Medium Afterhyperpolarization Research Articles

Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice.

Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10μM), an opener of Kv7/M channels, produced opposite effects to XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5μM) normalized CA1 PC excitability of Fmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5-10μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. KEY POINTS: Dorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. Kv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Kv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. A Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. Ex vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome.

Dysfunction of Small-Conductance Ca2+-Activated Potassium (SK) Channels Drives Amygdala Hyperexcitability and Neuropathic Pain Behaviors: Involvement of Epigenetic Mechanisms.

Neuroplasticity in the amygdala and its central nucleus (CeA) is linked to pain modulation and pain behaviors, but cellular mechanisms are not well understood. Here, we addressed the role of small-conductance Ca2+-activated potassium (SK) channels in pain-related amygdala plasticity. The facilitatory effects of the intra-CeA application of an SK channel blocker (apamin) on the pain behaviors of control rats were lost in a neuropathic pain model, whereas an SK channel activator (NS309) inhibited pain behaviors in neuropathic rats but not in sham controls, suggesting the loss of the inhibitory behavioral effects of amygdala SK channels. Brain slice electrophysiology found hyperexcitability of CeA neurons in the neuropathic pain condition due to the loss of SK channel-mediated medium afterhyperpolarization (mAHP), which was accompanied by decreased SK2 channel protein and mRNA expression, consistent with a pretranscriptional mechanisms. The underlying mechanisms involved the epigenetic silencing of the SK2 gene due to the increased DNA methylation of the CpG island of the SK2 promoter region and the change in methylated CpG sites in the CeA in neuropathic pain. This study identified the epigenetic dysregulation of SK channels in the amygdala (CeA) as a novel mechanism of neuropathic pain-related plasticity and behavior that could be targeted to control abnormally enhanced amygdala activity and chronic neuropathic pain.

AKAP150-anchored PKA regulates synaptic transmission and plasticity, neuronal excitability and CRF neuromodulation in the mouse lateral habenula

The scaffolding A-kinase anchoring protein 150 (AKAP150) is critically involved in kinase and phosphatase regulation of synaptic transmission/plasticity, and neuronal excitability. Emerging evidence also suggests that AKAP150 signaling may play a key role in brain’s processing of rewarding/aversive experiences, however its role in the lateral habenula (LHb, as an important brain reward circuitry) is completely unknown. Using whole cell patch clamp recordings in LHb of male wildtype and ΔPKA knockin mice (with deficiency in AKAP-anchoring of PKA), here we show that the genetic disruption of PKA anchoring to AKAP150 significantly reduces AMPA receptor-mediated glutamatergic transmission and prevents the induction of presynaptic endocannabinoid-mediated long-term depression in LHb neurons. Moreover, ΔPKA mutation potentiates GABAA receptor-mediated inhibitory transmission while increasing LHb intrinsic excitability through suppression of medium afterhyperpolarizations. ΔPKA mutation-induced suppression of medium afterhyperpolarizations also blunts the synaptic and neuroexcitatory actions of the stress neuromodulator, corticotropin releasing factor (CRF), in mouse LHb. Altogether, our data suggest that AKAP150 complex signaling plays a critical role in regulation of AMPA and GABAA receptor synaptic strength, glutamatergic plasticity and CRF neuromodulation possibly through AMPA receptor and potassium channel trafficking and endocannabinoid signaling within the LHb.

KCa 2.2 (KCNN2): A physiologically and therapeutically important potassium channel.

