An apamin-sensitive Ca2+-activated K+ current in hippocampal pyramidal neurons

In hippocampal and other cortical neurons, action potentials are followed by afterhyperpolarizations (AHPs) generated by the activation of small-conductance Ca2+-activated K+ channels (SK channels). By shaping the neuronal firing pattern, these AHPs contribute to the regulation of excitability and to the encoding function of neurons. Here we report that CA1 pyramidal neurons express an AHP current that is suppressed by apamin and is involved in the control of repetitive firing. This current presents distinct kinetic and pharmacological features, and it is modulated differently than the apamin-insensitive slow AHP current. Furthermore, our in situ hybridizations show that the apamin-sensitive SK subunits are expressed in CA1 pyramidal neurons, providing a potential molecular correlate to the apamin-sensitive AHP current. Altogether, these results clarify the discrepancy between the reported high density of apamin-binding sites in the CA1 region and the apparent lack of an apamin-sensitive current in CA1 pyramidal neurons, and they may explain the effects of this toxin on hippocampal synaptic plasticity and learning.     

Distinct potassium channels on pain-sensing neurons.

Differential expression of ion channels contributes functional diversity to sensory neuron signaling. We find nerve injury induced by the Chung model of neuropathic pain leads to striking reductions in voltage-gated K(+) (Kv) channel subunit expression in dorsal root ganglia (DRG) neurons, suggesting a potential molecular mechanism for hyperexcitability of injured nerves. Moreover, specific classes of DRG neurons express distinct Kv channel subunit combinations. Importantly, Kv1.4 is the sole Kv1 alpha subunit expressed in smaller diameter neurons, suggesting that homomeric Kv1.4 channels predominate in A delta and C fibers arising from these cells. These neurons are presumably nociceptors, because they also express the VR-1 capsaicin receptor, calcitonin gene-related peptide, and/or Na(+) channel SNS/PN3/Nav1.8. In contrast, larger diameter neurons associated with mechanoreception and proprioception express high levels of Kv1.1 and Kv1.2 without Kv1.4 or other Kv1 alpha subunits, suggesting that heteromers of these subunits predominate on large, myelinated afferent axons that extend from these cells.     

Elimination of fast inactivation in Kv4 A-type potassium channels by an auxiliary subunit domain

The Kv4 A-type potassium currents contribute to controlling the frequency of slow repetitive firing and back-propagation of action potentials in neurons and shape the action potential in heart. Kv4 currents exhibit rapid activation and inactivation and are specifically modulated by K-channel interacting proteins (KChIPs). Here we report the discovery and functional characterization of a modular K-channel inactivation suppressor (KIS) domain located in the first 34 aa of an additional KChIP (KChIP4a). Coexpression of KChIP4a with Kv4 alpha-subunits abolishes fast inactivation of the Kv4 currents in various cell types, including cerebellar granule neurons. Kinetic analysis shows that the KIS domain delays Kv4.3 opening, but once the channel is open, it disrupts rapid inactivation and slows Kv4.3 closing. Accordingly, KChIP4a increases the open probability of single Kv4.3 channels. The net effects of KChIP4a and KChIP1-3 on Kv4 gating are quite different. When both KChIP4a and KChIP1 are present, the Kv4.3 current shows mixed inactivation profiles dependent on KChIP4a/KChIP1 ratios. The KIS domain effectively converts the A-type Kv4 current to a slowly inactivating delayed rectifier-type potassium current. This conversion is opposite to that mediated by the Kv1-specific "ball" domain of the Kv beta 1 subunit. Together, these results demonstrate that specific auxiliary subunits with distinct functions actively modulate gating of potassium channels that govern membrane excitability.     

