Studying Synaptically Evoked Cortical Responses ex vivo With Combination of a Single Neuron Recording (Whole-Cell) and Population Voltage Imaging (Genetically Encoded Voltage Indicator)

In a typical electrophysiology experiment, synaptic stimulus is delivered in a cortical layer (1–6) and neuronal responses are recorded intracellularly in individual neurons. We recreated this standard electrophysiological paradigm in brain slices of mice expressing genetically encoded voltage indicators (GEVIs). This allowed us to monitor membrane voltages in the target pyramidal neurons (whole-cell), and population voltages in the surrounding neuropil (optical imaging), simultaneously. Pyramidal neurons have complex dendritic trees that span multiple cortical layers. GEVI imaging revealed areas of the brain slice that experienced the strongest depolarization on a specific synaptic stimulus (location and intensity), thus identifying cortical layers that contribute the most afferent activity to the recorded somatic voltage waveform. By combining whole-cell with GEVI imaging, we obtained a crude distribution of activated synaptic afferents in respect to the dendritic tree of a pyramidal cell. Synaptically evoked voltage waves propagating through the cortical neuropil (dendrites and axons) were not static but rather they changed on a millisecond scale. Voltage imaging can identify areas of brain slices in which the neuropil was in a sustained depolarization (plateau), long after the stimulus onset. Upon a barrage of synaptic inputs, a cortical pyramidal neuron experiences: (a) weak temporal summation of evoked voltage transients (EPSPs); and (b) afterhyperpolarization (intracellular recording), which are not represented in the GEVI population imaging signal (optical signal). To explain these findings [(a) and (b)], we used four voltage indicators (ArcLightD, chi-VSFP, Archon1, and di-4-ANEPPS) with different optical sensitivity, optical response speed, labeling strategy, and a target neuron type. All four imaging methods were used in an identical experimental paradigm: layer 1 (L1) synaptic stimulation, to allow direct comparisons. The population voltage signal showed paired-pulse facilitation, caused in part by additional recruitment of new neurons and dendrites. “Synaptic stimulation” delivered in L1 depolarizes almost an entire cortical column to some degree.

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Astrocytic modulation of information processing by layer 5 pyramidal neurons of the mouse visual cortex

10.1101/2020.07.09.190777 ◽

2020 ◽

Cited By ~ 2

Author(s):

Dimitri Ryczko ◽

Maroua Hanini-Daoud ◽

Steven Condamine ◽

Benjamin J. B. Bréant ◽

Maxime Fougère ◽

...

Keyword(s):

Visual Cortex ◽

Cortical Neurons ◽

Calcium Binding ◽

Pyramidal Neurons ◽

Binding Protein ◽

Contextual Information ◽

Dendritic Tree ◽

Cortical Layer ◽

List Type ◽

Ionic Environment

AbstractThe most complex cerebral functions are performed by the cortex which most important output is carried out by its layer 5 pyramidal neurons. Their firing reflects integration of sensory and contextual information that they receive. There is evidence that astrocytes influence cortical neurons firing through the release of gliotransmitters such as ATP, glutamate or GABA. These effects were described at the network and at the synaptic levels, but it is still unclear how astrocytes influence neurons input-output transfer function at the cellular level. Here, we used optogenetic tools coupled with electrophysiological, imaging and anatomical approaches to test whether and how astrocytic activation affected processing and integration of distal inputs to layer 5 pyramidal neurons (L5PN). We show that optogenetic activation of astrocytes near L5PN cell body prolonged firing induced by distal inputs to L5PN and potentiated their ability to trigger spikes. The observed astrocytic effects on L5PN firing involved glutamatergic transmission to some extent but relied on release of S100β, an astrocytic Ca2+-binding protein that decreases extracellular Ca2+ once released. This astrocyte-evoked decrease of extracellular Ca2+ elicited firing mediated by activation of Nav1.6 channels. Our findings suggest that astrocytes contribute to the cortical fundamental computational operations by controlling the extracellular ionic environment.Key Points SummaryIntegration of inputs along the dendritic tree of layer 5 pyramidal neurons is an essential operation as these cells represent the most important output carrier of the cerebral cortex. However, the contribution of astrocytes, a type of glial cell to these operations is poorly documented.Here we found that optogenetic activation of astrocytes in the vicinity of layer 5 in the mouse primary visual cortex induce spiking in local pyramidal neurons through Nav1.6 ion channels and prolongs the responses elicited in these neurons by stimulation of their distal inputs in cortical layer 1.This effect partially involved glutamatergic signalling but relied mostly on the astrocytic calcium-binding protein S100β, which regulates the concentration of calcium in the extracellular space around neurons.These findings show that astrocytes contribute to the fundamental computational operations of the cortex by acting on the ionic environment of neurons.