One group of the K+ ion channels, the small-conductance Ca2+ -activated potassium channels (KCa 2.x, also known as SK channels family), is widely expressed in neurons as well as the heart, endothelial cells, etc. They are named small-conductance Ca2+ -activated potassium channels (SK channels) due to their comparatively low single-channel conductance of about ~10 pS. These channels are insensitive to changes in membrane potential and are activated solely by rises in the intracellular Ca2+ . According to the phylogenic research done on the KCa 2.x channels family, there are three channels' subtypes: KCa 2.1, KCa 2.2, and KCa 2.3, which are encoded by KCNN1, KCNN2, and KCNN3 genes, respectively. The KCa 2.x channels regulate neuronal excitability and responsiveness to synaptic input patterns. KCa 2.x channels inhibit excitatory postsynaptic potentials (EPSPs) in neuronal dendrites and contribute to the medium afterhyperpolarization (mAHP) that follows the action potential bursts. Multiple brain regions, including the hippocampus, express the KCa 2.2 channel encoded by the KCNN2 gene on chromosome 5. Of particular interest, rat cerebellar Purkinje cells express KCa 2.2 channels, which are crucial for various cellular processes during development and maturation. Patients with a loss-of-function of KCNN2 mutations typically exhibit extrapyramidal symptoms, cerebellar ataxia, motor and language developmental delays, and intellectual disabilities. Studies have revealed that autosomal dominant neurodevelopmental movement disorders resembling rodent symptoms are caused by heterozygous loss-of-function mutations, which are most likely to induce KCNN2 haploinsufficiency. The KCa 2.2 channel is a promising drug target for spinocerebellar ataxias (SCAs). SCAs exhibit the dysregulation of firing in cerebellar Purkinje cells which is one of the first signs of pathology. Thus, selective KCa 2.2 modulators are promising potential therapeutics for SCAs.

Harmine exerts anxiolytic effects by regulating neuroinflammation and neuronal plasticity in the basolateral amygdala.

Increasing evidence indicates that an altered immune system is closely linked to the pathophysiology of anxiety disorders, and inhibition of neuroinflammation may represent an effective therapeutic strategy to treat anxiety disorders. Harmine, a beta-carboline alkaloid in various medicinal plants, has been widely reported to display anti-inflammatory and potentially anxiolytic effects. However, the exact underlying mechanisms are not fully understood. Our recent study has demonstrated that dysregulation of neuroplasticity in the basolateral amygdala (BLA) contributes to the pathological processes of inflammation-related anxiety. In this study, using a mouse model of anxiety challenged with Escherichia coli lipopolysaccharide (LPS), we found that harmine alleviated LPS-induced anxiety-like behaviors in mice. Mechanistically, harmine significantly prevented LPS-induced neuroinflammation by suppressing the expression of pro-inflammatory cytokines including IL-1β and TNF-α. Meanwhile, ex vivo whole-cell slice electrophysiology combined with optogenetics showed that LPS-induced increase of medial prefrontal cortex (mPFC)-driven excitatory but not inhibitory synaptic transmission onto BLA projection neurons, thereby alleviating LPS-induced shift of excitatory/inhibitory balance towards excitation. In addition, harmine attenuated the increased intrinsic neuronal excitability of BLA PNs by reducing the medium after-hyperpolarization. In conclusion, our findings provide new evidence that harmine may exert its anxiolytic effect by downregulating LPS-induced neuroinflammation and restoring the changes in neuronal plasticity in BLA PNs.

Roles of nucleus accumbens shell small-conductance calcium-activated potassium channels in the conditioned fear freezing

BackgroundPosttraumatic stress disorder (PTSD), a psychiatric disorder caused by stressful events, is characterized by long-lasting fear memory. The nucleus accumbens shell (NAcS) is a key brain region that regulates fear-associated behavior. Small-conductance calcium-activated potassium channels (SK channels) play a key role in regulating the excitability of NAcS medium spiny neurons (MSNs) but their mechanisms of action in fear freezing are unclear. MethodWe established an animal model of traumatic memory using conditioned fear freezing paradigm, and investigated the alterations in SK channels of NAc MSNs subsequent to fear conditioning in mice. We then utilized an adeno-associated virus (AAV) transfection system to overexpress the SK3 subunit and explore the function of the NAcS MSNs SK3 channel in conditioned fear freezing. ResultsFear conditioning activated NAcS MSNs with enhanced excitability and reduced the SK channel-mediated medium after-hyperpolarization (mAHP) amplitude. The expression of NAcS SK3 were also reduced time-dependently. The overexpression of NAcS SK3 impaired conditioned fear consolidation without affecting conditioned fear expression, and blocked fear conditioning-induced alterations in NAcS MSNs excitability and mAHP amplitude. Additionally, the amplitudes of mEPSC, AMPAR/NMDAR ratio, and membrane surface GluA1/A2 expression in NAcS MSNs was increased by fear conditioning and returned to normal levels upon SK3 overexpression, indicating that fear conditioning-induced decrease of SK3 expression caused postsynaptic excitation by facilitating AMPAR transmission to the membrane. ConclusionThese findings show that the NAcS MSNs SK3 channel plays a critical role in conditioned fear consolidation and that it may influence PTSD pathogenesis, making it a potential therapeutic target against PTSD.

