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• hNa v 1.9_C4 chimera in CHOK-1 cells encompasses the extracellular and transmembrane domain of hNa v 1.9. • Synthetic cannabinoid ACEA blocked hNa v 1.9_C4+ß1 currents causing a hyperpolarizing shift in inactivation. • hNa v 1.9_C4 channels display window currents, significantly reduced by ACEA. • Computationally, ACEA binds the local anaesthetic binding site. • The local anaesthetic profile of ACEA may be involved in cannabinoid analgesia. Cannabinoids regulate analgesia, which has aroused much interest in identifying new pharmacological therapies in the management of refractory pain. Voltage-gated Na+ channels (Na v s) play an important role in inflammatory and neuropathic pain. In particular, Na v 1.9 is involved in nociception and the understanding of its pharmacology has lagged behind because it is difficult to express in heterologous systems. Here, we utilized the chimeric channel hNa v 1.9_C4, that comprises the extracellular and transmembrane domains of hNa v 1.9, co-expressed with the ß1 subunit on CHO-K1 cells to characterize the electrophysiological effects of ACEA, a synthetic surrogate of the endogenous cannabinoid anandamide. ACEA induced a tonic block, decelerated the fast inactivation, markedly shifted steady-state inactivation in the hyperpolarized direction, decreasing the window current and showed use-dependent block, with a high affinity for the inactivated state (k i = 0.84 µM). Thus, we argue that ACEA possess a local anaesthetic-like profile. To provide a mechanistic understanding of its mode of action at the molecular level, we combined induced fit docking with Monte Carlo simulations and electrostatic complementarity. In agreement with the experimental evidence, our computer simulations revealed that ACEA binds Tyr1599 of the local anaesthetics binding site of the hNa v 1.9, contacting residues that bind cannabinol (CBD) in the Na v Ms channel. ACEA adopted a conformation remarkably similar to the crystallographic conformation of anandamide on a non-homologous protein, obstructing the Na+ permeation pathway below the selectivity filter to occupy a highly conserved binding pocket at the intracellular side. These results describe a mechanism of action, possibly involved in cannabinoid analgesia. [ABSTRACT FROM AUTHOR]

The presubiculum is part of the parahippocampal cortex and plays a fundamental role for orientation in space. Many principal neurons of the presubiculum signal head direction, and show persistent firing when the head of an animal is oriented in a specific preferred direction. GABAergic neurons of the presubiculum control the timing, sensitivity and selectivity of head directional signals from the anterior thalamic nuclei. However, the role of vasoactive intestinal peptide (VIP) expressing interneurons in the presubicular microcircuit has not yet been addressed. Here, we examined the intrinsic properties of VIP interneurons as well as their input connectivity following photostimulation of anterior thalamic axons. We show that presubicular VIP interneurons are more densely distributed in superficial than in deep layers. They are highly excitable. Three groups emerged from the unsupervised cluster analysis of their electrophysiological properties. We demonstrate a frequency dependent recruitment of VIP cells by thalamic afferences and facilitating synaptic input dynamics. Our data provide initial insight into the contribution of VIP interneurons for the integration of thalamic head direction information in the presubiculum., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

• I A K+ currents suppress the response of rNST neurons to afferent stimulation. • GABAergic inhibition also suppresses afferent-induced responses. • GABAergic inhibition and I A appear to operate independently. Responses in the rostral (gustatory) nucleus of the solitary tract (rNST) are modified by synaptic interactions within the nucleus and the constitutive membrane properties of the neurons themselves. The potassium current I A is one potential source of modulation. In the caudal NST, projection neurons with I A show lower fidelity to afferent stimulation compared to cells without. We explored the role of an A-type K+ current (I A) in modulating the response to afferent stimulation and GABA-mediated inhibition in the rNST using whole cell patch clamp recording in transgenic mice that expressed channelrhodopsin (ChR2 H134R) in GABAergic neurons. The presence of I A was determined in current clamp and the response to electrical stimulation of afferent fibers in the solitary tract was assessed before and after treatment with the specific Kv4 channel blocker AmmTX3. Blocking I A significantly increased the response to afferent stimulation by 53%. Using dynamic clamp to create a synthetic I A conductance, we demonstrated a significant 14% decrease in responsiveness to afferent stimulation in cells lacking I A. Because I A reduced excitability and is hyperpolarization-sensitive, we examined whether I A contributed to the inhibition resulting from optogenetic release of GABA. Although blocking I A decreased the percent suppression induced by GABA, this effect was attributable to the increased responsiveness resulting from AmmTX3, not to a change in the absolute magnitude of suppression. We conclude that rNST responses to afferent input are regulated independently by I A and GABA. [ABSTRACT FROM AUTHOR]

• Noise-induced hearing loss leads to reduced expression of PV, GAD65/67, and KV3.1. • PV expression in single neurons is correlated with GAD67 but not KV3.1 expression. • Spike frequency adaptation increases following noise-induced hearing loss. • Spike frequency adaption is correlated with KV3.1, but not PV or GAD67, expression. Parvalbumin-expressing (PV+) interneurons in the sensory cortex form powerful inhibitory synapses on the perisomatic compartments and axon initial segments of excitatory principal neurons (PNs), and perform diverse computational functions. Impaired PV+ interneuron functions have been reported in neural developmental and degenerative disorders. Expression of the unique marker parvalbumin (PV) is often used as a proxy of PV+ interneuron functions. However, it is not entirely clear how PV expression is correlated with PV+ interneuron properties such as spike firing and synaptic transmission. To address this question, we characterized electrophysiological properties of PV+ interneurons in the primary auditory cortex (AI) using whole-cell patch clamp recording, and analyzed the expression of several genes in samples collected from single neurons using the patch pipettes. We found that, after noise induced hearing loss (NIHL), the spike frequency adaptation increased, and the expression of PV, glutamate decarboxylase 67 (GAD67) and Shaw-like potassium channel (KV3.1) decreased in PV+ neurons. In samples prepared from the auditory cortical tissue, the mRNA levels of the target genes were all pairwise correlated. At the single neuron level, however, the expression of PV was significantly correlated with the expression of GAD67, but not KV3.1, maximal spike frequency, or spike frequency adaptation. The expression of KV3.1 was correlated with spike frequency adaptation, but not with the expression of GAD67. These results suggest separate transcriptional regulations of PV/GAD67 vs. KV3.1, both of which are modulated by NIHL. [ABSTRACT FROM AUTHOR]

• Prenatal nicotine alters development of acidosis response in medullary 5-HT neurons. • Prenatal nicotine blunts the acidosis response of cultured medullary 5-HT neurons. • Prenatal nicotine blunts the effects of acute nicotine on medullary 5-HT neurons. • Impaired function of 5-HT neurons through prenatal nicotine may increase SIDS risk. Perinatal exposure to nicotine produces ventilatory and chemoreflex deficits in neonatal mammals. Medullary 5-HT neurons are putative central chemoreceptors that innervate respiratory nuclei and promote ventilation, receive cholinergic input and express nicotinic acetylcholine receptors (nAChRs). Perforated patch clamp recordings were made from cultured 5-HT neurons dissociated from the medullary raphé of 0–3 day old mice expressing enhanced yellow fluorescent protein driven by the enhancer region for PET1 (ePet-EYFP). The effect of exposure to low (6 mg kg−1day−1) or high (60 mg kg−1day−1) doses of nicotine in utero (prenatal), in culture (postnatal), or both and the effect of acute nicotine exposure (10 μM), were examined on baseline firing rate (FR at 5% CO 2 , pH = 7.4) and the change in FR with acidosis (9% CO 2 , pH 7.2) in young (12–21 days in vitro , DIV) and older (≥22 DIV) acidosis stimulated 5-HT neurons. Nicotine exposed neurons exhibited ∼67% of the response to acidosis recorded in neurons given vehicle (p = 0.005), with older neurons exposed to high dose prenatal and postnatal nicotine, exhibiting only 28% of that recorded in the vehicle neurons (p < 0.01). In neurons exposed to low or high dose prenatal and postnatal nicotine , acute nicotine exposure led to a smaller increase in FR (∼+51% vs +168%, p = 0.026) and response to acidosis (+6% vs +67%, p = 0.014) compared to vehicle. These data show that exposure to nicotine during development reduces chemosensitivity of 5-HT neurons as they mature, an effect that may be related to the abnormal chemoreflexes reported in rodents exposed to nicotine in utero , and may cause a greater risk for sudden infant death syndrome (SIDS). [ABSTRACT FROM AUTHOR]

