T-type Calcium Current Research Articles

Integration of distinct cortical inputs to primary and higher order inhibitory cells of the thalamus.

The necortex regulates its own sensory input in part through top-down inhibitory mechanisms. Descending corticothalamic projections drive GABAergic neurons of the thalamic reticular nucleus (TRN), which then control thalamocortical cells via inhibition. Neurons in sensory TRN are organized into primary and higher order (HO) subpopulations, with separate intrathalamic connections and distinct genetic and functional properties. Here, we investigated top-down neocortical control over primary and HO neurons of somatosensory TRN. Projections from layer 6 of somatosensory cortex evoked stronger and more state-dependent activity in primary than in HO TRN, driven by more robust synaptic inputs and potent T-type calcium currents. However, HO TRN received additional, physiologically distinct, inputs from layer 5 of S1 and motor cortex. Thus, in a departure from the canonical focused sensory layer 6 innervation characteristic of primary TRN, HO TRN integrates broadly from multiple corticothalamic systems, with unique state-dependence, extending the range of mechanisms for top-down control.

Properties of rhythmogenic currents in spinal Shox2 interneurons across postnatal development.

Locomotor behaviors are performed by organisms throughout life, despite developmental changes in cellular properties, neural connectivity, and biomechanics. The basic rhythmic activity in the central nervous system that underlies locomotion is thought to be generated via a complex balance between network and intrinsic cellular properties. Within mature mammalian spinal locomotor circuitry, we have yet to determine which properties of spinal interneurons (INs) are critical to rhythmogenesis and how they change during development. Here, we combined whole cell patch clamp recordings, immunohistochemistry, and RNAscope targeting lumbar Shox2 INs in mice, which are known to be involved in locomotor rhythm generation. We focused on the properties of putatively rhythmogenic ionic currents and the expression of corresponding ion channels across postnatal time points in mice. We show that subsets of Shox2 INs display voltage-sensitive conductances, in addition to respective ion channels, which may contribute to or shape rhythmic bursting. Persistent inward currents, M-type potassium currents, slow afterhyperpolarization, and T-type calcium currents are enhanced with age. In contrast, the hyperpolarization-activated and A-type potassium currents were either found with low prevalence in subsets of neonatal, juvenile, and adult Shox2 INs or did not developmentally change. We show that Shox2 INs become more electrophysiologically diverse by juvenile and adult ages, when locomotor behavior is weight-bearing. These results suggest a developmental shift in the magnitude of rhythmogenic ionic currents and the expression of corresponding ion channels that may be important for mature, weight-bearing locomotor behavior.

Rhythm Generating Mechanisms in Rat Sinoatrial Node

Abstract Background: The major membrane currents responsible for sinoatrial (SA) rhythm generation are generally studied in isolated cardiac cells using electrophysiological tools. Such studies are resource and labor-intensive. Materials and Methods: Here, we have studied four major currents in isolated rat heart preparations, perfused in Langendorff mode, and demonstrate that this is a good preparation for such studies. Heart rates of isolated perfused rat hearts were recorded using surface electrocardiogram before and after perfusion with drugs and solutions that affect the four major currents responsible for SA rhythm generation. Results: The rates of whole isolated hearts beating with SA rhythm decreased with cesium and decreased by about half with ivabradine, both blockers of the funny current (If). Importantly, the rhythm was not abolished even with a high dose of ivabradine at which total blockade of If is expected. The rate was not affected by nickel, a blocker of T-type calcium current. The SA rhythm was abolished by the reduction or removal of sodium from the perfusate (interventions that inhibit the calcium-extrusive mode of the sodium-calcium exchanger) or by nifedipine, the L-type calcium channel blocker. Discussion: The inferences made based on these observations are (a) If contributes significantly to pacemaking, (b) ICaT does not play a role and (c) INCX and ICaL are obligatory rhythm-generating currents in the SA node. Cyclical calcium release from SR during diastole (the calcium clock), responsible for driving INCX in its forward mode is probably a phenomenon independent of membrane events, as total If blockade did not abolish rhythm generation. These results corroborate with published literature where most studies were done on single cells.

