Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating.

KEY POINTS: Sodium channels are critical for supporting fast action potentials in neurons; even mutations which cause small changes in sodium channel activity can have devastating consequences for the function of the nervous system. Alternative splicing also changes the activity of sodium channels, and while it is highly conserved, it is not known whether the functional role of this splicing is also conserved. Our data reveal that splicing has a highly conserved impact on the availability of sodium channels during trains of rapid stimulations, and suggest that in one mammalian channel, Nav1.1 encoded by SCN1A, the increased availability of one splice variant is detrimental. A model reproducing the effects of splicing on channel behaviour suggests that the voltage sensor in the first domain is a rate limiting step for release of the inactivation domain, and highlights the functional specialization of channel domains. ABSTRACT: Voltage-gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) that splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals.

Presynaptic muscarinic receptors reduce synaptic depression and facilitate its recovery at hippocampal GABAergic synapses.

Hippocampal gamma oscillation, involved in cognitive processes, can be induced by muscarinic acetylcholine receptors activation and depends in large part on the activation of γ-aminobutyric acidergic (GABAergic) interneurons. The precise role of the modulatory action of muscarinic receptors on GABAergic transmission still remains unclear due to the great heterogeneity of observed effects. We have examined the presynaptic and postsynaptic mechanisms involved. Methacholine induces a down-regulation of evoked inhibitory postsynaptic currents (eIPSCs) not associated with the change of postsynaptic receptors. The significant decrease in the paired-pulse depression strongly suggested a presynaptic mechanism of action. We have used cumulative amplitude profile analysis to show that the impairment of eIPSCs is not related to a decreased size of the readily releasable pool, but rather depends on the reduced release probability by a down-modulation of voltage-gated calcium channels. The decreased neurotransmitter release probability only partially accounts for the dramatic reduction in the rate of synaptic depression evoked by short- and long-lasting tetanic stimuli. This effect is accompanied by a significant enhancement in the rate of recovery from synaptic depression that demonstrates the reinforcement of the synaptic recycling processes. These results show that muscarinic modulation of hippocampal GABAergic synapses confers a greater resistance to sustain periods of intense synaptic activity in the gamma frequency range.

An adaptable, reusable, and light implant for chronic Neuropixels probes.

Electrophysiology has proven invaluable to record neural activity, and the development of Neuropixels probes dramatically increased the number of recorded neurons. These probes are often implanted acutely, but acute recordings cannot be performed in freely moving animals and the recorded neurons cannot be tracked across days. To study key behaviors such as navigation, learning, and memory formation, the probes must be implanted chronically. An ideal chronic implant should (1) allow stable recordings of neurons for weeks; (2) be light enough for use in mice; (3) allow reuse of the probes after explantation. Here, we present the "Apollo Implant", an open-source and editable device that meets these criteria and accommodates up to two Neuropixels 1.0 or 2.0 probes. The implant comprises a "payload" module that is attached to the probe and is recoverable, and a "docking" module that is cemented to the skull. The design is adjustable, making it easy to change the distance between probes, the angle of insertion, and the depth of insertion. We tested the implant across seven labs in head-fixed mice, freely moving mice, and freely moving rats. The number of neurons recorded across days was stable, even after repeated implantations of the same probe. The Apollo implant provides an inexpensive, lightweight, and flexible solution for reusable chronic Neuropixels recordings.

Gene therapy and editing: Novel potential treatments for neuronal channelopathies.

Pharmaceutical treatment can be inadequate, non-effective, or intolerable for many people suffering from a neuronal channelopathy. Development of novel treatment options, particularly those with the potential to be curative is warranted. Gene therapy approaches can permit cell-specific modification of neuronal and circuit excitability and have been investigated experimentally as a therapy for numerous neurological disorders, with clinical trials for several neurodegenerative diseases ongoing. Channelopathies can arise from a wide array of gene mutations; however they usually result in periods of aberrant network excitability. Therefore gene therapy strategies based on up or downregulation of genes that modulate neuronal excitability may be effective therapy for a wide range of neuronal channelopathies. As many channelopathies are paroxysmal in nature, optogenetic or chemogenetic approaches may be well suited to treat the symptoms of these diseases. Recent advances in gene-editing technologies such as the CRISPR-Cas9 system could in the future result in entirely novel treatment for a channelopathy by repairing disease-causing channel mutations at the germline level. As the brain may develop and wire abnormally as a consequence of an inherited or de novo channelopathy, the choice of optimal gene therapy or gene editing strategy will depend on the time of intervention (germline, neonatal or adult). This article is part of the Special Issue entitled 'Channelopathies.'