Cell‐type Specific Effects of AD Mutations on Hippocampal Intrinsic Firing Properties in Genetically Diverse Mice

AbstractBackgroundCognitive resilience to Alzheimer’s disease (AD) is a phenomenon whereby individuals with known AD mutations or high levels of pathology exhibit better than predicted cognitive performance. Identification of the factors governing resilience may lead to novel therapeutic interventions to treat AD. Analysis of hippocampal transcriptional profiles from susceptible and resilient AD‐BXD strains indicates that transcriptomic changes in excitatory neurons in CA1 and dentate gyrus (DG) are associated with resilience and we now seek to examine physiological changes.MethodWe adapted the Patch‐Seq method (Cadwell et al., 2017) to investigate cell type‐specific transcriptional and electrophysiological changes associated with AD using the AD‐BXD genetic reference panel (Neuner et al., 2019). Whole‐cell patch clamp was used to evaluate intrinsic excitability of neurons within the DG and CA1 of 14m old mice with cognitive deficits compared to age matched controls; their cellular contents, including the nucleus, were collected after each recording to characterize the transcriptome.ResultComparison of the intrinsic properties of DG and CA1 neurons of hippocampal slices from 5XFAD mice and their non‐transgenic (Ntg) counterparts revealed that DG granule cells from 5XFAD mice have a lower input resistance and an increased sag ratio in response to hyperpolarizing current injections. Pyramidal neurons from the CA1 of 5xFAD mice exhibit significantly more depolarized action potential thresholds, a significant increase in the medium afterhyperpolarization (mAHP) and sag ratio compared to Ntg controls. While these intrinsic properties are proxy measures for neuronal excitability, we did not observe a significant effect of genotype on firing rate in response to 1s and 15s depolarizing current steps.ConclusionOur findings newly demonstrate disease related changes to intrinsic firing properties of DG granule cells at 14mo indicating that these neurons may be hypoexcitable. Also found, CA1 neurons from AD mice exhibit an increased mAHP and changes to sag ratio and AP threshold, indicating they too become less excitable. Future work will add additional AD‐BXD strains characterized as resilient or susceptible to the AD transgene to discover changes in intrinsic properties that could underlie resilience. We will also use an integrated analysis of transcriptomic and electrophysiological data to identify relevant functional targets for validation.

Activation of histamine type 2 receptors enhances intrinsic excitability of medium spiny neurons in the nucleus accumbens.