Targeted dendrotomy reveals active and passive contributions of the dendritic tree to synaptic integration and neuronal output

Neurons typically function as transduction devices, converting patterns of synaptic inputs, received on the dendrites, into trains of output action potentials in the axon. This transduction process is surprisingly complex and has been proposed to involve a two-way dialogue between axosomatic and dendritic compartments that can generate mutually interacting regenerative responses. To manipulate this process, we have developed a new approach for rapid and reversible occlusion or amputation of the primary dendrites of individual neurons in brain slices. By applying these techniques to cerebellar Purkinje and layer 5 cortical pyramidal neurons, we show directly that both the active and passive properties of dendrites differentially affect firing in the axon depending on the strength of stimulation. For weak excitation, dendrites act as a passive electrical load, raising spike threshold and dampening axonal excitability. For strong excitation, dendrites contribute regenerative inward currents, which trigger burst firing and enhance neuronal excitability. These findings provide direct support for the idea that dendritic morphology and conductances act in concert to regulate the excitability of the neuron.     

Riluzole prevents soluble Aβ1–42 oligomers-induced perturbation of spontaneous discharge in the hippocampal CA1 region of rats

Abnormal accumulation of soluble amyloid beta (Aβ) is believed to cause malfunction of neurons in Alzheimer’s disease (AD). The hippocampus is one of the earliest affected brain regions in AD. However, little effort has been made to investigate the effects of soluble Aβ_1–42 oligomers on discharge properties of hippocampal neurons in vivo. This study was designed to examine the effects of soluble Aβ_1–42 oligomers on the discharge properties of hippocampal CA1 neurons using extracellular single-unit recordings in vivo. The protective effects of riluzole (RLZ) were also investigated for the prevention of soluble oligomers of Aβ_1–42-induced alterations in the spontaneous discharge of hippocampal neurons. The results showed that (1) the mean frequency of spontaneous discharge was increased by the local application of 100?μM Aβ_1–42 oligomers; (2) Aβ_1–42 oligomers also induced alterations of the neuronal firing patterns in the hippocampal CA1 region; and (3) pretreatment with 20?μM RLZ effectively inhibited the Aβ_1–42-induced enhancement of spontaneous discharge and alterations of neuronal firing patterns in CA1 neurons. Our study suggested that Aβ_1–42 oligomers induced hyperactivity and perturbed the firing patterns in hippocampal neurons. RLZ may provide neuroprotective effects on the Aβ_1–42-induced perturbation of neuronal activities in the hippocampal region of rats.     

Genotype–phenotype correlations in neonatal epilepsies caused by mutations in the voltage sensor of Kv7.2 potassium channel subunits

Mutations in the K_V7.2 gene encoding for voltage-dependent K⁺ channel subunits cause neonatal epilepsies with wide phenotypic heterogeneity. Two mutations affecting the same positively charged residue in the S₄ domain of K_V7.2 have been found in children affected with benign familial neonatal seizures (R213W mutation) or with neonatal epileptic encephalopathy with severe pharmacoresistant seizures and neurocognitive delay, suppression-burst pattern at EEG, and distinct neuroradiological features (R213Q mutation). To examine the molecular basis for this strikingly different phenotype, we studied the functional characteristics of mutant channels by using electrophysiological techniques, computational modeling, and homology modeling. Functional studies revealed that, in homomeric or heteromeric configuration with K_V7.2 and/or K_V7.3 subunits, both mutations markedly destabilized the open state, causing a dramatic decrease in channel voltage sensitivity. These functional changes were (i) more pronounced for channels incorporating R213Q- than R213W-carrying K_V7.2 subunits; (ii) proportional to the number of mutant subunits incorporated; and (iii) fully restored by the neuronal K_v7 activator retigabine. Homology modeling confirmed a critical role for the R213 residue in stabilizing the activated voltage sensor configuration. Modeling experiments in CA1 hippocampal pyramidal cells revealed that both mutations increased cell firing frequency, with the R213Q mutation prompting more dramatic functional changes compared with the R213W mutation. These results suggest that the clinical disease severity may be related to the extent of the mutation-induced functional K⁺ channel impairment, and set the preclinical basis for the potential use of K_v7 openers as a targeted anticonvulsant therapy to improve developmental outcome in neonates with K_V7.2 encephalopathy.     