Noradrenaline (NA) modulates the spinal motor networks for locomotion and facilitates neuroplasticity, possibly assisting neuronal network activation and neuroplasticity in the recovery phase of spinal cord injuries. However, neither the effects nor the mechanisms of NA on synaptic transmission and neuronal excitability in spinal ventral horn (VH) neurons are well characterized, especially in rats aged 7 postnatal days or older. To gain insight into NA regulation of VH neuronal activity, we used a whole-cell patch-clamp approach in late neonatal rats (postnatal day 7–15). In voltage-clamp recordings at − 70 mV, NA increased the frequency and amplitude of excitatory postsynaptic currents via the activation of somatic α 1 - and β-adrenoceptors of presynaptic neurons. Moreover, NA induced an inward current through the activation of postsynapticα 1 - and β-adrenoceptors. At a holding potential of 0 mV, NA also increased frequency and amplitude of both GABAergic and glycinergic inhibitory postsynaptic currents via the activation of somatic adrenoceptors in presynaptic neurons. In current-clamp recordings, NA depolarized resting membrane potentials and increased the firing frequency of action potentials in VH neurons, indicating that it enhances the excitability of these neurons. Our findings provide new insights that establish NA-based pharmacological therapy as an effective method to activate neuronal networks of the spinal VH in the recovery phase of spinal cord injuries. • The effects of noradrenaline (NA) on ventral horn neurons of rats were examined using a whole-cell patch-clamp technique. • NA facilitated excitatory synaptic transmission via the activation of α 1 - and β-adrenoceptors in presynaptic neurons. • NA induced an inward current via the activation of postsynaptic α 1 - and β-adrenoceptors. • NA enhanced inhibitory synaptic transmission via the activation of somatic adrenoceptors in presynaptic neurons. • NA depolarized the resting membrane potential and increased the firing frequency of action potentials. [ABSTRACT FROM AUTHOR]

Abstract The subfornical organ (SFO) is forebrain sensory circumventricular organ, characterized by lack of a blood–brain barrier. Neurons of the SFO can detect circulating molecules such as peptide hormones and communicate this information to regulatory centers behind the blood–brain barrier, thus playing a critical role in homeostatic processes including regulation of energy balance, hydromineral balance and cardiovascular control. The SFO contains two subregions defined by neuronal expression of molecular markers: the dorsolateral peripheral or shell SFO (sSFO) neurons express calretinin, and the ventromedial core (cSFO) neurons express calbindin D28K. Neurons from these two subregions project to different locations to subserve different roles in homeostatic regulation. It is unknown whether neurons from these two subregions exhibit unique or identifiable electrophysiological properties. This study used a gold nanoparticle-conjugated RNA fluorescent probe on dissociated SFO neuron cultures and patch clamp electrophysiology to characterize the intrinsic electrophysiological properties of cSFO and sSFO neurons. Our studies revealed that neurons originating from the core region exhibited significantly more action potential bursting, while neurons from non-core regions exhibited more tonic firing neurons, albeit at a higher overall frequency. The difference in activity is correlated with a more depolarized resting membrane potential and a higher density of voltage gated Na+ currents. Highlights • SFO neurons can be classified as core neurons (expressing CalB) or peripheral shell neurons (expressing CalR). • CalB expressing SFO neurons can be labeled with gold nanoparticles conjugated to antisense RNA and a fluorophore (SmartFlare™). • CalB(+) neurons were significantly more likely to be spontaneously active, but were bursty with an overall lower frequency. • Membranes of CalB(+) neurons showed significantly hyperpolarized membrane potential and less density of voltage gated Na + current. • Spontaneous electrical activity of SFO neurons may be important for maintaining tone on synaptic targets. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The olfactory and vomeronasal sensory neurons showed phasic and tonic firing under the patch-clamp condition. • The phasic firing of olfactory sensory neurons accurately encoded a stimulus changing within 200 ms. • The tonic firing of vomeronasal sensory neurons encoded smaller stimuli changing slowly with longer cycles of >500 ms. • Intrinsic channel kinetics determining firing patterns was demonstrated by a Hodgkin–Huxley style computation. Abstract The parallel processing of chemical signals by the main olfactory system and the vomeronasal system has been known to control animal behavior. The physiological significance of peripheral parallel pathways consisting of olfactory sensory neurons and vomeronasal sensory neurons is not well understood. Here, we show complementary characteristics of the information transfer of the olfactory sensory neurons and vomeronasal sensory neurons. A difference in excitability between the sensory neurons was revealed by patch-clamp experiments. The olfactory and vomeronasal sensory neurons showed phasic and tonic firing, respectively. Intrinsic channel kinetics determining firing patterns was demonstrated by a Hodgkin-Huxley-style computation. Our estimation of the information carried by action potentials during one cycle of sinusoidal stimulation with variable durations revealed distinct characteristics of information transfer between the sensory neurons. Phasic firing of the olfactory sensory neurons was suitable to carry information about rapid changes in a shorter cycle (<200 ms). In contrast, tonic firing of the vomeronasal sensory neurons was able to convey information about smaller stimuli changing slowly with longer cycles (>500 ms). Thus, the parallel pathways of the two types of sensory neurons can convey information about a wide range of dynamic stimuli. A combination of complementary characteristics of olfactory information transfer may enhance the synergy of the interaction between the main olfactory system and the vomeronasal system. [ABSTRACT FROM AUTHOR]

Phox2b encodes a paired-like homeodomain-containing transcription factor essential for development of the autonomic nervous system. Phox2b -expressing (Phox2b + ) neurons are present in the reticular formation dorsal to the trigeminal motor nucleus (RdV) as well as the nucleus of the solitary tract and parafacial respiratory group. However, the nature of Phox2b + RdV neurons is still unclear. We investigated the physiological and morphological properties of Phox2b + RdV neurons using postnatal day 2–7 transgenic rats expressing yellow fluorescent protein under the control of Phox2b . Almost all of Phox2b + RdV neurons were glutamatergic, whereas Phox2b -negative (Phox2b − ) RdV neurons consisted of a few glutamatergic, many GABAergic, and many glycinergic neurons. The majority (48/56) of Phox2b + neurons showed low-frequency firing (LF), while most of Phox2b − neurons (35/42) exhibited high-frequency firing (HF) in response to intracellularly injected currents. All, but one, Phox2b + neurons (55/56) did not fire spontaneously, whereas three-fourths of the Phox2b − neurons (31/42) were spontaneously active. K + channel and persistent Na + current blockers affected the firing of LF and HF neurons. The majority of Phox2b + (35/46) and half of the Phox2b − neurons (19/40) did not respond to stimulations of the mesencephalic trigeminal nucleus, the trigeminal tract, and the principal sensory trigeminal nucleus. Biocytin labeling revealed that about half of the Phox2b + (5/12) and Phox2b − RdV neurons (5/10) send their axons to the trigeminal motor nucleus. These results suggest that Phox2b + RdV neurons have distinct neurotransmitter phenotypes and firing properties from Phox2b − RdV neurons and might play important roles in feeding-related functions including suckling and possibly mastication. [ABSTRACT FROM AUTHOR]

