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Highlights • In IA-AVPNs, NMDA receptors, non-NMDA receptors, gap junction couplings and the type of α4β2 and α7 nAChRs involved in the rhythmic activation of synaptic currents during inspiratory bursts, while non-NMDA receptors constitute a major proportion. • CNQX, an antagonist of AMPA receptors, caused depolarization in some IA-AVPNs, indicating a weak agonistic effect of CNQX on AMPA receptors or some other excitatory pathways unveiled by CNQX. • In II-AVPNs, excitatory glutamatergic synaptic inputs presynaptically facilitated its inhibitory glycinergic inputs. Abstract The airway vagal preganglionic neurons (AVPNs) in the external formation of the nucleus ambiguus (eNA) can be separated into inspiratory-activated AVPNs (IA-AVPNs) and inspiratory-inhibited AVPNs (II-AVPNs). IA-AVPNs are activated by excitatory presynaptic inputs during inspiratory bursts, but the composition and the roles of these excitatory inputs still remain obscure. II-AVPNs are inhibited by inhibitory presynaptic inputs but whether these inhibitory inputs are regulated by excitatory inputs is also unclear. In the current study, AVPNs were retrogradely fluorescent labeled. The IA-AVPNs were discriminated from II-AVPNs by their different synaptic inputs during inspiratory bursts. The excitatory inputs to IA-AVPNs and the presynaptic regulation of II-AVPNs were examined by whole-cell patch clamping. Topical application of 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) to the recorded IA-AVPNs almost abolished the tonic EPSCs during inspiratory intervals, inhibited the phasic excitatory currents during inspiratory bursts and attenuated the phasic inspiratory inward currents (PIICs) driven by central inspiratory activity. Blockade of α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) respectively inhibited PIICs in some IA-AVPNs. Carbenoxolone, a gap junction uncoupler, partly inhibited the PIICs of IA-AVPNs. Focal application of CNQX to the II-AVPNs significantly inhibited the frequency, peak amplitude and area of the phasic inspiratory outward currents (PIOCs). These findings demonstrated that glutamatergic non-NMDA receptors played a predominant role in the excitatory drive to the IA-AVPNs, and that α4β2, α7 nAChRs and gap junctions were also rhythmically activated by central inspiratory activity. Additionally, glycinergic neurons making inhibitory inputs to the II-AVPNs were pre-synaptically facilitated by excitatory glutamatergic synaptic inputs. [ABSTRACT FROM AUTHOR]

Highlights • Functional TRPV1-L channels can be expressed in pyramidal cells of mouse neocortex using in utero electroporation. • IR (infrared) radiation induces membrane depolarization in TRPV1-L+ pyramidal neurons at near physiological temperatures. • Stronger IR radiation evokes a consistent spiking response in TRPV1-L+ neurons. Abstract Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of single neurons using thermosensitive cation channels and IR stimulation. The main advantage of IR stimulation compared to conventional visible light optogenetics is the depth of penetration (up to millimeters). Due to physiological limitations, thermogenetic molecular tools for mammalian brain stimulation remain poorly developed. Here, we tested the possibility of employment of this new technique for stimulation of neocortical neurons. The method is based on activation gating of TRPV1-L channels selectively expressed in specific cells. Pyramidal neurons of layer 2/3 of neocortex were transfected at an embryonic stage using a pCAG expression vector and electroporation in utero. Depolarization and spiking responses of TRPV1L+ pyramidal neurons to IR radiation were recorded electrophysiologically in acute brain slices of adult animals with help of confocal visualization. As TRPV1L-expressing neurons are not sensitive to visible light, there were no limitations of the use of this technique with conventional fluorescence imaging. Our experiments demonstrated that the TRPV1-L+ pyramidal neurons preserve their electrical excitability in acute brain slices, while IR radiation can be successfully used to induce single neuronal depolarization and spiking at near physiological temperatures. Obtained results provide important information for adaptation of thermogenetic technology to mammalian brain studies in vivo. [ABSTRACT FROM AUTHOR]

• Effects of menthol on neuronal excitability were examined in rat dural afferent neurons. • Menthol inhibited the leak K+ currents and depolarized membrane potentials. • Menthol at higher concentrations may excite dural afferent neurons by inhibiting K+ conductances. Menthol—a natural organic compound—is widely used for relieving various pain conditions including migraine. However, a high dose of menthol reportedly decreases pain thresholds and enhances pain responses. Accordingly, in the present study, we addressed the effect of menthol on the excitability of acutely isolated dural afferent neurons, which were identified with a fluorescent dye, using the whole-cell patch-clamp technique. Under a voltage-clamped condition, menthol altered the holding current levels in a concentration-dependent manner. The menthol-induced current (I Menthol) remained unaffected by the addition of selective transient receptor potential melastatin 8 antagonists. Moreover, the reversal potential of I Menthol was similar to the equilibrium potential of K+. I Menthol was accompanied by an increase in input resistance, thereby suggesting that menthol decreases the leak K+ conductance. Under a current-clamped condition, menthol caused depolarization of the membrane potential and decreased the threshold for the generation of action potential. While the I Menthol was substantially inhibited by 10 μM XE-991, a selective K V 7 blocker, the M-current mediated by K V 7 was not detected in the nociceptive neurons tested in the present study. Moreover, I Menthol decreased under acidic extracellular pH conditions or in the presence of 3 μM A-1899, a selective K2P3.1 and K2P9.1 blocker. The present results suggest that menthol inhibits leak K+ channels, possibly acid-sensitive two-pore domain K+ channels, thereby increasing the excitability of nociceptive sensory neurons. The resultant increase in neuron excitability may partially be responsible for the pronociceptive effect mediated by high menthol doses. [ABSTRACT FROM AUTHOR]