Pathogenic plasticity of Kv7.2/3 channel activity is essential for the induction of tinnitus

Tinnitus, the perception of phantom sound, is often a debilitating condition that affects many millions of people. Little is known, however, about the molecules that participate in the induction of tinnitus. In brain slices containing the dorsal cochlear nucleus, we reveal a tinnitus-specific increase in the spontaneous firing rate of principal neurons (hyperactivity). This hyperactivity is observed only in noise-exposed mice that develop tinnitus and only in the dorsal cochlear nucleus regions that are sensitive to high frequency sounds. We show that a reduction in Kv7.2/3 channel activity is essential for tinnitus induction and for the tinnitus-specific hyperactivity. This reduction is due to a shift in the voltage dependence of Kv7 channel activation to more positive voltages. Our in vivo studies demonstrate that a pharmacological manipulation that shifts the voltage dependence of Kv7 to more negative voltages prevents the development of tinnitus. Together, our studies provide an important link between the biophysical properties of the Kv7 channel and the generation of tinnitus. Moreover, our findings point to previously unknown biological targets for designing therapeutic drugs that may prevent the development of tinnitus in humans.     

Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in the hippocampal CA3 circuit

Homeostatic plasticity of intrinsic excitability goes hand in hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging, and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2 to 3 d induces an up-regulation of both synaptic transmission at CA3–CA3 connections and intrinsic excitability of CA3 pyramidal neurons. Intrinsic plasticity was found to be mediated by a reduction of Kv1.1 channel density at the axon initial segment. In activity-deprived circuits, CA3–CA3 synapses were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a reduction in presynaptic Kv1.1 function. Further support for the down-regulation of axonal Kv1.1 channels in activity-deprived neurons was the broadening of action potentials measured in the axon. We conclude that regulation of the axonal Kv1.1 channel constitutes a major mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

Chronic modulation of activity regimes in neuronal circuits induces homeostatic plasticity. This implicates a regulation of both intrinsic excitability (homeostatic plasticity of intrinsic excitability) and synaptic transmission (homeostatic plasticity of synaptic transmission) to maintain network activity within physiological bounds (1). In most cases, these two forms of homeostatic plasticity act synergistically but involve different molecular actors. Homeostatic intrinsic plasticity is associated with the regulation of voltage-gated ion channels (2–9), while homeostatic synaptic plasticity involves the regulation of postsynaptic receptors to neurotransmitters (10–17) or the regulation of the readily releasable pool of synaptic vesicles (18–20). However, the function of voltage-gated ion channels is not limited to the control of intrinsic excitability. Several studies point to the role of axonal voltage-gated channels in shaping presynaptic action potential (AP) waveform and subsequently controlling neurotransmitter release and synaptic transmission (21–35). Moreover, some studies describe homeostatic plasticity of the AP waveform via voltage-gated channel regulation (36–38), while other studies report an absence of this phenomenon (39).Kv1.1 channels are responsible for the fast-activating, slow-inactivating D-type current (I_D) in CA3 neurons. This current has been shown to create a delay in the onset of the first AP and to determine intrinsic excitability in various neuronal types, including CA1 and CA3 pyramidal neurons of the hippocampus (3, 40), L5 pyramidal neurons of the cortex (26, 34), and L2/3 fast-spiking interneurons of the somatosensory cortex (41, 42). Furthermore, Kv1.1 channels have been shown to control axonal AP width and subsequently presynaptic calcium entry and neurotransmitter release. In fact, pharmacological blockade of Kv1.1 channels broadens presynaptic APs and increases synaptic transmission at neocortical and hippocampal glutamatergic synapses and at cerebellar GABAergic synapses (21, 22, 26, 30, 32, 41, 43, 44). Moreover, Kv1.1 channels have been shown to be responsible for the phenomenon of depolarization-induced analog digital facilitation of synaptic transmission (d-ADF). In fact, at CA3–CA3 and L5–L5 synapses, a somatic subthreshold depolarization of the presynaptic cell leads to inactivation of axonal Kv1.1 channels, inducing the broadening of the presynaptic AP, an increase in spike-evoked calcium entry, and a facilitation of presynaptic glutamate release (26, 31, 33, 34, 45, 46). Therefore, Kv1.1 channels control both intrinsic excitability and glutamate release in CA3 pyramidal neurons.Kv1.1 channels have been shown to be involved in homeostatic regulation of neuronal excitability. Chronic activity enhancement by kainate application leads to an increase in I_D current and a decrease in excitability in dentate gyrus granule cells (47). Conversely, chronic sensory deprivation leads to Kv1.1 channel down-regulation and enhancement of excitability in the avian cochlear nucleus (48). In this study, we examined whether the increase in synaptic transmission could also be due to Kv1.1 channel down-regulation, which would possibly explain the observed synergy between homeostatic plasticity of excitability and synaptic transmission.We show here that chronic activity deprivation induced with an antagonist of ionotropic glutamate receptors (kynurenate) in hippocampal organotypic cultures provokes both an increase in CA3 pyramidal cells excitability and an enhancement of synaptic transmission at monosynaptically connected CA3 neurons. Deprived cultures display a decrease in Kv1.1 channel staining in the axon initial segment. Bath application of dendrotoxin-K (DTX-K), a selective blocker of Kv1.1 channels, leads to a larger excitability increase in control cultures than in deprived cultures. Focal puffing of DTX-K on the axon increases excitability in control but not in deprived cultures, showing that homeostatic plasticity of excitability in deprived cultures is partly due to the down-regulation of axonal Kv1.1 channels. In addition, we found that axonal Kv1.1 down-regulation in deprived cultures is responsible for a spike broadening in CA3 neurons, leading to elevated release probability at CA3–CA3 synapses. Consistent with these observations, d-ADF, a Kv1.1-dependent form of synaptic facilitation, is present in control cultures but not in deprived cultures. Altogether, these results show that chronic activity blockade of the hippocampal CA3 circuit induces the down-regulation of axonal Kv1.1 channels leading to a homeostatic increase in both excitability and presynaptic release probability.     