The intergeniculate leaflet (IGL) is a flat thalamic nucleus implicated in the modulation of circadian rhythmicity. In rat, two main GABAergic subpopulations can be distinguished in the IGL: neurons synthesizing neuropeptide Y (NPY), which directly innervates the suprachiasmatic nuclei, and enkephalinergic cells, which connect contralaterally located leaflets. The aim of this study was to evaluate possible effects of inner IGL neurotransmitters on the spontaneous and synaptic activity of IGL neurons. The data presented in this article provide evidence that enkephalin, and not NPY, could act upon the majority of IGL neurons. Moreover, we investigated the type of opioid receptor activated by enkephalin and showed that the μ-receptor is functionally predominant in the IGL. The application of met-enkephalin not only robustly hyperpolarized IGL neurons (both putatively NPY-synthesizing and putatively enkephalinergic neurons), but it also was able to inhibit GABAergic and glutamatergic synaptic transmission. Based on this and previous studies, we hypothesize that IGL enkephalinergic neurons may act as powerful interneurons that inhibit themselves and NPY-synthesizing neurons, also in the contralaterally located IGL. [ABSTRACT FROM AUTHOR]

This study examined the effect of neuron-endothelial coupling on the survival of neurons after ischemia and the possible mechanism underlying that effect. Whole-cell patch-clamp experiments were performed on cortical neurons cultured alone or directly cocultured with brain microvascular endothelial cells (BMEC). Propidium iodide (PI) and NeuN staining were performed to examine neuronal death following oxygen and glucose deprivation (OGD). We found that the neuronal transient outward potassium currents ( I A ) decreased in the coculture system, whereas the outward delayed-rectifier potassium currents ( I K ) did not. Sodium nitroprusside, a NO donor, enhanced BMEC-induced I A inhibition and nitro- l -arginine methylester, a NOS inhibitor, partially prevented this inhibition. Moreover, the neurons directly cocultured with BMEC showed more resistance to OGD-induced injury compared with the neurons cultured alone, and that neuroprotective effect was abolished by treatment with NS5806, an activator of the I A . These results indicate that vascular endothelial cells assist neurons to prevent hypoxic injury via inhibiting neuronal I A by production of NO in the direct neuron-BMEC coculture system. These results further provide direct evidence of functional coupling between neurons and vascular endothelial cells. This study clearly demonstrates that vascular endothelial cells play beneficial roles in the pathophysiological processes of neurons after hypoxic injury, suggesting that the improvement of neurovascular coupling or functional remodeling may become an important therapeutic target for preventing brain injury. [ABSTRACT FROM AUTHOR]

Hydrogen peroxide (H 2 O 2 ), a reactive oxygen species, is an important signaling molecule for synaptic and neuronal activity in the central nervous system; it is produced excessively in brain ischemia and spinal cord injury. Although H 2 O 2 -mediated modulations of synaptic transmission have been reported in ventral horn (VH) neurons of the rat spinal cord, the effects of H 2 O 2 on neuronal excitability and membrane properties remain poorly understood. Accordingly, the present study investigated such effects using a whole-cell patch-clamp technique. The bath-application of H 2 O 2 decreased neuronal excitability accompanied by decreased input resistance, firing frequency, and action potential amplitude and by increased rheobase. These H 2 O 2 -mediated changes were induced by activation of extrasynaptic, but not synaptic, GABA A receptors. Indeed, GABAergic tonic currents were enhanced by H 2 O 2 . On the other hand, the amplitude of medium and slow afterhyperpolarization (mAHP and sAHP), which plays important roles in controlling neuronal excitability and is mediated by small-conductance calcium-activated potassium (SK) channels, was significantly decreased by H 2 O 2 . When extrasynaptic GABA A receptors were completely blocked, these decreases of mAHP and sAHP persisted, and H 2 O 2 increased excitability, suggesting that H 2 O 2 per se might have the potential to increase neuronal excitability via decreased SK channel conductance. These findings indicate that activating extrasynaptic GABA A receptors or SK channels may attenuate acute neuronal damage caused by H 2 O 2 -induced hyperexcitability and therefore represent a novel therapeutic target for the prevention and treatment of H 2 O 2 -induced motor neuron disorders. [ABSTRACT FROM AUTHOR]

Emerging studies have shown that pharmacological activation of adenosine monophosphate-activated protein kinase (AMPK) produces potent analgesic effects in different animal pain models. Currently, the spinal molecular and synaptic mechanism by which AMPK regulates the pain signaling system remains unclear. To address this issue, we utilized the Cre-LoxP system to conditionally knockout the AMPKα1 gene in the nervous system of mice. We demonstrated that AMPKα1 is imperative for maintaining normal nociception, and mice deficient for AMPKα1 exhibit mechanical allodynia. This is concomitantly associated with increased glutamatergic synaptic activities in neurons located in the superficial spinal dorsal horn, which results from the increased glutamate release from presynaptic terminals and function of ligand-gated glutamate receptors at the postsynaptic neurons. Additionally, AMPKα1 knockout mice have increased activities of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinases (p38), as well as elevated levels of interleukin-1β (IL-1β), reactive oxygen species (ROS), and heme oxygenase 1 (HO-1) in the spinal dorsal horn. Systemic administration of a non-specific ROS scavenger (phenyl-N-tert-butylnitrone, PBN) or a HO-1 activator (Cobalt protoporphyrin IX, CoPP) attenuated allodynia in AMPKα1 knockout mice. Bath-perfusion of the ROS scavenger or HO-1 activator effectively attenuated the increased ROS levels and glutamatergic synaptic activities in the spinal dorsal horn. Our findings suggest that ROS are the key down-stream signaling molecules mediating the behavioral hypersensitivity in AMPKα1 knockout mice. Thus, targeting AMPKα1 may represent an effective approach for the treatment of pathological pain conditions associated with neuroinflammation at the spinal dorsal horn. [ABSTRACT FROM AUTHOR]

Retinal bipolar cells are second-order neurons in the visual system, which initiate multiple image feature-based neural streams. Among more than ten types of bipolar cells, type-5 cells are thought to play a role in motion detection pathways. Multiple subsets of type-5 cells have been reported; however, detailed characteristics of each subset have not yet been elucidated. Here, we found that they exhibit distinct morphological features as well as unique voltage-gated channel expression. We have conducted electrophysiological and immunohistochemical analysis of retinal bipolar cells. We defined type-5 cells by their axon terminal ramification in the inner plexiform layer between the border of ON/OFF sublaminae and the ON choline acetyltransferase (ChAT) band. We found three subsets of type-5 cells: XBCs had the widest axon terminals that stratified at a close approximation of the ON ChAT band as well as exhibiting large voltage-gated Na + channel activity, type-5-1 cells had compact terminals and no Na + channel activity, and type-5-2 cells contained umbrella-shaped terminals as well as large voltage-gated Na + channel activity. Hyperpolarization-activated cyclic nucleotide-gated (HCN) currents were also evoked in all type-5 bipolar cells. We found that XBCs and type-5-2 cells exhibited larger HCN currents than type-5-1 cells. Furthermore, the former two types showed stronger HCN1 expression than the latter. Our previous observations (Ichinose et al., 2014) match the current study: low temporal tuning cells that we named 5S corresponded to 5-1 in this study, while high temporal tuning 5f cells from the previous study corresponded to 5-2 cells. Taken together, we found three subsets of type-5 bipolar cells based on their morphologies and physiological features. [ABSTRACT FROM AUTHOR]

