the rise-time was 2.0 ± 0.7 ms at a temperature of 15 ± 1°C. The amplitude of these single-fiber EPSPs varied widely from 70 to 4,290 µV (528 ± 827 µV, 46 pairs). The propriospinal EPSPs were on average twice as large as reticulospinal EPSPs (856 vs. 406 µV) in agreement with findings from a previous study (Babalian and Shapovalov 1984). The difference between mean amplitude of single-fiber EPSPs recorded in two distant motoneurons was significant (2-tailed t-test; P < 0.05) in 20 of 21 cases (Figs. 1, 2, and 4). The ratio between mean amplitudes of such EPSPs was440 ± 124%. This difference in mean amplitudes can be due to the difference in number and location of synaptic contacts rather than to the difference in properties of individual synapses. To examine the latter possibility, we analyzed the modulation of single-fiber EPSPs by paired-pulse and tetanic stimulation (Fig. 2), which presumably affect presynaptic properties of the synapses.


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FIG. 1. Simultaneous triple intracellular recording from descending axon (top trace) and 2 lumbar motoneurons (2 bottom traces). Spike in presynaptic axon A was evoked by 0.5 ms × 2 nA current pulse. Axon was identified as reticulospinal by showing short-latency spike after stimulation of reticular formation (12 V). The motoneurons (M) in this experiment were located in the rostral and caudal parts of the 9th segment (2nd and 3rd traces, respectively). Note the difference in peak amplitude and shape of excitatory postsynaptic potentials (EPSPs). Each trace is an average of 50 sweeps. Temperature was 15.5° C. Data from experiment No. 208.


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FIG. 2. Difference in short-term use-dependent modulation of amplitude of EPSPs in 2 synaptic connections formed by a single propriospinal axon on 2 motoneurons. A: paired-pulse stimulation with interstimulus interval of 40 ms. Each curve is an average of 50 sweeps. B: 20 stimuli were applied at 50 Hz (black curve); a single shock applied 3 s later evoked a potentiated EPSP (gray curve). Average of 30 sweeps. Note that paired-pulse facilitation and posttetanic potentiation were significantly stronger in motoneuron located in 9th segment. The axon was identified as propriospinal by not responding to stimulation of reticular formation (50 V). Temperature was 15°C. Data from experiment No. 199.


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FIG. 4. Difference in long-term use-dependent modulation of amplitude of EPSPs in synaptic connections formed by a single propriospinal axon on 2 motoneurons. A: paired-pulse stimulation was not affected by theta-burst stimulation. B: two periods when sweeps were averaged; 2 episodes of theta-burst stimulation (down-arrow ) did not evoke significant changes in 1 synapse but produced long-term depression in another. Amplitude was measured as difference between peak amplitude and baseline and normalized to 100% before the 1st theta-burst stimulation. Each point represents an average of 25 amplitudes ± SE. Each episode of theta-burst stimulation consisted of 300 bursts applied at 5 Hz, 5 stimuli at 100 Hz in each burst. Resting membrane potential was -73 and -84 mV in motoneurons located in the 10th and 9th segments, respectively. It did not change by >2 mV during experiment. After the end of recording of EPSP, the antidromic spike was >90 mV in both motoneurons. Note that despite considerable depression of peak amplitude of EPSPs in motoneuron located in 9th segment, amplitude of electrical component (<= ) did not change. Data from experiment No. 238.

The ratio between the second and first amplitudes of EPSPs evoked by paired-pulse stimulation (40-ms interstimulus interval) varied from 72 to 179% in different synapses (118 ± 23%; 36 pairs). Similar amplitude changes were found after tetanic stimulation (20 stimuli applied at 50 Hz); the ratio of the amplitudes of EPSPs recorded both before and 3 s after the train varied from 90 to 187% (129 ± 25%; 30 pairs). There was no difference between reticulospinal and propriospinal fibers in this respect. Interestingly, in 5 of 19 triplets the ratio between amplitudes of the second and the first EPSPs evoked by paired-pulse stimulation was significantly different in two motoneurons contacted by a single fiber. In 4 of 15 triplets to which tetanic stimulation was applied, we also found significant differences between the connections in short-term posttetanic potentiation produced by the same fiber. The degree of the difference in paired-pulse modulation recorded in two motoneurons after stimulation of a single axon was significantly correlated with the difference in short-term posttetanic modulation, (PTM; r = 0.57, P < 0.05; 15 triplets). However, only the difference in PTM in two motoneurons was significantly negatively correlated with the difference in mean release probability P (Fig. 3A).