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Cell type-specific effects of isoflurane on two distinct afferent inputs to cortical layer 1

10.1101/2020.05.18.102913 ◽

2020 ◽

Author(s):

Caitlin A. Murphy ◽

Matthew I. Banks

Keyword(s):

Ex Vivo ◽

Brain Slices ◽

Pyramidal Cells ◽

Cellular Level ◽

Cortical Layer ◽

Cell Type ◽

Specific Effects ◽

Primary Mouse ◽

Cell Type Specific ◽

Synaptic Responses

ABSTRACTBackgroundWhile their behavioral effects are well-characterized, the mechanisms by which anaesthetics induce loss of consciousness are largely unknown. Anaesthetics may disrupt integration and propagation of information in corticothalamic networks. Recent studies have shown that isoflurane diminishes synaptic responses of thalamocortical (TC) and corticocortical (CC) afferents in a pathway-specific manner. However, whether the synaptic effects of isoflurane observed in extracellular recordings persist at the cellular level has yet to be explored.MethodsHere, we activate TC and CC layer 1 inputs in non-primary mouse neocortex in ex vivo brain slices and explore the degree to which isoflurane modulates synaptic responses in pyramidal cells and in two inhibitory cell populations, somatostatin-positive (SOM+) and parvalbumin-positive (PV+) interneurons.ResultsWe show that the effects of isoflurane on synaptic responses and intrinsic properties of these cells varies among cell type and by cortical layer. Layer 1 inputs to L4 pyramidal cells were suppressed by isoflurane at both TC and CC synapses, while those to L2/3 pyramidal cells and PV+ interneurons were not. TC inputs to SOM+ cells were rarely observed at all, while CC inputs to SOM+ interneurons were robustly suppressed by isoflurane.ConclusionsThese results suggest a mechanism by which isoflurane disrupts integration and propagation of thalamocortical and intracortical signals.

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The antihelminthic moxidectin enhances tonic GABA currents in rodent hippocampal pyramidal neurons

Journal of Neurophysiology ◽

10.1152/jn.00587.2017 ◽

2018 ◽

Vol 119 (5) ◽

pp. 1693-1698

Author(s):

Jay Spampanato ◽

Anne Gibson ◽

F. Edward Dudek

Keyword(s):

Central Nervous System ◽

Nervous System ◽

Gaba Receptors ◽

Pyramidal Neurons ◽

Ex Vivo ◽

Companion Animals ◽

Brain Slices ◽

Dependent Manner ◽

Hippocampal Pyramidal Neurons ◽

Mammalian Central Nervous System

Macrocyclic lactones (MLs) are commonly used treatments for parasitic worm and insect infections in humans, livestock, and companion animals. MLs target the invertebrate glutamate-activated chloride channel that is not present in vertebrates. MLs are not entirely inert in vertebrates, though; they have been reported to have activity in heterologous expression systems consisting of ligand-gated ion channels that are present in the mammalian central nervous system (CNS). However, these compounds are typically not able to reach significant concentrations in the CNS because of the activity of the blood-brain barrier P-glycoprotein extrusion system. Despite this, these compounds are able to reach low levels in the CNS that may be useful in the design of novel “designer” ligand-receptor systems that can be used to directly investigate neuronal control of behavior in mammals and have potential for use in treating human neurological diseases. To determine whether MLs might affect neurons in intact brains, we investigated the activity of the ML moxidectin (MOX) at native GABA receptors. Specifically, we recorded tonic and phasic miniature inhibitory postsynaptic currents (mIPSCs) in ex vivo brain slices. Our data show that MOX potentiated tonic GABA currents in a dose-dependent manner but had no concomitant effects on phasic GABA currents (i.e., MOX had no effect on the amplitude, frequency, or decay kinetics of mIPSCs). These studies indicate that behavioral experiments that implement a ML-based novel ligand-receptor system should take care to control for potential effects of the ML on native tonic GABA receptors.NEW & NOTEWORTHY We have identified a novel mechanism of action in the mammalian central nervous system for the antihelminthic moxidectin, commonly prescribed to animals worldwide and currently being evaluated for use in humans. Specifically, moxidectin applied to rodent brain slices selectively enhanced the tonic GABA conductance of hippocampal pyramidal neurons.