Targeting the voltage sensor of Kv7.2 voltage-gated K+ channels with a new gating-modifier

The pore and gate regions of voltage-gated cation channels have been often targeted with drugs acting as channel modulators. In contrast, the voltage-sensing domain (VSD) was practically not exploited for therapeutic purposes, although it is the target of various toxins. We recently designed unique diphenylamine carboxylates that are powerful Kv7.2 voltage-gated K⁺ channel openers or blockers. Here we show that a unique Kv7.2 channel opener, NH29, acts as a nontoxin gating modifier. NH29 increases Kv7.2 currents, thereby producing a hyperpolarizing shift of the activation curve and slowing both activation and deactivation kinetics. In neurons, the opener depresses evoked spike discharges. NH29 dampens hippocampal glutamate and GABA release, thereby inhibiting excitatory and inhibitory postsynaptic currents. Mutagenesis and modeling data suggest that in Kv7.2, NH29 docks to the external groove formed by the interface of helices S1, S2, and S4 in a way that stabilizes the interaction between two conserved charged residues in S2 and S4, known to interact electrostatically, in the open state of Kv channels. Results indicate that NH29 may operate via a voltage-sensor trapping mechanism similar to that suggested for scorpion and sea-anemone toxins. Reflecting the promiscuous nature of the VSD, NH29 is also a potent blocker of TRPV1 channels, a feature similar to that of tarantula toxins. Our data provide a structural framework for designing unique gating-modifiers targeted to the VSD of voltage-gated cation channels and used for the treatment of hyperexcitability disorders.     

Electrical properties of T-type Ca channels

Neurons possess multiple types of voltage-dependent calcium channels. These channels are classified as either low-voltage-activated (LVA/T-type) or high-voltage-activated (HVA) consisting of L, N, P, Q and R subtypes. T-type Ca channels can be opened by small depolarization that are under the threshold for action potential generation. These T-type Ca channels may be involved in the sub threshold depolarizations that underlies neuronal bursting activities in neurons. Such burst firing might originate in the postsynaptic membrane of the neuronal dendrites.     

