Bruce Walmsley

Counting quanta: Direct measurements of transmitter release at a central synapse

Contradictory hypotheses regarding the nature of synaptic transmission in the CNS have arisen from indirect methods of quantal analysis. In this study, we directly count the quanta released following nerve stimulation to examine synaptic transmission at a fast glutamatergic synapse in the mammalian auditory brainstem. Our results demonstrate the relationship between spontaneous and nerve-evoked synaptic events, indicate that asynchronous transmitter release governs the time course of evoked transmission, and show that the stochastic quantal release process, as originally proposed at the neuromuscular junction, is highly conserved at this central synapse.

A Novel Presynaptic Inhibitory Mechanism Underlies Paired Pulse Depression at a Fast Central Synapse

Several distinct mechanisms may cause synaptic depression, a common form of short-term synaptic plasticity. These include postsynaptic receptor desensitization, presynaptic depletion of releasable vesicles, or other presynaptic mechanisms depressing vesicle release. At the endbulb of Held, a fast central calyceal synapse in the auditory pathway, cyclothiazide (CTZ) abolished marked paired pulse depression (PPD) by acting presynaptically to enhance transmitter release, rather than by blocking postsynaptic receptor desensitization. PPD and its response to CTZ were not altered by prior depletion of the releasable vesicle pool but were blocked by lowering external calcium concentration, while raising external calcium enhanced PPD. We conclude that a major component of PPD at the endbulb is due to a novel, transient depression of release, which is dependent on the level of presynaptic calcium entry and is CTZ sensitive.

Synaptic transmission in the auditory brainstem of normal and congenitally deaf mice

The deafness (dn/dn) mutant mouse provides a valuable model of human congenital deafness. We investigated the properties of synaptic transmission in the anteroventral cochlear nucleus (AVCN) of normal and congenitally deaf dn/dn mice. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation of single auditory nerve fibres, and measured by whole‐cell recordings from neurones in AVCN slices (mean postnatal age = P13). Absolute amplitudes of both AMPA‐ and NMDA‐mediated components of evoked EPSCs were greater (170 %) in deaf versus control animals. Enhanced transmission in deaf mice was due to a presynaptic mechanism. Variance‐mean analysis revealed that the probability of transmitter release was significantly greater in deaf (Pr= 0.8) versus control animals (Pr= 0.5). Following high frequency stimulation, deaf mice showed a greater depression of evoked EPSCs, and a significant increase in the frequency of delayed‐release (asynchronous) miniature EPSCs (aEPSCs) (deaf 100 Hz vs. control 7 Hz). The acetoxymethyl ester of EGTA (EGTA‐AM) blocked the increase in miniature aEPSCs and returned tetanic depression to control values. In deaf mice, reduction of mean Pr using cadmium caused an expected increase in paired‐pulse ratio (PPR). However, in the same cells, a similar reduction in release by EGTA‐AM did not result in a change in PPR, demonstrating that a change may occur in Pr without a concomitant change in PPR. In many respects, transmission in deaf mice was found to be remarkably similar to control mice, implying that many parameters of synaptic transmission develop normally in these animals. The two significant differences (higher Pr and asynchronous release in deaf mice) could both be reversed by the addition of EGTA‐AM, suggesting that endogenous calcium buffering may be impaired or undeveloped in the presynaptic terminals of the auditory nerve in deaf mice.

Diversity of structure and function at mammalian central synapses

Our appreciation of the relationship between synaptic structure and function, and in particular our understanding of quantal synaptic transmission, is derived from classical studies on the neuromuscular junction. However, physiological studies of quantal transmission at mammalian CNS synapses have produced a variety of results, and thus no consensus of opinion has emerged. This variability could be due, in part, to experimental and analytical limitations or to differences in the structural and functional features of central synapses, or both. Some of the experimental limitations have recently been overcome by the use of novel preparations that permit direct measurement of quantal synaptic events in the CNS. Although these studies reveal similarities between the synaptic mechanisms of the neuromuscular junction and CNS synapses, important differences and specializations are also evident. The purpose of this review is to highlight the structural and functional diversity of synapses in the mammalian CNS, and to discuss the potential relevance of structural features to synaptic function.