Tuberoinfundibular dopaminergic (TIDA) neurons in the arcuate nucleus (ARC) of the hypothalamus play a role in inhibiting prolactin (PRL) secretion from the anterior pituitary. PRL is involved in a variety of behaviors, including feeding. Consequently, we hypothesized that fasting might reduce the activity of TIDA neurons, which might alter PRL secretion. However, direct examinations of TIDA neuron activity are difficult. Recently, transgenic mice were generated that expressed green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene. We first determined that GFP in the dorsomedial ARC was a reliable marker of TIDA neurons. Then, we performed electrophysiology and immunocytochemistry in GFP-labeled TIDA neurons to examine whether different feeding conditions could change their activity. Eight-week-old male mice were fed or fasted for 24 h. After sacrifice, we prepared acutely isolated brain slices for conducting whole-cell voltage-clamp recordings. TIDA neurons were identified with fluorescence microscopy. The mean amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced in fasting mice compared to fed mice, but different feeding conditions did not affect the mean mEPSC intervals. This result suggested that fasting reduced the number of excitatory synaptic inputs to TIDA neurons. To determine whether a reduction in excitatory synaptic inputs would cause a reduction in TIDA neuron activity, we examined the effect of 24-h fasting on c-Fos expression in the ARC. We found that fasting significantly reduced the number of Fos-positive TIDA neurons. In addition, serum PRL levels were significantly increased. Taken together, the present findings suggested that short-term fasting attenuated TIDA neuron activity. [ABSTRACT FROM AUTHOR]

Perampanel is an antiepileptic drug that is used to treat partial-onset seizures and generalized tonic-clonic seizures. It is a highly selective AMPA receptor allosteric antagonist. However, published data on perampanel activity vary in different studies. In the present work we studied the inhibition of native calcium-permeable and calcium-impermeable AMPA receptors in rat brain neurons by perampanel using whole-cell patch clamp technique. We found that inhibitory activity and kinetics of perampanel action do not differ between calcium-permeable AMPA receptors of rat giant striatum interneurons and calcium-impermeable receptors of hippocampal CA1 pyramidal neurons (the IC 50 value about 60 nM). Also, perampanel caused the same inhibition of steady-state currents induced by kainate and glutamate. From the other side perampanel-induced inhibition was markedly reduced in the presence of cyclothiazide (IC 50 value increased to 1.2 ± 0.2 μM). We demonstrated that perampanel competes with GYKI-52466 for binding site. [ABSTRACT FROM AUTHOR]

1,8-Cineole is a terpenoid present in many essential oil of plants with several pharmacological and biological effects, including antinociceptive, smooth muscle relaxant and ion channel activation. Also, 1,8-cineole blocked action potentials, reducing excitability of peripheral neurons. The objective of this work was to investigate effects of 1,8-cineole on Na + currents ( I Na + ) in dissociated superior cervical ganglion neurons (SCG). Wistar rats of both sexes were used (10–12 weeks old, 200–300 g). SCG’s were dissected and neurons were enzymatically treated. To study 1,8-cineole effect on I Na + , the patch-clamp technique in whole-cell mode was employed. 1,8-Cineole (6.0 mM) partially blocked I Na + in SCG neurons. The effect stabilized within ∼150 s and there was a partial recovery of I Na + after washout. Current density was reduced from −105.8 to −83.7 pA/pF, corresponding to a decrease to ∼20% of control. 1,8-Cineole also reduced the time-to-peak of I Na + activation and the amplitude and decay time constants of I Na + inactivation. Current–voltage plots revealed that 1,8-cineole left-shifted the V 1/2 of both activation and inactivation curves by ∼10 and ∼20 mV, respectively. In conclusion, we demonstrate that 1,8-cineole directly affects Na + channels of the SCG by modifying several gating parameters that are likely to be the major cause of excitability blockade. [ABSTRACT FROM AUTHOR]

The roles of persistent Na + currents ( I NaP ) in intrinsic membrane properties were examined in rat substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis using a conventional whole-cell patch clamp technique. In a voltage-clamp mode, riluzole inhibited the slow voltage ramp-induced I NaP but had little effect on the peak amplitude of transient Na + currents in SG neurons. In a current-clamp mode, most SG neurons exhibited spontaneous action potentials and tonic firing pattern. Riluzole reduced both spontaneous and elicited action potentials in a concentration-dependent manner. The present results suggest that the riluzole-sensitive I NaP plays an important role in the excitability of SG neurons and are thus, likely to contribute to the modulation of nociceptive transmission from the orofacial tissues. [ABSTRACT FROM AUTHOR]

Maternal inflammation is a known risk factor for schizophrenia and autism. Since the visual processing has shown abnormalities in these disorders, we explored whether neuropathologic changes can be caused in the primary visual cortex in offsprings due to the maternal inflammation induced by a single lipopolysaccharide (LPS) injection in pregnant mouse dams. The morphology and electrophysiological properties of layer II pyramidal cells (L2PC) in the primary visual cortex were investigated with whole-cell patch-clamping recording and 3D neuron reconstruction techniques. Although the composition of two L2PC types was unchanged, a reorganization of the dendritic architecture was found in both L2PC_A and L2PC_B types, predominantly in the L2PC_A type, of the mice prenatally exposed to LPS. Moreover, prenatal LPS exposure differentially altered intrinsic electrophysiological properties of the two L2PC types. L2PC_A neurons showed reduced excitability as featured by a hyperpolarization of the resting membrane potential, whereas L2PC_B neurons showed enhanced excitability as featured by a decrease in cellular input resistance at resting membrane potential. These significant changes in neuronal morphological and electrophysiological properties might contribute to the dysfunctions of pyramidal neurons after maternal inflammation. [ABSTRACT FROM AUTHOR]

