Ilya A. Fleidervish

Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea-pig neocortical neurones in slices.

1. Spike adaptation of neocortical pyramidal neurones was studied with sharp electrode recordings in slices of guinea‐pig parietal cortex and whole‐cell patch recordings of mouse somatosensory cortex. Repetitive intracellular stimulation with 1 s depolarizing pulses delivered at intervals of < 5 s caused slow, cumulative adaptation of spike firing, which was not associated with a change in resting conductance, and which persisted when Co2+ replaced Ca2+ in the bathing medium. 2. Development of slow cumulative adaptation was associated with a gradual decrease in maximal rates of rise of action potentials, a slowing in the post‐spike depolarization towards threshold, and a positive shift in the threshold voltage for the next spike in the train; maximal spike repolarization rates and after‐hyperpolarizations were unchanged. 3. The data suggested that slow adaptation reflects use‐dependent removal of Na+ channels from the available pool by an inactivation process which is much slower than fast, Hodgkin‐Huxley‐type inactivation. 4. We therefore studied the properties of Na+ channels in layer II‐III mouse neocortical cells using the cell‐attached configuration of the patch‐in‐slice technique. These had a slope conductance of 18 +/‐ 1 pS and an extrapolated reversal potential of 127 +/‐ 6 mV above resting potential (Vr) (mean +/‐ S.E.M.; n = 5). Vr was estimated at ‐72 +/‐ 3 mV (n = 8), based on the voltage dependence of the steady‐state inactivation (h infinity) curve. 5. Slow inactivation (SI) of Na+ channels had a mono‐exponential onset with tau on between 0.86 and 2.33 s (n = 3). Steady‐state SI was half‐maximal at ‐43.8 mV and had a slope of 14.4 mV (e‐fold)‐1. Recovery from a 2 s conditioning pulse was bi‐exponential and voltage dependent; the slow time constant ranged between 0.45 and 2.5 s at voltages between‐128 and ‐68 mV. 6. The experimentally determined parameters of SI were adequate to simulate slow cumulative adaptation of spike firing in a single‐compartment computer model. 7. Persistent Na+ current, which was recorded in whole‐cell configuration during slow voltage ramps (35 mV s‐1), also underwent pronounced SI, which was apparent when the ramp was preceded by a prolonged depolarizing pulse.

Na+ imaging reveals little difference in action potential-evoked Na+ influx between axon and soma

In cortical pyramidal neurons, the axon initial segment (AIS) is pivotal in synaptic integration. It has been asserted that this is because there is a high density of Na+ channels in the AIS. However, we found that action potential–associated Na+ flux, as measured by high-speed fluorescence Na+ imaging, was about threefold larger in the rat AIS than in the soma. Spike-evoked Na+ flux in the AIS and the first node of Ranvier was similar and was eightfold lower in basal dendrites. At near-threshold voltages, persistent Na+ conductance was almost entirely axonal. On a time scale of seconds, passive diffusion, and not pumping, was responsible for maintaining transmembrane Na+ gradients in thin axons during high-frequency action potential firing. In computer simulations, these data were consistent with the known features of action potential generation in these neurons.

Functionally Distinct NMDA Receptors Mediate Horizontal Connectivity within Layer 4 of Mouse Barrel Cortex

In sensory areas of neocortex, thalamocortical afferents project primarily onto the spiny stellate neurons of Layer 4. Anatomical evidence indicates that these cells receive most of their excitatory input from other cortical neurons, including other spiny stellate cells. Although this local network must play an important role in sensory processing, little is known about the properties of the neurons and synapses involved. We have produced a slice preparation of mouse barrel cortex that isolates Layer 4. We report that excitatory interaction between spiny stellate neurons is largely via N-methyl-D-aspartate receptors (NMDARs) and that a given neuron contains more than one type of NMDAR, as distinguished by voltage dependence. Thus, spiny stellate cells act as effective integrators of powerful and persistent NMDAR-mediated recurrent excitation.

Spatial mismatch between the Na+ flux and spike initiation in axon initial segment

It is widely believed that, in cortical pyramidal cells, action potentials (APs) initiate in the distal portion of axon initial segment (AIS) because that is where Na+ channel density is highest. To investigate the relationship between the density of Na+ channels and the spatiotemporal pattern of AP initiation, we simultaneously recorded Na+ flux and action currents along the proximal axonal length. We found that functional Na+ channel density is approximately four times lower in the AP trigger zone than in the middle of the AIS, where it is highest. Computational analysis of AP initiation revealed a paradoxical mismatch between the AP threshold and Na+ channel density, which could be explained by the lopsided capacitive load imposed on the proximal end of the AIS by the somatodendritic compartment. Favorable conditions for AP initiation are therefore achieved in the distal AIS portion, close to the edge of myelin, where the current source–load ratio is highest. Our findings suggest that cable properties play a central role in determining where the AP starts, such that small plastic changes in the local AIS Na+ channel density could have a large influence on neuronal excitability as a whole.