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Vasopressin acts as a synapse organizer in limbic regions by boosting PSD95 and GluA1 expression

10.1101/2020.11.07.373027 ◽

2020 ◽

Author(s):

Limei Zhang ◽

Teresa Padilla-Flores ◽

Vito S. Hernández ◽

Elba Campos-Lira ◽

Mario A. Zetter ◽

...

Keyword(s):

Neuronal Excitability ◽

Pyramidal Neurons ◽

Ex Vivo ◽

Hippocampal Slices ◽

Brain Slices ◽

Synaptic Proteins ◽

Scaffolding Protein ◽

Synaptic Organization ◽

Limbic Regions

AbstractHypothalamic arginine vasopressin (AVP)-containing magnocellular neurosecretory neurons (AVPMNN) emit collaterals to synaptically innervate limbic regions influencing learning, motivational behavior and fear responses. The purpose of the present work is to characterize the dynamics of expression changes of two postsynaptic density (PSD) proteins, AMPAR subunit GluA1 and PSD scaffolding protein 95 (PSD95), which are known to be key determinants for synaptic strength, in response to in vivo and ex vivo manipulations of AVPMNN neuronal activation state, or exposure to exogenous AVP, metabolites and some signaling pathway inhibitors. Both long term water deprivation in vivo, which powerfully upregulates AVPMNN activity, and exogenous APV application ex vivo in brain slices, increased GluA1 and PSD95 expression and enhanced neuronal excitability in ventral hippocampal CA1 pyramidal neurons. Involvement of PI3k signaling in AVP-dependent plasticity is suggested by blockade of both AVP-induced protein up-regulation and enhanced neuronal excitability by the PI3k blocker wortmannin in hippocampal slices. We interpret these results as part of vasopressin’s central effects on synaptic organization in limbic regions modulating the strength of a specific set of synaptic proteins in hypothalamic-limbic circuits.Supported by grantsUNAM-DGAPA-PAPIIT-IN216918 & CONACYT-CB-238744.

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Optimizing computer models of corticospinal neurons to replicate in vitro dynamics

Journal of Neurophysiology ◽

10.1152/jn.00570.2016 ◽

2017 ◽

Vol 117 (1) ◽

pp. 148-162 ◽

Cited By ~ 13

Author(s):

Samuel A. Neymotin ◽

Benjamin A. Suter ◽

Salvador Dura-Bernal ◽

Gordon M. G. Shepherd ◽

Michele Migliore ◽

...

Keyword(s):

Motor Cortex ◽

Pyramidal Neurons ◽

Computer Models ◽

Cortical Layer ◽

Cortical Layers ◽

Electrophysiological Properties ◽

Full Reconstruction ◽

Resonance Properties ◽

Oscillation Frequencies

Corticospinal neurons (SPI), thick-tufted pyramidal neurons in motor cortex layer 5B that project caudally via the medullary pyramids, display distinct class-specific electrophysiological properties in vitro: strong sag with hyperpolarization, lack of adaptation, and a nearly linear frequency-current ( F– I) relationship. We used our electrophysiological data to produce a pair of large archives of SPI neuron computer models in two model classes: 1) detailed models with full reconstruction; and 2) simplified models with six compartments. We used a PRAXIS and an evolutionary multiobjective optimization (EMO) in sequence to determine ion channel conductances. EMO selected good models from each of the two model classes to form the two model archives. Archived models showed tradeoffs across fitness functions. For example, parameters that produced excellent F– I fit produced a less-optimal fit for interspike voltage trajectory. Because of these tradeoffs, there was no single best model but rather models that would be best for particular usages for either single neuron or network explorations. Further exploration of exemplar models with strong F– I fit demonstrated that both the detailed and simple models produced excellent matches to the experimental data. Although dendritic ion identities and densities cannot yet be fully determined experimentally, we explored the consequences of a demonstrated proximal to distal density gradient of Ih, demonstrating that this would lead to a gradient of resonance properties with increased resonant frequencies more distally. We suggest that this dynamical feature could serve to make the cell particularly responsive to major frequency bands that differ by cortical layer. NEW & NOTEWORTHY We developed models of motor cortex corticospinal neurons that replicate in vitro dynamics, including hyperpolarization-induced sag and realistic firing patterns. Models demonstrated resonance in response to synaptic stimulation, with resonance frequency increasing in apical dendrites with increasing distance from soma, matching the increasing oscillation frequencies spanning deep to superficial cortical layers. This gradient may enable specific corticospinal neuron dendrites to entrain to relevant oscillations in different cortical layers, contributing to appropriate motor output commands.