The modulation of GABAergic transmission by metabotropic glutamate (mGlu) receptors was examined in histaminergic neurons using a conventional whole-cell patch clamp technique. DHPG, a selective group I mGlu receptor agonist, had no effect on GABAergic inhibitory postsynaptic currents (IPSCs). However, DCG-IV (1 μМ) and L-AP4 (1 μМ), selective group II or III mGlu receptor agonists, respectively, decreased the amplitude of GABAergic IPSCs and simultaneously increased the paired-pulse ratio. The inhibitory effect of DCG-IV was completely blocked by 200 nM LY341495, a group II and III mGlu receptor antagonist, and the inhibitory effect of L-AP4 was completely blocked by 10 μМ LY341495. These results suggest that multiple mGlu receptors are involved in regulating the excitability of histaminergic neurons. [ABSTRACT FROM AUTHOR]

Recently, evidence has accumulated that the vomeronasal system cooperates with the main olfactory system to process volatile cues that regulate the animal's behavior. This is contradictory to the traditional view that the vomeronasal system is quite different from the main olfactory system in the time scale of information processing. Particularly, the firing rate of mitral/tufted cells in the accessory olfactory bulb (MTAOB) is known to be significantly lower than that of mitral cells in the main olfactory bulb (MCMOB). To address this question of whether the low-frequency firing in MTAOB carries less information than the high-frequency firing in MCMOB in the early stages of stimulation, we compared MTAOB and MCMOB for their firing mechanisms and information transfer characteristics. A model computation demonstrated that the inherent channel kinetics of MTAOB was responsible for their firing at a lower frequency than MCMOB. Nevertheless, our analysis suggested that MTAOB were comparable to MCMOB in both the amount and speed of information transfer about depolarizing current intensity immediately after current injection onset (<200ms). Our results support a hypothesis of simultaneous processing of common cues in both systems. [ABSTRACT FROM AUTHOR]

Highlights: [•] We demonstrate how peripheral administration of cannabinoids can produce analgesia. [•] Currents mediated by TRPV1 are increased by nerve growth factor in sensory neurons. [•] A CB1 cannabinoid agonist attenuates sensitization of TRPV1 by nerve growth factor. [Copyright &y& Elsevier]

Abstract: Firing regularity has long been an issue of firing dynamics in the vestibular circuitry. Spike frequency adaption (SFA) is ubiquitous in neuronal activity and can modulate neural coding, which may disrupt the regularity or accuracy of firing. We previously observed different characteristics of intrinsic excitability in Purkinje cells (PCs) of lobule X (vestibulocerebellum) compared to lobules III–V (spinocerebellum). However, systematic comparison of the extent of SFA in PCs of different lobules has not yet been made. In this study we examined the degree of SFA and compared the firing regularity by measuring interspike interval (ISI). During the course of low-frequency spike trains, PCs in lobules III–V showed gradual lengthening of ISI due to SFA. In contrast, ISI showed little change during the propagation of spikes in lobule X PCs. In high-frequency firing, PCs in lobules III–V exhibited gradual SFA, whereas lobule X neurons showed dramatic increase in ISI during the first four spikes and then stayed unchanged. The coefficient of variation of ISI of lobule X PCs was significantly lower in lobules III–V PCs during low-frequency firing. The comparison of duration of action potential showed no significant difference between lobules III–V and lobule X PCs during SFA even in low-frequency firing. The lack of SFA in lobule X PCs, as a part of vestibulocerebellum, might be involved in a consistent and regular coordination of vestibular function by the cerebellar cortex in response to low vestibular stimulation. However, the difference of SFA between lobules may be explained by other mechanisms than those which have been reported to be responsible for the SFA formation. [Copyright &y& Elsevier]

Abstract: Mutations of the voltage gated sodium channel gene (SCN4A) are responsible for non-dystrophic myotonia including hyperkalemic periodic paralysis, paramyotonia congenita, and sodium channel myotonia, as well as congenital myasthenic syndrome. In vitro functional analyses have demonstrated the non-dystrophic mutants to show a gain-of-function defect of the channel; a disruption of fast inactivation, an enhancement of activation, or both, while the myasthenic mutation presents a loss-of function defect. This report presents a case of non-dystrophic myotonia that is incidentally accompanied with acquired myasthenia. The patient presented a marked warm-up phenomenon of myotonia but the repeated short exercise test suggested mutations of the sodium channel. The genetic analysis identified a novel mutation, G1292D, of SCN4A. A functional study of the mutant channel revealed marked enhancement of activation and slight impairment of fast inactivation, which should induce muscle hyperexcitability. The effects of the alteration of channel function to the myasthenic symptoms were explored by using stimulation of repetitive depolarization pulses. A use-dependent channel inactivation was reduced in the mutant in comparison to normal channel, thus suggesting an opposing effect to myasthenia. [Copyright &y& Elsevier]

Abstract: To clarify the vulnerability of spinal motoneurons to excitotoxicity, we analyzed the agonal current induced by experimental ischemia in ventral lamina IX neurons of spinal cord slices from neonatal rats by using whole-cell patch-clamp. Ischemia was simulated in vitro by oxygen/glucose deprivation. Superfusion with ischemia-simulating medium elicited an agonal inward current, which was initially slow and then became rapid. We compared 8-, 9-, 10-, 11-, and 12-day postnatal rats and found age-dependent shortening of the latency of the rapid inward current. Furthermore, the membrane capacitance (Cm) and resting membrane potential (RMP) of the lamina IX neurons demonstrated significant negative correlations with the latency of the rapid inward current. Logistic regression analysis showed that postnatal age, Cm, and RMP were independent contributing factors to ischemic vulnerability. These results suggest that not only cell volume and ionic balance but also early postnatal maturation of the intracellular environment is vital for developing vulnerability to excitotoxicity. [Copyright &y& Elsevier]

