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, 591 (7), 1771-91

A Defined Heteromeric KV1 Channel Stabilizes the Intrinsic Pacemaking and Regulates the Output of Deep Cerebellar Nuclear Neurons to Thalamic Targets

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A Defined Heteromeric KV1 Channel Stabilizes the Intrinsic Pacemaking and Regulates the Output of Deep Cerebellar Nuclear Neurons to Thalamic Targets

Saak V Ovsepian et al. J Physiol.

Abstract

The output of the cerebellum to the motor axis of the central nervous system is orchestrated mainly by synaptic inputs and intrinsic pacemaker activity of deep cerebellar nuclear (DCN) projection neurons. Herein, we demonstrate that the soma of these cells is enriched with K(V)1 channels produced by mandatory multi-merization of K(V)1.1, 1.2 α and KV β2 subunits. Being constitutively active, the K(+) current (IK(V)1) mediated by these channels stabilizes the rate and regulates the temporal precision of self-sustained firing of these neurons. Placed strategically, IK(V)1 provides a powerful counter-balance to prolonged depolarizing inputs, attenuates the rebound excitation, and dampens the membrane potential bi-stability. Somatic location with low activation threshold render IK(V)1 instrumental in voltage-dependent de-coupling of the axon initial segment from the cell body of projection neurons, impeding invasion of back-propagating action potentials into the somato-dendritic compartment. The latter is also demonstrated to secure the dominance of clock-like somatic pacemaking in driving the regenerative firing activity of these neurons, to encode time variant inputs with high fidelity. Through the use of multi-compartmental modelling and retro-axonal labelling, the physiological significance of the described functions for processing and communication of information from the lateral DCN to thalamic relay nuclei is established.

