RESUMEN
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 â¼5%. 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 â¼5%.
Asunto(s)
Calcio/fisiología , Hipocampo/fisiología , Proteínas Luminiscentes/fisiología , Células Piramidales/fisiología , Animales , Señalización del Calcio , Masculino , RatonesRESUMEN
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.
Asunto(s)
Región CA1 Hipocampal/fisiología , Calcio/metabolismo , Dendritas/fisiología , Retículo Endoplásmico/metabolismo , Plasticidad Neuronal , Células de Lugar/fisiología , Potenciales de Acción , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Señalización del Calcio , Citosol/metabolismo , Electroporación , Femenino , Masculino , Ratones , Optogenética , Análisis de la Célula Individual , Navegación EspacialRESUMEN
Hippocampal place cells underlie spatial navigation and memory. Remarkably, CA1 pyramidal neurons can form new place fields within a single trial by undergoing rapid plasticity. However, local feedback circuits likely restrict the rapid recruitment of individual neurons into ensemble representations. This interaction between circuit dynamics and rapid feature coding remains unexplored. Here, we developed "all-optical" approaches combining novel optogenetic induction of rapidly forming place fields with 2-photon activity imaging during spatial navigation in mice. We find that induction efficacy depends strongly on the density of co-activated neurons. Place fields can be reliably induced in single cells, but induction fails during co-activation of larger subpopulations due to local circuit constraints imposed by recurrent inhibition. Temporary relief of local inhibition permits the simultaneous induction of place fields in larger ensembles. We demonstrate the behavioral implications of these dynamics, showing that our ensemble place field induction protocol can enhance subsequent spatial association learning.
Asunto(s)
Hipocampo , Células de Lugar , Animales , Región CA1 Hipocampal/fisiología , Retroalimentación , Hipocampo/fisiología , Ratones , Neuronas/fisiología , Células Piramidales/fisiologíaRESUMEN
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.
Asunto(s)
Interneuronas , Ácido gamma-Aminobutírico , Animales , Axones/fisiología , Hipocampo/fisiología , Interneuronas/fisiología , Ratones , Células Piramidales/fisiología , Sinapsis/fisiología , Ácido gamma-Aminobutírico/fisiologíaRESUMEN
Synaptic connectivity within adult circuits exhibits a remarkable degree of cellular and subcellular specificity. We report that the axon guidance receptor Robo2 plays a role in establishing synaptic specificity in hippocampal CA1. In vivo, Robo2 is present and required postsynaptically in CA1 pyramidal neurons (PNs) for the formation of excitatory (E) but not inhibitory (I) synapses, specifically in proximal but not distal dendritic compartments. In vitro approaches show that the synaptogenic activity of Robo2 involves a trans-synaptic interaction with presynaptic Neurexins, as well as binding to its canonical extracellular ligand Slit. In vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in awake behaving mice shows that preventing Robo2-dependent excitatory synapse formation cell autonomously during development alters place cell properties of adult CA1 PNs. Our results identify a trans-synaptic complex linking the establishment of synaptic specificity to circuit function.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Células Piramidales/metabolismo , Receptores Inmunológicos/metabolismo , Sinapsis/metabolismo , Animales , Región CA1 Hipocampal/citología , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Potenciales Postsinápticos Excitadores , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Moléculas de Adhesión de Célula Nerviosa/genética , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Células de Lugar/metabolismo , Receptores Inmunológicos/genética , Proteínas RoundaboutRESUMEN
The 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 modeling could explain both functional and structural data with two strikingly different nanotopographical motifs: strong synapses are composed of SVs that are tightly coupled (â¼10 nm) to VGCC clusters, whereas at weak synapses VGCCs were excluded from the vicinity (â¼50 nm) of docked vesicles. The distinct VGCC-SV topographical motifs also confer differential sensitivity to neuromodulation. Thus, VGCC-SV arrangements are not canonical, and their diversity could underlie functional heterogeneity across CNS synapses.
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Canales de Calcio/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
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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Algoritmos , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Programas Informáticos , Sinapsis/metabolismo , Animales , HumanosRESUMEN
The 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.