Highlights d Number of presynaptic calcium channels (Ca V ) does not correlate with synaptic strength d Weak synapses are more sensitive to competition with exogenous Ca 2+ chelators d EM immunogold labeling of Ca V 2.1 and Munc13-1 shows synapse-specific nanotopographies d Different nanoscale Ca V -synaptic vesicle arrangements explain functional differences

SummaryThe strength and variability of electrical synaptic connections between GABAergic interneurons are key determinants of spike synchrony within neuronal networks. However, little is known about how electrical coupling strength is determined due to the inaccessibility of gap junctions on the dendritic tree. We investigated the properties of gap junctions in cerebellar interneurons by combining paired somato-somatic and somato-dendritic recordings, anatomical reconstructions, immunohistochemistry, electron microscopy, and modeling. By fitting detailed compartmental models of Golgi cells to their somato-dendritic voltage responses, we determined their passive electrical properties and the mean gap junction conductance (0.9 nS). Connexin36 immunofluorescence and freeze-fracture replica immunogold labeling revealed a large variability in gap junction size and that only 18% of the 340 channels are open in each plaque. Our results establish that the number of gap junctions per connection is the main determinant of both the strength and variability in electrical coupling between Golgi cells.

The axon initial segment of hippocampal pyramidal cells is a key subcellular compartment for action potential generation, under GABAergic control by the ''chandelier'' or axo-axonic cells (AACs). Although AACs are the only cellular source of GABA targeting the initial segment, their in vivo activity patterns and influence over pyramidal cell dynamics are not well understood. We achieved cell-type-specific genetic access to AACs in mice and show that AACs in the hippocampal area CA1 are synchronously activated by episodes of locomotion or whisking during rest. Bidirectional intervention experiments in head-restrained mice performing a random foraging task revealed that AACs inhibit CA1 pyramidal cells, indicating that the effect of GABA on the initial segments in the hippocampus is inhibitory in vivo. Finally, optogenetic inhibition of AACs at specific track locations induced remapping of pyramidal cell place fields. These results demonstrate brain-state-specific dynamics of a critical inhibitory controller of cortical circuits.

Dendritic calcium signaling is central to neural plasticity mechanisms that allow animals to adapt to the environment. Intracellular calcium release (ICR) from the endoplasmic reticulum has long been thought to shape these mechanisms. However, ICR has not been investigated in mammalian neurons in vivo. We combined electroporation of single CA1 pyramidal neurons, simultaneous imaging of dendritic and somatic activity during spatial navigation, optogenetic place field induction, and acute genetic augmentation of ICR cytosolic impact to reveal that ICR supports the establishment of dendritic feature selectivity and shapes integrative properties determining output-level receptive fields. This role for ICR was more prominent in apical than in basal dendrites. Thus, ICR cooperates with circuit-level architecture in vivo to promote the emergence of behaviorally relevant plasticity in a compartment-specific manner.

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Key points The amplitude of unitary, single action potential‐evoked [Ca2+] transients negatively correlates with GCaMP6f expression, but displays large variability among hippocampal pyramidal cells with similarly low expression levels. The summation of fluorescence signals is frequency‐dependent, supralinear and also shows remarkable cell‐to‐cell variability. The main source of spike inference error is variability in the peak amplitude, and not in the decay or supralinearity. We developed two procedures to estimate the peak amplitudes of unitary [Ca2+] transients and show that spike inference performed with MLspike using these unitary amplitude estimates in weakly GCaMP6f‐expressing cells results in error rates of ∼5%. Abstract 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%.

SUMMARYThe nanoscale topographical arrangement of voltage-gated calcium channels (VGCC) and synaptic vesicles (SVs) determines synaptic strength and plasticity, but whether distinct spatial distributions underpin diversity of synaptic function is unknown. We performed single bouton Ca2+ imaging, Ca2+ chelator competition, immunogold electron microscopic (EM) localization of VGCCs and the active zone (AZ) protein Munc13-1, at two cerebellar synapses. Unexpectedly, we found that weak synapses exhibited 3-fold more VGCCs than strong synapses, while the coupling distance was 5-fold longer. Reaction-diffusion modelling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (∼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (∼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus VGCC-SV arrangements are not canonical across CNS synapses and their diversity could underlie functional heterogeneity.

Highlights d The axon guidance receptors Robo1/2 can function as synaptogenic cues d Postsynaptic Robo forms a trans-synaptic complex with Slit and presynaptic Neurexin d Robo2 is required for formation of a subset of excitatory CA3 to CA1 synapses d Sparse deletion of Robo2 in CA1 pyramidal neurons impairs emergence of place cells