Histaminergic neurons are exclusively located in the hypothalamic tuberomammillary nucleus, from where they project to many brain areas including the nucleus accumbens (NAc), a brain area that integrates diverse monoaminergic inputs to coordinate motivated behaviours. While the NAc expresses various histamine receptor subtypes, the mechanisms by which histamine modulates NAc activity are still poorly understood. Using whole‐cell patch‐clamp recordings, we found that pharmacological activation of histamine 2 (H2) receptors elevates the excitability of NAc medium spiny neurons (MSNs), while activation of H1 receptors failed to significantly affect MSN excitability. The evoked firing of MSNs increased after seconds of local H2 agonist administration and remained elevated for minutes. H2 receptor (H2R) activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential afterhyperpolarization and increased the action potential half‐width. The increased excitability was protein kinase A‐dependent and associated with decreased A‐type K+ currents. In addition, selective pharmacological inhibition of the Kv4.2 channel, the main molecular determinant of A‐type K+ currents in MSNs, mimicked and occluded the increased excitability induced by H2R activation. Our results indicate that histaminergic transmission in the NAc increases MSN intrinsic excitability through H2R‐dependent modulation of Kv4.2 channels. Activation of H2R will significantly alter spike firing in MSNs in vivo, and this effect could be an important mechanism by which these receptors mediate certain aspects of goal‐induced behaviours.Key points Histamine is synthesized and released by hypothalamic neurons of the tuberomammillary nucleus and serves as a general modulator for whole‐brain activity including the nucleus accumbens.Histamine receptors type 2 (HR2), which are expressed in the nucleus accumbens, couple to Gαs/off proteins which elevate cyclic adenosine monophosphate levels and activate protein kinase A.Whole‐cell patch‐clamp recordings revealed that H2R activation increased the evoked firing in medium spiny neurons of the nucleus accumbens via protein kinase A‐dependent mechanisms.HR2 activation accelerated subthreshold depolarization in response to current injection, reduced the latency to fire, diminished action potential medium after‐hyperpolarization and increased the action potential half‐width. HR2 activation also reduced A‐type potassium current.Selective pharmacological inhibition of the Kv4.2 channel mimicked and occluded the increased excitability induced by H2R activation.

The Molecular Basis for the Calcium-Dependent Slow Afterhyperpolarization in CA1 Hippocampal Pyramidal Neurons.

Neuronal signal transmission depends on the frequency, pattern, and timing of spike output, each of which are shaped by spike afterhyperpolarizations (AHPs). There are classically three post-spike AHPs of increasing duration categorized as fast, medium and slow AHPs that hyperpolarize a cell over a range of 10 ms to 30 s. Intensive early work on CA1 hippocampal pyramidal cells revealed that all three AHPs incorporate activation of calcium-gated potassium channels. The ionic basis for a fAHP was rapidly attributed to the actions of big conductance (BK) and the mAHP to small conductance (SK) or Kv7 potassium channels. In stark contrast, the ionic basis for a prominent slow AHP of up to 30 s duration remained an enigma for over 30 years. Recent advances in pharmacological, molecular, and imaging tools have uncovered the expression of a calcium-gated intermediate conductance potassium channel (IK, KCa3.1) in central neurons that proves to contribute to the slow AHP in CA1 hippocampal pyramidal cells. Together the data show that the sAHP arises in part from a core tripartite complex between Cav1.3 (L-type) calcium channels, ryanodine receptors, and IK channels at endoplasmic reticulum-plasma membrane junctions. Work on the sAHP in CA1 pyramidal neurons has again quickened pace, with identified contributions by both IK channels and the Na-K pump providing answers to several mysteries in the pharmacological properties of the sAHP.

Secondhand Smoke Decreased Excitability and Altered Action Potential Characteristics of Cardiac Vagal Neurons in Mice.

Background: Secondhand smoke (SHS), a major indoor pollutant, is a significant risk factor for cardiovascular morbidity and mortality including arrhythmias and sudden cardiac death. Exposure to SHS can produce autonomic imbalance, as evidenced by reduced heart rate variability (HRV)—a clinical metric of cardiac vagal regulation. Currently, the mechanisms through which SHS changes the vagal preganglionic neuronal inputs to the heart to produce this remains unknown.Objectives: To characterize the effect of SHS on both the excitability and action potential (AP) characteristics of anatomically identified cardiac vagal neurons (CVNs) in the nucleus ambiguus and examine whether SHS alters small conductance calcium-activated potassium (SK) channel activity of these CVNs.Methods: Adult male mice were exposed to four weeks of filtered air or SHS (3 mg/m3) 6 h/day, 5 day/week. Using patch-clamp recordings on identified CVNs in brainstem slices, we determined neuronal excitability and AP characteristics with depolarizing step- and ramp-current injections.Results: Four weeks of SHS exposure reduced spiking responses to depolarizing current injections and increased AP voltage threshold in CVNs. Perfusion with apamin (20 nM) magnified these SHS-induced effects, suggesting reduced SK channel activity may serve to minimize the SHS-induced decreases in CVNs excitability. Medium afterhyperpolarization (a measurement of SK channel activity) was smaller in the SHS group, further supporting a lower SK channel activity. AP amplitude, rise rate, fast afterhyperpolarization amplitude (a measurement of voltage-gated channel activity), and decay rate were higher in the SHS group at membrane voltages more positive to 0 mV, suggesting altered inactivation properties of voltage-dependent channels underlying APs.Discussion: SHS exposure reduced neuronal excitability of CVNs with compensatory attenuation of SK channel activity and altered AP characteristics. Neuroplasticity of CVNs could blunt regulatory cardiac vagal signaling and contribute to the cardiovascular consequences associated with SHS exposure, including reduced HRV.