Abstract P3148: Wnt/β-catenin Signaling Inhibits T-type Calcium Current And Causes Sinoatrial Node Dysfunction

Background: The T-type Ca 2+ channels (I Ca,T ) are expressed in the fetal and neonatal cardiomyocytes, but in the adult heart they are exclusively expressed in the cardiac conduction system, including the sinoatrial node (SAN). In contrast, the L-type Ca 2+ channels (I Ca,L ) are expressed ubiquitously in the adult heart and are important for cardiac contraction. The Wnt/β-catenin signaling (Wnt signaling) is activated in various heart disease and we and others have recently demonstrated that Wnt signaling inhibits cardiac Na + current. In the present study, we investigated if calcium channels are also regulated by Wnt signaling. Methods and Results: Because neonatal rat ventricular myocytes (NRVMs) express both I Ca,L and I Ca,T , we investigated the effect of Wnt signaling on NRVMs by treatment with either Wnt3a protein or CHIR-99021 (CHIR) (two different activators of the Wnt signaling). PCR array and RNA sequencing studies showed that Wnt signaling did not affect the mRNA of Cacna1c (encoding the α subunit of I Ca,L , Ca v 1.2), but the mRNA of Cacna1g (encoding the α subunit of I Ca,T , Ca v 3.1) was reduced in NRVMs (p<0.05, n=3) after treatment with either Wnt3a or CHIR. These observations were verified by qRT-PCR which showed dose-dependent reductions (p<0.05, n=4) in Cacna1g mRNA after CHIR treatment. Western blot and patch-clamp also demonstrated reductions (p<0.01, n=5) in channel protein and ionic current density of T-type, but not L-type, Ca 2+ channels after treatment with Wnt3a or CHIR. These observations suggest that Wnt signaling selectively inhibits T-type Ca 2+ channels in cardiomyocytes. Consistent with a critical role of T-type Ca 2+ channels in the pacemaker activity of SAN (the primary pacemaker of the heart), the spontaneous firing rates of isolated rat and mouse SAN tissues were reduced by ML-218 (an I Ca,T blocker) in a dose-dependent manner (p<0.01, n=5). In addition, activation of Wnt signaling in adult rat SAN tissues by CHIR led to slower and irregular beating, indicative of SAN dysfunction (p<0.01, n=5). Conclusions: These observations are consistent with the conclusions that Wnt/β-catenin signaling selectively inhibits T-type calcium current and may play a role in sinoatrial node dysfunction.

Estradiol enhances T-type calcium channel activation in human myometrium telocytes.

Uterine peristalsis is essential for gamete transport and embryo implantation. It shares the characteristics of spontaneity, rhythmicity, and directivity with gastrointestinal peristalsis. Telocytes, the "interstitial Cajal-like cells" outside the digestive canal, are also located in the uterus and may act as pacemakers. To investigate the possible origin and regulatory mechanism of periodic uterine peristalsis in the human menstrual cycle, telocytes in the myometrium were studied to determine the effect of estradiol on T-type calcium channel regulation. In this study, biopsies of the human myometrium were obtained for cell culture, and double-labeling immunofluorescence screening was used to identify telocytes and T-type calcium channel expression. Intracellular calcium signal measurements and patch-clamp recordings were used to investigate the role of T-type calcium channels in regulating calcium currents with or without estradiol. Our study demonstrates that telocytes exist in the human uterus and express T-type calcium channels. The intracellular Ca2+ fluorescence intensity marked by Fluo-4AM was dramatically decreased by NNC 55-0396, a highly selective T-type calcium channel blocker, but enhanced by estradiol. T-type calcium current amplitude increased in telocytes incubated with estradiol in a dose-dependent manner compared to the control group. In conclusion, our study demonstrated that telocytes exist in the human myometrium, expressing T-type calcium channels and estradiol-enhanced T-type calcium currents, which may be a reasonable explanation for the origin of uterine peristalsis. The role of telocytes in the human uterus as pacemakers and message transfer stations in uterine peristalsis may be worth further investigation.

