Mechanisms of transmitter release and synaptic plasticity at excitatory synapses in the hippocampus

The work presented in this thesis addresses two distinct questions: What is the distribution and role of presynaptic calcium channels involved in the release of glutamate at excitatory synapses in hippocampal cultures? What is the locus of expression of long-term potentiation at the perforant path-granule cell synapse in slices of the dentate gyrus? A number of different subtypes of Ca2+ channels are known to support the release of glutamate at excitatory synapses. The pattern of co-localisation of these subtypes on presynaptic terminals in hippocampal cultures was investigated. N-type (co-conotoxin GVIA-sensitive, co-CTx) or P/Q-type (co-agatoxin IVA-sensitive, co-Aga) Ca2+ channels were selectively blocked and the reduction in transmitter release probability (Pr) was measured using MK-801. The toxins completely blocked release at some terminals, reduced Pr at others, and failed to affect the remainder. In contrast, nonselective reduction of presynaptic Ca2+ influx by adding Cd2+ or lowering external Ca2+ reduced Pr uniformly at all terminals. It was concluded from these results that the mixture of N-type and P/Q-type channels varies markedly between terminals on the same afferent. A model was developed incorporating a non-uniform distribution of Ca2+ channel subtypes across presynaptic terminals. The model suggests that about 10% of terminals have only N-type channels, about 45% of terminals have only P/Qtype channels, and the remaining 45% have a mixture of subtypes. This non-uniform distribution may enable terminal-specific modulation of synaptic function by neuromodulators that differentially affect a particular Ca2+ channel subtype. The relationship between extracellular Ca2+ concentration and EPSC amplitude was investigated at excitatory autapses on cultured hippocampal neurons. This relationship was steeply non-linear, implicating the cooperative involvement of several Ca2+ ions in the release of each vesicle of transmitter. The cooperativity was estimated to be 3.1 using a power function fit, and 3.3 using a Hill equation fit. However, simulations suggest that these values underestimate the true cooperativity. The role of different Ca2+ channel subtypes in shaping the Ca2+ dose-response relationship was studied using the selective Ca2+ channel blockers co-Aga (P/Q-type channels) and co-CTx (Ntype channels). Both toxins broadened the dose-response relationship, and the Hill coefficient was reduced to 2.5 by co-Aga and to 2.6 by co-CTx. This broadening is consistent with a non-uniform distribution of Ca2+ channel subtypes across presynaptic terminals. The similar Hill coefficients in co-Aga or co-CTx suggest that there was no difference in the degree of cooperativity for transmitter release mediated via N-type or P/Q-type Ca2+ channels. A model of calcium's role in transmitter release was developed. It is based on a modified Dodge-Rahamimoff equation that includes a non­ linear relationship between extraand intracellular Ca2+ concentration, has a cooperativity of 4, and incorporates a non-uniform distribution of Ca2+ channel subtypes across presynaptic terminals. The model predictions are consistent with all of the results reported in this study. Long-term potentiation (LTP) of synaptic transmission is the putative mechanism underlying certain forms of learning and memory. Despite intensive study, it remains controversial whether LTP is expressed at a preor postsynaptic locus. A novel approach was used to investigated this question at excitatory synapses onto granule cells in acute slices of the dentate gyrus. The variance of the evoked synaptic amplitude was plotted against mean synaptic amplitude at several different Cd2+ concentrations. The slope of the variance-mean plot estimates the average amplitude of the response following the release of a single vesicle of transmitter (Qav). The variance-mean technique was tested by applying the analysis before and after three different synaptic modulations: (i) a reduction in Qav by the addition of the glutamate receptor antagonist, CNQX, (ii) a reduction in the average probability of transmitter release (Pr) by the addition of baclofen, and (iii) an increase in the number of active synaptic terminals (N) by increasing the stimulus strength. CNQX reduced the average synaptic amplitude and Qav to the same extent, consistent with a postsynaptic action. In contrast, neither a change in N or Pr altered Qav. This confirmed that the variancemean technique can distinguish between a preand a postsynaptic site of modulation. The induction of LTP increased the synaptic amplitude and Qav to the same extent, strongly supporting a postsynaptic locus of LTP expression in the dentate gyrus. A postsynaptic increase in synaptic efficacy could be due to the insertion of additional AMPA receptors into the postsynaptic membrane, the unmasking of AMPA receptors already present in the membrane or a change in the single channel conductance of the AMPA receptor.