The purpose of the present study was to characterize the properties of A-type GABA receptor (GABA A receptor) currents in human sensory neurons. Neurons were obtained from adult organ donors. GABA A currents were recorded in isolated neurons. Both large inactivating low-affinity currents and smaller persistent high-affinity currents were present in all of the 129 neurons studied from 15 donors. The kinetics of human GABA A currents were slower than those in rat sensory neurons. GABA currents were completely blocked by bicuculline (10 μM), and persistent currents were activated by the δ-subunit-preferring agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). The GABA current equilibrium potential was ∼20 mV more hyperpolarized than in rat neurons. Both low- and high-affinity currents were increased by inflammatory mediators but via different second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool to test compounds prior to use in humans. [ABSTRACT FROM AUTHOR]

Orexins/hypocretins (OXA and OXB) are two hypothalamic peptides involved in the regulation of many physiological processes including the sleep-wake cycle, food intake and arousal. The orexinergic system of the lateral hypothalamus is considered a non-specific peptidergic system, and its nerve fibers innervate numerous brain areas. Among many targets of orexinergic neurons is the intergeniculate leaflet (IGL) of the thalamus – a small but important structure of the mammalian biological clock. In rats, the IGL consists of GABAergic cells which also synthesize different neuropeptides. One group of neurons produces neuropeptide Y (NPY) and sends its axons to the master biological clock known as the suprachiasmatic nuclei. Another neuronal group produces enkephalin and is known to connect contralateral IGLs. This study evaluated the effects of orexins on identified IGL neurons revealing that 58% of the recorded neurons were sensitive to OXA (200 nM) and OXB (200 nM) administration. Both NPY-positive and -negative neurons were depolarized by these neuropeptides. Experiments using selective orexin receptor antagonists (SB-334867, 10 μM and TCS-OX2-29, 10 μM) suggested that both orexin receptors participate in the recorded OXA effects. In addition, IGL neurons were either directly depolarized by OXA or their activity was altered by changes in presynaptic inputs. We observed an increase of GABA release onto the investigated IGL neuron after OXA application, consistent with a presynaptic localization of the orexin receptors. An increase in miniature excitatory postsynaptic current frequency was not observed within the IGL. Our findings reinforce the connection between circadian clock physiology and the orexinergic system. [ABSTRACT FROM AUTHOR]

We recently indicated that the vascular endothelial growth factor (VEGF) protects neurons against hypoxic death via enhancement of tyrosine phosphorylation of Kv1.2, an isoform of the delayed-rectifier potassium channels through activation of the phosphatidylinositol 3-kinase (PI3-K) signaling pathway. The present study investigated whether VEGF could attenuate ischemia-induced increase of the potassium currents in the hippocampal pyramidal neurons of rats after ischemic injury. Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion (MCAO) to induce brain ischemia. The whole-cell patch-clamp technique was used to record the potassium currents of hippocampal neurons in brain slices from the ischemically injured brains of the rats 24 h after MCAO. We detected that transient MCAO caused a significant increase of voltage-gated potassium currents ( Kv ) and outward delayed-rectifier potassium currents ( I K ), but not outward transient potassium currents ( I A ), in the ipsilateral hippocampus compared with the sham. Moreover, we found that VEGF could acutely, reversibly and voltage-dependently inhibit the ischemia-induced I K increase. This inhibitory effect of VEGF could be completely abolished by wortmannin, an inhibitor of PI3-K. Our data indicate that VEGF attenuates the ischemia-induced increase of I K via activation of the PI3-K signaling pathway. [ABSTRACT FROM AUTHOR]

Both central and peripheral sympathetic nervous systems contribute to the cardiovascular effects of dexmedetomidine (DMED), a highly selective and widely used a 2 -adrenoceptor agonist for sedation, analgesia, and stress management. The central sympatholytic effects are augmented by peripheral inhibition of sympathetic ganglion transmission. The mechanism is not clear. In this research, using conventional patch-clamp recordings we investigated the direct effects of DMED on sodium (Na + ) channel currents ( I Na ) and nicotinic acetylcholine (ACh) receptor (nAChRs) channel currents ( I ACh ) in rat superior cervical ganglion (SCG) neurons to explore the possible mechanisms of sympathetic ganglion transmission inhibition by DMED. DMED voltage-dependently suppressed I Na with half maximal inhibitory concentration (IC 50 ) values of 67.2 ± 9.6 μM and 26.1 ± 5.3 μM at holding potentials of −80 mV and −60 mV, respectively. The inhibition of Na + channels by DMED was also frequency dependent. 100 μM DMED shifted the Na + channel inactivation curves to the hyperpolarizing direction by 9.8 mV ( P < 0.01) and slowed the recovery from inactivation by 8.9 ms ( P < 0.01), but no effects were seen on the shape of the current–voltage relationship or Na + channels activation curves. DMED dose-dependently inhibited I ACh with an IC 50 value of 5.5 ± 2.4 μM in SCG neurons, and this inhibition was voltage-independent. DMED pretreatment followed by fast co-application of DMED and ACh produced a significantly larger I ACh inhibition than without DMED pretreatment. Yohimbine, phentolamine, and atropine pretreatment did not alter the inhibitory effects of DMED on I Na and I ACh . In conclusion, DMED dose-dependently inhibits I Na and I ACh in rat SCG neurons by preferential binding to the inactivated state of the Na + channels and the closed state (resting) of nAChR channels respectively. Both inhibitions are a 2 -adrenoceptor independent. Furthermore, the nAChR channels in rat SCG neurons are much more sensitive to inhibition by DMED than Na + channels. [ABSTRACT FROM AUTHOR]

To evaluate physiological roles of the large, second cytoplasmic loops (C2) situated between the M3 and M4 transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits. We have constructed chimeric β2 (β2χ) and β4 (β4χ) subunits in which the “nested” C2 domains (but not the “proximal” sequences of ∼14 residues immediately adjacent to the M3 or M4 domains) of these β subunits were replaced by the corresponding sequence from the serotonin 5-HT 3A receptor subunit. We previously reported that heterologously expressed nAChR containing α4 and β2χ subunits displayed a faster whole-cell current decay in its agonist response compared to responses of all-wild-type α4β2-nAChR. This suggests an unexpected, functional role for the C2 domain of the β2 subunit in α4β2-nAChR acute desensitization. Here we report that there also is faster desensitization of α4β4χ-nAChR relative to α4β4-nAChR stably and heterologously expressed in the human SH-EP1 cell-line. In addition, cell-attached, single-channel recording shows that both acetylcholine-activated α4β2χ- and α4β4χ-nAChR have a significantly lower mean open probability, shorter mean open-time, and a longer mean closed-time than their fully wild-type counterparts while not having different conductance amplitudes. These findings reveal microscopic bases for the faster desensitization of α4 ∗ -nAChR containing chimeric instead of wild-type β subunits. Our findings also remain consistent with novel and unexpected roles of β subunit-nested C2 domains in modulation of α4 ∗ -nAChR function. [ABSTRACT FROM AUTHOR]