Figures

Figure 1
Figure 1. Expression and distribution of KV1.1/1.2 α and KVβ2 subunits in neurons of the lateral nucleus
Aa, fluorescence micrographs of the lateral nuclear area labelled individually for Kv1.1 or 1.2 α subunits (top, middle) and merged (bottom). Ab, sequential confocal optical sections in Z-plane obtained from a representative neuron (top to bottom). Punctuate surface labelling (yellow) corresponds to clustered KV1.1 and 1.2 subunits (arrows) on the plasmalemma. Note the stronger intracellular KV1.1 (green) and significant presence of KV1.2 (red) in surrounding neuropile. Ba, fluorescence micrographs of the lateral nucleus stained individually for Kv1.1 α or KVβ2 subunits (top and middle) and merged (bottom). Sequential confocal sections in Z-plane obtained from a representative neuron (Bb); yellow punctuates on the surface correspond to co-localized KV1.1 and β2 subunits. Directional arrows L and D: lateral and dorsal, respectively. MN, medial nucleus (equivalent to human fastigial nucleus); LN, lateral nucleus (equivalent to human dentate nucleus); IN, interpositus nucleus (equivalent to human emboliform and globose nuclei combined). Calibration scales: 75 μm (Aa and Ba) and 10 μm (Ab and Bb).
Figure 2
Figure 2. Hetero-multimeric KV1 channels containing Kv1.1–1.2 α and KVβ2 subunits are expressed in neurons of the lateral nucleus
A and B, confocal micrographs of KV1 channel-enriched surface clusters illustrate strong overlap between KV1.1 and 1.2 (A) or KV1.1 and KVβ2 (B) subunits. Arrows indicate the interior of the neuron. C, immuno-absorption of KV1.X subunits associated with Kv1.1. After reacting with the detergent extract, DCN tissue with resin containing immobilised anti-Kv1.1 antibody, the resultant eluate (C) proved positive for Kv1.1 and 1.2 (lanes: 3 and 5, respectively) with the flow-through devoid of Kv1.1 (2) but containing residual Kv1.2 (4); Kv1.1 detected in the total extract (6); lane 1 represents protein markers (C). D, the same procedure was applied using anti-Kv1.2 antibody attached to the resin; the eluate showed immuno-reactivity for Kv1.2 and 1.1 (2 and 4, respectively); the flow-through lacked Kv1.2 (1) but possessed a trace of Kv1.1 (3); Kv1.2 in the total extracts (5). E, using immobilized anti-β2 IgGs, the flow-through revealed some Kv1.2 and 1.1 (1 and 3, respectively) but the eluate gave stronger staining for Kv1.2 and 1.1 (2 and 4, respectively); β2 in the total extract (5). Arrows indicate the bands of interest.
Figure 3
Figure 3. IKV1 mediated by KV1.1- and/or 1.2-containing channels stabilizes pacemaking of lateral DCN neurons
Aa and b, DTXK accelerates and renders irregular the constitutive firing of nuclear neurons: Aa, representative cell-attached patch recordings. Top, before (left) and in the presence of DTXK (right). Bottom, graph illustrating the time course of the toxin's effect on instantaneous (Inst.) firing rate (FR). Dashed line here and below indicates toxin application period. Ab, inter-spike interval (ISI) distribution histograms of the same neuron before and after treatment with DTXK (black and red, respectively). Ba and b, representative example of the effect of TsTX-Kα on a spontaneous firing neuron. Ba, cell-attached recordings before and after toxin treatments (top) with graph of instantaneous firing rate (bottom). Bb, corresponding ISI distribution histograms. C, acceleration of spontaneous firing produced by co-application of DTXK with TsTX-Kα. Note further addition of TsTXα (red horizontal bar) after the effect of DTXK had reached plateau (dashed line) did not cause further increase in the rate of spontaneous firing (bottom). Representative cell-attached recordings before and after toxin treatments are presented as above. D, a summary plot of the increase in firing rate caused by the blockade of IKV1 with selective blockers of KV1.1 or 1.2 α subunits (DTXK with TsTX-Kα applied individually or co-applied; here and below concentrations (C, in nm) shown below bars). Asterisks here and below highlight statistically significant differences compared to controls.
Figure 4
Figure 4. IKv1 attenuates the excitability and counter-balances strong depolarizing membrane potential in DCN neurons
A, typical recordings of membrane responses to sub-threshold (bottom), supra-threshold (middle) and strong (>5 times the threshold, top) stimuli before (left) and after (right) DTXK treatment. Note the lower threshold current (bottom left compared to right), higher discharge rate (middle) and greater spike adaptation with depolarization block produced by strong stimulus (top) in the presence of DTXK. Horizontal arrows (top) indicate the level of minimal inter-spike potential before application of DTXK; insets (bottom left panel) illustrate DTXK-induced augmentation of rebound discharge in the same neuron (−80 pA, 500 ms): traces obtained before (black) and after (red) toxin treatment with overlay. B and C, relation of stimulus intensity to onset delay of action potential (B) and evoked initial firing rate (C). Note, while DTXK lowers the current threshold for evoked firing and accelerates the onset of spikes at supra-threshold stimulus intensities, the time to the first spike evoked by threshold stimulus intensity in the presence of DTXK is longer. Top insets (B and C) illustrate that DTXK accelerated the spike onset in response to supra-threshold stimulus (B) and enhanced membrane excitability (emergence of slow depolarizing drive) (C, arrow). D and E, DTXK augmented sub-threshold membrane depolarization (before INaV and ICaV blockade by TTX and CoCl2) and enhanced depolarization induced plateau membrane responses (after INaV and ICaV blockade). Horizontal arrows point to the sub-threshold depolarization potential (minimal inter-spike voltage) (D) and sustained (E) potentials before (left) and after (right) application of DTXK. F, summary plot of the estimated membrane resistance (to 2.6 timesthreshold depolarizing current pulse stimuli). Note that both DTXK and TsTX-Kα applied individually and together notably increase the membrane resistance due to removal of voltage-activated K+ conductance.
Figure 5
Figure 5. Rebound discharge and membrane potential bi-stability are controlled by IKV1 in DCN neurons
