Indira M. Raman

Altered Subthreshold Sodium Currents and Disrupted Firing Patterns in Purkinje Neurons of Scn8a Mutant Mice

Sodium currents and action potentials were characterized in Purkinje neurons from ataxic mice lacking expression of the sodium channel Scn8a. Peak transient sodium current was approximately 60% of that in normal mice, but subthreshold sodium current was affected much more. Steady-state current elicited by voltage ramps was reduced to approximately 30%, and resurgent sodium current, an unusual transient current elicited on repolarization following strong depolarizations, was reduced to 8%-18%. In jolting mice, with a missense mutation in Scn8a, steady-state and resurgent current were also reduced, with altered voltage dependence and kinetics. Both spontaneous firing and evoked bursts of spikes were diminished in cells from null and jolting mice. Evidently Scn8a channels carry most subthreshold sodium current and are crucial for repetitive firing.

Potentiation of Mossy Fiber EPSCs in the Cerebellar Nuclei by NMDA Receptor Activation followed by Postinhibitory Rebound Current

Behavioral and computational studies predict that synaptic plasticity of excitatory mossy fiber inputs to cerebellar nuclear neurons is required for associative learning, but standard tetanization protocols fail to potentiate nuclear cell EPSCs in mouse cerebellar slices. Nuclear neurons fire action potentials spontaneously unless strongly inhibited by Purkinje neurons, raising the possibility that plasticity-triggering signals in these cells differ from those at classical Hebbian synapses. Based on predictions of neuronal activity during delay eyelid conditioning, we developed quasi-physiological induction protocols consisting of high-frequency mossy fiber stimulation and postsynaptic hyperpolarization. Robust, NMDA receptor-dependent potentiation of nuclear cell EPSCs occurred with protocols including a 150-250 ms hyperpolarization in which mossy fiber stimulation preceded a postinhibitory rebound depolarization. Mossy fiber stimulation potentiated EPSCs even when postsynaptic spiking was prevented by voltage-clamp, as long as rebound current was evoked. These data suggest that Purkinje cell inhibition guides the strengthening of excitatory synapses in the cerebellar nuclei.

Inactivation and Recovery of Sodium Currents in Cerebellar Purkinje Neurons: Evidence for Two Mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.

Purkinje neuron synchrony elicits time-locked spiking in the cerebellar nuclei

An unusual feature of the cerebellar cortex is that its output neurons, Purkinje cells, release GABA (γ-aminobutyric acid). Their high intrinsic firing rates (50 Hz) and extensive convergence predict that their target neurons in the cerebellar nuclei would be largely inhibited unless Purkinje cells pause their spiking, yet Purkinje and nuclear neuron firing rates do not always vary inversely. One indication of how these synapses transmit information is that populations of Purkinje neurons synchronize their spikes during cerebellar behaviours. If nuclear neurons respond to Purkinje synchrony, they may encode signals from subsets of inhibitory inputs. Here we show in weanling and adult mice that nuclear neurons transmit the timing of synchronous Purkinje afferent spikes, owing to modest Purkinje-to-nuclear convergence ratios (∼40:1), fast inhibitory postsynaptic current kinetics (τdecay = 2.5 ms) and high intrinsic firing rates (∼90 Hz). In vitro, dynamically clamped asynchronous inhibitory postsynaptic potentials mimicking Purkinje afferents suppress nuclear cell spiking, whereas synchronous inhibitory postsynaptic potentials entrain nuclear cell spiking. With partial synchrony, nuclear neurons time-lock their spikes to the synchronous subpopulation of inputs, even when only 2 out of 40 afferents synchronize. In vivo, nuclear neurons reliably phase-lock to regular trains of molecular layer stimulation. Thus, cerebellar nuclear neurons can preferentially relay the spike timing of synchronized Purkinje cells to downstream premotor areas.

Relative Contributions of Axonal and Somatic Na Channels to Action Potential Initiation in Cerebellar Purkinje Neurons