Ethanol exposure in early adolescence inhibits intrinsic neuronal plasticity via sigma-1 receptor activation in hippocampal CA1 neurons

Background: We demonstrated previously that rats exposed to chronic intermittent ethanol (CIE) vapors in early adolescence show increased magnitudes of long‐term potentiation (LTP) of excitatory transmission when recorded at dendritic synapses in hippocampus. Large amplitude LTP following CIE exposure is mediated by sigma‐1 receptors; however, not yet addressed is the role of sigma‐1 receptors in modulating the intrinsic properties of neurons to alter their action potential firing during LTP. Methods: Activity‐induced plasticity of spike firing was investigated using rat hippocampal slice recordings to measure changes in both field excitatory postsynaptic potentials (fEPSPs) and population spikes (pop. spikes) concomitantly at dendritic inputs and soma of CA1 pyramidal neurons, respectively. Results: We observed unique modifications in plasticity of action potential firing in hippocampal slices from CIE exposed adolescent rats, where the induction of large amplitude LTP by 100 Hz stimulations was accompanied by reduced CA1 neuronal excitability––reflected as decreased pop. spike efficacy and impaired activity‐induced fEPSP‐to‐spike (E‐S) potentiation. In contrast, LTP induction in ethanol‐naïve control slices resulted in increased spike efficacy and robust E‐S potentiation. E‐S potentiation impairments emerged at 24 hours after CIE treatment cessation, but not before the alcohol withdrawal period, and were restored with bath‐application of the sigma‐1 receptor selective antagonist BD1047, but not the NMDA receptor antagonist d ‐AP5. Further evidence revealed a significantly shortened somatic fEPSP time course in adolescent CIE‐withdrawn hippocampal slices during LTP; however, paired‐pulse data show no apparent correspondence between E‐S dissociation and altered recurrent feedback inhibition. Conclusions: Results here suggest that acute withdrawal from adolescent CIE exposure triggers sigma‐1 receptors that act to depress the efficacy of excitatory inputs in triggering action potentials during LTP. Such withdrawal‐induced depression of E‐S plasticity in hippocampus probably entails sigma‐1 receptor modulation of 1 or several voltage‐gated ion channels controlling the neuronal input–output dynamics.     

Characterization of the CA1 pyramidal neurons in rat model of hepatic cirrhosis: insights into their electrophysiological properties

Although the key contributors of altering neurological function in hepatic encephalopathy are relatively well known, the electrophysiological mechanism of CA1 damage, a key vulnerable area during hyperammonemia, have not yet been defined. Therefore, here we focus on the electrophysiological mechanisms of cognitive impairments following bile duct ligation (BDL). We performed patch-clamp recordings from the CA1 pyramidal neurons in hippocampus of male Wistar rats, which underwent sham or BDL surgery. A striking electrophysiological change of hippocampal neurons in experimental model of BDL was observed in the present study. Spontaneous firing frequency and rate of action potential (AP) rebound was decreased and afterhyperpolarization amplitude (AHP) was increased significantly in hippocampal cells of BDL animals compared to sham group. Together, the results suggest that altered intrinsic properties of the hippocampal neurons may contribute to the cognitive abnormalities during hepatic encephalopathy (HE), highlighting the electrophysiological mechanisms for providing new treatments against HE.

     

Melatonin ameliorates hippocampal nitric oxide production and large conductance calcium-activated potassium channel activity in chronic intermittent hypoxia

Melatonin protects against hippocampal injury induced by intermittent hypoxia (IH). IH-induced oxidative stress is associated with decreases in constitutive production of nitric oxide (NO) and in the activity of large conductance calcium-activated potassium (BK) channels in hippocampal neurons. We tested the hypothesis that administration of melatonin alleviates the NO deficit and impaired BK channel activity in the hippocampus of IH rats. Sprague-Dawley rats were injected with melatonin (10 mg/kg, i.p.) or vehicle before daily IH exposure for 8 hr for 7 days. The NO and intracellular calcium ([Ca2+]i) levels in the CA1 region of hippocampal slices were measured by electrochemical microsenor and spectrofluorometry, respectively. The activity of BK channels was recorded by patch-clamping electrophysiology in dissociated CA1 neurons. Malondialdehyde levels were increased in the hippocampus of hypoxic rats and were lowered by the melatonin treatment. Levels of NO under resting and hypoxic conditions, and the protein expression of neuronal NO synthase (nNOS) were significantly reduced in the CA1 neurons of hypoxic animals compared with the normoxic controls. These deficits were mitigated in the melatonin-treated hypoxic rats with an improved [Ca2+]i response to acute hypoxia. The open probability of BK channels was decreased in the hypoxic rats and was partially restored in the melatonin-treated animals, without alterations in the expression of channel subunits and unitary conductance. Acute treatment of melatonin had no significant effects on the BK channel activity or on the [Ca2+]i response to hypoxia. Collectively, these results suggest that melatonin ameliorates the constitutive NO production and BK channel activity via an antioxidant mechanism against an IH-induced down-regulation of nNOS expression in hippocampal neurons.     