Oxidative stress and the production of reactive oxygen radicals play a key role in neuronal cell damage. This paper describes an in vitro study that explores the neuronal responses to oxidative stress focusing on changes in neuronal excitability and functional membrane properties. This study was carried out in pyramidal cells of the motor cortex by applying whole-cell patch-clamp techniques on brain slices from young adult rats. Oxygen-derived free radical formation was induced by bath application of 10 μM cumene hydroperoxide (CH) for 30 min. CH produced marked changes in the electrophysiological properties of neurons ( n = 30). Resting membrane potential became progressively depolarized, as well as depolarization voltage, with no variations in voltage threshold. Membrane resistance showed a biphasic behavior, increasing after 5 min of drug exposure and then it started to decrease, even under control values, after 15 and 30 min. At the same time, changes in membrane resistance produced compensatory variations in the rheobase. The amplitude of the action potentials diminished and the duration increased progressively over time. Some of the neurons under study also lost their ability to discharge action potentials in a repetitive way. Most of the neurons, however, kept their repetitive discharge even though their maximum frequency and gain decreased. Furthermore, cancelation of the repetitive firing discharge took place at intensities that decreased with time of exposure to CH, which resulted in a narrower working range. We can conclude that oxidative stress compromises both neuronal excitability and the capability of generating action potentials, and so this type of neuronal functional failure could precede the neuronal death characteristics of many neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Highlights: [•] Neonatal injury alters the passive membrane properties of adult dorsal horn neurons. [•] Early incision reduces intrinsic membrane excitability in the mature dorsal horn. [•] Kir channels regulate membrane excitability in adult mouse dorsal horn cells. [•] Neonatal incision up-regulates Kir currents selectively in adult GABAergic neurons. [ABSTRACT FROM AUTHOR]

Highlights: [•] Tipepidine inhibited DA D2 receptor-mediated GIRK channel currents in VTA neurons. [•] Tipepidine activated single VTA neurons inhibited by dopamine. [•] Tipepidine increased double immunopositive cells for TH and c-Fos in VTA. [•] The results show a part of mechanisms for novel antidepressant effect of tipepidine. [Copyright &y& Elsevier]

Abstract: Neuropathy is often seen in uncontrolled diabetes and the mechanisms involved for neuropathic pain are poorly understood. Hyperglycemia is a consequence of chronic uncontrolled diabetes and it is postulated to produce neuropathic pain. Therefore, in this study, we have investigated the effects of hyperglycemia on Na+ channel kinetics in cultured dorsal root ganglion (DRG) neurons from neonatal rats using whole-cell patch-clamp technique. Hyperglycemia-induced increase in density of tetrodotoxin resistant (TTXr) Na+ currents was increased in time- and concentration-dependent manner. The increase was maximal with 60mM and 24h. There was no change Na+ current density in time-matched control neurons. The conductance curve of TTXr Na+ current shifted leftward after 24h exposure to 45mM glucose. Carbamazepine (CBZ, 100μM) depressed TTXr Na+ current in neurons incubated with control (17.26), 45 and 60mM of glucose. The depression observed with CBZ in the presence of high glucose, i.e., 45mM (86.5±4.9%) was significantly greater than control (61.6±1.8%). Hyperglycemia also increased reactive oxygen species (ROS) activity and was attenuated by CBZ. These results suggest that short-term exposure of DRG neurons to high glucose concentrations enhance the Na+ channel activity, and were attenuated by CBZ via ROS-dependent mechanisms. [Copyright &y& Elsevier]

Abstract: The ventral tegmental area (VTA) plays an important role in reward and motivational processes that facilitate the development of drug addiction. Glutamatergic inputs into the VTA contribute to dopamine (DA) neuronal activation related to reward and response-initiating effects in drug abuse. Previous investigations indicate that alpha1-adrenoreceptors (α1-ARs) are primarily localized at presynaptic elements in the ventral midbrain. Studies from several brain regions have shown that presynaptic α1-AR activation enhances glutamate release. Therefore, we hypothesized that glutamate released onto VTA-DA neurons is modulated by pre-synaptic α1-AR. Recordings were obtained from putative VTA-DA cells of male Sprague–Dawley rats (28–50days postnatal) using voltage clamp techniques. Phenylephrine (10μM) and methoxamine (80μM), both α1-AR agonists, increased AMPA receptor-mediated excitatory postsynaptic currents’ (EPSCs) amplitude evoked by electrical stimulation of afferent fibers (p <0.05). This effect was blocked by the α1-AR antagonist prazosin (1μM). Phenylephrine decreased the paired-pulse ratio (PPR) and increased spontaneous EPSCs’ frequencies but not their amplitudes suggesting a presynaptic locus of action. No changes in miniature EPSCs (0.5μM, tetrodotoxin [TTX]) were observed after phenylephrine’s application which suggests that α1-AR effect was action potential dependent. Normal extra- and intracellular Ca2+ concentration seems necessary for the α1-AR effect since phenylephrine in low Ca2+ artificial cerebrospinal fluid (ACSF) and depletion of intracellular Ca2+ stores with thapsigargin (10μM) failed to increase the AMPA EPSCs’ amplitude. Chelerythrine (1μM, protein kinase C (PKC) inhibitor) but not Rp-cAMPS (11μM, PKA inhibitor) blocked the α1-AR activation effect on AMPA EPSCs, indicating that a PKC intracellular pathway is required. These results demonstrated that presynaptic α1-AR activation modulates glutamatergic inputs that affect VTA-DA neuronal excitability. α1-AR action might be heterosynaptically localized at glutamatergic fibers terminating onto VTA-DA neurons. It is suggested that drug-induced changes in α1-AR could be part of the neuroadaptations occurring in the mesocorticolimbic circuitry during the addiction process. [Copyright &y& Elsevier]

Abstract: Enhanced nociceptive firing in trigeminal ganglion neurons is a likely reason for migraine pain. In experimental migraine-like conditions induced by the calcitonin gene-related peptide (CGRP), P2X3 receptors abundantly expressed in trigeminal neurons are highly responsive to the excitatory action of extracellular ATP. In this study, we tested whether naproxen, a common antimigraine medicine, could affect the function of P2X3 receptors in the presence or absence of the algogen nerve growth factor (NGF), the level of which is elevated in patients with chronic migraine. We used calcium imaging and patch clamp recordings from rat trigeminal neurons, which were activated by a relative specific P2X3 agonist α,β-meATP or by high potassium-induced depolarization. In the absence of NGF, naproxen dose-dependently (0.1–1 mM) reduced intracellular calcium transients elicited by α,β-meATP. Naproxen also led to a slight, but significant, reduction in calcium transients induced by potassium ions, indicating the involvement of voltage-gated calcium channels. The inhibitory action of 1 mM naproxen was enhanced after NGF pretreatment, suggesting that P2X3 receptors in sensitized neurons are more susceptible to inhibition by high doses of this nonsteroidal anti-inflammatory drug (NSAID). Using patch clamp recordings from HEK293 cells expressing P2X3 receptors, we tested the direct action of naproxen on P2X3 receptor-mediated membrane currents. In clinically relevant concentrations of 0.5 mM, naproxen produced a use-dependent blocking effect on ATP receptors. Kinetic analysis suggests that naproxen inhibited P2X3 receptors via facilitation of fast desensitization, which determines current decay in the continuous presence of the agonist. In summary, we present a novel fast mechanism for the antimigraine action of naproxen, which can act in synergy with the cyclooxygenase inhibition to attenuate headaches. [Copyright &y& Elsevier]

