The hyperpolarization-activated cyclic nucleotide-gated 4 channel as a potential anti-seizure drug target.

BACKGROUND AND PURPOSE: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are encoded by four genes (HCN1-4) with distinct biophysical properties and functions within the brain. HCN4 channels activate slowly at robust hyperpolarizing potentials, making them more likely to be engaged during hyperexcitable neuronal network activity seen during seizures. HCN4 channels are also highly expressed in thalamic nuclei, a brain region implicated in seizure generalization. Here, we assessed the utility of targeting the HCN4 channel as an anti-seizure strategy using pharmacological and genetic approaches. EXPERIMENTAL APPROACH: The impact of reducing HCN4 channel function on seizure susceptibility and neuronal network excitability was studied using an HCN4 channel preferring blocker (EC18) and a conditional brain specific HCN4 knockout mouse model. KEY RESULTS: EC18 (10 mg·kg-1 ) and brain-specific HCN4 channel knockout reduced seizure susceptibility and proconvulsant-mediated cortical spiking recorded using electrocorticography, with minimal effects on other mouse behaviours. EC18 (10 µM) decreased neuronal network bursting in mouse cortical cultures. Importantly, EC18 was not protective against proconvulsant-mediated seizures in the conditional HCN4 channel knockout mouse and did not reduce bursting behaviour in AAV-HCN4 shRNA infected mouse cortical cultures. CONCLUSIONS AND IMPLICATIONS: These data suggest the HCN4 channel as a potential pharmacologically relevant target for anti-seizure drugs that is likely to have a low side-effect liability in the CNS.

Using a Multiplex Nucleic Acid in situ Hybridization Technique to Determine HCN4 mRNA Expression in the Adult Rodent Brain.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels carry a non-selective cationic conductance, I h , which is important for modulating neuron excitability. Four genes (HCN1-4) encode HCN channels, with each gene having distinct expression and biophysical profiles. Here we use multiplex nucleic acid in situ hybridization to determine HCN4 mRNA expression within the adult mouse brain. We take advantage of this approach to detect HCN4 mRNA simultaneously with either HCN1 or HCN2 mRNA and markers of excitatory (VGlut-positive) and inhibitory (VGat-positive) neurons, which was not previously reported. We have developed a Fiji-based analysis code that enables quantification of mRNA expression within identified cell bodies. The highest HCN4 mRNA expression was found in the habenula (medial and lateral) and the thalamus. HCN4 mRNA was particularly high in the medial habenula with essentially no co-expression of HCN1 or HCN2 mRNA. An absence of I h -mediated "sag" in neurons recorded from the medial habenula of knockout mice confirmed that HCN4 channels are the predominant subtype in this region. Analysis in the thalamus revealed HCN4 mRNA in VGlut2-positive excitatory neurons that was always co-expressed with HCN2 mRNA. In contrast, HCN4 mRNA was undetectable in the nucleus reticularis. HCN4 mRNA expression was high in a subset of VGat-positive cells in the globus pallidus external. The majority of these neurons co-expressed HCN2 mRNA while a smaller subset also co-expressed HCN1 mRNA. In the striatum, a small subset of large cells which are likely to be giant cholinergic interneurons co-expressed high levels of HCN4 and HCN2 mRNA. The amygdala, cortex and hippocampus expressed low levels of HCN4 mRNA. This study highlights the heterogeneity of HCN4 mRNA expression in the brain and provides a morphological framework on which to better investigate the functional roles of HCN4 channels.

Synapse Type-Dependent Expression of Calcium-Permeable AMPA Receptors.

Calcium-permeable (CP) AMPA-type glutamate receptors (AMPARs) are known to mediate synaptic plasticity in several different interneuron (IN) types. Recent evidence suggests that CP-AMPARs are synapse-specifically expressed at excitatory connections onto a subset of IN types in hippocampus and neocortex. For example, CP-AMPARs are found at connections from pyramidal cells (PCs) to basket cells (BCs), but not to Martinotti cells (MCs). This synapse type-specific expression of CP-AMPARs suggests that synaptic dynamics as well as learning rules are differentially implemented in local circuits and has important implications not just in health but also in disease states such as epilepsy.

Ion Channels in Genetic Epilepsy: From Genes and Mechanisms to Disease-Targeted Therapies.

Epilepsy is a common and serious neurologic disease with a strong genetic component. Genetic studies have identified an increasing collection of disease-causing genes. The impact of these genetic discoveries is wide reaching-from precise diagnosis and classification of syndromes to the discovery and validation of new drug targets and the development of disease-targeted therapeutic strategies. About 25% of genes identified in epilepsy encode ion channels. Much of our understanding of disease mechanisms comes from work focused on this class of protein. In this study, we review the genetic, molecular, and physiologic evidence supporting the pathogenic role of a number of different voltage- and ligand-activated ion channels in genetic epilepsy. We also review proposed disease mechanisms for each ion channel and highlight targeted therapeutic strategies.