A and B, DTXK augments and accelerates anode break induced rebound firing of these neurons. A, anode break induced (hyperpolarizing current 2.5 times thresholds) rebound discharge before and after DTXK (left and middle) and overlay of both (right). Insets below illustrate the initial firing at an expanded time scale. Note acceleration of discharge onset and prolongation of the membrane up-state associated with enhanced rebound firing caused by DTXK. B, relation of initial (estimated over first 5 action potentials, inset below) rebound discharge rate – rebound discharge delay: measurements represent average estimates of these parameters pooled from individual neurons before and after application of DTXK. Top inset illustrates the stimulus with a representative evoked response. C and D, DTXK accelerated the spontaneous firing and enhanced anode break-induced excitation. Note toxin-induced increase in pre- and post-stimulus firing with prolongation of accelerated rebound firing after the turn-off of the hyperpolarizing ramp stimulus. Raster plots (C, top) from 3 representative neurons collected before and after toxin treatment (left and right, respectively). D, summary graph illustrating DTXK-induced acceleration of intrinsic and rebound firing with delayed post-stimulus firing rate recovery. Inset graph illustrates the rate of the rebound firing frequency recovery in a representative neuron before and after treatment with DTXK. E, DTXK enhances the membrane bi-stability of DCN neurons. Evoked firing induced by brief depolarizing ramp before (left) and after (middle and right) DTXK treatment. Note robust prolongation of the membrane potential ‘up-state’ caused by sustained post-stimulus depolarization with repetitive firing (right) or plateau potential devoid of action potentials (middle). Left and middle insets illustrate the acceleration of the membrane potential discharge caused by DTXK (black and red, control and DTXK, respectively). Inset on the right (arrow) illustrates rapidly adapting action potentials at expanded time scale with the onset of complete depolarization-induced block of firing. F, summary plot of toxin effect on the duration of depolarizing ramp-evoked firing transients (‘up-states’ highlighted by arrows in E, left). Note significant prolongation of the up-state duration after DTXK treatment within the weaker stimulus ranges.
Figure 6
Figure 6. Attenuation of axon-derived membrane potential fluctuations by IKV1 promotes clock-like somatic firing in DCN neurons
A and B, continuous current-clamp recordings of membrane potential from a representative DCN neuron. Top, spontaneous firing activity before (A, Control) and after exposure of the same neuron to DTXK (B, DTXK). Middle and bottom, recordings of the membrane potential from the same neuron after silencing by injection of incremented hyperpolarizing current. Note emergence of low amplitude membrane potential oscillations (A, middle) at less hyperpolarized potentials which were abolished by further hyperpolarization of the soma (A, bottom). In the presence of DTXK, the membrane potential fluctuations become more robust (abortive spikes) and occasionally reached to the action potential generation threshold, triggering full somatic spikes. C, a brief recording episode illustrating somatic and IS generated action potentials in the same neuron with inset below showing the content of the dashed rectangular box at extended time scale. D, superimposed recordings of spontaneous activity of DTXK-treated DCN neuron from hyperpolarized potentials revealing of occasions both IS and IS–SD spikes. For illustrative purpose, traces are aligned to reveal the prolonged quiescent phase after the SD spike (also illustrated in the inset raster diagram).
Figure 7
Figure 7. IKV1 impedes somatic invasion of antidromic spikes and lowers the fidelity of information transfer at somato-axonal junctions
A and B, two-photon micrographs of representative lateral nuclear projection neurons: reconstructed 3D images: sub-panels a and b, areas indicated in panel A enlarged to illustrate the axon (A, arrows on a) and dendritic segment decorated with rare spine-like elements (A, arrowheads on b). B, neuron with clearly visible axon branching local collaterals (arrows) before exiting into the peri-nuclear white matter. C, examples of antidromic spikes with voltage-dependent IS→SD transition before (left) and after (right) the blockade of IKV1 with DTXK. Above each trace the membrane potentials at the spike foot are indicated. For illustrative clarity stimulus artifacts are truncated. D, a summary graph illustrating the relation of antidromic spike amplitude and pre-stimulus somatic potential with representative spikes at decrementing negative somatic potentials (top). E, examples of second antidromic spikes evoked by paired-pulse stimulation protocol at incrementing inter-stimulus intervals before (left) and after (right) blockade of IKV1 with DTXK. Inter-stimulus intervals are indicated above each trace. F, a summary graph illustrating the dependence of antidromic spike amplitude on inter-pulse interval and the effect of DTXK on this parameter. Inset (top) illustrates examples of spikes evoked by paired-pulse stimulation. In the graph, the amplitude of the second spike is expressed as a fraction of the amplitude of the first spike, calculated using the formula (P2P1)/P1, where P1 and P2 are the first and second antidromic action potentials, respectively.
Figure 8
Figure 8. IKv1 controls the output of neurons in lateral nucleus to the anterior thalamus
A, schematic diagram of retrograde labelling experiments (left) with a micrograph of live neurons from lateral nucleus (right). Latex beads exhibiting punctuate fluorescence were excited with a two-photon laser (730 nm). B, cell-attached patch recordings from typical projection neuron. Top, before (left) and in DTXK (right). Bottom, graph illustrating the time course of the effect of toxin on instantaneous firing rate and regularity. Dashed line indicates the time window of DTXK application. C, typical recordings of evoked firing from identified projection neuron before and after treatment with DTXK: stimulus intensities 3.8 times threshold current. Note that DTXK at this stimulus intensity caused robust augmentation of spike adaptation (inset, right) with upward shift of the sub-threshold membrane potential and depolarization block. Inset on the left shows the acceleration of the onset of evoked firing by the same treatment. D, DTXK prolonged the anodal break-induced rebound discharge in the identified projection neuron: control rebound firing (left) and after DTXK treatment (right). Inset: summary plot of RD duration before and after treatment of neurons with DTXK. E–G, summary histograms illustrating acceleration of the spontaneous firing rate (E), ISI CV increase (F) and reduction in the intensity of threshold current for evoked firing activity (G) produced by individually applied DTXK or TsTXα or when co-applied.
Figure 9
Figure 9. Delayed rectifying K+ currents regulate excitability, rebound responses and antidromic action potential invasion in a computational model of a DCN neuron
In the simulations shown in panels AD, the conductance densities of the fast and slow delayed rectifier K+ (Kdr) current were varied between 60 and 130% of their default values in the model. A, relationship between Kdr conductance and firing rate. B, relationship between Kdr conductance and coefficient of variation (CV). C, relationship between Kdr conductance and maximum spike rate in the rebound response that followed a −120 pA current injection for 1 s. D, relationship between Kdr conductance and latency of the first spike in the rebound response after offset of the −120 pA current injection. E, relationship between input current amplitude and initial spike rate (see text and Methods) for 100% (filled symbols) and 70% (open symbols) of the Kdr conductance density. F, relationship between prespike voltage and somatic spike voltage (see text and Methods) for 100% (filled symbols) and 70% (open symbols) of the Kdr conductance density.

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