Early life stress induces anxiety-like behavior during adulthood through dysregulation of neuronal plasticity in the basolateral amygdala

AimsEarly life stress (ELS) increases the risk of psychiatric diseases such as anxiety disorders and depression in later life. Hyperactivation of the basolateral amygdala (BLA) neurons plays a pivotal role in the pathogenesis of stress-related diseases. However, the functional roles of BLA neurons in ELS-induced anxiety disorders are not completely understood. Main methodsMice were subjected to maternal separation (MS) during postnatal days 3 to 21 to mimic ELS. Anxiety-like behavior was tested by open field test (OFT), elevated plus maze (EPM), and novelty suppressed feeding (NSF). Then, c-fos expression, a proxy for neuronal activity, was evaluated by immunofluorescence. Finally, synaptic transmission and intrinsic excitability were measured by whole-cell patch-clamp recordings. Key findingsMS significantly increased anxiety-like behavior in adulthood, as indicated by less time spent in the center area of the OFT, less time spent in and fewer entries to the open arms of the EPM, and increased latency to feed in NSF. Mechanistically, MS increased the expression of c-fos in BLA. MS enhanced the excitatory, but not inhibitory, synaptic transmission onto BLA projection neurons (PNs), which was caused by enhanced presynaptic glutamate release. Moreover, MS also markedly increased the intrinsic neuronal excitability of BLA PNs, probably due to the reduced medium afterhyperpolarization (mAHP) in BLA PNs. SignificanceOur results suggest that the changes of neuronal activity and synaptic transmission in the BLA PNs may play a crucial role in ELS-induced anxiety-like behavior, and these findings provide new insights into the pathological mechanisms of stress-related anxiety disorders.

Physiology and Therapeutic Potential of SK, H, and M Medium AfterHyperPolarization Ion Channels.

SK, HCN, and M channels are medium afterhyperpolarization (mAHP)-mediating ion channels. The three channels co-express in various brain regions, and their collective action strongly influences cellular excitability. However, significant diversity exists in the expression of channel isoforms in distinct brain regions and various subcellular compartments, which contributes to an equally diverse set of specific neuronal functions. The current review emphasizes the collective behavior of the three classes of mAHP channels and discusses how these channels function together although they play specialized roles. We discuss the biophysical properties of these channels, signaling pathways that influence the activity of the three mAHP channels, various chemical modulators that alter channel activity and their therapeutic potential in treating various neurological anomalies. Additionally, we discuss the role of mAHP channels in the pathophysiology of various neurological diseases and how their modulation can alleviate some of the symptoms.

BK Channel Regulation of Afterpotentials and Burst Firing in Cerebellar Purkinje Neurons.