Differential Regulation of Evoked and Spontaneous Release by Presynaptic NMDA Receptors.

Presynaptic NMDA receptors (preNMDARs) control synaptic release, but it is not well understood how. Rab3-interacting molecules (RIMs) provide scaffolding at presynaptic active zones and are involved in vesicle priming. Moreover, c-Jun N-terminal kinase (JNK) has been implicated in regulation of spontaneous release. We demonstrate that, at connected layer 5 pyramidal cell pairs of developing mouse visual cortex, Mg2+-sensitive preNMDAR signaling upregulates replenishment of the readily releasable vesicle pool during high-frequency firing. In conditional RIM1αß deletion mice, preNMDAR upregulation of vesicle replenishment was abolished, yet preNMDAR control of spontaneous release was unaffected. Conversely, JNK2 blockade prevented Mg2+-insensitive preNMDAR signaling from regulating spontaneous release, but preNMDAR control of evoked release remained intact. We thus discovered that preNMDARs signal differentially to control evoked and spontaneous release by independent and non-overlapping mechanisms. Our findings suggest that preNMDARs may sometimes signal metabotropically and support the emerging principle that evoked and spontaneous release are distinct processes. VIDEO ABSTRACT.

Synapse-specific expression of calcium-permeable AMPA receptors in neocortical layer 5.

KEY POINTS: In the hippocampus, calcium-permeable AMPA receptors have been found in a restricted subset of neuronal types that inhibit other neurons, although their localization in the neocortex is less well understood. In the present study, we looked for calcium-permeable AMPA receptors in two distinct populations of neocortical inhibitory neurons: basket cells and Martinotti cells. We found them in the former but not in the latter. Furthermore, in basket cells, these receptors were associated with particularly fast responses. Computer modelling predicted (and experiments verified) that fast calcium-permeable AMPA receptors enable basket cells to respond rapidly, such that they promptly inhibit neighbouring cells and shut down activity. The results obtained in the present study help our understanding of pathologies such as stroke and epilepsy that have been associated with disordered regulation of calcium-permeable AMPA receptors. ABSTRACT: AMPA-type glutamate receptors (AMPARs) lacking an edited GluA2 subunit are calcium-permeable (CP) and contribute to synaptic plasticity in several hippocampal interneuron types, although their precise role in the neocortex is not well described. We explored the presence of CP-AMPARs at pyramidal cell (PC) inputs to Martinotti cells (MCs) and basket cells (BCs) in layer 5 of the developing mouse visual cortex (postnatal days 12-21). GluA2 immunolabelling was stronger in MCs than in BCs. A differential presence of CP-AMPARs at PC-BC and PC-MC synapses was confirmed electrophysiologically, based on measures of spermine-dependent rectification and CP-AMPAR blockade by 1-naphtyl acetyl spermine using recordings from synaptically connected cell pairs, NPEC-AMPA uncaging and miniature current recordings. In addition, CP-AMPAR expression in BCs was correlated with rapidly decaying synaptic currents. Computer modelling predicted that this reduces spike latencies and sharpens suprathreshold responses in BCs, which we verified experimentally using the dynamic clamp technique. Thus, the synapse-specific expression of CP-AMPARs may critically influence both plasticity and information processing in neocortical microcircuits.

Target-specific expression of presynaptic NMDA receptors in neocortical microcircuits.

Traditionally, NMDA receptors are located postsynaptically; yet, putatively presynaptic NMDA receptors (preNMDARs) have been reported. Although implicated in controlling synaptic plasticity, their function is not well understood and their expression patterns are debated. We demonstrate that, in layer 5 of developing mouse visual cortex, preNMDARs specifically control synaptic transmission at pyramidal cell inputs to other pyramidal cells and to Martinotti cells, while leaving those to basket cells unaffected. We also reveal a type of interneuron that mediates ascending inhibition. In agreement with synapse-specific expression, we find preNMDAR-mediated calcium signals in a subset of pyramidal cell terminals. A tuned network model predicts that preNMDARs specifically reroute information flow in local circuits during high-frequency firing, in particular by impacting frequency-dependent disynaptic inhibition mediated by Martinotti cells, a finding that we experimentally verify. We conclude that postsynaptic cell type determines presynaptic terminal molecular identity and that preNMDARs govern information processing in neocortical columns.