Neuronal excitability is likely to be regulated by the site of action potential initiation, the location on a neuron that crosses threshold first. Although initiation is axonal in many neurons, in Purkinje cells, somatic conductances can generate spontaneous action potentials, suggesting that the perisomatic region (soma and/or initial segment) contributes to spike initiation and may regulate firing. To identify directly the cellular regions at which Na channel modulation significantly influences firing, we measured spontaneous and evoked action potentials in Purkinje cells in cerebellar slices from postnatal day 14–28 mice while applying drugs locally to either the soma/initial segment or the first node of Ranvier. Na currents were decreased by tetrodotoxin (TTX) or increased by β-pompilidotoxin (β-PMTX). Dual somatic and axonal recordings indicated that spike thresholds and input–output curves were sensitive to TTX or β-PMTX at the perisomatic region but were unchanged by either drug at the first node. When perisomatic Na channel availability was reduced with subsaturating TTX, however, the input–output curve became shallower during additional TTX block of nodal channels, revealing a latent role for nodal Na channels in facilitating firing. In perisomatic TTX, axons failed to generate spontaneous or evoked spike trains. In contrast, choline block of the initial segment alone altered normal input–output curves. The data suggest that, although the first node reliably follows action potentials, spike initiation in Purkinje neurons occurs in the initial segment. Moreover, Purkinje cell output depends on the density, availability, and kinetics of perisomatic Na channels, a characteristic that may distinguish spontaneously firing from quiescent neurons.

Axonal Propagation of Simple and Complex Spikes in Cerebellar Purkinje Neurons

In cerebellar Purkinje neurons, the reliability of propagation of high-frequency simple spikes and spikelets of complex spikes is likely to regulate inhibition of Purkinje target neurons. To test the extent to which a one-to-one correspondence exists between somatic and axonal spikes, we made dual somatic and axonal recordings from Purkinje neurons in mouse cerebellar slices. Somatic action potentials were recorded with a whole-cell pipette, and the corresponding axonal signals were recorded extracellularly with a loose-patch pipette. Propagation of spontaneous and evoked simple spikes was highly reliable. At somatic firing rates of ∼200 spikes/sec, <10% of spikes failed to propagate, with failures becoming more frequent only at maximal somatic firing rates (∼260 spikes/sec). Complex spikes were elicited by climbing fiber stimulation, and their somatic waveforms were modulated by tonic current injection, as well as by paired stimulation to depress the underlying EPSCs. Across conditions, the mean number of propagating action potentials remained just above two spikes per climbing fiber stimulation, but the instantaneous frequency of the propagating spikes changed, from ∼375 Hz during somatic hyperpolarizations that silenced spontaneous firing to ∼150 Hz during spontaneous activity. The probability of propagation of individual spikelets could be described quantitatively as a saturating function of spikelet amplitude, rate of rise, or preceding interspike interval. The results suggest that ion channels of Purkinje axons are adapted to produce extremely short refractory periods and that brief bursts of forward-propagating action potentials generated by complex spikes may contribute transiently to inhibition of postsynaptic neurons.

Regulation of Persistent Na Current by Interactions between β Subunits of Voltage-Gated Na Channels

The β subunits of voltage-gated Na channels (Scnxb) regulate the gating of pore-forming α subunits, as well as their trafficking and localization. In heterologous expression systems, β1, β2, and β3 subunits influence inactivation and persistent current in different ways. To test how the β4 protein regulates Na channel gating, we transfected β4 into HEK (human embryonic kidney) cells stably expressing NaV1.1. Unlike a free peptide with a sequence from the β4 cytoplasmic domain, the full-length β4 protein did not block open channels. Instead, β4 expression favored open states by shifting activation curves negative, decreasing the slope of the inactivation curve, and increasing the percentage of noninactivating current. Consequently, persistent current tripled in amplitude. Expression of β1 or chimeric subunits including the β1 extracellular domain, however, favored inactivation. Coexpressing NaV1.1 and β4 with β1 produced tiny persistent currents, indicating that β1 overcomes the effects of β4 in heterotrimeric channels. In contrast, β1C121W, which contains an extracellular epilepsy-associated mutation, did not counteract the destabilization of inactivation by β4 and also required unusually large depolarizations for channel opening. In cultured hippocampal neurons transfected with β4, persistent current was slightly but significantly increased. Moreover, in β4-expressing neurons from Scn1b and Scn1b/Scn2b null mice, entry into inactivated states was slowed. These data suggest that β1 and β4 have antagonistic roles, the former favoring inactivation, and the latter favoring activation. Because increased Na channel availability may facilitate action potential firing, these results suggest a mechanism for seizure susceptibility of both mice and humans with disrupted β1 subunits.