BK calcium-activated potassium channels have complex kinetics because they are activated by both voltage and cytoplasmic calcium. The timing of BK activation and deactivation during action potentials determines their functional role in regulating firing patterns but is difficult to predict a priori. We used action potential clamp to characterize the kinetics of voltage-dependent calcium current and BK current during action potentials in Purkinje neurons from mice of both sexes, using acutely dissociated neurons that enabled rapid voltage clamp at 37°C. With both depolarizing voltage steps and action potential waveforms, BK current was entirely dependent on calcium entry through voltage-dependent calcium channels. With voltage steps, BK current greatly outweighed the triggering calcium current, with only a brief, small net inward calcium current before Ca-activated BK current dominated the total Ca-dependent current. During action potential waveforms, although BK current activated with only a short (∼100 μs) delay after calcium current, the two currents were largely separated, with calcium current flowing during the falling phase of the action potential and most BK current flowing over several milliseconds after repolarization. Step depolarizations activated both an iberiotoxin-sensitive BK component with rapid activation and deactivation kinetics and a slower-gating iberiotoxin-resistant component. During action potential firing, however, almost all BK current came from the faster-gating iberiotoxin-sensitive channels, even during bursts of action potentials. Inhibiting BK current had little effect on action potential width or a fast afterhyperpolarization but converted a medium afterhyperpolarization to an afterdepolarization and could convert tonic firing of single action potentials to burst firing.SIGNIFICANCE STATEMENT BK calcium-activated potassium channels are widely expressed in central neurons. Altered function of BK channels is associated with epilepsy and other neuronal disorders, including cerebellar ataxia. The functional role of BK in regulating neuronal firing patterns is highly dependent on the context of other channels and varies widely among different types of neurons. Most commonly, BK channels are activated during action potentials and help produce a fast afterhyperpolarization. We find that in Purkinje neurons BK current flows primarily after the fast afterhyperpolarization and helps to prevent a later afterdepolarization from producing rapid burst firing, enabling typical regular tonic firing.

Alterations of Electrophysiological Properties and Ion Channel Expression in Prefrontal Cortex of a Mouse Model of Schizophrenia.

Maternal immune activation (MIA) and juvenile social isolation (SI) are two most prevalent and widely accepted environmental insults that could increase the propensity of psychiatric illnesses. Using a two-hit mouse model, we examined the impact of the combination of these two factors on animal behaviors, neuronal excitability and expressions of voltage-gated sodium (Nav) and small conductance calcium-activated potassium (SK) channels in the prefrontal cortex (PFC). We found that MIA-SI induced a number of schizophrenia-related behavioral deficits. Patch clamp recordings revealed alterations in electrophysiological properties of PFC layer-5 pyramidal cells, including hyperpolarized resting membrane potential (RMP), increased input resistance and enhanced medium after-hyperpolarization (mAHP). MIA-SI also increased the ratio of the maximal slope of somatodendritic potential to the peak slope of action potential upstroke, indicating a change in perisomatic Nav availability. Consistently, MIA-SI significantly increased the expression level of Nav1.2 and SK3 channels that contribute to the somatodendritic potential and the mAHP, respectively. Together, these changes may alter neuronal signaling in the PFC and behavioral states, representing a molecular imprint of environmental insults associated with neuropsychiatric illnesses.

Balanced cholinergic modulation of spinal locomotor circuits via M2 and M3 muscarinic receptors

Neuromodulation ensures that neural circuits produce output that is flexible whilst remaining within an optimal operational range. The neuromodulator acetylcholine is released during locomotion to regulate spinal motor circuits. However, the range of receptors and downstream mechanisms by which acetylcholine acts have yet to be fully elucidated. We therefore investigated metabotropic acetylcholine receptor-mediated modulation by using isolated spinal cord preparations from neonatal mice in which locomotor-related output can be induced pharmacologically. We report that M2 receptor blockade decreases the frequency and amplitude of locomotor-related activity, whilst reducing its variability. In contrast, M3 receptor blockade destabilizes locomotor-related bursting. Motoneuron recordings from spinal cord slices revealed that activation of M2 receptors induces an outward current, decreases rheobase, reduces the medium afterhyperpolarization, shortens spike duration and decreases synaptic inputs. In contrast, M3 receptor activation elicits an inward current, increases rheobase, extends action potential duration and increases synaptic inputs. Analysis of miniature postsynaptic currents support that M2 and M3 receptors modulate synaptic transmission via different mechanisms. In summary, we demonstrate that M2 and M3 receptors have opposing modulatory actions on locomotor circuit output, likely reflecting contrasting cellular mechanisms of action. Thus, intraspinal cholinergic systems mediate balanced, multimodal control of spinal motor output.

Paradoxical Excitatory Impact of SK Channels on Dendritic Excitability.