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FIG. 3. Summary of experiments. A: difference in posttetanic modulation (PTM2 - PTM1) in 2 synapses formed by a single fiber showed negative correlation with difference in mean release probabilities, P2 - P1. B: difference in properties of synapses formed either by a single axon on 2 motoneurons (1st, 2nd, and 4th groups of columns) or by 2 axons on 1 motoneuron (3rd group of columns). In the latter case, pairs were selected in which both axons were tentatively identified as propriospinal or reticulospinal. In 1st group of columns, motoneurons were separated by 1-2 mm whereas separation was <200 µm in the 2nd group. In 1st 3 groups, differences between parameters measured in pairs of motoneurons were calculated after special sorting; value measured in motoneuron having the smaller mean amplitude was subtracted from value obtained for motoneuron having the larger amplitude. In the last group the values obtained for motoneurons located in the 10th spinal segment were subtracted from values obtained for the corresponding motoneurons in the 9th segment. The following parameters were measured: mean amplitude (A1); coefficient of variation (CV); binomial parameters: release probability P, maximal number of quantal units N, and quantal size Q; ratio between amplitudes of the 2nd and 1st EPSPs in paired-pulse experiments (A2/A1); and posttetanic modulation (PTM). Log, difference of logarithms was used as a measure of differentiation; abs, absolute difference was calculated; N, sample size.

The results of analysis obtained using the CV and binomial model parameters (Fig. 3B, 1st group of columns) indicate that the difference in mean amplitude of single-fiber EPSPs recorded from different motoneurons is due mainly to presynaptic differences. First, we found a substantial difference in log CV of the amplitude of EPSPs evoked in two motoneurons. It was significantly different from 0 (2-tailed t-test; P < 0.0001) and strongly correlated with the difference in mean amplitudes (r = -0.62, P < 0.01). Because the mean EPSP amplitude is the product of mean release probability maximal number of quanta released times mean quantal size, it was convenient to analyze the difference of the logarithms of the mean amplitudes measured in pairs of motoneurons. This result should be the sum of the differences of these three parameters estimated for these motoneurons. This method gave us a convenient way to estimate the relative contributions of these parameters in the difference between mean amplitudes (Fig. 3B). It appeared that most of the difference between connections was due to differences in the number of quanta released (2-tailed t-test; P < 0.00001). A smaller but significant (P < 0.001) contribution arose from the difference in release probabilities. The difference in quantal size was estimated to be minimal, despite the fact that the smaller EPSPs had longer rise-time and half-width than the larger EPSP in two-thirds of the cases. The mean differences calculated for these two parameters were 0.31 ± 0.13 ms (different from 0 with P < 0.01; t-test, 19 triplets) and 5.6 ± 3.1 ms (P < 0.05), respectively. The values of CV and estimates of binomial parameters N and P were very similar for propriospinal and reticulospinal EPSPs (mean ± SE: CVs of 31 ± 4% vs. 32 ± 5%, N of 11 ± 1.2 vs. 11 ± 1.3; P of 0.58 ± 0.03 vs. 0.57 ± 0.04, respectively), but values of quantal size were larger for propriospinal than for reticulospinal connections (130 ± 34 vs. 65 ± 62 µV; P < 0.05, Wilcoxon's test).

To answer the question of whether the differences found in presynaptic properties of nerve terminals are random or can be related to the identity of the postsynaptic cells, we compared the present data on triples to two groups of experiments in which a single axon contacting a single motoneuron was studied. In these experiments, either a common axon was connected to two adjacent motoneurons that were impaled and studied in succession or two axons recorded one after another were found to connect to a common motoneuron. The expectations of the mean EPSP amplitude, CV, and binomial parameters in these three groups were not different (analysis of variance, P > 0.1). However, the difference in logarithms of mean amplitude, CV, and binomial N and P in pairs of compared connections was significantly smaller (t-test, P < 0.05) when two motoneurons were located close to one another in the same segment (the 2nd group of columns in Fig. 3B) than in the case when the motoneurons were located in two adjacent segments and/or were separated by >1 mm (the 1st group of columns in Fig. 3B). The difference in paired-pulse modulation was also two times smaller (t-test; P = 0.1). The differences in mean amplitudes and binomial N estimated in the pairs of connections formed by different axons on one motoneuron were significantly smaller than those calculated for triplets consisting of one axon and two widely separated motoneurons. However, the difference in synaptic modulation by paired-pulse stimulation was similar in both groups.

To employ the advantages of triple recording, we evaluated how independent the fluctuation of EPSP amplitudes are in motoneurons contacted by the same fiber; we also examined the possibility of evoking different forms of long-term modulation in two synapses formed by the same axon. The coefficients of correlation between amplitudes of EPSPs evoked in two motoneurons were not significantly different from 0 in all 21 triplets. This result implies that quantal release occurs independently in well-separated synapses. In two of five experiments, where two episodes of theta-burst stimulation were used to modulate synaptic transmission, the amplitude of EPSPs evoked in one of the motoneurons was transiently potentiated for 15 min. In the other motoneuron there was no modulation or there was a depression to 60% of control amplitude. In three other experiments, no long-lasting modulation was observed in one motoneuron but a long-term plateaulike depression was observed in the other (Fig. 4). The depression lasted for the duration of each recording (15, 25, and 55 min) and reached 80, 30, and 70%, respectively, of the control value at the end of each test.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