Mechanisms of Potentiation of Mossy Fiber EPSCs in the Cerebellar Nuclei by Coincident Synaptic Excitation and Inhibition

Neurons of the cerebellar nuclei receive synaptic excitation from cerebellar mossy fibers. Unlike in many principal neurons, coincident presynaptic activity and postsynaptic depolarization do not generate long-term potentiation at these synapses. Instead, EPSCs are potentiated by high-frequency trains of presynaptic activity applied with postsynaptic hyperpolarization, in patterns resembling mossy-fiber-mediated excitation and Purkinje-cell-mediated inhibition that are predicted to occur during delay eyelid conditioning. Here, we have used electrophysiology and Ca imaging to test how synaptic excitation and inhibition interact to generate long-lasting synaptic plasticity in nuclear cells in cerebellar slices. We find that the extent of plasticity varies with the relative timing of synaptic excitation and hyperpolarization. Potentiation is most effective when synaptic stimuli precede the postinhibitory rebound by ∼400 ms, whereas with longer intervals, or with a reverse sequence, EPSCs tend to depress. When basal intracellular Ca is raised by spontaneous firing or reduced by voltage clamping at subthreshold potentials, potentiation is induced as long as the synaptic-rebound temporal sequence is maintained, suggesting that plasticity does not require Ca levels to exceed a threshold or attain a specific concentration. Although rebound and spike-dependent Ca influx are global, potentiation is synapse specific, and is disrupted by inhibitors of calcineurin or Ca-calmodulin-dependent protein kinase II, but not PKC. When IPSPs replace the hyperpolarizing step in the induction protocol, potentiation proceeds normally. These results lead us to propose that synaptic and inhibitory/rebound stimuli initiate separate processes, with local NMDA receptor-mediated Ca influx “priming” synapses, and Ca changes from the inhibition and rebound “triggering” potentiation at recently activated synapses.

Control of transient, resurgent, and persistent current by open-channel block by Na channel β4 in cultured cerebellar granule neurons

Voltage-gated Na channels in several classes of neurons, including cells of the cerebellum, are subject to an open-channel block and unblock by an endogenous protein. The NaVβ4 (Scn4b) subunit is a candidate blocking protein because a free peptide from its cytoplasmic tail, the β4 peptide, can block open Na channels and induce resurgent current as channels unblock upon repolarization. In heterologous expression systems, however, NaVβ4 fails to produce resurgent current. We therefore tested the necessity of this subunit in generating resurgent current, as well as its influence on Na channel gating and action potential firing, by studying cultured cerebellar granule neurons treated with siRNA targeted against Scn4b. Knockdown of Scn4b, confirmed with quantitative RT-PCR, led to five electrophysiological phenotypes: a loss of resurgent current, a reduction of persistent current, a hyperpolarized half-inactivation voltage of transient current, a higher rheobase, and a decrease in repetitive firing. All disruptions of Na currents and firing were rescued by the β4 peptide. The simplest interpretation is that NaVβ4 itself blocks Na channels of granule cells, making this subunit the first blocking protein that is responsible for resurgent current. The results also demonstrate that a known open-channel blocking peptide not only permits a rapid recovery from nonconducting states upon repolarization from positive voltages but also increases Na channel availability at negative potentials by antagonizing fast inactivation. Thus, NaVβ4 expression determines multiple aspects of Na channel gating, thereby regulating excitability in cultured cerebellar granule cells.

Production of Resurgent Current in NaV1.6-Null Purkinje Neurons by Slowing Sodium Channel Inactivation with β-Pompilidotoxin

Voltage-gated tetrodotoxin-sensitive sodium channels of Purkinje neurons produce “resurgent” current with repolarization, which results from relief of an open-channel block that terminates current flow at positive potentials. The associated recovery of sodium channels from inactivation is thought to facilitate the rapid firing patterns characteristic of Purkinje neurons. Resurgent current appears to depend primarily on NaV1.6 α subunits, because it is greatly reduced in “med” mutant mice that lack NaV1.6. To identify factors that regulate the susceptibility of α subunits to open-channel block, we voltage clamped wild-type and med Purkinje neurons before and after slowing conventional inactivation with β-pompilidotoxin (β-PMTX). β-PMTX increased resurgent current in wild-type neurons and induced resurgent current in med neurons. In med cells, the resurgent component of β-PMTX-modified sodium currents could be selectively abolished by application of intracellular alkaline phosphatase, suggesting that, like in NaV1.6-expressing cells, the open-channel block of NaV1.1 and NaV1.2 subunits is regulated by constitutive phosphorylation. These results indicate that the endogenous blocker exists independently of NaV1.6 expression, and conventional inactivation regulates resurgent current by controlling the extent of open-channel block. In Purkinje cells, therefore, the relatively slow conventional inactivation kinetics of NaV1.6 appear well adapted to carry resurgent current. Nevertheless, NaV1.6 is not unique in its susceptibility to open-channel block, because under appropriate conditions, the non-NaV1.6 subunits can produce robust resurgent currents.