Dynamic spike threshold reveals a mechanism for synaptic coincidence detection in cortical neurons in vivo

Cortical neurons are sensitive to the timing of their synaptic inputs. They can synchronize their firing on a millisecond time scale and follow rapid stimulus fluctuations with high temporal precision. These findings suggest that cortical neurons have an enhanced sensitivity to synchronous synaptic inputs that lead to rapid rates of depolarization. The voltage-gated currents underlying action potential generation may provide one mechanism to amplify rapid depolarizations. We have tested this hypothesis by analyzing the relations between membrane potential fluctuations and spike threshold in cat visual cortical neurons recorded intracellularly in vivo. We find that visual stimuli evoke broad variations in spike threshold that are caused in large part by an inverse relation between spike threshold and the rate of membrane depolarization preceding a spike. We also find that spike threshold is inversely related to the rate of rise of the action potential upstroke, suggesting that increases in spike threshold result from a decrease in the availability of Na(+) channels. By using a simple neuronal model, we show that voltage-gated Na(+) and K(+) conductances endow cortical neurons with an enhanced sensitivity to rapid depolarizations that arise from synchronous excitatory synaptic inputs. Thus, the basic mechanism responsible for action potential generation also enhances the sensitivity of cortical neurons to coincident synaptic inputs.     

Synaptic plasticity by antidromic firing during hippocampal network oscillations

Learning and other cognitive tasks require integrating new experiences into context. In contrast to sensory-evoked synaptic plasticity, comparatively little is known of how synaptic plasticity may be regulated by intrinsic activity in the brain, much of which can involve nonclassical modes of neuronal firing and integration. Coherent high-frequency oscillations of electrical activity in CA1 hippocampal neurons [sharp-wave ripple complexes (SPW-Rs)] functionally couple neurons into transient ensembles. These oscillations occur during slow-wave sleep or at rest. Neurons that participate in SPW-Rs are distinguished from adjacent nonparticipating neurons by firing action potentials that are initiated ectopically in the distal region of axons and propagate antidromically to the cell body. This activity is facilitated by GABA_A-mediated depolarization of axons and electrotonic coupling. The possible effects of antidromic firing on synaptic strength are unknown. We find that facilitation of spontaneous SPW-Rs in hippocampal slices by increasing gap-junction coupling or by GABA_A-mediated axon depolarization resulted in a reduction of synaptic strength, and electrical stimulation of axons evoked a widespread, long-lasting synaptic depression. Unlike other forms of synaptic plasticity, this synaptic depression is not dependent upon synaptic input or glutamate receptor activation, but rather requires L-type calcium channel activation and functional gap junctions. Synaptic stimulation delivered after antidromic firing, which was otherwise too weak to induce synaptic potentiation, triggered a long-lasting increase in synaptic strength. Rescaling synaptic weights in subsets of neurons firing antidromically during SPW-Rs might contribute to memory consolidation by sharpening specificity of subsequent synaptic input and promoting incorporation of novel information.     

