GABAA Channels Research Articles

Human-Induced Pluripotent Stem Cell (iPSC)-Derived GABAergic Neuron Differentiation in Bipolar Disorder.

Bipolar disorder (BP) is a recurring psychiatric condition characterized by alternating episodes of low energy (depressions) followed by manias (high energy). Cortical network activity produced by GABAergic interneurons may be critical in maintaining the balance in excitatory/inhibitory activity in the brain during development. Initially, GABAergic signaling is excitatory; with maturation, these cells undergo a functional switch that converts GABAA channels from depolarizing (excitatory) to hyperpolarizing (inhibitory), which is controlled by the intracellular concentration of two chloride transporters. The earliest, NKCC1, promotes chloride entry into the cell and depolarization, while the second (KCC2) stimulates movement of chloride from the neuron, hyperpolarizing it. Perturbations in the timing or expression of NKCC1/KCC2 may affect essential morphogenetic events including cell proliferation, migration, synaptogenesis and plasticity, and thereby the structure and function of the cortex. We derived induced pluripotent stem cells (iPSC) from BP patients and undiagnosed control (C) individuals, then modified a differentiation protocol to form GABAergic interneurons, harvesting cells at sequential stages of differentiation. qRT-PCR and RNA sequencing indicated that after six weeks of differentiation, controls transiently expressed high levels of NKCC1. Using multi-electrode array (MEA) analysis, we observed that BP neurons exhibit increased firing, network bursting and decreased synchrony compared to C. Understanding GABA signaling in differentiation may identify novel approaches and new targets for treatment of neuropsychiatric disorders such as BP.

Bidirectional Regulation of GABAA Reversal Potential in the Adult Brain: Physiological and Pathological Implications.

In physiological conditions, the intracellular chloride concentration is much lower than the extracellular. As GABAA channels are permeable to anions, the reversal potential of GABAA is very close to that of Cl-, which is the most abundant free anion in the intra- and extracellular spaces. Intracellular chloride is regulated by the activity ratio of NKCC1 and KCC2, two chloride-cation cotransporters that import and export Cl-, respectively. Due to the closeness between GABAA reversal potential and the value of the resting membrane potential in most neurons, small changes in intracellular chloride have a major functional impact, which makes GABAA a uniquely flexible signaling system. In most neurons of the adult brain, the GABAA reversal potential is slightly more negative than the resting membrane potential, which makes GABAA hyperpolarizing. Alterations in GABAA reversal potential are a common feature in numerous conditions as they are the consequence of an imbalance in the NKCC1-KCC2 activity ratio. In most conditions (including Alzheimer's disease, schizophrenia, and Down's syndrome), GABAA becomes depolarizing, which causes network desynchronization and behavioral impairment. In other conditions (neonatal inflammation and neuropathic pain), however, GABAA reversal potential becomes hypernegative, which affects behavior through a potent circuit deactivation.

Multi-faceted Anti-obesity Effects of N-Methyl-D-Aspartate (NMDA) Receptor Modulators: Central-Peripheral Crosstalk.

Hypothalamus is central to food intake and satiety. Recent data unveiled the expression of N-methyl-D-aspartate receptors (NMDAR) on hypothalamic neurons and their interaction with GABAA and serotoninergic neuronal circuits. However, the precise mechanisms governing energy homeostasis remain elusive. Notably, in females, the consumption of progesterone-containing preparations, such as hormonal replacement therapy and birth control pills, has been associated with hyperphagia and obesity-effects mediated through the hypothalamus. To elucidate this phenomenon, we employed the progesterone-induced obesity model in female Swiss albino mice. Four NMDAR modulators were selected viz. dextromethorphan (Dxt), minocycline, d-aspartate, and cycloserine. Obesity was induced in female mice by progesterone administration for 4 weeks. Mice were allocated into 7 groups, group-1 as vehicle control (arachis oil), group-2 (progesterone + arachis oil), and group-3 as positive-control (progesterone + sibutramine); other groups were treated with test drugs + progesterone. Various parameters were recorded like food intake, thermogenesis, serum lipids, insulin, AST and ALT levels, organ-to-body weight ratio, total body fat, adiposity index, brain serotonin levels, histology of liver, kidney, and sizing of fat cells. Dxt-treated group has shown a significant downturn in body weight (p < 0.05) by a decline in food intake (p < 0.01), organ-to-liver ratio (p < 0.001), adiposity index (p < 0.01), and a rise in body temperature and brain serotonin level (p < 0.001). Dxt demonstrated anti-obesity effects by multiple mechanisms including interaction with hypothalamic GABAA channels and anti-inflammatory and free radical scavenging effects, improving the brain serotonin levels, and increasing insulin release from the pancreatic β-cells.