SGLT2 Inhibitor Empagliflozin Modulates Ion Channels in Adult Zebrafish Heart.

Empagliflozin, an inhibitor of sodium-glucose co-transporter 2 (iSGLT2), improves cardiovascular outcomes in patients with and without diabetes and possesses an antiarrhythmic activity. However, the mechanisms of these protective effects have not been fully elucidated. This study aimed to explore the impact of empagliflozin on ion channel activity and electrophysiological characteristics in the ventricular myocardium. The main cardiac ionic currents (INa, ICaL, ICaT, IKr, IKs) and action potentials (APs) were studied in zebrafish. Whole-cell currents were measured using the patch clamp method in the isolated ventricular cardiomyocytes. The conventional sharp glass microelectrode technique was applied for the recording of APs from the ventricular myocardium of the excised heart. Empagliflozin pretreatment compared to the control group enhanced potassium IKr step current density in the range of testing potentials from 0 to +30 mV, IKr tail current density in the range of testing potentials from +10 to +70 mV, and IKs current density in the range of testing potentials from −10 to +20 mV. Moreover, in the ventricular myocardium, empagliflozin pretreatment shortened AP duration APD as shown by reduced APD50 and APD90. Empagliflozin had no influence on sodium (INa) and L- and T-type calcium currents (ICaL and ICaT) in zebrafish ventricular cardiomyocytes. Thus, we conclude that empagliflozin increases the rapid and slow components of delayed rectifier K+ current (IKr and IKs). This mechanism could be favorable for cardiac protection.

Electrophysiological and Morphological Features of Rebound Depolarization Characterized Interneurons in Rat Superficial Spinal Dorsal Horn.

Substantia gelatinosa (SG) neurons, which are located in the spinal dorsal horn (lamina II), have been identified as the “central gate” for the transmission and modulation of nociceptive information. Rebound depolarization (RD), a biophysical property mediated by membrane hyperpolarization that is frequently recorded in the central nervous system, contributes to shaping neuronal intrinsic excitability and, in turn, contributes to neuronal output and network function. However, the electrophysiological and morphological properties of SG neurons exhibiting RD remain unclarified. In this study, whole-cell patch-clamp recordings were performed on SG neurons from parasagittal spinal cord slices. RD was detected in 44.44% (84 out of 189) of the SG neurons recorded. We found that RD-expressing neurons had more depolarized resting membrane potentials, more hyperpolarized action potential (AP) thresholds, higher AP amplitudes, shorter AP durations, and higher spike frequencies in response to depolarizing current injection than neurons without RD. Based on their firing patterns and morphological characteristics, we propose that most of the SG neurons with RD mainly displayed tonic firing (69.05%) and corresponded to islet cell morphology (58.82%). Meanwhile, subthreshold currents, including the hyperpolarization-activated cation current (Ih) and T-type calcium current (IT), were identified in SG neurons with RD. Blockage of Ih delayed the onset of the first spike in RD, while abolishment of IT significantly blunted the amplitude of RD. Regarding synaptic inputs, SG neurons with RD showed lower frequencies in both spontaneous and miniature excitatory synaptic currents. Furthermore, RD-expressing neurons received either Aδ- or C-afferent-mediated monosynaptic and polysynaptic inputs. However, RD-lacking neurons received afferents from monosynaptic and polysynaptic Aδ fibers and predominantly polysynaptic C-fibers. These findings demonstrate that SG neurons with RD have a specific cell-type distribution, and may differentially process somatosensory information compared to those without RD.