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Analyzing Branch‐specific Dendritic Spikes Using an Ultrafast Laser Scalpel

Frontiers in Physics ◽

10.3389/fphy.2020.600971 ◽

2020 ◽

Vol 8 ◽

Author(s):

Michael L. Castañares ◽

Hans-A. Bachor ◽

Vincent R. Daria

Keyword(s):

Cortical Neurons ◽

Pyramidal Neurons ◽

Dendritic Tree ◽

Cortical Layer ◽

Ultrafast Laser ◽

Laser Scalpel ◽

Dendritic Spikes ◽

Dendritic Spike ◽

Neuronal Computation

Dendritic spikes facilitate neuronal computation and they have been reported to occur in various regions of the dendritic tree of cortical neurons. Spikes that occur only on a select few branches are particularly difficult to analyze especially in complex and intertwined dendritic arborizations where highly localized application of pharmacological blocking agents is not feasible. Here, we present a technique based on highly targeted dendrotomy to tease out and study dendritic spikes that occur in oblique branches of cortical layer five pyramidal neurons. We first analyze the effect of cutting dendrites in silico and then confirmed in vitro using an ultrafast laser scalpel. A dendritic spike evoked in an oblique branch manifests at the soma as an increase in the afterdepolarization (ADP). The spikes are branch-specific since not all but only a few oblique dendrites are observed to evoke spikes. Both our model and experiments show that cutting certain oblique branches, where dendritic spikes are evoked, curtailed the increase in the ADP. On the other hand, cutting neighboring oblique branches that do not evoke spikes maintained the ADP. Our results show that highly targeted dendrotomy can facilitate causal analysis of how branch-specific dendritic spikes influence neuronal output.

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Impaired state-dependent potentiation of GABAergic synaptic currents triggers seizures in an idiopathic generalized epilepsy model

10.1101/2020.05.10.087114 ◽

2020 ◽

Author(s):

Chun-Qing Zhang ◽

Mackenzie A. Catron ◽

Li Ding ◽

Caitlyn M. Hanna ◽

Martin J. Gallagher ◽

...

Keyword(s):

Slow Wave ◽

Cortical Neurons ◽

Pyramidal Neurons ◽

Slow Wave Sleep ◽

Ex Vivo ◽

Brain Slices ◽

Idiopathic Generalized Epilepsy ◽

Synaptic Currents ◽

Generalized Epilepsy

AbstractIdiopathic generalized epilepsy(IGE) patients have genetic causes and their seizure onset mechanisms particularly during sleep remain elusive. Here we proposed that sleep-like slow-wave oscillations(0.5 Hz SWOs) potentiated excitatory or inhibitory synaptic currents in layer V cortical pyramidal neurons from wild-type(wt) mouse ex vivo brain slices. In contrast, SWOs potentiated excitatory, not inhibitory, currents in cortical neurons from heterozygous(het) knock-in(KI) IGE mice(GABAA receptor γ2 subunit Gabrg2Q390X mutation), creating an imbalance between evoked excitatory and inhibitory currents to effectively prompt neuronal action potentials. Similarly, more physiologically similar up/down-state(present during slow-wave sleep) induction in cortical neurons could potentiate excitatory synaptic currents within slices from wt/het Gabrg2Q390X KI mice. Consequently, SWOs or up/down-state induction in vivo (using optogenetic method) could trigger epileptic spike-wave discharges(SWDs) in het Gabrg2Q390X KI mice. To our knowledge, this is the first operative mechanism to explain why epileptic SWDs preferentially happen during non-REM sleep or quiet-wakefulness in human IGE patients.

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