The differential in vivo contribution of spinal α2A- and α2C-adrenoceptors in tonic and acute evoked nociception in the rat.

Spinal α2-adrenoceptor induces analgesia by neuronal inhibition of primary afferent fibers. This family receptor coupled to G i/o proteins can be subdivided into three functional subtypes: α2A, α2B, and α2C-adrenoceptors, and current evidence on spinal analgesia supports the relevance of α2A and seems to exclude the role of α2B, but the functional contribution of α2C-adrenoceptors remains elusive. The present study was designed to pharmacologically dissect the contribution of spinal α2-adrenoceptor subtypes modulating tonic or acute peripheral nociception. Using male Wistar rats, we analyzed the effect of spinal clonidine (a non-selective α2A/α2B/α2C-adrenoceptor agonist) and/or selective subtype α2-adrenoceptor antagonists on: 1) tonic nociception induced by subcutaneous formalin (flinching behavior) or 2) acute nociception induced by peripheral electrical stimulus in in vivo extracellular recordings of spinal dorsal horn second-order wide dynamic range (WDR) neurons. Clonidine inhibited the nocifensive behavior induced by formalin, an effect blocked by BRL 44408 (α2A-adrenoceptor antagonist) but not by imiloxan (α2B-adrenoceptor antagonist) or JP 1302 (α2C-adrenoceptor antagonist). Similarly, spinal BRL 44408 reversed the clonidine-induced inhibition of nociceptive WDR activity. Interestingly, spinal JP 1302 per se produced behavioral antinociception (an effect blocked by bicuculline, a preferent GABAA channel blocker), but no correlation was found with the electrophysiological experiments. These data imply that, at the spinal level, 1) presynaptic α2A-adrenoceptor activation produces antinociception during acute or tonic nociceptive stimuli; and 2) under tonic nociceptive (inflammatory) input, spinal α2C-adrenoceptors are pronociceptive, probably by the inactivation of GABAergic transmission. This result supports a differential role of α2A and α2C-adrenoceptors modulating nociception.

Subunit-dependent interaction of propoxazepam and its metabolite with the -aminobuturic acid type A receptor

Benzodiazepines (BDZ) are widely used in clinics in the treatment of psychiatric disorders, and their main action is considered to be determined by more selective binding with α1, α2, α3 or α5 subunits of GABA receptor.&#x0D; The aim of this work was studying of the molecular mechanism of action of new analgesic – propoxazepam and its metabolite (3-hydroxypropoxazepam) on α1, α2, α3, α4 or α5 subunits containing GABAA channels.&#x0D; Materials and methods GABA ha1b3g2, ha2b3g2, ha3b3g2, ha4b3g2 and ha5b3g2 ionotropic GABAARs expressed in HEK293 were used on the automated SP384PE Patch Clamp system. In addition, Propoxazepam, 3-hydroxypropoxazepam, diazepam (positive allosteric modulator) and GABA (positive control) were administered at concentrations 0.001–300 nM to determine the EC50 and Emax for corresponding substances.&#x0D; Results The α subunit plays a significantl role in determining the receptor’s affinity for propoxazepam and 3-hydroxypropoxazepam. The rank order of decreasing EC50 are α1 = α5&gt; α2 &gt; α3 &gt; α4 (propoxazepam) and α1&gt; α2&gt; α5 &gt; α3 &gt; α4 (3-hydroxypropoxazepam), and for Emax α3 &gt; α2 &gt;α5 &gt; α1 &gt; α4 (propoxazepam), α3 &gt; α1 &gt; α2&gt; α5 &gt; α4 (3-hydroxypropoxazepam).&#x0D; The data, transformed to Emax/EC50, show that propoxazepam exhibits tenfold (compared to diazepam) activity (taking into account the magnitude of the maximum effect) to the α3 subunit, which distinguishes it from 3-hydroxypropoxazepam.&#x0D; Conclusion Due to the determined selectivity of propoxazepam for binding with different α subunit-containing GABAA-receptors (mostly α3 and α2 types), it has the potential to provide analgesia with less sedation than non-selective BDZ.