Different roles of T-type calcium channel isoforms in hypnosis induced by an endogenous neurosteroid epipregnanolone

BackgroundMany neuroactive steroids induce sedation/hypnosis by potentiating γ-aminobutyric acid (GABAA) currents. However, we previously demonstrated that an endogenous neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] (EpiP) exerts potent peripheral analgesia and blocks T-type calcium currents while sparing GABAA currents in rat sensory neurons. This study seeks to investigate the behavioral effects elicited by systemic administration of EpiP and to characterize its use as an adjuvant agent to commonly used general anesthetics (GAs). MethodsHere, we utilized electroencephalographic (EEG) recordings to characterize thalamocortical oscillations, as well as behavioral assessment and mouse genetics with wild-type (WT) and different knockout (KO) models of T-channel isoforms to investigate potential sedative/hypnotic and immobilizing properties of EpiP. ResultsConsistent with increased oscillations in slower EEG frequencies, EpiP induced an hypnotic state in WT mice when injected alone intra-peritoneally (i.p.) and effectively facilitated anesthetic effects of isoflurane (ISO) and sevoflurane (SEVO). The CaV3.1 (Cacna1g) KO mice demonstrated decreased sensitivity to EpiP-induced hypnosis when compared to WT mice, whereas no significant difference was noted between CaV3.2 (Cacna1h), CaV3.3 (Cacna1i) and WT mice. Finally, when compared to WT mice, onset of EpiP-induced hypnosis was delayed in CaV3.2 KO mice but not in CaV3.1 and CaV3.3 KO mice. ConclusionWe posit that EpiP may have an important role as novel hypnotic and/or adjuvant to volatile anesthetic agents. We speculate that distinct hypnotic effects of EpiP across all three T-channel isoforms is due to their differential expression in thalamocortical circuitry.

TRPC1/5-Ca V 3 Complex Mediates Leptin-Induced Excitability in Hypothalamic Neurons.

Leptin regulates hypothalamic POMC+ (pro-opiomelanocortin) neurons by inducing TRPC (Transient Receptor Potential Cation) channel-mediate membrane depolarization. The role of TRPC channels in POMC neuron excitability is clearly established; however, it remains unknown whether their activity alone is sufficient to trigger excitability. Here we show that the right-shift voltage induced by the leptin-induced TRPC channel-mediated depolarization of the resting membrane potential brings T-type channels into the active window current range, resulting in an increase of the steady state T-type calcium current from 40 to 70% resulting in increased intrinsic excitability of POMC neurons. We assessed the role and timing of T-type channels on excitability and leptin-induced depolarization in vitro in cultured mouse POMC neurons. The involvement of TRPC channels in the leptin-induced excitability of POMC neurons was corroborated by using the TRPC channel inhibitor 2APB, which precluded the effect of leptin. We demonstrate T-type currents are indispensable for both processes, as treatment with NNC-55-0396 prevented the membrane depolarization and rheobase changes induced by leptin. Furthermore, co-immunoprecipitation experiments suggest that TRPC1/5 channels and CaV3.1 and CaV3.2 channels co-exist in complex. The functional relevance of this complex was corroborated using intracellular Ca2+ chelators; intracellular BAPTA (but not EGTA) application was sufficient to preclude POMC neuron excitability. However, leptin-induced depolarization still occurred in the presence of either BAPTA or EGTA suggesting that the calcium entry necessary to self-activate the TRPC1/5 complex is not blocked by the presence of BAPTA in hypothalamic neurons. Our study establishes T-type channels as integral part of the signaling cascade induced by leptin, modulating POMC neuron excitability. Leptin activation of TRPC channels existing in a macromolecular complex with T-type channels recruits the latter by locally induced membrane depolarization, further depolarizing POMC neurons, triggering action potentials and excitability.

Fast A-type currents shape a rapidly adapting form of delayed short latency firing of excitatory superficial dorsal horn neurons that express the neuropeptide Y Y1 receptor.