Release probability modulates short-term plasticity at a rat giant terminal

1 Modulation of release probability is a major factor underlying short‐term synaptic plasticity in the central nervous system. We have investigated the relationship between release probability ((Pr) and paired‐pulse modulation at a large auditory calyceal synapse containing many transmitter release sites. Whole‐cell patch electrode recordings were made of excitatory postsynaptic currents (EPSCs), evoked by stimulation of auditory nerve fibres giving rise to the endbulbs of Held. 2 Quantitative estimates of Pr and quantal amplitude were obtained using the recently developed variance‐mean analysis technique. Release probability conditions were modulated by bath application of cadmium, elevated calcium and protein kinase C activation by phorbol esters. 3 Our results show that, under physiological conditions, most sites released neurotransmitter following a single presynaptic nerve impulse, with a mean Pr of 0·6. The mean quantal amplitude was 44 pA, which was consistent with the mean amplitude of miniature EPSCs (47 pA). 4 Under high release probability conditions with elevated calcium or phorbol esters, Pr at all sites approached 1·0. At these high Pr values, variance‐mean analysis indicated a significant postsynaptic contribution to paired‐pulse depression. The miniature EPSC amplitudes were decreased following stimulation in elevated calcium, confirming a postsynaptic component of paired‐pulse depression at this glutamatergic connection. 5 A notable feature was the large variability between neurons in the relationship between paired‐pulse ratio and Pr. Based on current models of vesicle release and ultrastructural evidence, we suggest that this variability may be partly due to morphological differences between endbulb specializations, particularly in the ratio of fusion‐ready to reserve populations of vesicles at endbulb release sites.

Developmental changes in EPSC quantal size and quantal content at a central glutamatergic synapse in rat

1 Developmental changes in amplitude and time course of single‐fibre‐evoked and spontaneous EPSCs mediated by AMPA and NMDA receptors at the endbulb‐bushy cell synapse of rats from 4 to 22 days of age were recorded using whole‐cell patch‐clamp methods in in vitro slices of cochlear nucleus. 2 The mean conductance of the AMPA component of evoked EPSCs increased by 66 %, while that of the NMDA component decreased by 61 %, for 12‐ to 18‐day‐old rats cf. 4‐ to 11‐day‐old rats. 3 The mean AMPA spontaneous EPSC conductance increased by 54 %, while mean NMDA spontaneous EPSC conductance decreased by 83 %, for 12‐ to 22‐day‐old rats cf. 4‐ to 11‐day‐old rats. The mean number of quanta contributing to peak evoked AMPA conductance also increased by 78 % in the older age group, after correction for the asynchrony of evoked quantal release. 4 The decay time constant of spontaneous AMPA EPSCs showed a small decrease in older animals, while the decay time constant of spontaneous NMDA EPSCs was markedly decreased in older animals. The decay time constants of evoked NMDA EPSCs showed a quantitatively similar decrease to that of spontaneous NMDA EPSCs. This suggests that AMPA receptor subunit composition is unlikely to undergo developmental change, while NMDA receptor subunit composition may be substantially altered during synaptic maturation. 5 These data are consistent with a developmentally increased efficacy of AMPA receptor‐mediated synaptic transmission at the endbulb‐bushy cell synapse, due to an increase in underlying AMPA‐mediated quantal size and content during the same period as a transient co‐localization of NMDA receptors.

GABA mediates presynaptic inhibition at glycinergic synapses in a rat auditory brainstem nucleus

1 Many inhibitory nerve terminals in the mammalian anteroventral cochlear nucleus (AVCN) contain both glycine and GABA, but the reason for the co‐localization of these two inhibitory neurotransmitters in the AVCN is unknown. We have investigated the roles of glycine and GABA at synapses on bushy cells in the rat AVCN, using receptor immunohistochemistry and electrophysiology. 2 Our immunohistochemical results show prominent punctate labelling of postsynaptic clusters of glycine receptors and of the receptor clustering protein gephyrin over the surface of bushy cells. In contrast, weak diffuse membrane immunolabelling of GABAA receptors was observed. 3 Whole‐cell recordings from bushy cells in AVCN slices demonstrated that evoked inhibitory postsynaptic currents (IPSCs) were predominantly (81%) glycinergic, based on the decrease in amplitude of the IPSCs in bicuculline (10 μm). This observation was supported by the effect of strychnine (1 μm), which was to decrease the evoked IPSC (to 10% of control IPSC amplitude) and to produce a greater than 90% block of spontaneous miniature IPSCs. 4 These results suggest a minor role for postsynaptic GABAA receptors in bushy cells, despite a high proportion of GABA‐containing terminals on these cells. Therefore, a role for metabotropic GABAB receptors was investigated. Activation of GABAB receptors with baclofen revealed a significant attenuation of evoked glycinergic IPSCs. The effect of baclofen was presynaptic, as indicated by a lack of change in the mean amplitude of spontaneous IPSCs. 5 Significantly, the decrease in the amplitude of evoked glycinergic IPSCs observed following repetitive nerve stimulation was reduced in the presence of the GABAB antagonist, CGP 35348. This indicates that synaptically released GABA can activate presynaptic GABAB receptors to reduce transmitter release at glycinergic synapses. Our results suggest specific pre‐ versus postsynaptic physiological roles for GABA and glycine in the AVCN.