Abstract: The GABAA receptors are ligand-gated chloride channels which are the targets for many clinically used sedatives, including the barbiturates. The barbiturate pentobarbital acts through multiple sites on the GABAA receptor. At low concentrations (μM), it acts as a positive allosteric modulator while at higher concentrations it can directly activate the receptor. This agonist action is influenced by the subunit composition of the receptor, and pentobarbital is a more effective agonist than GABA only at receptors containing an α6 subunit. The conformational change that translates GABA binding into channel opening is known to involve a lysine residue located in an extracellular domain between the 2nd and 3rd transmembrane domains. Mutations of this residue disrupt activation of the channel by GABA and have been linked to inherited epilepsy. Pentobarbital binds to the receptor at a different agonist site than GABA, but could use a common signal transduction mechanism to gate the channel. To address this question, we compared the effect of a mutating the homologous lysine residue in the α1 or α6 subunits (K278 or K277, respectively) to methionine on direct activation of recombinant GABAA receptors by GABA or pentobarbital. We found that this mutation reduced GABA sensitivity for both α1 and α6 subunits, but affected pentobarbital sensitivity only for the α1 subunit. This suggests that pentobarbital acts through a distinct signal transduction pathway at the α6 subunit, which may account for its greater efficacy compared to GABA at receptors containing this subunit. [Copyright &y& Elsevier]

Abstract: Postmitotic neurons were generated from the human NT2 teratocarcinoma cell line in a novel cell aggregate differentiation procedure. The NT2 model neurons express punctate immunoreactivity for synapsin and for cell markers related to GABAergic and glutamatergic neurotransmission. Using the outside-out patch-clamp configuration, we characterized the kinetics of currents elicited by a rapid application of the amino acid neurotransmitters. Moreover, we detected spontaneous postsynaptic currents in glia free cell cultures that may result from the firing activity of glutamatergic and GABAergic NT2 neurons. These cultured spontaneously active networks may be a useful tool to analyze factors that modulate the formation and efficacy of synapses between human neurons. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the existence of functional TRPV1 receptor by substantia gelatinosa (SG) neurons of the trigeminal subnucleus caudalis (Vc), which is implicated in the processing of nociceptive information from orofacial regions. The direct membrane effects of a TRPV1 receptor agonist, capsaicin, were examined by gramicidin-perforated patch clamp recording using a trigeminal brainstem slice preparation containing Vc from immature mice. Capsaicin (1–2μM) induced a membrane depolarization in 58 out of 71 SG neurons tested (82%). Capsaicin-induced depolarization was maintained in 20 out of 32 (63%) SG neurons in the presence of amino acid and voltage-dependent sodium channel blockers (10μM CNQX, 20μM AP-5, 0.5μM TTX, 50μM picrotoxin and 2μM strychnine). In addition, capsaicin-induced depolarization was maintained in the presence of L-732,138 (1μM), an NK1 receptor antagonist, in 14 out of 17 neurons (82%) tested. The capsaicin-induced depolarizing effects were blocked by a TRPV1 receptor antagonist, capsazepine (10μM). These results indicate that a sub-population of SG neurons in the Vc express functional TRPV1 receptors, and that capsaicin can directly activate the TRPV1 receptor on the postsynaptic membrane of SG neurons. [Copyright &y& Elsevier]

Abstract: Whole-cell patch clamp technique was used to study the modulatory effect of Cholecystokin-8S (CCK-8S), a neuropeptide, on NMDA-activated current (I NMDA) in cultured rat hippocampal neurons under condition of normal and ethanol exposure. Our study showed that CCK-8S inhibited I NMDA in a non-concentration and non-competition dependent manner. Ethanol significantly inhibited I NMDA, and CCK-8S further inhibited I NMDA. Our results suggest that CCK-8S is an inhibitory modulator of NMDA receptors under normal and ethanol exposure conditions. [Copyright &y& Elsevier]

Abstract: Transient receptor potential melastatin 7 (TRPM7) channels are widely expressed in the nervous system; however, their function and regulation are largely unknown. This study aimed to explore whether the current mediated by TRPM7-like channels in rat CA1 hippocampal neurons could be modulated by nerve growth factor (NGF). Using whole-cell patch clamp techniques, we identified an outward-rectifying TRPM7-like current in hippocampal neurons freshly isolated from postnatal (10-day-old) rats. The outward component of this current was reversibly reduced by NGF in dose- and time-dependent manners, and this effect was substantially blocked by K252a, an inhibitor of TrkA. In addition, NGF-induced reduction of the TRPM7-like current was abolished by U73122, a phopholipase C inhibitor. In light of the abundance of NGF in hippocampus that express both TrkA and TRPM7, these results suggest that the function of TRPM7-like channels in hippocampal neurons may be regulated by NGF. [Copyright &y& Elsevier]