Spontaneous seizure and memory loss in mice expressing an epileptic encephalopathy variant in the calmodulin-binding domain of Kv7.2

Epileptic encephalopathy (EE) is characterized by seizures that respond poorly to antiseizure drugs, psychomotor delay, and cognitive and behavioral impairments. One of the frequently mutated genes in EE is KCNQ2, which encodes the K_v7.2 subunit of voltage-gated K_v7 potassium channels. K_v7 channels composed of K_v7.2 and K_v7.3 are enriched at the axonal surface, where they potently suppress neuronal excitability. Previously, we reported that the de novo dominant EE mutation M546V in human K_v7.2 blocks calmodulin binding to K_v7.2 and axonal surface expression of K_v7 channels via their intracellular retention. However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral comorbidities remains unknown. Here, we report conditional transgenic cKcnq2^+/M547V mice, in which expression of mouse K_v7.2-M547V (equivalent to human K_v7.2-M546V) is induced in forebrain excitatory pyramidal neurons and astrocytes. These mice display early mortality, spontaneous seizures, enhanced seizure susceptibility, memory impairment, and repetitive behaviors. Furthermore, hippocampal pathology shows widespread neurodegeneration and reactive astrocytes. This study demonstrates that the impairment in axonal surface expression of K_v7 channels is associated with epileptic seizures, cognitive and behavioral deficits, and neuronal loss in KCNQ2-related EE.

Epileptic encephalopathies (EEs) are a collection of heterogeneous disorders in which early-onset severe seizures contribute to developmental delay and progressive cognitive and behavioral impairments (1). Current treatments for EEs have limited efficacy in alleviating seizures and comorbidities (2), posing an urgent need to understand the etiology of EEs and find new therapeutic targets. Recent discoveries of epilepsy-related genes in multiple laboratories and through the large Epi4K, EpiPM, and EuroEPINOMICS-RES consortia have identified a diverse array of proteins that may contribute to epileptogenesis (3–5). Among them, dominant variants associated with benign familial neonatal epilepsy (BFNE) and EE have been found in KCNQ2 and KCNQ3 genes, which encode the K_v7.2 and K_v7.3 subunits of voltage-gated potassium (K⁺) channel subfamily Q (K_v7) (https://www.rikee.org; ClinVar Database, National Center for Biotechnology Information [NCBI]).Neuronal K_v7 channels are mostly heterotetrameric channels of K_v7.2 and K_v7.3 subunits (6), which have overlapping distribution in the central nervous system including the cerebral cortex and hippocampus (7). In neurons, they are preferentially localized to the axonal plasma membrane with the highest concentration at the axonal initial segment (AIS) (7, 8), where the action potential (AP) initiates (9). They give rise to the slowly activating and noninactivating outward K⁺ current termed M current (I_M) (6). Because they open at subthreshold potentials (6), I_M potently suppresses AP firing (6, 10, 11), underscoring their critical roles in reducing neuronal excitability. By contrast, activation of G_q-coupled receptors, including muscarinic acetylcholine receptors, inhibits I_M by depleting the lipid cofactor PIP₂, resulting in enhanced AP firing (12).To date, 193 dominant variants in KCNQ2 and 2 variants in KCNQ3 have been identified in patients with EE (https://www.rikee.org; ClinVar Database, NCBI). EE variants are clustered at the functional domains of K_v7.2 important for voltage-dependent opening of K_v7 channels (13) and typically decrease the function of heterotetrameric channels by 20 to 75% (13–17). EE variants are also enriched at helices A and B in the intracellular C-terminal tail of K_v7.2 (13, 14, 16), which mediate calmodulin (CaM) binding critical for axonal enrichment of K_v7 channels (18). Among these variants, a mutation of methionine at amino acid position 546 to valine (M546V) was found in a male patient who displayed drug-resistant neonatal tonic-clonic seizures and later developed profound intellectual and language disability, spasticity, and autistic behavior (15). While this mutation in helix B abolishes current expression of homomeric but not heteromeric channels in heterologous cells (14, 17), it severely reduces CaM binding and axonal surface expression of heteromeric channels in cultured hippocampal neurons (14). This mutation also induces ubiquitination and proteasomal degradation of K_v7.2, whereas the presence of K_v7.3 blocks this degradation and accumulates ubiquitinated K_v7.2 (14). However, whether these pathogenic mechanisms underlie epileptic seizures and behavioral deficits in EE remains unknown.In this study, we investigated the contribution of the EE variant M546V by generating conditional transgenic mice in which heterozygous expression of mouse K_v7.2-M547V was induced in forebrain excitatory pyramidal neurons. M546 in the human K_v7.2 is conserved in the mouse K_v7.2 at amino acid position 547. These mice showed widespread neurodegeneration and reactive astrogliosis in the hippocampus and cortex, and displayed spontaneous seizures and cognitive deficit, providing a causal link between M546V-mediated disruption of axonal surface expression of K_v7 channels and KCNQ2-associated EE.     

