Target cell type-dependent differences in Ca2+ channel function underlie distinct release probabilities at hippocampal glutamatergic terminals

Target cell type-dependent differences in presynaptic release probability (Pr) and short-term plasticity are intriguing features of cortical microcircuits that increase the computational power of neuronal networks. Here we tested the hypothesis that different voltage-gated Ca2+ channel densities in presynaptic active zones (AZs) underlie different Pr Two-photon Ca2+ imaging, triple immunofluorescent labeling and three-dimensional electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed approximately 1.7 - 1.9-times higher Ca2+inflow per AZ area in high Pr boutons synapsing onto parvalbumin positive interneurons than in low Pr boutons synapsing onto mGluR1alpha positive interneurons. EM replica immunogold labeling, however, demonstrated only 1.15-times larger Cav2.1 and Cav2.2 subunit densities in high Pr AZs. Our results indicate target cell type-specific modulation of voltage-gated Ca2+ channel function or different subunit composition as possible mechanisms underlying the functional differences. In addition, high Pr synapses are also characterized by a higher density of docked vesicles, suggesting that a concerted action of these mechanisms underlies the functional differences. SIGNIFICANCE STATEMENT: Target cell type-dependent variability in presynaptic properties is an intriguing feature of cortical synapses. When a single cortical pyramidal cell establishes a synapse onto a somatostatin-expressing interneuron (IN), the synapse releases glutamate with low probability, whereas the next bouton of the same axon has high release probability when its postsynaptic target is a parvalbumin-expressing IN. Here we used combined molecular, imaging and anatomical approaches to investigate the mechanisms underlying these differences. Our functional experiments implied a approximately 2-fold larger Ca2+ channel density in high release probability boutons whereas freeze-fracture immunolocalization demonstrated only a 15% difference in Ca2+ channel subunit densities. Our results point toward a postsynaptic target cell type-dependent regulation of Ca2+ channel function or different subunit composition as the underlying mechanism

Improved spike inference accuracy by estimating the peak amplitude of unitary [Ca 2+ ] transients in weakly GCaMP6f expressing hippocampal pyramidal cells

Investigating neuronal activity using genetically encoded Ca2+ indicators in behaving animals is hampered by inaccuracies in spike inference from fluorescent tracers. Here we combine two‐photon [Ca2+] imaging with cell‐attached recordings, followed by post hoc determination of the expression level of GCaMP6f, to explore how it affects the amplitude, kinetics and temporal summation of somatic [Ca2+] transients in mouse hippocampal pyramidal cells (PCs). The amplitude of unitary [Ca2+] transients (evoked by a single action potential) negatively correlates with GCaMP6f expression, but displays large variability even among PCs with similarly low expression levels. The summation of fluorescence signals is frequency‐dependent, supralinear and also shows remarkable cell‐to‐cell variability. We performed experimental data‐based simulations and found that spike inference error rates using MLspike depend strongly on unitary peak amplitudes and GCaMP6f expression levels. We provide simple methods for estimating the unitary [Ca2+] transients in individual weakly GCaMP6f‐expressing PCs, with which we achieve spike inference error rates of ∼5%