Abstract: The neurosteroid 3α-hydroxy-5α-pregnan-20-one (allopregnanolone or ALLO) positively modulates GABAA receptors, an action that may contribute to the anxiolytic effects of ALLO. Recent evidence suggests that ALLO''s anxiolytic effects appear to be mediated by the amygdala, a key neural structure for emotional and cognitive behaviors. However, little is known regarding ALLO effects on amygdala physiology. We therefore explored ALLO effects on GABA neurotransmission in the central nucleus (Ce) of the amygdala, a major output nucleus involved in fear and anxiety. We recorded evoked GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs) in Ce neurons using whole-cell patch clamp techniques. We observed that ALLO significantly reduced the amplitude of evoked GABAA receptor-mediated IPSCs. However, the effect of ALLO was occluded by the NMDA receptor antagonist D-APV. D-APV alone also reduced evoked IPSCs in Ce neurons. These results suggest that ALLO-induced reduction of GABAergic transmission in Ce appears to depend on neural network activity, possibly involving an NMDA receptor-mediated mechanism. These ALLO effects on GABAergic transmission in the central amygdala may play a role in mediating its anxiolytic actions. [Copyright &y& Elsevier]

Abstract: Iptakalim hydrochloride, a novel cardiovascular ATP-sensitive K+ (KATP) channel opener, has shown remarkable antihypertensive and neuroprotective effects in a variety of studies using in vivo and in vitro preparations. We recently found that iptakalim blocked human α4-containing nicotinic acetylcholine receptors (nAChRs) heterologously expressed in the human SH-EP1 cell line. In the present study, we examined the effects of iptakalim on several neurotransmitter-induced current responses in single DA neurons freshly dissociated from rat substantia nigra pars compacta (SNc), using perforated patch-clamp recordings combined with a U-tube rapid drug application. In identified DA neurons under voltage-clamp configuration, glutamate-, NMDA-, and GABA-induced currents were insensitive to co-application with iptakalim (100μM), while whole-cell currents induced by ACh (1mM+1μM atropine) or an α4β2 nicotinic acetylcholine receptors relatively selective agonist, RJR-2403 (300μM), were eliminated by iptakalim. Iptakalim inhibited RJR-2403-induced current in a concentration-dependent manner, and reduced maximal RJR-2403-induced currents at the highest agonist concentration, suggesting a non-competitive block. In current-clamp mode, iptakalim failed to affect resting membrane potential and spontaneous action potential firing, but abolished RJR-2403-induced neuronal firing acceleration. Together, these results indicate that in dissociated SNc DA neurons, α4-containing nAChRs, rather than ionotropic glutamate receptors, GABAA receptors or perhaps K-ATP channels are the sensitive targets to mediate iptakalim''s pharmacological roles. [Copyright &y& Elsevier]

Abstract: Whole-cell patch clamp recordings were made from ICC neurons in brain slices of 9–16 day old rats. Postsynaptic currents were evoked by electrical stimulation of the lemniscal inputs. Excitatory postsynaptic currents (EPSCs) were isolated pharmacologically by blocking GABAA and glycine receptors. EPSCs were further dissected into AMPA and NMDA receptor-mediated responses by adding the receptor antagonists, APV and CNQX, respectively. The internal solution in the recording electrodes contained CsF and TEA to block K+ channels that might be activated by postsynaptic GABAB receptors. The modulatory effects of GABAB receptors on EPSCs in ICC neurons were examined by bath application of the GABAB receptor agonist, baclofen, and the antagonist, CGP 35348. The amplitudes of EPSCs in ICC neurons were reduced to 34.4±3.2% of the control by baclofen (5–10μM). The suppressive effect by baclofen was concentration-dependent. The reduction of the EPSC amplitude was reversed by CGP35348. The ratio of the 2nd to 1st EPSCs evoked by paired-pulse stimulation was significantly increased after application of baclofen. These results suggest that glutamatergic excitation in the ICC can be modulated by presynaptic GABAB receptors. In addition, baclofen reduced NMDA EPSCs more than AMPA EPSCs. The GABAB receptor-mediated modulation of glutamatergic excitation in the ICC provides a likely mechanism for preventing overstimulation and/or regulating the balance of excitation and inhibition involved in processing auditory information. [Copyright &y& Elsevier]

Abstract: The role of voltage-dependent potassium channel currents in glutamate-treated rat hippocampal neurons was investigated. Cell viability was evaluated by MTT reduction assay and morphological changes. Apoptosis was detected by Hoechst33342 staining with fluorescent microscopy and propidium iodide staining with flow cytometry. Membrane potassium channel currents were recorded with whole-cell patch clamp recordings. Results showed that after shortly exposed to glutamate, about 25 and 50% cells died in 3h and 24h, respectively. Meanwhile, the enhancement of I K was observed within 6h after the glutamate insult. TEA selectively blocked I K and significantly reduced cell apoptosis. I A did not change in the insult though 4-AP, the blocker of this current, showed a protective effect against the injury. These data were in consistent with the hypothesis that K+ efflux contributed to glutamate-triggered apoptosis and I K channels might have a therapeutic effect on the treatment of cerebral ischemia. [Copyright &y& Elsevier]

Abstract: The heterogeneous population of mammalian taste cells includes several cellular subtypes specializing in distinct physiological functions. They are poorly understood at the single cell level because the available physiological data have generally been obtained from unidentified taste cells. We recorded them from individual taste cells isolated from circumvallate, foliate, and fungiform papilla of the mouse, employing the patch clamp technique, and tried to elucidate whether universal electrophysiological criteria may be established for the identification of functionally different cellular subpopulations. It was found that irrespective of the papillae type, most (∼96%) of robust taste cells could be categorized into three distinct subgroups on the basis of families of whole-cell (WC) currents exhibited in response to membrane polarization. The validity of this quite simple criterion was further confirmed by using different voltage clamp protocols, ion substitutions, and channel blockers to record different ionic currents, including voltage-gated (VG) Ca2+, inward-rectifying K+, and hyperpolarization-activated currents. Given that our findings are based on the statistically significant number of recordings, we believe that the electrophysiological identification of taste cells presented here may be effective for further studies on single taste cell physiology, including taste transduction. [Copyright &y& Elsevier]