Effects of estrogen on neuronal excitability in the hippocampal-septal-hypothalamic system.

The effects of electrical stimulation of the medial preoptic area (MPO) upon unit firing in the periventricular arcuate nucleus, and that of the dorsal hippocampus (DHPC) on medial septo-preoptic and arcuate (ARC) neurons, were investigated in Wistar and Sprague-Dawley female rats throughout the 4-day estrous cycle. Unit activity was recorded using stereotaxically-oriented tungsten microelectrodes under light urethane anesthesia. Repetitive stimulation of monophasic square waves varying only in current intensity was used. The following results were obtained: (a) An increase in activity of all ARC neurons recorded was induced by MPO stimulation on each day of the estrous cycle. (b) The minimum current (threshold) effective in increasing activity in the ARC neurons varied throughout the estrous cycle; the lowest threshold was observed in proestrus and the highest on the first day of diestrus. Also, the threshold current of MPO stimulation required in increase ARC activity was found to be elevated after ovariectomy and markedly reduced to the levels of the proestrous animal by estrogen treatment. (c) Stimulation of the DHPC (field CA3) increased activity in the medial septum but decreased activity in the MPO. Two pools of neurons, one increased and one decreased by DHPC stimulation, were observed in the ARC. (d) Variation in the threshold hippocampal stimulation during the estrous cycle was observed in the response of MPO and ARC neurons (the stimulation was effective only in proestrus and estrus); but not in that of medial septal neurons. (e) In addition, the spontaneous activity of septal; MPO; and ARC neurons was increased at proestrus or after estrogen injection. The present results suggest that plasma levels of estrogen play an essential role in the cyclic process of the regulation of ovulation by way of the selective facilitation of neuronal excitability in specific functional neural pathways. Furthermore, the results support the existence of a hippocampal inhibitory projection originating in field CA3 and terminating in the final common pathway, MPO to ARC.     

The effects of apelin on the electrical activity of hypothalamic magnocellular vasopressin and oxytocin neurons and somatodendritic Peptide release

Apelin, a novel peptide originally isolated from bovine stomach tissue extracts, is widely but selectively distributed throughout the nervous system. Vasopressin and oxytocin are synthesized in the magnocellular neurons of the hypothalamic supraoptic nucleus (SON) and paraventricular nucleus, which are apelin-rich regions in the central nervous system. We made extracellular electrophysiological recordings from the transpharyngeally exposed SON of urethane-anaesthetized rats to assess the role of apelin in the control of the firing activity of identified magnocellular vasopressin and oxytocin neurons in vivo. Apelin-13 administration onto SON neurons via microdialysis revealed cell-specific responses; apelin-13 increased the firing rates of vasopressin cells but had no effect on the firing rate of oxytocin neurons. A direct excitatory effect of apelin-13 on vasopressin cell activity is also supported by our in vitro studies showing depolarization of membrane potential and increase in action potential firing. To assess the effects of apelin-13 on somatodendritic peptide release, we used in vitro release studies from SON explants in combination with highly sensitive and specific RIA. Apelin-13 decreases basal (by 78%; P < 0.05; n = 6) and potassium-stimulated (by 57%; P < 0.05; n = 6) vasopressin release but had no effect on somatodendritic oxytocin release. Taken together, our data suggest a local autocrine feedback action of apelin on magnocellular vasopressin neurons. Furthermore, these data show a marked dissociation between axonal and dendritic vasopressin release with a decrease in somatodendritic release but an increase in electrical activity at the cell bodies, indicating that release from these two compartments can be regulated wholly independently.