Neuropeptide Y Y1 receptor-expressing neurons in the dorsal horn of the spinal cord contribute to chronic pain. For the first time, we characterized the firing patterns of Y1-expressing neurons in Y1eGFP reporter mice. Under hyperpolarized conditions, most Y1eGFP neurons exhibited fast A-type potassium currents and delayed, short-latency firing (DSLF). Y1eGFP DSLF neurons were almost always rapidly adapting and often exhibited rebound spiking, characteristics of spinal pain neurons under the control of T-type calcium channels. These results will inspire future studies to determine whether tissue or nerve injury downregulates the channels that underlie A-currents, thus unmasking membrane hyperexcitability in Y1-expressing dorsal horn neurons, leading to persistent pain. Neuroanatomical and behavioural evidence indicates that neuropeptide Y Y1 receptor-expressing interneurons (Y1-INs) in the superficial dorsal horn (SDH) are predominantly excitatory and contribute to chronic pain. Using an adult ex vivo spinal cord slice preparation from Y1eGFP reporter mice, we characterized firing patterns in response to steady state depolarizing current injection of GFP-positive cells in lamina II, the great majority of which expressed Y1 mRNA (88%). Randomly sampled (RS) and Y1eGFP neurons exhibited five firing patterns: tonic, initial burst, phasic, delayed short-latency <180ms (DSLF) and delayed long-latency >180ms (DLLF). When studied at resting membrane potential, most RS neurons exhibited delayed firing, while most Y1eGFP neurons exhibited phasic firing. A preconditioning membrane hyperpolarization produced only subtle changes in the firing patterns of RS neurons, but dramatically shifted Y1eGFP neurons to DSLF (46%) and DLLF (24%). In contrast to RS DSLF neurons, which rarely exhibited spike frequency adaptation, Y1eGFP DSLF neurons were almost always rapidly adapting, a characteristic of nociceptive-responsive SDH neurons. Rebound spiking was more prevalent in Y1eGFP neurons (6% RS vs. 32% Y1eGFP), indicating enrichment of T-type calcium currents. Y1eGFP DSLF neurons exhibited fast A-type potassium currents that are known to delay or limit action potential firing and exhibited smaller current density as compared to RS DSLF neurons. Our results will inspire future studies to determine whether tissue or nerve injury downregulates channels that contribute to A-currents, thus potentially unmasking T-type calcium channel activity and membrane hyperexcitability in Y1-INs, leading to persistent pain.

Abstract 16959: A Youthful Cardiomyocyte Phenotype Drives Enhanced Cardiac Recovery in Adult Spiny Mice

Introduction: Adult mammals, including humans, experience limited recovery following myocardial infarction (MI). Recently, our group has uncovered a unique ability for enhanced cardiac recovery in adult spiny mice ( Acomys ). Methods and Results: Our preliminary studies have established appropriate similarities in heart structure and coronary anatomy between Acomys and Mus . Following the same permanent left artery descending (LAD) ligation surgery, Acomys and Mus showed a similar degree of early cardiac injury. Echocardiography and histological analyses showed more robust cardiac recovery and smaller scar area in Acomys . Moreover, Acomys exhibited a higher survival rate compared to Mus with no evidence of cardiac rupture or hypertrophy (measured by increasing heart/body weight) after MI. To investigate the mechanisms underlying this phenomenon, we isolated and quantified the size, nuclei number, and ploidy of adult cardiomyocytes (CMs) in both species (n=5 animals/group) using the Langendorff perfusion technique. Acomys CMs are significantly smaller in size compared to Mus and Acomys hearts have higher percentage of mononucleated cardiomyocytes. Using flow cytometry, we found that Acomys possess a higher percentage of diploid CMs compared to Mus . A closer examination of cardiomyocytes using electrophysiology revealed the presence of a T-type calcium current in adult Acomys CMs; a characteristic which is usually found in highly proliferative embryonic ventricular cardiomyocytes. Lastly, Acomys CMs demonstrated higher proliferative rates after injury compared to Mus . Conclusion: Taken together, our data provide strong evidence for enhanced cardiac repair in an adult mammalian model. Our results strongly suggest that Acomys maintain proliferative adult cardiomyocytes as adults and thus provide a cellular mechanism for their enhanced cardiac recovery after AMI.

Temperature effects on synaptic transmission and neuronal function in the visual thalamus.

