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

Target cell type-dependent differences in presynaptic release probability (P r ) 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 Ca 2ϩ channel densities in presynaptic active zones (AZs) underlie different P r values. Two-photon Ca 2ϩ imaging, triple immunofluorescent labeling, and 3D electron microscopic (EM) reconstruction of rat CA3 pyramidal cell axon terminals revealed ϳ1.7-1.9 times higher Ca 2ϩ inflow per AZ area in high P r boutons synapsing onto parvalbumin-positive interneurons (INs) than in low P r boutons synapsing onto mGluR1␣-positive INs. EM replica immunogold labeling, however, demonstrated only 1.15 times larger Cav2.1 and Cav2.2 subunit densities in high P r AZs. Our results indicate target cell type-specific modulation of voltage-gated Ca 2ϩ channel function or different subunit composition as possible mechanisms underlying the functional differences. In addition, high P r synapses are also characterized by a higher density of docked vesicles, suggesting that a concerted action of these mechanisms underlies the functional differences.

Potassium channels comprise the most diverse family of ion channels and play critical roles in a large variety of physiological and pathological processes. In addition to their molecular diversity, variations in their distributions and densities on the axo-somato-dendritic surface of neurons are key parameters in determining their functional impact. Despite extensive electrophysiological and anatomical investigations, the exact location and densities of most K+ channels in small subcellular compartments are still unknown. Here we aimed at providing a quantitative surface map of two delayed-rectifier (Kv1.1 and Kv2.1) and one G-protein-gated inwardly rectifying (Kir3.2) K+ channel subunits on hippocampal CA1 pyramidal cells (PCs). Freeze-fracture replica immunogold labelling was employed to determine the relative densities of these K+ channel subunits in 18 axo-somato-dendritic compartments. Significant densities of the Kv1.1 subunit were detected on axon initial segments (AISs) and axon terminals, with an approximately eight-fold lower density in the latter compartment. The Kv2.1 subunit was found in somatic, proximal dendritic and AIS plasma membranes at approximately the same densities. This subunit has a non-uniform plasma membrane distribution; Kv2.1 clusters are frequently adjacent to, but never overlap with, GABAergic synapses. A quasi-linear increase in the Kir3.2 subunit density along the dendrites of PCs was detected, showing no significant difference between apical dendritic shafts, oblique dendrites or dendritic spines at the same distance from the soma. Our results demonstrate that each subunit has a unique cell-surface distribution pattern, and predict their differential involvement in synaptic integration and output generation at distinct subcellular compartments.

The release of GABA from cholecystokinin-containing interneurons is modulated by type-1 cannabinoid receptors (CB1). Here we tested the hypothesis that the strength of CB1-mediated modulation of GABA release is related to the CB1 content of axon terminals. Basket cell boutons have on average 78% higher CB1 content than those of dendritic-layer-innervating (DLI) cells, a consequence of larger bouton surface and higher CB1 density. The CB1 antagonist AM251 caused a 54% increase in action potential-evoked [Ca2+] in boutons of basket, but not in DLI cells. However, the effect of AM251 did not correlate with CB1 immunoreactivity of individual boutons. Moreover, a CB1 agonist decreased [Ca2+] in a cell type- and CB1 content-independent manner. Replica immunogold labeling demonstrated the colocalization of CB1 with the Cav2.2 Ca2+ channel subunit. Our data suggest that only a subpopulation of CB1s, within nanometer distances from their target Cav2.2 channels, are responsible for endocannabinoid-mediated modulation of GABA release.

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.

Nanoscale distribution of molecules within small subcellular compartments of neurons critically influences their functional roles. Although, numerous ways of analyzing the spatial arrangement of proteins have been described, a thorough comparison of their effectiveness is missing. Here we present an open source software, GoldExt, with a plethora of measures for quantification of the nanoscale distribution of proteins in subcellular compartments (e.g. synapses) of nerve cells. First, we compared the ability of five different measures to distinguish artificial uniform and clustered patterns from random point patterns. Then, the performance of a set of clustering algorithms was evaluated on simulated datasets with predefined number of clusters. Finally, we applied the best performing methods to experimental data, and analyzed the nanoscale distribution of different pre- and postsynaptic proteins, revealing random, uniform and clustered sub-synaptic distribution patterns. Our results reveal that application of a single measure is sufficient to distinguish between different distributions.

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We have recently identified that Figure 8F of our article included an incorrect dataset (Munc-Munc cluster distance, green), a mislabeled axis, and an incorrect depiction of how the dataset in the panel was analyzed. In order to minimize errors due to incomplete labeling efficiency, we intended to show the minimum NND between Munc-Cav2.1 and Munc-Munc clusters per bouton. The corrected panel (Figure 8F) now shows that the minimum NND between the edge of Munc13-1 clusters and Cav2.1 gold particles is still substantially smaller (mean ± SEM.: 27 ± 3 nm, median: 20 nm, n = 36) than the minimum NNDs between the edges of Munc13-1 clusters (mean ± SEM.: 81 ± 11 nm, median: 61 nm, n = 35; p < 0.001, Mann-Whitney test) in stellate cell boutons. This corrected panel supports the original conclusions of the study that the Cav-SV topography in stellate cell boutons can be explained by a perimeter-coupled model. We would like to reiterate that the minimum NND should not be considered as a direct estimate of coupling distance. Finally, we deeply regret this error and apologize for any inconvenience. F Figure 8. Nanotopographical Arrangement of VGCC and Munc13-1 Gold Particles in SC Boutons Is Compatible with PRM (F) Cumulative distributions of the minimum NND (per bouton) from the edge of Munc13-1 clusters to the nearest Cav2.1 gold particle (blue) and between Munc13-1 cluster edges (n = 36 boutons, p < 0.001, Mann-Whitney test).