Abstract: Recent analysis of current responses to exogenous GABA applications recorded from excised patches indicated that membrane voltage affected the GABAA receptor gating mainly by altering desensitization and binding [M. Pytel, K. Mercik, J.W. Mozrzymas, Membrane voltage modulates the GABAA receptor gating in cultured rat hippocampal neurons, Neuropharmacology, in press]. In order investigate the impact of such voltage effect on GABAA receptors in conditions of synaptic transmission, mIPSCs and current responses to rapid GABA applications were recorded from the same culture of rat hippocampal neurons. We found that I–V relationship for mIPSCs amplitudes showed a clear outward rectification while for current responses an inward rectification was seen, except for very low GABA concentrations. A clear shift in amplitude cumulative distributions indicated that outward rectification resulted from the voltage effect on the majority of mIPSCs. Moreover, the decaying phase of mIPSCs was clearly slowed down at positive voltages and this effect was represented by a shift in cumulative distributions of weighted decaying time constants. In contrast, deactivation of current responses was only slightly affected by membrane depolarization. These data indicate that the mechanisms whereby the membrane voltage modulates synaptic and extrasynaptic receptors are qualitatively different but the mechanism underlying this difference is not clear. [Copyright &y& Elsevier]

Abstract: To explore the ionic mechanisms of memory deficits induced by cholinergic lesion, whole-cell patch clamp recording techniques in combination with single-cell RT-PCR were used to characterize delayed rectifier potassium currents (IK) in acutely isolated hippocampal pyramidal neurons of scopolamine-induced cognitive impairment rats. Scopolamine could induce deficits in spatial memory of rats. The peak amplitude and current density of IK measured in hippocampal pyramidal neurons were increased from 1.2±0.6nA and 38±19pA/pF of the control group (n=12) to 1.8±0.5nA and 62±24pA/pF (n=48, P<0.01) of the scopolamine-treated group. The steady-state activation curve of IK was shifted about 8mV (P<0.01) in the direction of hyperpolarization in scopolamine-treated rats. The mRNA level of Kv2.1 was increased (P<0.01) in the scopolamine-treated group, but there was no significant change of Kv1.5 mRNA level. The present study demonstrated for the first time that IK was enhanced significantly in hippocampal pyramidal neurons of scopolamine-induced cognitive impairment rats. The increase of Kv2.1 mRNA expression in hippocampal pyramidal cells might be responsible for the enhancement of IK and could be the ionic basis of the memory deficits induced by scopolamine. [Copyright &y& Elsevier]

The effects of salicylate (a tinnitus inducer) were studied on the transient outward potassium current (IK(A)) and the delayed rectifier potassium current (IK(DR)) in acutely dissociated rat inferior colliculus neurons by the whole-cell voltage-clamp method. Salicylate''s inhibition of the amplitude of IK(A) and IK(DR) was concentration-dependent. The IC50 values for the blocking action of salicylate on IK(A) and IK(DR) were 2.27 and 0.80mM, respectively. At a concentration of 1mM, salicylate significantly shifted the activation and inactivation curves of IK(DR) negatively by approximately 11 and 24mV, respectively, but did not shift the IK(A) curves. In conclusion, salicylate inhibits both IK(A) and IK(DR) in rat inferior colliculus neurons but only significantly affects the activation and inactivation kinetics of IK(DR). Depression of IK(DR) by salicylate may play an important role in salicylate-induced tinnitus. [Copyright &y& Elsevier]

Noxious heat above ∼45 °C applied on cold spots evokes a paradoxical cold sensation by activating cold fibers. It remains unresolved whether cold receptors respond to heat as well, or whether noxious-heat receptors and cold receptors coexist in the same fiber. Recently, noxious heat receptors (TRPV1) and cold receptors (TRPM8) have been cloned. It is controversial, however, whether TRPV1 and TRPM8 coexist in the same sensory neuron. Here, we investigate colocalization of these receptors in dorsal root ganglion (DRG) of rats. TRPV1 was expressed in 29% of TRPM8-positive cells in DRG sections. In Ca2+ imaging, noxious heat excited many of the cold-sensitive cells in culture. In a whole-cell current-clamp mode, noxious heat, capsaicin, cooling and menthol all evoked receptor potentials and impulses in a subset of DRG neurons. This colocalization of TRPV1 and TRPM8 in a DRG neuron may be the basis for the paradoxical cold sensation. [Copyright &y& Elsevier]

Modification of voltage-gated Ca2+ channels by hydrogen peroxide, a membrane-permeable form of reactive oxygen species, in cultured dentate granule cells was examined using the whole cell patch clamp technique. Pretreatment with hydrogen peroxide (1 and 10 μM) for 2 h enhanced the Ca2+ current without affecting its voltage dependence. The enhancement was completely cancelled by 1 mM glutathione, an antioxidant, and 2 μM nifedipine, an L-type Ca2+ channel blocker. In contrast, the enhancement of the Ca2+ current was not mimicked by pretreatment with 10 μg/ml tunicamycin, an endoplasmic reticulum stressor. These results suggest that oxidative stress induced by hydrogen peroxide selectively regulates the activity of L-type Ca2+ channels. [Copyright &y& Elsevier]

Recently we found that subfornical organ (SFO) neurons were activated through nicotinic receptors as well as muscarinc. In this study, the preferential responses of single SFO neurons to both muscarine and nicotine were examined by using rat slice preparations, and current and voltage clamp recordings. When the amplitudes of the depolarizations and inward currents by muscarine and nicotine in single SFO neurons were compared, some neurons showed a higher sensitivity to muscarine than to nicotine while others showed vice versa. These data indicate that acetylcholine activates SFO neurons preferentially through either muscarinic or nicotinic receptors and suggest a diversity of cholinergic responses in the SFO. [Copyright &y& Elsevier]