Numerous neuronal properties including the synaptic vesicle release process, neurotransmitter receptor complement, and postsynaptic ion channels are involved in transforming synaptic inputs into postsynaptic spiking. Temperature is a significant influencer of neuronal function and synaptic integration. Changing temperature can affect neuronal physiology in a diversity of ways depending on how it affects different members of the cell’s ion channel complement. Temperature’s effects on neuronal function are critical for pathological states such as fever, which can trigger seizure activity, but are also important in interpreting and comparing results of experiments conducted at room vs physiological temperature. The goal of this study was to examine the influence of temperature on synaptic properties and ion channel function in thalamocortical (TC) relay neurons in acute brain slices of the dorsal lateral geniculate nucleus, a key synaptic target of retinal ganglion cells in the thalamus. Warming the superfusate in patch clamp experiments with acutely-prepared brain slices led to an overall inhibition of synaptically-driven spiking behavior in TC neurons in response to a retinal ganglion cell spike train. Further study revealed that this was associated with an increase in presynaptic synaptic vesicle release probability and synaptic depression and altered passive and active membrane properties. Additionally, warming the superfusate triggered activation of an inwardly rectifying potassium current and altered the voltage-dependence of voltage-gated Na+ currents and T-type calcium currents. This study highlights the importance of careful temperature control in ex vivo physiological experiments and illustrates how numerous properties such as synaptic inputs, active conductances, and passive membrane properties converge to determine spike output.

A low voltage activated Ca2+ current found in a subset of human ventricular myocytes

ABSTRACT Low voltage activated (ICa-LVA) calcium currents including Cav1.3 and T-type calcium current (ICa-T) have not been reported in adult human left ventricular myocytes (HLVMs). We tried to examine their existence and possible correlation with etiology and patient characteristics in a big number of human LVMs isolated from explanted terminally failing (F) hearts, failing hearts with left ventricular assist device (F-LVAD) and nonfailing (NF) human hearts. LVA (ICa-LVA) was determined by subtracting L-type Ca2+ current (ICa-L) recorded with the holding potential of −50 mV from total Ca2+ current recorded with the holding potential of −90 mV or −70 mV. ICa- LVA was further tested with its sensitivity to 100 µM CdCl2 and tetrodotoxin. Three HLVMs (3 of 137 FHLVMs) from 2 (N = 30 hearts) failing human hearts, of which one was idiopathic and the other was due to primary pulmonary hypertension, were found with ICa-LVA. ICa-LVA in one FHLVM was not sensitive to 100 µM CdCl2 while ICa-LVA in another two FHLVMs was not sensitive to tetrodotoxin. It peaked at the voltage of −40~-20 mV and had a time-dependent decay faster than ICa-L but slower than sodium current (INa). ICa-LVA was not found in any HLVMs from NF (75 HLVMs from 17 hearts) or F-LVAD hearts (82 HLVMs from 18 hearts) but a statistically significant correlation could not be established. In conclusion, ICa-LVA was detected in some HLVMs of a small portion of human hearts that happened to be nonischemic failing hearts.

Adult loss of Cacna1a in mice recapitulates childhood absence epilepsy by distinct thalamic bursting mechanisms.

Inborn errors of CACNA1A-encoded P/Q-type calcium channels impair synaptic transmission, producing early and lifelong neurological deficits, including childhood absence epilepsy, ataxia and dystonia. Whether these impairments owe their pathologies to defective channel function during the critical period for thalamic network stabilization in immature brain remains unclear. Here we show that mice with tamoxifen-induced adult-onset ablation of P/Q channel alpha subunit (iKOp/q) display identical patterns of dysfunction, replicating the inborn loss-of-function phenotypes and, therefore demonstrate that these neurological defects do not rely upon developmental abnormality. Unexpectedly, unlike the inborn model, the adult-onset pattern of excitability changes believed to be pathogenic within the thalamic network is non-canonical. Specifically, adult ablation of P/Q channels does not promote Cacna1g-mediated burst firing or T-type calcium current (IT) in the thalamocortical relay neurons; however, burst firing in thalamocortical relay neurons remains essential as iKOp/q mice generated on a Cacna1g deleted background show substantially diminished seizure generation. Moreover, in thalamic reticular nucleus neurons, burst firing is impaired accompanied by attenuated IT. Interestingly, inborn deletion of thalamic reticular nucleus-enriched, human childhood absence epilepsy-linked gene Cacna1h in iKOp/q mice reduces thalamic reticular nucleus burst firing and promotes rather than reduces seizure, indicating an epileptogenic role for loss-of-function Cacna1h gene variants reported in human childhood absence epilepsy cases. Together, our results demonstrate that P/Q channels remain critical for maintaining normal thalamocortical oscillations and motor control in the adult brain, and suggest that the developmental plasticity of membrane currents regulating pathological rhythmicity is both degenerate and age-dependent.