Fisetin decreases the duration of ictal-like discharges in mouse hippocampal slices.

There is an increasing interest in the biological and therapeutic effects of fisetin, a natural phenolic compound. Fisetin has affinity on some neuronal targets and may have the potential to modulate neuronal activity. In this study the effects of acute application of fisetin on synchronized events were evaluated electro-physiologically. Besides, interaction of fisetin with closely related channels were investigated in silico. Acute horizontal hippocampal slices were obtained from 32- to 36-day-old C57BL/6 mice. Extracellular field potentials were recorded from CA3 region of the hippocampus. Bath application of 4 aminopyridine (4AP, 100µM) initiated ictal- and interictal-like synchronized epileptiform discharges in the brain slices. Fifty micromolar fisetin was applied to the recording chamber during the epileptiform activity. The duration and frequencies of both ictal-like and interictal-like activities were calculated from the electrophysiological records. Molecular docking was performed to reveal interaction of fisetin on GABA-A, NMDA, AMPA receptors, and HCN2 channel, which are neuronal structures directly involved in recorded activity. Although fisetin does not affect basal neuronal activity in brain slice, it reduced the duration of ictal-like discharges significantly. Molecular docking results indicated that fisetin has no effect on GABA-A, NMDA, and AMPA receptors. However, fisetin binds to the (5JON) HCN2 channel strongly with the binding energy of -7.66kcal/mol. Reduction on the duration of 4AP-induced ictal-like discharges can be explained as HCN channels can cause an inhibitory effect via enhancing M-type K + channels which increase K outward currents.

Positive allosteric γ-aminobutyric acid type A receptor modulation prevents lipotoxicity-induced injury in hepatocytes in vitro.

To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABAA ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury. Allosteric modulation of the GABAA receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABAA channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting. Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABAA channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAA receptor. In HepG2 cells, which expressed the main GABAA receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI. HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.

Maturation Delay of Human GABAergic Neurogenesis in Fragile X Syndrome Pluripotent Stem Cells.

Fragile X Syndrome (FXS), the leading monogenic cause of intellectual disability and autism spectrum disorder, is caused by expansion of a CGG trinucleotide repeat in the 5ʹ-UTR of the Fragile X Mental Retardation-1 (FMR1) gene. Epigenetic silencing of FMR1 results in loss of the Fragile X Mental Retardation Protein (FMRP). Although most studies to date have focused on excitatory neurons, recent evidence suggests that GABAergic inhibitory networks are also affected. To investigate human GABAergic neurogenesis, we established a method to reproducibly derive inhibitory neurons from multiple FXS and control human pluripotent stem cell (hPSC) lines. Electrophysiological analyses suggested that the developing FXS neurons had a delay in the GABA functional switch, a transition in fetal development that converts the GABAA channel’s function from depolarization to hyperpolarization, with profound effects on the developing brain. To investigate the cause of this delay, we analyzed 14 400 single-cell transcriptomes from FXS and control cells at 2 stages of GABAergic neurogenesis. While control and FXS cells were similar at the earlier time point, the later-stage FXS cells retained expression of neuroblast proliferation-associated genes and had lower levels of genes associated with action potential regulation, synapses, and mitochondria compared with controls. Our analysis suggests that loss of FMRP prolongs the proliferative stage of progenitors, which may result in more neurons remaining immature during the later stages of neurogenesis. This could have profound implications for homeostatic excitatory-inhibitory circuit development in FXS, and suggests a novel direction for understanding disease mechanisms that may help to guide therapeutic interventions.