Neuronal γ-aminobutyric acid (GABA)A receptors are extremely heterogeneous and therefore GABAergic currents represent responses of unknown mixture of receptor subtypes. Expression of recombinant receptors in foreign cells allows to investigate a defined receptor subtype but its properties can be altered due to, e.g., differences in the endogenous modulators. In the present study the α1β2γ2 receptors were expressed in HEK293 and QT-6 cells and current responses to ultrafast GABA applications were recorded. Rise time and rapid deactivation component were faster in responses recorded from QT-6 cells. Moreover, in QT-6 cells desensitization was faster and more profound. Recovery in the paired pulse experiments was faster in HEK293 cells. In conclusion, we provide evidence that recombinant receptors may show functional differences when expressed in different cells. [Copyright &y& Elsevier]

Dorsal horn neurons expressing receptor for substance P (SP), the neurokinin 1 (NK1) receptor, play an important role in transmission and processing of nociceptive stimuli. To identify and study these neurons in the rat spinal cord slice preparation, we used fluorescence-conjugated SP to label NK1 receptor-expressing neurons. Labeled neurons in lamina I and III/IV were patch clamped and the vanilloid receptor 1 (TRPV1) agonist, capsaicin, was applied to evoke glutamate release from central terminals of peripheral nociceptors. Capsaicin induced an increase in the frequency of miniature excitatory postsynaptic currents in 73% of lamina I and 43% of lamina III/IV neurons expressing NK1-receptor indicating that these neurons receive direct input from capsaicin and heat sensitive nociceptors. [Copyright &y& Elsevier]

To elucidate whether the methamphetamine (MAP)-induced hypersensitivity to dopamine occurs in the ventral tegmental area (VTA), patch clamp studies were performed using brain slice preparations. MAP or physiological saline was administered to 8–10-day-old rats daily for 5 days. On day 5, patch clamp studies were performed on VTA dopamine neurons which were identified by morphological and electrophysiological characteristics. Dopamine (1–100 μM) and talipexole (a dopamine D2 receptor agonist, 1–100 μM) produced a dose-dependent hyperpolarization in these neurons; treatment with SKF 38393 or PD128907 (Dl and D3 receptor agonists, respectively) had no effect. The extent of hyperpolarization was significantly greater in the MAP group compared to the saline controls, suggesting that repeated MAP administration causes a hypersensitivity to dopamine that is D2 receptor-dependent. This hypersensitivity reduces the excitability of VTA dopamine neurons, thus decreasing dopamine release in the nucleus accumbens area. This may compensate for the MAP-induced elevation of dopamine levels and modulate the dopamine-induced signal transduction cascades, leading to reverse tolerance in nucleus accumbens neurons. [Copyright &y& Elsevier]

We investigated characteristics of identified GABAergic neurons in slices of the superficial superior colliculus (sSC) using transgenic mice in which green fluorescent protein (GFP) is specifically expressed in GABAergic neurons. Whole-cell patch clamp recordings and intracellular staining with biocytin revealed electrophysiological and morphological properties of GFP-positive neurons. GFP-positive neurons had a tangentially elongated wide dendritic field. In response to depolarizing current pulses, most of the GFP-positive neurons showed either of two firing properties; 55% (n=36/65) generated successive spikes with a constant firing frequency and 22% (n=14/65) showed transient burst firing at a threshold level for spike generation. These results elucidate a functional heterogeneity of GABAergic neurons, and provide fundamental information to understand the function of the GABAergic system in the sSC. [Copyright &y& Elsevier]

Inhibitory glycine receptor (GlyR) and GABAA receptor (GABAAR)-mediated synaptic transmission was examined in two strains of the GlyR mutant mouse spastic and the respective wild types. The mutants display a mild and a severe neurological phenotype. Electrically evoked postsynaptic whole-cell currents were recorded from α-motoneurons in lumbar spinal cord slices. Amplitudes of GlyR-mediated IPSCs were significantly reduced in the severe phenotype in comparison to the respective wild type and the mild phenotype mutants. Surprisingly, amplitudes of GABAAR-mediated IPSCs were also significantly reduced in both mutants. Fast time constants of the decay phase of IPSCs were slightly reduced for the GlyR-mediated IPSCs and significantly larger for the GABAAR-mediated IPSCs in both mutant strains. [Copyright &y& Elsevier]

To investigate synaptic formation of neurons grown on three-dimensional (3D) collagen gels, neurons dissociated from embryonic rat cerebral cortices were seeded onto type-I collagen gels and cultured in serum-free medium for up to 2 weeks. Double-immunostaining for mitogen-activated protein-2 (MAP-2), a neuronal cell body and dendritic marker, and synapsin I, a synaptic vesicle antigen, was carried out to identify pre- and postsynaptic structural specializations, respectively. MAP-2+ neuronal soma and dendrites were found to be surrounded by numerous puncta of synapsin I in a 1 week-old culture. Whole-cell patch clamp experiments demonstrated that the neurons grown on 3D gels exhibit sodium and potassium currents similar to those seen in 2D culture. Spontaneous action potentials were found in neurons that had been in culture for 8–12 days. In addition, spontaneous, bicuculline-sensitive gamma-aminobutyric acidergic postsynaptic currents were also present. This is the first demonstration of functional synapse formation among neurons grown on 3D collagen gels, suggesting that type-I collagen can be a promising material for neuronal regeneration. [Copyright &y& Elsevier]