Abstract: l-PGlu-(2-propyl)-l-His-l-ProNH2 (NP-647) is a CNS active thyrotropin-releasing hormone (TRH) analog with potential application in various CNS disorders including seizures. In the present study, mechanism of action for protective effect of NP-647 was explored by studying role of NP-647 on epileptiform activity and sodium channels by using patch-clamp methods. Epileptiform activity was induced in subicular pyramidal neurons of hippocampal slice of rat by perfusing 4-aminopyridine (4-AP) containing Mg+2-free normal artificial cerebrospinal fluid (nACSF). Increase in mean firing frequency was observed after perfusion of 4-AP and zero Mg+2 (2.10±0.47 Hz) as compared with nACSF (0.12±0.08 Hz). A significant decrease in mean firing frequency (0.61±0.22 Hz), mean frequency of epileptiform events (0.03±0.02 Hz vs. 0.22±0.05 Hz of 4-AP+0 Mg), and average number of action potentials in paroxysmal depolarization shift-burst (2.54±1.21 Hz vs. 8.16±0.88 Hz of 4-AP+0 Mg) was observed. A significant reduction in peak dV/dt (246±19 mV ms−1 vs. 297±18 mV ms−1 of 4-AP+0 Mg) and increase (1.332±0.018 ms vs. 1.292±0.019 ms of 4-AP+0 Mg) in time required to reach maximum depolarization were observed indicating role of sodium channels. Concentration-dependent depression of sodium current was observed after exposure to dorsal root ganglion neurons to NP-647. NP-647 at different concentrations (1, 3, and 10 μM) depressed sodium current (15±0.5%, 50±2.6%, and 75±0.7%, respectively). However, NP-647 did not show change in the peak sodium current in CNa18 cells. Results of present study demonstrated potential of NP-647 in the inhibition of epileptiform activity by inhibiting sodium channels indirectly. [Copyright &y& Elsevier]

Abstract: The BK channel is a Ca2+ and voltage-gated conductance responsible for shaping action potential waveforms in many types of neurons. Type II BK channels are differentiated from type I channels by their pharmacology and slow gating kinetics. The β4 accessory subunit confers type II properties on BK α subunits. Empirically derived properties of BK channels, with and without the β4 accessory subunit, were obtained using a heterologous expression system under physiological ionic conditions. These data were then used to study how BK channels alone (type I) and with the accessory β4 subunit (type II) modulate action potential properties in biophysical neuron models. Overall, the models support the hypothesis that it is the slower kinetics provided by the β4 subunit that endows the BK channel with type II properties, which leads to broadening of action potentials and, secondarily, to greater recruitment of SK channels reducing neuronal excitability. Two regions of parameter space distinguished type II and type I effects; one where the range of BK-activating Ca2+ was high (>20 μM) and the other where BK-activating Ca2+ was low (∼0.4–1.2 μM). The latter required an elevated BK channel density, possibly beyond a likely physiological range. BK-mediated sharpening of the spike waveform associated with the lack of the β4 subunit was sensitive to the properties of voltage-gated Ca2+ channels due to electrogenic effects on spike duration. We also found that depending on Ca2+ dynamics, type II BK channels may have the ability to contribute to the medium AHP, a property not generally ascribed to BK channels, influencing the frequency–current relationship. Finally, we show how the broadening of action potentials conferred by type II BK channels can also indirectly increase the recruitment of SK-type channels decreasing the excitability of the neuron. [Copyright &y& Elsevier]

Abstract: The supraoptic nuclei (SON), the hypothalamic release site of vasopressin and oxytocin, receive a non-glutamatergic, excitatory input from the caudal medulla that uses noradrenaline and ATP as neurotransmitters. Here, we studied the actions of extracellular ATP on SON neurons in hypothalamic slices isolated from the brains of 16- to 24-day-old rats. Whole-cell current clamp recordings performed 1–6 h after isolation showed that exogenous ATP application increased the frequency of action potentials and induced the depolarization of resting membranes. Voltage clamp recordings showed that ATP increased the frequency of GABAergic or glutamatergic spontaneous synaptic currents without changing their amplitude and evoked inward current (126±13 pA) in about 80% of SON neurons. The application of ATPγS and 2MeSATP mimicked the effects of ATP, but 2MeSADP, 2MeSAMP and αβmeATP had no effect. The P2X7 receptor agonist, BzATP, did not induce an inward current, but it increased intracellular calcium concentration in non-neuronal SON cells in slices. Suramin and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) inhibited ATP-induced currents, whereas pH 6.5 and ivermectin, a specific allosteric modulator of the P2X4 receptor, potentiated ATP-induced currents. The P2Y1-selective antagonist, 2′-deoxy-N6-methyladenosine 3′,5′-bisphosphate tetrasodium salt (MRS 2179), had no effect on ATP-induced responses. Quantitative real-time PCR showed that P2X2>P2X7>P2X4 purinergic receptor mRNAs were expressed in the SON tissue, but the levels of P2X1, P2X3, P2X5, P2X6, P2Y1, P2Y2 and P2Y12 mRNA were minor. These results show that SON neurons express functional presynaptic and extrasynaptic P2X2 and P2X4 receptors that modulate glutamate and GABA release and control the electrical excitability of SON neurons. [Copyright &y& Elsevier]

Abstract: Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K+ channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1–10 μM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Kα (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Kα and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 μM). Single-cell RT-PCR revealed mRNA expression for the α-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory β-subunits was detected for Kvβ2 in all SPN with differential expression of mRNA for KChIP1, Kvβ1 and Kvβ3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the β-subunit Kvβ2. Differential expression of the accessory β subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons. [Copyright &y& Elsevier]

Abstract: Cholinergic neurons of the pontine laterodorsal tegmentum (LDT) play a critical role in regulation of behavioral state. Therefore, elucidation of mechanisms that control their activity is vital for understanding of how switching between wakefulness, sleep and anesthetic states is effectuated. In vivo studies suggest that GABAergic mechanisms within the pons play a critical role in behavioral state switching. However, the postsynaptic, electrophysiological actions of GABA on LDT neurons, as well as the identity of GABA receptors present in the LDT mediating these actions is virtually unexplored. Therefore, we studied the actions of GABA agonists and antagonists on cholinergic LDT cells by performing patch clamp recordings in mouse brain slices. Under conditions where detection of Cl− -mediated events was optimized, GABA induced gabazine (GZ)-sensitive inward currents in the majority of LDT neurons. Post-synaptic location of GABAA receptors was demonstrated by persistence of muscimol-induced inward currents in TTX and low Ca2+ solutions. THIP, a selective GABAA receptor agonist with a preference for δ-subunit containing GABAA receptors, induced inward currents, suggesting the existence of extrasynaptic GABAA receptors. LDT cells also possess GABAB receptors as baclofen-activated a TTX- and low Ca2+-resistant outward current that was attenuated by the GABAB antagonists CGP 55845 and saclofen. The tertiapin sensitivity of baclofen-induced outward currents suggests that a GIRK mediated this effect. Further, outward currents were never additive with those induced by application of carbachol, suggesting that they were mediated by activation of GABAB receptors linked to the same GIRK activated in these cells by muscarinic receptor stimulation. Activation of GABAB receptors inhibited Ca2+ increases induced by a depolarizing voltage step shown previously to activate VOCCs in cholinergic LDT neurons. Baclofen-mediated reductions in depolarization-induced Ca2+ were unaltered by prior emptying of intracellular Ca2+ stores, but were abolished by low extracellular Ca2+ and pre-application of nifedipine, indicating that activation of GABAB receptors inhibits influx of Ca2+ involving L-type Ca2+ channels. Presence of GABAC receptors is suggested by the induction of inward current by (E)-4- amino-2-butenoic acid (TACA) and its inhibition by 1,2,5,6-tetrahydropyridine-4-ylmethylphosphinic (TPMPA), a relatively selective agonist and antagonist, respectively, of GABAC receptors. All of these GABA-mediated actions were found to occur in histochemically-identified cholinergic neurons. Taken together, these data indicate for the first time that cholinergic neurons of the LDT exhibit functional GABAA, B and C receptors, including extrasynaptically located GABAA receptors, which may be tonically activated by synaptic overflow of GABA. Accordingly, the activity of cholinergic LDT neurons is likely to be significantly affected by GABAergic tone within the nucleus, and so, demonstrated effects of GABA on behavioral state may be mediated, in part, via direct actions on cholinergic neurons in the LDT. [Copyright &y& Elsevier]