A potential role for T-type calcium channels in homocysteinemia-induced peripheral neuropathy.

Homocysteinemia is a metabolic condition characterized by abnormally high level of homocysteine in the blood and is considered to be a risk factor for peripheral neuropathy. However, the cellular mechanisms underlying toxic effects of homocysteine on the processing of peripheral nociception have not yet been investigated comprehensively. Here, using a rodent model of experimental homocysteinemia, we report the causal association between homocysteine and the development of mechanical allodynia. Homocysteinemia-induced mechanical allodynia was reversed on pharmacological inhibition of T-type calcium channels. In addition, our in vitro studies indicate that homocysteine enhances recombinant T-type calcium currents by promoting the recycling of Cav3.2 channels back to the plasma membrane through a protein kinase C-dependent signaling pathway that requires the direct phosphorylation of Cav3.2 at specific loci. Altogether, these results reveal an unrecognized signaling pathway that modulates the expression of T-type calcium channels, and may potentially contribute to the development of peripheral neuropathy associated with homocysteinemia.

Biophysical modeling of C. elegans neurons: Single ion currents and whole-cell dynamics of AWCon and RMD.

C. elegans neuronal system constitutes the ideal framework for studying simple, yet realistic, neuronal activity, since the whole nervous system is fully characterized with respect to the exact number of neurons and the neuronal connections. Most recent efforts are devoted to investigate and clarify the signal processing and functional connectivity, which are at the basis of sensing mechanisms, signal transmission, and motor control. In this framework, a refined modelof whole neuron dynamics constitutes a key ingredient to describe the electrophysiological processes, both at thecellular and at the network scale. In this work, we present Hodgkin-Huxley-based models of ion channels dynamics black, built on data available both from C. elegans and from other organisms, expressing homologous channels. We combine these channel models to simulate the electrical activity oftwo among the most studied neurons in C. elegans, which display prototypical dynamics of neuronal activation, the chemosensory AWCON and the motor neuron RMD. Our model properly describes the regenerative responses of the two cells. We analyze in detail the role of ion currents, both in wild type and in in silico knockout neurons. Moreover, we specifically investigate the behavior of RMD, identifying a heterogeneous dynamical response which includes bistable regimes and sustained oscillations. We are able to assess the critical role of T-type calcium currents, carried by CCA-1 channels, and leakage currents in the regulation of RMD response. Overall, our results provide new insights in the activity of key C. elegans neurons. The developed mathematical framework constitute a basis for single-cell and neuronal networks analyses, opening new scenarios in the in silico modeling of C. elegans neuronal system.

Effect of heat stress and Hsp90 inhibition on T-type calcium currents and voltage-dependent potassium currents in leydig cells.

Heat can trigger testicular damage and impair fertility. Leydig cells produce testosterone in response to stimulation by luteinizing hormone (LH), which induces Ca2+ entry and K+ efflux through ion channels in their plasma membrane. Considering that mechanisms coordinating the Leydig cell responses to hyperthermic stress remain unclear; the present study analyzed the effects of heat stress (HS, 43°C, 15min) and inhibition of Hsp90 on T-type calcium currents and voltage-dependent potassium currents (VKC) in mice Leydig cells. Results show that HS reduced the VKC steady state currents at +80mV (45.3%) and maximum conductance (71.5%), as well as increased the activation time constant (31.7%) and the voltage for which half the channels are open (30%). Hsp90 inhibition did not change the VKC currents. T-type calcium currents were not affected by HS or Hsp90 inhibition. In conclusion, HS can slow the activation, reduce the currents and voltage dependence of the VKC, suggesting a possible role of these currents in the response to hyperthermic stress in Leydig cells.