Dendritic excitability regulates how neurons integrate synaptic inputs and thereby influences neuronal output. As active dendritic events are associated with significant calcium influx they are likely to be modulated by calcium-dependent processes, such as calcium-activated potassium channels. Here we investigate the impact of small conductance calcium-activated potassium channels (SK channels) on dendritic excitability in male and female rat cortical pyramidal neurons in vitro and in vivo Using local applications of the SK channel antagonist apamin in vitro, we show that blocking somatic SK channels enhances action potential output, whereas blocking dendritic SK channels paradoxically reduces the generation of dendritic calcium spikes and associated somatic burst firing. Opposite effects were observed using the SK channel enhancer NS309. The effect of apamin on dendritic SK channels was occluded when R-type calcium channels were blocked, indicating that the inhibitory impact of apamin on dendritic calcium spikes involved R-type calcium channels. Comparable effects were observed in vivo Intracellular application of apamin via the somatic whole-cell recording pipette reduced the medium afterhyperpolarization and increased action potential output during UP states. In contrast, extracellular application of apamin to the cortical surface to block dendritic SK channels shifted the distribution of action potentials within UP states from an initial burst to a more distributed firing pattern, while having no impact on overall action potential firing frequency or UP and DOWN states. These data indicate that somatic and dendritic SK channels have opposite effects on neuronal excitability, with dendritic SK channels counter-intuitively promoting rather than suppressing neuronal output.SIGNIFICANCE STATEMENT Neurons typically receive input from other neurons onto processes called dendrites, and use electrical events such as action potentials for signaling. As electrical events in neurons are usually associated with calcium influx they can be regulated by calcium-dependent processes. One such process is through the activation of calcium-dependent potassium channels, which usually act to reduce action potential signaling. Although this is the case for calcium-dependent potassium channels found at the cell body, we show here that calcium-dependent potassium channels in dendrites of cortical pyramidal neurons counter-intuitively promote rather than suppress action potential output.

Different Effects of 5-HT1 and 5-HT2 Receptor Agonists on Excitability Modulation of Motoneurons in Frog Spinal Cord

Effects of 5-HT1 and 5-HT2 receptor agonists and antagonists on membrane properties of motoneurons in the isolated lumbar segment of the frog spinal cord were investigated using intracellular recordings. Application of a 5-HT2A,B,C receptor agonist α-Me-5-HT evoked depolarization of the motoneuronal membrane. The co-application of α-Me-5-HT and a specific 5-HT2B receptor antagonist SB 206553 did not result in depolarization. α-Me-5-HT reduced the amplitude of medium afterhyperpolarization and increased the number of antidromic action potentials (APs). The application of an antagonist SB 206553 abolished these effects. A 5-HT1A/7 receptor agonist 8-OH-DPAT had a time-dependent effect on the number of antidromic APs, evoking an initial short-term excitation followed by an inhibition. The data obtained in our experiments indicate the presence of 5-HT1A/7 and 5-HT2B,C receptors on the postsynaptic membrane of motoneurons. We suggest a possible co-modulation of the accommodative properties of motoneurons by the two types of serotonin receptors, 5-HT2B,C and 5-HT1A.

Nucleus accumbens shell small conductance potassium channels underlie adolescent ethanol exposure-induced anxiety

Alcohol use typically begins in adolescence, increasing the likelihood of adult mental disorders such as anxiety. However, the cellular mechanisms underlying the consequences of adolescent alcohol exposure as well as the behavioral consequences remain poorly understood. We examined the effects of adolescent or adult chronic intermittent ethanol (CIE) exposure on intrinsic excitability of striatal medium-sized spiny neurons (MSNs) and anxiety levels. Rats underwent one of the following procedures: (1) light–dark transition (LDT) and open-field (OF) tests to evaluate anxiety levels and general locomotion; (2) whole-cell patch clamp recordings and biocytin labeling to assess excitability of striatal MSNs, as well as morphological properties; and (3) western blot immunostaining to determine small conductance (SK) calcium-activated potassium channel protein levels. Three weeks, but not 2 days, after CIE treatment, adolescent CIE-treated rats showed shorter crossover latency from the light to dark side in the LDT test and higher MSN excitability in the nucleus accumbens shell (NAcS). Furthermore, the amplitude of the medium afterhyperpolarization (mAHP), mediated by SK channels, and SK3 protein levels in the NAcS decreased concomitantly. Finally, increased anxiety levels, increased excitability, and decreased amplitude of mAHP of NAcS MSNs were reversed by SK channel activator 1-EBIO and mimicked by the SK channel blocker apamin. Thus, adolescent ethanol exposure increases adult anxiety-like behavior by downregulating SK channel function and protein expression, which leads to an increase of intrinsic excitability in NAcS MSNs. SK channels in the NAcS may serve as a target to treat adolescent alcohol binge exposure-induced mental disorders, such as anxiety in adulthood.