Abstract: Pregnenolone sulfate (PS) acts as an excitatory neuromodulator and has a variety of neuropharmacological actions, such as memory enhancement and convulsant effects. In the present study, we investigated the effect of PS on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in acutely isolated dentate gyrus (DG) hilar neurons by use of a conventional whole-cell patch-clamp technique. PS significantly increased sEPSC frequency in a concentration-dependent manner without affecting the current amplitude, suggesting that PS acts presynaptically to increase the probability of spontaneous glutamate release. However, known molecular targets of PS, such as α7 nicotinic ACh, NMDA, σ1 receptors and voltage-dependent Ca2+ channels, were not responsible for the PS-induced increase in sEPSC frequency. In contrast, the PS-induced increase in sEPSC frequency was completely occluded in a Ca2+-free external solution, and was significantly reduced by either the depletion of presynaptic Ca2+ stores or the blockade of ryanodine receptors, suggesting that PS elicits Ca2+-induced Ca2+ release (CICR) within glutamatergic nerve terminals. In addition, the PS-induced increase in sEPSC frequency was completely occluded by transient receptor potential (TRP) channel blockers. These data suggest that PS increases spontaneous glutamate release onto acutely isolated hilar neurons via presynaptic CICR, which was triggered by the influx of Ca2+ through presynaptic TRP channels. The PS-induced modulation of excitatory transmission onto hilar neurons could have a broad impact on the excitability of hilar neurons and affect the pathophysiological functions mediated by the hippocampus. [ABSTRACT FROM AUTHOR]

Abstract: (R)-roscovitine (Ros) is a cyclin-dependent kinase inhibitor that also has been shown to have direct agonist and antagonist actions on Cav2.1 (P/Q-type) and Cav 2.2 (N-type) families of voltage-gated calcium channels. These kinase-independent effects represent a novel opportunity to advance our understanding of calcium channel function and calcium-triggered neurotransmitter release. Furthermore, such actions on calcium channels may direct the development of Ros derivatives as new therapeutic agents. We used patch clamp recordings to characterize mechanisms that underlie the agonist effects of Ros on unitary N-type calcium channel gating. We found that N-type channels normally gate with either a short or long mean open time, that Ros significantly prolonged the mean open time of the long gating component and increased the probability of observing channels that gated with a long open time, but had no effect on single channel conductance. Using Monte Carlo simulations of a single channel kinetic model and Ros interactions, we were able to reproduce our experimental results and investigate the model''s microscopic dynamics. In particular, our simulations predicted that the longer open times generated by Ros were due to the appearance of a long open state combined with an increased amount of time spent in transitions between open states. Our results suggest a mechanism for agonist effects of Ros at the level of single channels, and provide a mechanistic explanation for previously reported agonist effects on whole cell calcium currents. [Copyright &y& Elsevier]

Abstract: The subthalamic nucleus (STN) modulates the activity of globus pallidus (GP), entopeduncular nucleus (EP) and substantia nigra pars reticulata (SNr) neurons via its direct glutamatergic projections. To investigate the mechanism by which STN affects activity in these structures and whether STN induced activity is comparable among STN target neurons, we performed patch clamp recordings in a tilted, parasagittal, basal ganglia slice (BGS) that preserves these functional connections. We report that single, brief stimulation of the STN generates a brief monosynaptic AMPA-mediated excitatory postsynaptic current (EPSC) in GP, EP and SNr neurons. A higher intensity, supra-threshold activation evokes a compound EPSC consisting of an early monosynaptic component followed by a slow inward NMDA-mediated current with an overlying barrage of AMPA-mediated EPSCs. These late EPSCs were polysynaptic and gave rise to bursts of spikes that lasted several hundreds of milliseconds. They were eliminated by surgical removal of the STN from the BGS slice, indicating that the STN is required for their generation. Reconstruction of biocytin-filled STN neurons revealed that a third of STN neurons project intra-STN axon collaterals that may underlie polysynaptic activity. We propose that activation of the STN yields comparable long lasting excitations in its target neurons by means of a polysynaptic network. [Copyright &y& Elsevier]

Abstract: Neurotransmitter release from the basolateral surface of auditory and vestibular hair cells is mediated by Ca2+ influx through voltage-gated Ca2+ channels. Co-localization of large-conductance Ca2+-activated K+ (BK) channels at the active zones of these cells affords them with an optimal location to act as reporters of the Ca2+ concentration changes at active zones of transmitter release. In this report we use BK channels in frog (Rana pipiens) hair cells to monitor dynamic changes in intracellular Ca2+ concentration during transient influxes of Ca2+, showing that BK current magnitude and delay to onset are correlated with the rate and duration of Ca2+ entry through Ca2+ channels. We also show that BK channels exhibit a much higher Ca2+ binding affinity in the open state than in the closed state. [Copyright &y& Elsevier]

Abstract: The responsiveness of the rat anterior substantia nigra pars reticulata (SNR) GABAergic neurons to GABAAergic drugs changes with age and gender, altering its role in seizure control. To determine whether maturational and gender-specific differences in the properties of spontaneous GABAARs-mediated inhibitory postsynaptic currents (sIPSCs) underlie these events, we studied sIPSCs at baseline and after application of the α1 GABAARs subunit selective agonist zolpidem, at postnatal days (PN) 5–9, PN12–15, and PN28–32. Results were correlated with the α1 and α3 GABAARs subunit immunoreactivity (-ir) at PN5, PN15, and PN30, using immunochemistry. The mean frequency, amplitude and charge transfer increased whereas the 10–90% rise time and decay time accelerated with age in both genders. The faster sIPSC kinetics in older rats were paralleled by increased α1-ir and decreased α3-ir. At PN5–9, males had more robust sIPSCs (frequency, amplitude, charge carried per event and charge transfer) than females. At PN28–32, males exhibited higher amplitudes and faster kinetics than females. The zolpidem-induced increase of decay times, amplitude and charge transfer and α1-ir expression were the lowest in PN5–9 males but increased with age, in both genders. Our findings demonstrate that alterations in GABAARs subunit expression partially underlie age- and gender-specific sIPSC changes in SNR neurons. However, the observation of gender differences in sIPSC kinetics that cannot be attributed to changes in perisomatic α1 expression suggests the existence of additional gender-specific factors that control the sIPSC kinetics in rat SNR. [Copyright &y& Elsevier]

