Synaptic vesicle pool size, release probability and synaptic depression are sensitive to Ca2+ buffering capacity in the developing rat calyx of Held

The calyx of Held, a specialized synaptic terminal in the medial nucleus of the trapezoid body, undergoes a series of changes during postnatal development that prepares this synapse for reliable high frequency firing. These changes reduce short-term synaptic depression during tetanic stimulation and thereby prevent action potential failures during a stimulus train. We measured presynaptic membrane capacitance changes in calyces from young postnatal day 5-7 (p5-7) or older (p10-12) rat pups to examine the effect of calcium buffer capacity on vesicle pool size and the efficiency of exocytosis. Vesicle pool size was sensitive to the choice and concentration of exogenous Ca2+ buffer, and this sensitivity was much stronger in younger animals. Pool size and exocytosis efficiency in p5-7 calyces were depressed by 0.2 mM EGTA to a greater extent than with 0.05 mM BAPTA, even though BAPTA is a 100-fold faster Ca2+ buffer. However, this was not the case for p10-12 calyces. With 5 mM EGTA, exocytosis efficiency was reduced to a much larger extent in young calyces compared to older calyces. Depression of exocytosis using pairs of 10-ms depolarizations was reduced by 0.2 mM EGTA compared to 0.05 mM BAPTA to a similar extent in both age groups. These results indicate a developmentally regulated heterogeneity in the sensitivity of different vesicle pools to Ca2+ buffer capacity. We propose that, during development, a population of vesicles that are tightly coupled to Ca2+ channels expands at the expense of vesicles more distant from Ca2+ channels.

Presynaptic Mechanism Underlying Regulation of Transmitter Release by G Protein Coupled Receptors

A variety of G protein coupled receptors (GPCRs) are expressed in the presynaptic terminals of central and peripheral synapses and play regulatory roles in transmitter release. The patch-clamp whole-cell recording technique, applied to the calyx of Held presynaptic terminal in brainstem slices of rodents, has made it possible to directly examine intracellular mechanisms underlying the GPCR-mediated presynaptic inhibition. At the calyx of Held, bath-application of agonists for GPCRs such as GABAB receptors, group III metabotropic glutamate receptors (mGluRs), adenosine A1 receptors, or adrenaline alpha2 receptors, attenuate evoked transmitter release via inhibiting voltage-activated Ca2+ currents without affecting voltage-activated K+ currents or inwardly rectifying K+ currents. Furthermore, inhibition of voltage-activated Ca2+ currents fully explains the magnitude of GPCR-mediated presynaptic inhibition, indicating no essential involvement of exocytotic mechanisms in the downstream of Ca2+ influx. Direct loadings of G protein beta gamma subunit (G beta gamma) into the calyceal terminal mimic and occlude the inhibitory effect of a GPCR agonist on presynaptic Ca2+ currents (IpCa), suggesting that G beta gammamediates presynaptic inhibition by GPCRs. Among presynaptic GPCRs glutamate and adenosine autoreceptors play regulatory roles in transmitter release during early postnatal period when the release probability (p) is high, but these functions are lost concomitantly with a decrease in p during postnatal development.