Two components of transmitter release at a central synapse.

Abstract

After the arrival of a presynaptic nerve impulse at an excitatory synapse in hippocampal neurons, the rate of neurotransmitter release increases rapidly and then returns to low levels with a biphasic decay. The two kinetically distinct components are differentially affected when Sr2+ is substituted for Ca2+ ions. Our rmdings are comparable to those of the classical studies for the frog neuromuscular junction, and thus the basic aspects of Ca2+-activated transmitter release machinery appear to be conserved in central synapses. The method we have used, in addition, permits us to estimate the average neurotransmitter release rate for a single bouton. The observation of differential Ca2+/Sr2+ sensitivity is consistent with a release mechanism mediated by two Ca2+ sensors with distinct Ca2+ affinities: the low-affinity Ca2+ sensor facilitates the fast synchronous phase of release, whereas the high-affinity sensor sustains the slow asynchronous phase of release.Classical electrophysiological studies of synaptic transmission, most notably at the frog neuromuscular junction, have demonstrated the possibility of multiple mechanisms of Ca2+-dependent transmitter release based on distinct phases of exocytotic rates and pharmacology: the rate of transmitter release after the initial rapid rise appeared to decay biphasically (1), and the two components of release exhibited differential sensitivity toward the external concentrations of Ca2+, Mg2+, and Sr2+ (2, 3). In another study, the increase in the size of evoked transmitter release elicited by a repetitive stimulation decayed with four kinetically distinct components that again showed differential sensitivity toward various divalent cations (4).Although fundamental aspects of synaptic transmission in neuromuscular junctions are thought to be conserved in central synapses, some important differences between peripheral and central transmission have been recognized in recent studies (5). We have therefore initiated a characterization of the stimulus-evoked transmitter release in a central synapse, using cultured hippocampal neurons as a model system. MATERIALS AND METHODSCell Culture. Dissociated hippocampal neurons were prepared from embryonic day 15-17 C57BL/6 mice (HarlanSprague-Dawley) and cultured as described (6-8). Cell cultures were used for recording 8-13 days after plating.Electrophysiology. Dual whole-cell patch clamp recordings from paired cells were carried out as described (8). Concentrations of external CaCl2, SrCl2, and MgCl2 were varied as indicated. Microperfusion experiments were done as outlined (8), and boutons were visualized by synapsin I immunofluorescence (9). RESULTSTo examine transmitter release in central neurons, we monitored synaptic transmission between pairs of hippocampal pyramidal neurons in primary culture. Synaptic responses measured from a pair of neurons were averaged across stimulation trials and then time averaged over 10-ms bins to create histograms that estimate the quantal release rate as function of time (see Appendix). Suc...

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

“…In addition, cleavage of Syb2 by TeNT was shown to modify the coupling of intracellular calcium and releasecompetent vesicles (32), suggesting that removal of TeNT-sensitive v-SNAREs allows for the expression of a secretory machinery that may be hard to observe otherwise. Interestingly, Sr ϩ preferentially stimulates asynchronous release (33), and different synaptotagmin isoforms show different sensitivities to Ca 2ϩ and Sr ϩ (34). For instance, Synaptotagmin 1 and 7 have different sensitivities to calcium (35), the latter interacting with TI-VAMP in fibroblasts (36) and showing biochemical properties suitable for a Ca 2ϩ sensor for asynchronous release (35,37,38).…”

Characterization and reversal of synaptic defects in the amygdala in a mouse model of fragile X syndrome

“…LA cells held were at −70 mV and thalamic afferents were stimulated in the presence of 100 μM picrotoxin and 0.5 μM TTX. 2 mM Ca 2+ in the aCSF was replaced with 2 mM Sr 2+ and the initial synchronous EPSC decreased in size in parallel with an appearance of SrEPSCs (38,39). EPSC amplitude stabilized in ≈15 min, and stimulation frequency was 2 Hz for 10 traces, repeated every 20 s (25).…”

Differential dependence of phasic transmitter release on synaptotagmin 1 at GABAergic and glutamatergic hippocampal synapses

“…Synaptotagmins are differentially expressed in central neurons (16,19,20) and differ in kinetic properties (21). Thus, it is possible that differential expression of synaptotagmins determines the time course of transmitter release and the proportion of synchronous and asynchronous release at different synapses (6,(22)(23)(24). However, this hypothesis has not been directly tested.…”