The differences in mean amplitudes of EPSPs evoked in two motoneurons by a single axon appear in large part to be due to differences in the number of quanta released. This finding and the correlation of difference in PTM magnitude with difference in release probabilities are in good agreement with previous findings (Cooper et al. 1995; Govind et al. 1994; Katz et al. 1993; Koerber and Mendell 1991; Laurent and Sivaramakrishnan 1992). Significantly larger differences in efficacy were found in the triplets when the motoneurons were widely separated than in experiments in which they were near to one another in the cord. The differences between EPSPs produced by synapses in the 9th and 10th spinal segments suggest that position along the rostro-caudal axis could be a factor determining the properties of connections (Lüscher and Clamann 1992; Lüscher and Vardar 1989). However, it must be remarked that triplets located in the eighth and the ninth segments or in rostral and caudal parts of the ninth segment showed no such systematic differences in mean EPSP amplitude. We therefore suggest that functional identity of motoneurons, rather than separation distance per se, could be an important factor that accounts for differences in efficacy. Because motoneuron pools may extend over a rostro-caudal length of a spinal segment or more, particularly in the eighth and ninth segments in the frog, they significantly overlap, making it difficult to reveal a systematic difference between motoneurons or their synapses. An overlap is less likely to have occurred in the 9th and 10th segments, in which the motor columns of individual muscles have a shorter rostro-caudal extent and motoneurons are more densely packed (Székely and Czéh 1976). Thus the probability that the motoneurons located in the 9th and 10th segments innervate different muscles (proximal vs. distal, respectively) is very high and one can expect some differences in functional tasks or properties of motoneurons that could retrogradely induce differentiation of the nerve terminals. Additional experiments with triple recording from identified motoneurons should be done to check this point. The isolation of the brain stem and spinal cord along with nerves innervating the legs recently was achieved in this laboratory and allows one to identify motoneurons in vitro.

The results of binomial analysis must be considered to be tentative because of possible nonuniformity of release probabilities shown in the spinal cord (Dityatev et al. 1992; Walmsley et al. 1988) and in the hippocampus (Dobrunz and Stevens 1997; Murthy et al. 1997). If this factor contributes significantly and there are many quantal units of low efficacy, the values of mean release probability given in this work will be overestimated and the values of the binomial parameter N will underestimate the actual number of quantal units. Nevertheless, the binomial approximation could still provide a useful measure; binomial N corresponds to the number of quantal units when the release probability is higher than 0.2 in sensory-motor synapses of the frog (Dityatev et al. 1992). Variability of quantal size (Isaacson and Walmsley 1995) could also bias estimates of quantal parameters. However, we have carried out simulations (not shown) that indicate that even a 30% variability of Q would not affect the results presented in this paper.

The recent work of Shigemoto et al. (1996) suggests that regulation of the probability of transmitter release can be achieved by differential expression of presynaptic metabotropic glutamate receptors (mGluRs) that are influenced by the postsynaptic target. It is known that mGluRs are able to modulate the amplitude and paired-pulse facilitation in synapses between descending fibers and motoneurons of the frog (Gotani et al. 1995; Nakamura et al. 1992, 1993). Our data show that blocks of group III mGluRs that are negatively coupled with adenylate cyclase are important for the induction of long-term presynaptic modulations of synaptic transmission in these synapses (Z. H. Melian, V. M. Kozhanov, A. E. Dityatev, and H. P. Clamann, unpublished data). The data from that study allow the interpretation that in the present work long-term changes in EPSP amplitude were induced in synapses with a low number of a type of mGluRs that, when present in higher numbers, play the role of stabilizer of synaptic efficacy in other synapses.

Recently, triple whole cell recordings from cultured hippocampal neurons were used to detect lateral pre- and postsynaptic propagation of long-term depression (Fitzsimonds et al. 1997). Triple recording from motoneurons and descending axons used in our study can be used to reveal the extent of this propagation in less artificial conditions.

    ACKNOWLEDGEMENTS

  We acknowledge the cooperation of Dr. N. M. Chmykhova.

  This work was supported by Swiss National Fund Grants 31-36508.92 and 31-45729.95.

    FOOTNOTES

   Present address of A. E. Dityatev: Institute for Biosynthesis of Neural Structures, Zentrum für Molekulare Neurobiologie Hamberg, Universitäts-Krankenhaus Eppendorf, University of Hamburg, Martinistr. 52, 20246 Hamburg, Germany.

  Address for reprint requests: H. P. Clamann, Institute of Physiology, University of Bern, Bühlplatz 5, CH-3012, Bern, Switzerland.

  Received 27 June 1997; accepted in final form 17 October 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society




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