Is neuronal inhibition or excitability controlled by Na+K+2CL– transporters?

Is neuronal inhibition or excitability controlled by Na⁺K⁺2CL^– transporters?

Neuropathic Injury-Induced Plasticity of GABAergic System in Peripheral Sensory Ganglia.

GABA is a major inhibitory neurotransmitter in the mammalian central nervous system (CNS). Inhibitory GABAA channel circuits in the dorsal spinal cord are the gatekeepers of the nociceptive input from the periphery to the CNS. Weakening of these spinal inhibitory mechanisms is a hallmark of chronic pain. Yet, recent studies have suggested the existence of an earlier GABAergic “gate” within the peripheral sensory ganglia. In this study, we performed systematic investigation of plastic changes of the GABA-related proteins in the dorsal root ganglion (DRG) in the process of neuropathic pain development. We found that chronic constriction injury (CCI) induced general downregulation of most GABAA channel subunits and the GABA-producing enzyme, glutamate decarboxylase, consistent with the weakening of the GABAergic inhibition at the periphery. Strikingly, the α5 GABAA subunit was consistently upregulated. Knock-down of the α5 subunit in vivo moderately alleviated neuropathic hyperalgesia. Our findings suggest that while the development of neuropathic pain is generally accompanied by weakening of the peripheral GABAergic system, the α5 GABAA subunit may have a unique pro-algesic role and, hence, might represent a new therapeutic target.

Unraveling the mechanism and the risk behind seizure liability of lead compounds in a neuroscience project

IntroductionSeveral compounds from a neuroscience project induced convulsions in animals, at low exposure levels via a hypothetical off-target mechanism. A set of in vitro and in vivo experiments were conducted in order to 1) identify the mechanism behind convulsions; 2) characterize the convulsions, 3) detect premonitory signs that could be monitored clinically, and 4) assess the development of tolerance after repeat dosing. MethodsPatch clamp assays were conducted on 12 different ion channels (e.g. sodium, potassium, calcium, AMPA, NMDA, GABAA and purinergic receptors) known to be associated with seizures, to identify the off-target culprit. A multiphase study was conducted with UCB-A and UCB-B in Beagle dogs telemetered for video EEG/EMG monitoring to further characterize the convulsive pattern. First, both compounds were administered by intravenous constant infusion (dose: 5 mg/kg/h) over 2 h. Thereafter, the same dogs received a daily oral administration of UCB-A (8 mg/kg/day) for 7 days. ResultsCompounds inducing convulsions showed strong inhibitory activity on GABAA channels (IC50 values <10 μM), whereas compounds with partial or no inhibitory effect on these channels did not induce seizures. In EEG experiments, convulsions were preceded by premonitory clinical signs (e.g. tremors, myoclonic jerks) and morphological EEG abnormalities (e.g. sharp waves, spike and wave patterns), confirming their CNS origin. No attenuation of the seizurogenic effects was observed over the 7-day treatment period. DiscussionA well-designed set of experiments including electrophysiological assays on seizure-related ion channels and EEG/EMG assessment in telemetered dogs allowed a proper seizure liability risk assessment, leading to a rapid no go decision for the two most advanced leads.

Learning-induced enduring changes in inhibitory synaptic transmission in lateral amygdala are mediated by p21-activated kinase.