Differential TGF-β Signaling in Glial Subsets Underlies IL-6–Mediated Epileptogenesis in Mice

TGF-β1 is a master cytokine in immune regulation, orchestrating both pro- and anti-inflammatory reactions. Recent studies show that whereas TGF-β1 induces a quiescent microglia phenotype, it plays a pathogenic role in the neurovascular unit and triggers neuronal hyperexcitability and epileptogenesis. In this study, we show that, in primary glial cultures, TGF-β signaling induces rapid upregulation of the cytokine IL-6 in astrocytes, but not in microglia, via enhanced expression, phosphorylation, and nuclear translocation of SMAD2/3. Electrophysiological recordings show that administration of IL-6 increases cortical excitability, culminating in epileptiform discharges in vitro and spontaneous seizures in C57BL/6 mice. Intracellular recordings from layer V pyramidal cells in neocortical slices obtained from IL-6–treated mice show that during epileptogenesis, the cells respond to repetitive orthodromic activation with prolonged after-depolarization with no apparent changes in intrinsic membrane properties. Notably, TGF-β1–induced IL-6 upregulation occurs in brains of FVB/N but not in brains of C57BL/6 mice. Overall, our data suggest that TGF-β signaling in the brain can cause astrocyte activation whereby IL-6 upregulation results in dysregulation of astrocyte–neuronal interactions and neuronal hyperexcitability. Whereas IL-6 is epileptogenic in C57BL/6 mice, its upregulation by TGF-β1 is more profound in FVB/N mice characterized as a relatively more susceptible strain to seizure-induced cell death.

Optogenetic control of mitochondrial metabolism and Ca2+ signaling by mitochondria-targeted opsins

Significance Mitochondrial functions depend on the steep H+ electrochemical gradient (ΔμH+) across their inner membrane. The available tools for controlling this gradient are essentially limited to inhibitors of the respiratory chain or of the H+ ATPase or to uncouplers, poisons plagued by important side effects and that lack both cell and spatial specificity. We show here that, by transfecting cells with the cDNA encoding channelrhodopsins specifically targeted to the inner mitochondrial membrane, we can obtain an accurate and spatially confined, light-dependent control of mitochondrial membrane potential and, as a consequence, of a series of mitochondrial activities ranging from electron transport to ATP synthesis and Ca2+ signaling. Key mitochondrial functions such as ATP production, Ca2+ uptake and release, and substrate accumulation depend on the proton electrochemical gradient (ΔμH+) across the inner membrane. Although several drugs can modulate ΔμH+, their effects are hardly reversible, and lack cellular specificity and spatial resolution. Although channelrhodopsins are widely used to modulate the plasma membrane potential of excitable cells, mitochondria have thus far eluded optogenetic control. Here we describe a toolkit of optometabolic constructs based on selective targeting of channelrhodopsins with distinct functional properties to the inner mitochondrial membrane of intact cells. We show that our strategy enables a light-dependent control of the mitochondrial membrane potential (Δψm) and coupled mitochondrial functions such as ATP synthesis by oxidative phosphorylation, Ca2+ dynamics, and respiratory metabolism. By directly modulating Δψm, the mitochondria-targeted opsins were used to control complex physiological processes such as spontaneous beats in cardiac myocytes and glucose-dependent ATP increase in pancreatic β-cells. Furthermore, our optometabolic tools allow modulation of mitochondrial functions in single cells and defined cell regions.

Functional implications of axon initial segment cytoskeletal disruption in stroke

Axon initial segment (AIS) is the proximal part of the axon, which is not covered with a myelin sheath and possesses a distinctive, specialized assembly of voltage-gated ion channels and associated proteins. AIS plays critical roles in synaptic integration and action potential generation in central neurons. Recent evidence shows that stroke causes rapid, irreversible calpain-mediated proteolysis of the AIS cytoskeleton of neurons surrounding the ischemic necrotic core. A better understanding of the molecular mechanisms underlying this “non-lethal” neuronal damage might provide new therapeutic strategies for improving stroke outcome. Here, we present a brief overview of the structure and function of the AIS. We then discuss possible mechanisms underlying stroke-induced AIS damage, including the roles of calpains and possible sources of Ca2+ ions, which are necessary for the activation of calpains. Finally, we discuss the potential functional implications of the loss of the AIS cytoskeleton and ion channel clusters for neuronal excitability.