Topographic Organization in the Auditory Brainstem of Juvenile Mice is Disrupted in Congenital Deafness

There is an orderly topographic arrangement of neurones within auditory brainstem nuclei based on sound frequency. Previous immunolabelling studies in the medial nucleus of the trapezoid body (MNTB) have suggested that there may be gradients of voltage‐gated currents underlying this tonotopic arrangement. Here, our electrophysiological and immunolabelling results demonstrate that underlying the tonotopic organization of the MNTB is a combination of medio‐lateral gradients of low‐and high‐threshold potassium currents and hyperpolarization‐activated cation currents. Our results also show that the intrinsic membrane properties of MNTB neurones produce a topographic gradient of time delays, which may be relevant to sound localization, following previous demonstrations of the importance of the timing of inhibitory input from the MNTB to the medial superior olive (MSO). Most importantly, we demonstrate that, in the MNTB of congenitally deaf mice, which exhibit no spontaneous auditory nerve activity, the normal tonotopic gradients of neuronal properties are absent. Our results suggest an underlying mechanism for the observed topographic gradient of neuronal firing properties in the MNTB, show that an intrinsic neuronal mechanism is responsible for generating a topographic gradient of time‐delays, and provide direct evidence that these gradients rely on spontaneous auditory nerve activity during development.

Ultrastructural basis of synaptic transmission between endbulbs of Held and bushy cells in the rat cochlear nucleus

Auditory nerve fibres make large excitatory synaptic contacts, the endbulbs of Held, with bushy cells in the anteroventral cochlear nucleus (AVCN). We have used serial‐section electron microscopy to reconstruct seven endbulbs of Held in contact with three different AVCN bushy cells from a 25‐day‐old rat, as a basis for interpreting our previous physiological results at this connection. Four endbulbs of Held contacting the same bushy cell were completely reconstructed. The number of separate synaptic specializations within these endbulbs varied from 85 to 217, with a mean of 155. Detailed measurements were obtained from high magnification segments of four endbulbs contacting three different bushy cells. Large variability was found in the size of synaptic specializations within an individual endbulb. The size of postsynaptic densities (PSDs) varied between endbulbs (mean PSD area 0.03, 0.07, 0.07 and 0.18 μm2; n= 4 endbulbs). The number of morphologically docked vesicles at individual specializations within the same endbulb varied considerably (between 1 and 102). The mean number of morphologically docked vesicles per specialization differed between endbulbs (mean numbers of docked vesicles per specialization = 2.1, 3.7, 5.3, 14.8; n= 4 endbulbs). Despite these large differences, the density of docked vesicles per square micron of PSD was similar between endbulbs (54, 80, 81, 83 docked vesicles per μm2; n= 4 endbulbs). Within an endbulb, a linear relationship was found between the number of docked vesicles and PSD area, and between PSD area and the number of undocked vesicles within 150 nm of the active zone. The ratio of undocked vesicles (< 150 nm) to docked vesicles ranged from 2 to 5 in different endbulbs (n= 4 endbulbs). These structural observations are discussed in relation to the functional properties of synaptic transmission between endbulbs of Held and bushy cells in the AVCN.

Quantal size is correlated with receptor cluster area at glycinergic synapses in the rat brainstem

1 Whole‐cell patch electrode recordings of glycinergic miniature inhibitory postsynaptic currents (mIPSCs) were obtained in neurons of the rat anteroventral cochlear nucleus (AVCN). Mean mIPSC peak amplitude was found to vary considerably between AVCN neurons (range, ‐19·1 to ‐317·9 pA; mean ± s.d., ‐159·1 ± 100·7 pA; 14 cells). 2 Immunolabelling of glycinergic receptor clusters in AVCN neurons was performed using antibodies against the glycine receptor clustering protein gephyrin. Measurements of the area of gephyrin immunoreactive clusters were obtained using confocal fluorescence microscopy. These measurements showed a large variability in cluster area, not only in the same cell (mean coefficient of variation, c.v., 0·66 ± 0·18; 16 cells), but also in mean cluster area between cells (range, 0·21‐0·84 μm2; 16 cells). 3 A possible relationship between mIPSC amplitude and receptor cluster area was investigated in a further series of experiments, in which mIPSCs recordings and immunolabelling of glycine receptor clusters were obtained for the same cells. In these experiments, AVCN neurons were identified using intracellular labelling with neurobiotin. Successful results using a combination of whole‐cell recordings, neurobiotin identification and immunolabelling were obtained for a total of 10 AVCN neurons. Analysis of the results revealed a positive, statistically significant correlation between mean receptor cluster size and mean mIPSC amplitude (P < 0·05, 10 cells, Spearmans correlation test). 4 These results provide direct experimental evidence supporting a hypothesis of central glycinergic transmission in which synaptic strength may be regulated by changes in the size of the postsynaptic receptor region.