Novel neurosteroid hypnotic blocks T-type calcium channel-dependent rebound burst firing and suppresses long-term potentiation in the rat subiculum

BackgroundHypnotics and general anaesthetics impair memory by altering hippocampal synaptic plasticity. We recently reported on a neurosteroid analogue with potent hypnotic activity [(3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; 3β-OH], which does not cause developmental neurotoxicity in rat pups. Here, we investigated the effects of 3β-OH on neuronal excitability in the subiculum, the major output structure of the hippocampal formation, and synaptic plasticity at two key hippocampal synapses in juvenile rats. MethodsBiophysical properties of isolated T-type calcium currents (T-currents) in the rat subiculum were investigated using acute slice preparations. Subicular T-type calcium channel (T-channel) subtype mRNA expression was compared using qRT–PCR. Using electrophysiological recordings, we examined the effects of 3β-OH and an endogenous neuroactive steroid, allopregnanolone (Allo), on T-currents and burst firing properties of subicular neurones, and on the long-term potentiation (LTP) in CA3-CA1 and CA1-subiculum pathways. ResultsBiophysical and molecular studies confirmed that CaV3.1 channels represent the dominant T-channel isoform in the subiculum of juvenile rats. 3β-OH and Allo inhibited rebound burst firing by decreasing the amplitude of T-currents in a voltage-dependent manner with similar potency, with 30–80% inhibition. Both neurosteroids suppressed LTP at the CA1-subiculum, but not at the CA3-CA1 Schaffer collateral synapse. ConclusionsNeurosteroid effects on T-channels modulate hippocampal output and provide possible molecular mechanisms for the amnestic action of the novel hypnotic 3β-OH. Effects on T-channels in the subiculum provide a novel target for amnestic effects of hypnotics.

Adrenergic agonist induces rhythmic firing in quiescent cardiac preganglionic neurons in nucleus ambiguous via activation of intrinsic membrane excitability.

Cholinergic vagal nerves projecting from neurons in the brain stem nucleus ambiguus (NAm) play a predominant role in cardiac parasympathetic pacemaking control. Central adrenergic signaling modulates the tone of this vagal output; however, the exact excitability mechanisms are not fully understood. We investigated responses of NAm neurons to adrenergic agonists using in vitro mouse brain stem slices. Preganglionic NAm neurons were identified by ChAT-tdTomato fluorescence in young adult transgenic mice, and their cardiac projection was confirmed by retrograde dye tracing. Juxtacellular recordings detected sparse or absent spontaneous action potentials (AP) in NAm neurons. However, bath application of epinephrine or norepinephrine strongly and reversibly activated most NAm neurons regardless of their basal firing rate. Epinephrine was more potent than norepinephrine, and this activation largely depends on α1-adrenoceptors. Interestingly, adrenergic activation of NAm neurons does not require an ionotropic synaptic mechanism, because postsynaptic excitatory or inhibitory receptor blockade did not occlude the excitatory effect, and bath-applied adrenergic agonists did not alter excitatory or inhibitory synaptic transmission. Instead, adrenergic agonists significantly elevated intrinsic membrane excitability to facilitate generation of recurrent action potentials. T-type calcium current and hyperpolarization-activated current are involved in this excitation pattern, although not required for spontaneous AP induction by epinephrine. In contrast, pharmacological blockade of persistent sodium current significantly inhibited the adrenergic effects. Our results demonstrate that central adrenergic signaling enhances the intrinsic excitability of NAm neurons and that persistent sodium current is required for this effect. This central balancing mechanism may counteract excessive peripheral cardiac excitation during increased sympathetic tone.NEW & NOTEWORTHY Cardiac preganglionic cholinergic neurons in the nucleus ambiguus (NAm) are responsible for slowing cardiac pacemaking. This study identified that adrenergic agonists can induce rhythmic action potentials in otherwise quiescent cholinergic NAm preganglionic neurons in brain stem slice preparation. The modulatory influence of adrenaline on central parasympathetic outflow may contribute to both physiological and deleterious cardiovascular regulation.

Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision

ABSTRACTVoltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5β-reduced neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC50 of 3.3 μM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.