The earliest neuronal responses to hypoxia in the neocortical circuit are glutamate-dependent

Soon after exposure to hypoxia or ischemia, neurons in cortical tissues undergo massive anoxic depolarization (AD). This precipitous event is preceded by more subtle neuronal changes, including enhanced excitatory and inhibitory synaptic transmitter release. Here, we have used patch-in-slice techniques to identify the earliest effects of acute hypoxia on the synaptic and intrinsic properties of Layer 5 neurons, to determine their time course and to evaluate the role of glutamate receptors in their generation. Coronal slices of mouse somatosensory cortex were maintained at 36°C in an interface chamber and challenged with episodes of hypoxia. In recordings with cell-attached electrodes, the open probability of Ca(2+)-dependent BK channels began to increase within seconds of hypoxia onset, indicating a sharp rise in [Ca(2+)]i just beneath the membrane. By using a high concentration of K(+) in the pipette, we simultaneously monitored the membrane potential and showed that the [Ca(2+)]i rise was not associated with membrane depolarization. The earliest hypoxia-induced synaptic disturbance was a marked increase in the frequency of sPSCs, which also began soon after the removal of oxygen and long before AD. This synaptic effect was accompanied by depletion of the readily releasable transmitter pools, as demonstrated by a decreased response to hyperosmotic solutions. The early [Ca(2+)]i rise, the early increase in transmitter release and the subsequent AD itself were all prevented by bathing in a cocktail containing blockers of ionotropic glutamate receptors. We found no evidence for involvement of pannexin hemichannels or TRPM7 channels in the early responses to hypoxia in this experimental preparation. Our data indicate that the earliest cellular consequences of cortical hypoxia are triggered by activation of glutamate-gated channels.

M-current inhibition rapidly induces a unique CK2-dependent plasticity of the axon initial segment

Significance The axon initial segment (AIS), the region where neurons generate spikes, was recently shown to be a highly dynamic structure, exhibiting plasticity over wide timescales. Here we triggered a unique form of AIS plasticity in hippocampal pyramidal neurons by selectively targeting the AIS M-type K+ channels. We uncovered the mechanisms whereby sustained cholinergic activation or direct M-channel block rapidly trigger a unique form of AIS plasticity. Minutes to hours of sustained M-current depression resulted in a compensatory reduction in intrinsic excitability associated with distal shift of the axonal spike trigger zone and distal relocation of both Na+ and M-channels. These fast homeostatic changes, necessary to stabilize network excitability, were dependent on the crucial AIS protein, protein kinase CK2. Alterations in synaptic input, persisting for hours to days, elicit homeostatic plastic changes in the axon initial segment (AIS), which is pivotal for spike generation. Here, in hippocampal pyramidal neurons of both primary cultures and slices, we triggered a unique form of AIS plasticity by selectively targeting M-type K+ channels, which predominantly localize to the AIS and are essential for tuning neuronal excitability. While acute M-current inhibition via cholinergic activation or direct channel block made neurons more excitable, minutes to hours of sustained M-current depression resulted in a gradual reduction in intrinsic excitability. Dual soma–axon patch-clamp recordings combined with axonal Na+ imaging and immunocytochemistry revealed that these compensatory alterations were associated with a distal shift of the spike trigger zone and distal relocation of FGF14, Na+, and Kv7 channels but not ankyrin G. The concomitant distal redistribution of FGF14 together with Nav and Kv7 segments along the AIS suggests that these channels relocate as a structural and functional unit. These fast homeostatic changes were independent of l-type Ca2+ channel activity but were contingent on the crucial AIS protein, protein kinase CK2. Using compartmental simulations, we examined the effects of varying the AIS position relative to the soma and found that AIS distal relocation of both Nav and Kv7 channels elicited a decrease in neuronal excitability. Thus, alterations in M-channel activity rapidly trigger unique AIS plasticity to stabilize network excitability.

The Outwardly Rectifying Current of Layer 5 Neocortical Neurons that was Originally Identified as "Non-Specific Cationic" Is Essentially a Potassium Current.

In whole-cell patch clamp recordings from layer 5 neocortical neurons, blockade of voltage gated sodium and calcium channels leaves a cesium current that is outward rectifying. This current was originally identified as a “non-specific cationic current”, and subsequently it was hypothesized that it is mediated by TRP channels. In order to test this hypothesis, we used fluorescence imaging of intracellular sodium and calcium indicators, and found no evidence to suggest that it is associated with influx of either of these ions to the cell body or dendrites. Moreover, the current is still prominent in neurons from TRPC1-/- and TRPC5-/- mice. The effects on the current of various blocking agents, and especially its sensitivity to intracellular tetraethylammonium, suggest that it is not a non-specific cationic current, but rather that it is generated by cesium-permeable delayed rectifier potassium channels.