β3-Adrenergic receptor-dependent modulation of the medium afterhyperpolarization in rat hippocampal CA1 pyramidal neurons.

Action potential firing in hippocampal pyramidal neurons is regulated by generation of an afterhyperpolarization (AHP). Three phases of AHP are recognized, with the fast AHP regulating action potential firing at the onset of a burst and the medium and slow AHPs supressing action potential firing over hundreds of milliseconds and seconds, respectively. Activation of β-adrenergic receptors suppresses the slow AHP by a protein kinase A-dependent pathway. However, little is known regarding modulation of the medium AHP. Application of the selective β-adrenergic receptor agonist isoproterenol suppressed both the medium and slow AHPs evoked in rat CA1 hippocampal pyramidal neurons recorded from slices maintained in organotypic culture. Suppression of the slow AHP was mimicked by intracellular application of cAMP, with the suppression of the medium AHP by isoproterenol still being evident in cAMP-dialyzed cells. Suppression of both the medium and slow AHPs was antagonized by the β-adrenergic receptor antagonist propranolol. The effect of isoproterenol to suppress the medium AHP was mimicked by two β3-adrenergic receptor agonists, BRL37344 and SR58611A. The medium AHP was mediated by activation of small-conductance calcium-activated K+ channels and deactivation of H channels at the resting membrane potential. Suppression of the medium AHP by isoproterenol was reduced by pretreating cells with the H-channel blocker ZD7288. These data suggest that activation of β3-adrenergic receptors inhibits H channels, which suppresses the medium AHP in CA1 hippocampal neurons by utilizing a pathway that is independent of a rise in intracellular cAMP. This finding highlights a potential new target in modulating H-channel activity and thereby neuronal excitability. NEW & NOTEWORTHY The noradrenergic input into the hippocampus is involved in modulating long-term synaptic plasticity and is implicated in learning and memory. We demonstrate that activation of functional β3-adrenergic receptors suppresses the medium afterhyperpolarization in hippocampal pyramidal neurons. This finding provides an additional mechanism to increase action potential firing frequency, where neuronal excitability is likely to be crucial in cognition and memory.

A Mutant Sk Channel Rescued locomotion Defects in C. elegans ALS Model

Small-conductance Ca2+-activated K+(SK) channels mediate medium afterhyperpolarization in the neurons which limits the firing frequency of action potentials and, thus, play a key role in the regulation of neuronal excitability. Given their importance in neurons, SK channels are potential drug targets for movement disorders, including ataxia and Amyotrophic Lateral Sclerosis (ALS). The SK channels are activated exclusively by the Ca2+-bound calmodulin. Here, we report that substitution of a valine to phenylalanine (V407F) in the SK2 channel caused a ∼6 fold increase in the Ca2+sensitivity. Subsequent tests with equivalent valine to phenylalanine substitutions in SK1 and SK3 channels also exhibited Ca2+hypersensitivity. Additionally, an equivalent phenylalanine substitution in the Caenorhabditis elegans (C. elegans) SK2 ortholog kcnl-2 partially rescued locomotion defects in an existing C. elegans ALS model, in which human SOD1G85R is expressed at high levels in neurons. This supports the idea that the phenylalanine substitution impacts SK channel function in vivo. This work for the first time provides a criticalreagent for future studies: an SK channel that is hypersensitive toCa2+concentrations with increased activity in vivo.