Abstract: Accumulating evidence show that chemokines can modulate the activity of neurons through various mechanisms. Recently, we demonstrated that CCR2, the main receptor for the chemokine CCL2, is constitutively expressed in dopamine neurons in the rat substantia nigra. Here we show that unilateral intranigral injections of CCL2 (50 ng) in freely moving rats increase extracellular concentrations of dopamine and its metabolites and decrease dopamine content in the ipsilateral dorsal striatum. Furthermore, these CCL2 injections are responsible for an increase in locomotor activity resulting in contralateral circling behavior. Using patch-clamp recordings of dopaminergic neurons in slices of the rat substantia nigra, we observed that a prolonged exposure (>8 min) to 10 nM CCL2 significantly increases the membrane resistance of dopaminergic neurons by closure of background channels mainly selective to potassium ions. This leads to an enhancement of dopaminergic neuron discharge in pacemaker or burst mode necessary for dopamine release. We provide here the first evidence that application of CCL2 on dopaminergic neurons increases their excitability, dopamine release and related locomotor activity. [Copyright &y& Elsevier]

Abstract: Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone that are illicitly self-administered for enhancement of performance and body image, but which also have significant effects on the brain and on behavior. While the stereotypical AAS user is an adult male, AAS abuse in women is rapidly increasing, yet few studies have examined AAS effects in female subjects. We have assessed the effects in female mice of a combination of commonly abused AAS on neuronal activity and neurotransmission mediated by GABA type A (GABAA) receptors in the medial preoptic nucleus (MPN); a nexus in the circuits of the hypothalamus and forebrain that are critical for the expression of social behaviors known to be altered in AAS abuse. Our data indicate that chronic exposure to AAS resulted in androgen receptor (AR)–dependent upregulation of α5, β3 and δ subunit mRNAs. Acute application of the α5 subunit-selective inverse agonist, L-655,708 (L6), indicated that a significant fraction of the synaptic current is carried by α5-containing receptors and that AAS treatment may enhance expression of α5-containing receptors contributing to synaptic, but not tonic, currents in the MPN. AAS treatment also resulted in a significant decrease in action potential frequency in MPN neurons that was also correlated with an increased sensitivity to L-655,708. Our data demonstrate that chronic exposure to multiple AAS elicits significant changes in GABAergic transmission and neuronal activity that are likely to reflect changes in the expression of α5-containing synaptic receptors within the MPN. [Copyright &y& Elsevier]

Abstract: In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect of l-arginine, a precursor for NO synthesis, and Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 μM) and dl-2-amino-5-phosphonovaleric acid (dl-AP5) (30 μM) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 μM) and saclofen (30 μM) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 μM l-arginine decreased the firing rate from 3.7±0.6 Hz to 1.3±0.3 Hz. Conversely, 100 μM l-NAME increased the firing rate from 3.0±0.3 Hz to 5.8±0.4 Hz. All points histogram analysis showed changes in resting potential from −58.1±0.8 mV to −62.2±1.1 mV in the presence of l-arginine and from −59.8±0.7 mV to −56.9±0.8 mV by l-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4±1.2 mV to 16.8±0.7 mV by l-arginine, but without significant changes by l-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation. [Copyright &y& Elsevier]

Abstract: ATP-sensitive potassium (K atp ) channels play an important role in controlling insulin secretion and vascular tone as well as protecting neurons under metabolic stress. We have previously demonstrated that stimulation of the K atp channel by nitric oxide (NO) requires activation of Ras- and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) family. However, the mechanistic link between ERK and the K atp channel remained unknown. To investigate how ERK modulates the function of K atp channels, we performed single-channel recordings in combination with site-directed mutagenesis. The Kir6.2/SUR1 channel, a neuronal K atp channel isoform, was expressed in human embryonic kidney (HEK) 293 cells by transient transfection. Direct application of the activated ERK2 to the cytoplasmic surface of excised, inside-out patches markedly enhanced the single-channel activity of Kir6.2/SUR1 channels. The normalized open probability (NPo) and opening frequency were significantly increased, whereas the mean closed duration was reduced. The single-channel conductance level was not affected. The ERK2-induced stimulation of Kir6.2/SUR1 channels was prevented by heat-inactivation of the enzyme. Furthermore, alanine substitutions of T341 and S385 to disrupt the potential ERK phosphorylation sites present in the Kir6.2 subunit significantly abrogated the stimulatory effects of ERK2, while aspartate substitutions of T341 and S385 to mimic the (negative) charge effect of phosphorylation rendered a small yet significant reduction in the ATP sensitivity of the channel. Taken together, here we report for the first time that ERK2/MAPK activates neuronal-type K atp channels, and this stimulation requires ERK phosphorylation of the Kir6.2 subunit at T341 and S385 residues. The ERK2-induced K atp channel stimulation can be accounted for by changes in channel gating that destabilize the closed states and by reduction in the ATP sensitivity. As Kir6.2 is the pore-forming subunit of K atp channels, ERK2-mediated phosphorylation may represent a common mechanism for K atp channel regulation in different tissues. [Copyright &y& Elsevier]

Abstract: The subthalamic nucleus (STN) plays a pivotal role in normal and abnormal motor function. We used patch pipettes to study effects of 5-HT on synaptic currents evoked in STN neurons by focal electrical stimulation of rat brain slices. 5-HT (10 μM) reduced glutamate-mediated excitatory postsynaptic currents (EPSCs) by 35±4%. However, a much higher concentration of 5-HT (100 μM) was required to inhibit GABA-mediated inhibitory postsynaptic currents (IPSCs) to a comparable extent. Concentration–response curves showed that the 5-HT inhibitory concentration 50% (IC50) for inhibition of IPSCs (20.2 μM) was more than fivefold greater than the IC50 for inhibition of EPSCs (3.4 μM). The 5-HT-induced reductions in EPSCs and IPSCs were accompanied by increases in paired-pulse ratios, indicating that 5-HT acts presynaptically to inhibit synaptic transmission. The 5-HT1B receptor antagonist NAS-181 significantly antagonized 5-HT-induced inhibitions of EPSCs and IPSCs. These studies show that 5-HT inhibits synaptic transmission in the STN by activating presynaptic 5-HT1B receptors. [Copyright &y& Elsevier]

Abstract: We have recently reported that most of NG2 glycoprotein expressing glial cells, or NG2 glia, in rat hippocampus persistently express sodium channel currents (I Na) during development, but little is known about its function. We report here that hippocampal NG2 glia recorded in either acute slices or freshly isolated preparations from postnatal days (P) 7–21 rats express low density I Na (9.5–15.7 pA/pF) that is characterized by a fast activation and rapid inactivation kinetics with a tetrodotoxin (TTX) IC50 value of 39.3 nM. The I Na expression correlated with a ∼25 mV more depolarized resting membrane potential (RMP) as compared with non-I Na-expressing GLAST(+) astrocytes in situ at the same age. In the presence of the sodium channel blocker TTX (0.1 μM), these depolarized RMPs were negatively shifted by an average of 19 mV and 16 mV for I Na-expressing glia recordings from in situ and freshly isolated preparations, respectively. The I Na expressing glia actually showed a positive RMP (+12 mV) in the absence of potassium conductance that was inhibited to 0 mV by 0.1 μM TTX. Analysis of the I Na activation/inactivation curves yields an I Na “window current” at −40±20 mV, implying a persistent I Na component being active around the NG2 glia RMP of ∼−45 mV. According to the constant-field equation analysis, this active I Na component leads to a pNa/pK ratio of 0.14 at RMP which is ∼threefold higher than astrocytes (0.05). These results indicate that a TTX sensitive I Na component in NG2 glia contributes significantly to the depolarized NG2 glia RMP in the developing brain. [Copyright &y& Elsevier]