In this study we explored whether learning leads to enduring changes in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that olfactory discrimination (OD) learning in rats led to a long-lasting increase in postsynaptic GABAA channel-mediated miniature inhibitory postsynaptic currents (mIPSCs) in LA. Olfactory fear conditioning, but not auditory fear conditioning, also led to enduring enhancement in GABAA-mediated mIPSCs. Auditory fear conditioning, but not olfactory fear conditioning or OD learning, induced an enduring reduction in the frequency but not the current of mIPSC events. We found that p21-activated kinase (PAK) activity is needed to maintain OD and olfactory fear conditioning learning-induced enduring enhancement of mIPSCs. Further analysis revealed that OD led to an increase in GABAA channel conductance whereas olfactory fear conditioning increased the number of GABAA channels. These alterations in GABAA channels conductance and level are controlled by PAK activity. Our study shows that the learning-induced increase in postsynaptic inhibitory transmission in LA is specific to the sensory modality. However, the mechanism that mediates the increase in inhibitory transmission, namely the increase in the conductance or in the level of GABAA channel, is learning specific.NEW & NOTEWORTHY Here we studied whether learning leads to long-lasting alterations in inhibitory synaptic transmission in lateral amygdala (LA). We revealed that learning led to enduring changes in inhibitory synaptic transmission in LA that are affected by the sensory modality (auditory or olfaction) used during learning. However, the mechanism that mediated the changes in inhibitory transmission (alterations in GABAA channel level or conductance) depended on the type of learning. These long-lasting alterations are maintained by p21-activated kinase.

Opposite responses to aversive stimuli in lateral habenula neurons.

Appropriate behavioural strategies to cope with unexpected salient stimuli require synergistic neuronal responses in diverse brain regions. Among them, the epithalamic lateral habenula (LHb) plays a pivotal role in processing salient stimuli of aversive valence. Integrated in the complex motivational circuit, LHb neurons are indeed excited by aversive stimuli, including footshock (Fs). However, whether such excitation is a common feature represented throughout the LHb remains unclear. Here, we combined single-unit extracellular recordings in anaesthetized mice with juxtacellular labelling to describe the nature, location and pharmacological properties of Fs-driven responses within the LHb. We find that, along with Fs-excited cells, about 10% of LHb neurons display Fs-mediated inhibitory responses. Such inhibited neuronal population, in contrast to Fs-excited neurons, display regular and high frequency activity at baseline and is clustered in the medial portion of the LHb. Juxtacellular labelling of Fs-excited and inhibited neurons unravels that both populations are of glutamatergic type, as they co-localized with the EAAC1 glutamatergic transporter but not with the GAD67 GABAergic marker. Moreover, while the excitatory responses to Fs require both AMPA and NMDA receptors, the inhibitory responses rely instead on GABAA channels. Taken together, our results indicate that two functionally and partly segregated LHb neuronal ensembles encode Fs in an opposite fashion. This highlights the neuronal complexity in the LHb for processing aversive external stimuli.

De novo synthesis of the sedative valerenic acid in Saccharomyces cerevisiae

Valeriana officinalis (Valerian) root extracts have been used by European and Asian cultures for millennia for their anxiolytic and sedative properties. However, the efficacy of these extracts suffers from variable yields and composition, making these extracts a prime candidate for microbial production. Recently, valerenic acid, a C15 sesquiterpenoid, was identified as the active compound that modulates the GABAA channel. Although the first committed step, valerena-4,7(11)-diene synthase, has been identified and described, the complete valerenic acid biosynthetic pathway remains to be elucidated. Sequence homology and tissue-specific expression profiles of V. officinalis putative P450s led to the discovery of a V. officinalis valerena-4,7(11)-diene oxidase, VoCYP71DJ1, which required coexpression with a V. officinalis alcohol dehydrogenase and aldehyde dehydrogenase to complete valerenic acid biosynthesis in yeast. Further, we demonstrated the stable integration of all pathway enzymes in yeast, resulting in the production of 140 mg/L of valerena-4,7(11)-diene and 4 mg/L of valerenic acid in milliliter plates. These findings showcase Saccharomyces cerevisiae's potential as an expression platform for facilitating multiply-oxidized medicinal terpenoid pathway discovery, possibly paving the way for scale up and FDA approval of valerenic acid and other active compounds from plant-derived herbal medicines.

Design, synthesis, in vivo and in silico evaluation of phenacyl triazole hydrazones as new anticonvulsant agents

A series of phenacyl triazole hydrazones 3 have been designed based on the hybridization of (arylalkly)triazole and aroyl hydrazone scaffolds as new anticonvulsant agents. The target compounds 3 were easily synthesized from appropriate phenacyl triazoles and aryl acid hydrazides and characterized by IR, NMR and Mass spectroscopy. The in vivo anticonvulsant evaluation of synthesized compounds by using MES and PTZ tests revealed that they are more effective in MES model respect to PTZ test. All compounds showed 33–100% protection against MES-induced seizures at the dose of 100 mg/kg. However, the isonicotinic acid hydrazide derivative 3h showed the best profile of activity in both models. Molecular docking studies of compound 3h with different targets (NMDA, AMPA, GABAA and sodium channel), postulated that the compound acts mainly via GABAA receptors. In silico molecular properties predictions indicated that all compounds have favourable oral bioavailability and BBB permeability.

Quercetin Exerts Anti-convulsant Effects in Animal Model of Grand Mal Epilepsy: Modulation of GABA and Glycinergic Pathways

Quercetin is low molecular weight flavonoid having multiple neuropharmacological actions. Recently, its anticonvulsant effect was reported in rats using electrical kindling models. However, earlier cell culture studies have documented antagonistic action of quercetin on GABAA, GABAC and Glycine channels, which is contrary to recent findings. Hence, the present study aimed at characterizing the effects of quercetin administration in various experimental models of epilepsy. Dose-dependent effects of quercetin on maximal-electroshock seizure (MES) were determined. Further, the effective dose was tested in pentylenetetrazol-induced seizures (PTZ), and strychnine-induced seizures to study the involvement of GABA and Glycinergic receptors. The results revealed a potent anticonvulsant effect of quercetin in MES induced seizures at a dose of 5 and 10 mg/kg. This effect was found to be retained in case of PTZ-induced convulsions and strychnine-induced convulsions (quercetin 10 mg/kg). The present finding warrants further substantiation along with their correlation to molecular mechanism of action.

Learning-induced modulation of the effect of endocannabinoids on inhibitory synaptic transmission.

are key modulators that regulate central brain functions and behaviors, including learning and memory. At the cellular and molecular levels, endocannabinoids are potent modulators of excitatory and inhibitory synaptic function. Most effects of cannabinoids are thought to be mediated via G protein-coupled cannabinoid receptors. In particular, cannabinoids released from postsynaptic neurons are suggested to act as retrograde messengers, activating presynaptic type-1 cannabinoid receptors (CB1Rs), thereby inducing suppression of synaptic release. Another central mechanism of cannabinoid-induced action requires activation of astroglial CB1Rs. CB1Rs are also implicated in self-modulation of cortical neurons. Rats that are trained in a particularly difficult olfactory-discrimination task show a dramatic increased ability to acquire memories of new odors. The memory of the acquired high-skill acquisition, termed "rule learning" or "learning set," lasts for many months. Using this behavioral paradigm, we show a novel function of action for CB1Rs, supporting long-term memory by maintaining persistent enhancement of inhibitory synaptic transmission. Long-lasting enhancement of inhibitory synaptic transmission is blocked by a CB1R inverse agonist. This effect is mediated by a novel purely postsynaptic mechanism, obtained by enhancing the single GABAA channel conductance that is PKA dependent. The significant role that CB1R has in maintaining learning-induced long-term strengthening of synaptic inhibition suggests that endocannabinoids have a key role in maintaining long-term memory by enhancing synaptic inhibition. NEW & NOTEWORTHY In this study we show a novel function and mechanism of action for cannabinoids in neurons, mediated by activation of type-1 cannabinoid receptors, supporting long-term memory by maintaining persistent enhancement of inhibitory synaptic transmission on excitatory neurons. This effect is mediated by a novel purely postsynaptic mechanism, obtained by enhancing the single GABAA channel conductance that is PKA dependent. Thus we report for the first time that endocannabinoids have a key role maintaining learning-induced synaptic modification.

Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion.

Gonadotrophs are basophilic cells of the anterior pituitary gland specialized to secrete gonadotropins in response to elevation in intracellular calcium concentration. These cells fire action potentials (APs) spontaneously, coupled with voltage-gated calcium influx of insufficient amplitude to trigger gonadotropin release. The spontaneous excitability of gonadotrophs reflects the expression of voltage-gated sodium, calcium, potassium, non-selective cation-conducting, and chloride channels at their plasma membrane (PM). These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the PM, as well as GABAA, nicotinic, and purinergic P2X channels gated by γ-aminobutyric acid (GABA), acetylcholine (ACh), and ATP, respectively. Activation of these channels leads to initiation or amplification of the pacemaking activity, facilitation of calcium influx, and activation of the exocytic pathway. Gonadotrophs also express calcium-conducting channels at the endoplasmic reticulum membranes gated by inositol trisphosphate and intracellular calcium. These channels are activated potently by hypothalamic gonadotropin-releasing hormone (GnRH) and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate cyclase-activating peptides, endothelins, ACh, vasopressin, and oxytocin. Activation of these channels causes oscillatory calcium release and a rapid gonadotropin release, accompanied with a shift from tonic firing of single APs to periodic bursting type of electrical activity, which accounts for a sustained calcium signaling and gonadotropin secretion. This review summarizes our current understanding of ion channels as signaling molecules in gonadotrophs, the role of GnRH and paracrine agonists in their gating, and the cross talk among channels.

Targeting brain-derived neurotrophic factor in the medial thalamus for the treatment of central poststroke pain in a rodent model.

Approximately 7% to 10% of patients develop a chronic pain syndrome after stroke. This chronic pain condition is called central poststroke pain (CPSP). Recent studies have observed an abnormal increase in the secretion of brain-derived neurotrophic factor (BDNF) in spinal cord tissue after spinal cord injury. An animal model of CPSP was established by an intrathalamus injection of collagenase. Mechanical and thermal allodynia was induced after lesions of the thalamic ventral basal complex in rats. Four weeks after the injection, the number of neurons decreased, the number of astrocytes, microglia, and P2X4 receptors increased, and BDNF mRNA expression increased in the brain lesion area. Nociceptive activity in the medial thalamus (MT) and the coherence coefficient of spontaneous field potential oscillations in the anterior cingulate cortex were enhanced in CPSP animals, and these enhancements were blocked by an acute injection of TrkB-Fc and TrkB antagonist Tat Cyclotraxin-B. Instead of being inhibited by the γ-aminobutyric acid (GABA) system in normal rats, multiunit activity in the MT was enhanced after a microinjection of muscimol, a GABAA receptor agonist, in CPSP animals. After CPSP, BDNF expression was enhanced in the MT, whereas the expression of GABAA channels and the cotransporter KCC2 decreased in the same area. These findings suggest that neuronal plasticity in the MT that was induced by BDNF overexpression after the thalamic lesion was a key factor in CPSP.

Imaging and structural studies of DNA-protein complexes and membrane ion channels.

In bio-imaging by electron microscopy, damage of the sample and limited contrast are the two main hurdles for reaching high image quality. We extend a new preparation method based on nanofabrication and super-hydrophobicity to the imaging and structural studies of nucleic acids, nucleic acid-protein complexes (DNA/Rad51 repair protein complex) and neuronal ion channels (gap-junction, K+ and GABAA channels) as paradigms of biological significance and increasing complexity. The preparation method is based on the liquid phase and is compatible with physiological conditions. Only in the very last stage, samples are dried for TEM analysis. Conventional TEM and high-resolution TEM (HRTEM) were used to achieve a resolution of 3.3 and 1.5 Å, respectively. The EM dataset quality allows the determination of relevant structural and metrological information on the DNA structure, DNA-protein interactions and ion channels, allowing the identification of specific macromolecules and their structure.