We examined the effect of 5-hydroxyindole (5-HI) on the potentiation of 5-hydroytryptamine (5-HT)3 receptor function by ethanol (EtOH) so as to study whether EtOH potentiates channel function through increasing activation or blocking desensitization. We measured 5-HT3 receptor current using a whole-cell voltage clamp technique with a method of rapid drug application in NCB-20 neuroblastoma cells. The 5-HI, an agent that block receptor desensitization, increased the decay time constant (τ), not the peak of 5-HT3 receptor-mediated currents induced by 10 μM 5-HT. EtOH did not change the peak amplitude and τ of the current induced by 10 μM 5-HT. Coapplication of EtOH and 5-HI with 5-HT caused no increase in the peak currents, but τ was further increased. Therefore, a further block in desensitization could be induced by 5-HI, despite the presence of EtOH. These results indicate that EtOH potentiates 5-HT3 receptor function, with these effects due at least in part by increasing channel activation rather than by blocking desensitization. [Copyright &y& Elsevier]

The patch-clamp method was used to measure transmembrane Na+ and K+ currents of the action potential in SH-SY5Y neuroblastoma cells exposed to static magnetic fields of 1, 5, and 75 G, 60 Hz fields of 1 and 5 G, and to combined static and low-frequency fields tuned for resonance of Na+ and K+. The maximum currents and their inactivation rates, and the activation rate of the Na+ current were measured. Application of the magnetic fields did not result in detectable changes in any of the parameters of the action potential chosen for study. The occurrence of effects due to the fields could be excluded down to at least one part in 1000. The results suggest that magnetic fields of the type studied do not affect the cellular mechanisms responsible for generating the action potential. [Copyright &y& Elsevier]

Local anesthetics (LAs) block Na+ channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na+ channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site. [Copyright &y& Elsevier]

Galantamine is an acetylcholinesterase inhibitor in Alzheimer''s disease therapy. In the present study, we investigated the effects of galantamine on delayed rectifier potassium current (IK(DR)) and transient outward potassium current (IK(A)) in acutely dissociated rat hippocampal pyramidal neurons by using whole-cell patch clamp technique. IK(DR) was inhibited by galantamine in a concentration-dependent manner, while IK(A) remained unaffected. The IC50 value for the blocking action of galantamine on IK(DR) was calculated as 2.0 μM. At the concentration of 10 μM, galantamine inhibited IK(DR) by 40.2% at +40 mV when depolarized from −50 mV, and shifted the activation curve and inactivation curve of IK(DR) to negative potential by −3.8 mV and −11.0 mV, respectively. In conclusion, galantamine potently inhibits IK(DR) but not IK(A) in rat hippocampal pyramidal neurons. [Copyright &y& Elsevier]

The effects of rises in external K+ (Kext) on Ih were investigated in CA1 pyramidal cells of rat hippocampal slices using the whole-cell patch clamp technique. At the basal Kext level (2.5 mM), hyperpolarization-activated cation current (Ih) had a maximal amplitude of −350±60 pA which was enhanced by ∼60 and ∼95% at 5 and 7.5 mM Kext, respectively. The midpoint activation voltage was significantly shifted from −80 mV in the negative direction to about −87 mV at both 5 and 7.5 mM Kext, without appreciable alterations of the current kinetics. The maximal conductance was ∼2.4 nS under control conditions and significantly increased to ∼3.3 and ∼5.6 nS at 5 and 7.5 mM Kext, respectively. The reversal potential was shifted in the positive direction, from a control value of ∼−30 mV by ∼6 and ∼14 mV at 5 and 7.5 mM Kext, respectively. Our data demonstrate that even moderate changes in Kext have a substantial effect on the properties of Ih. [Copyright &y& Elsevier]

The entorhinal cortex (EC) is an essential relay station in neocortical – hippocampal information transfer and memory functions. Layer II stellate and layer III pyramidal neurons show specific damage in Alzheimer''s disease and epilepsy, respectively. Using whole cell patch clamp recording in rat brain slices we here demonstrate that high voltage activated Ca2+-currents (ICa2+) are about 1.6-fold bigger in stellate cells than in pyramidal neurons while current density is equal in both cell types. In stellate cells ICa2+ shows stronger inactivation with depolarization, block of ICa2+ by Ni2+ (300 and 600 μM) is more effective, and this block decreases more for currents evoked from a less negative holding potential than in layer III pyramidal neurons. These data indicate distinct molecular composition of Ca2+-channels and can partially explain stronger increases of [Ca2+]i during 10 Hz firing activity in EC pyramidal versus stellate neurons. [Copyright &y& Elsevier]

It has been reported that GABAB receptors are abundantly expressed in the superficial superior colliculus (sSC). To elucidate the action of GABAB receptors in the sSC, we tested the effect of a specific GABAB receptor agonist baclofen on sSC neurons in slices obtained from rats (16–22 days postnatal) using the whole-cell patch clamp technique. Bath application of baclofen (10–30 μM) elicited hyperpolarization associated with an increase in potassium conductance in morphologically different types of neurons including putative excitatory and inhibitory neurons. In addition, baclofen presynaptically suppressed electrically induced excitatory postsynaptic currents and inhibitory postsynaptic currents. These effects of baclofen were antagonized by a specific GABAB receptor antagonist CGP55845A (1 μM). The results suggest that the GABAB receptors have an inhibitory action at both post- and pre-synaptic sites in the sSC. [Copyright &y& Elsevier]

The main targets of the insecticide imidacloprid are neuronal nicotinic acetylcholine receptors (nAChRs) within the insect brain. We tested the effects of imidacloprid on ligand-gated ion channels of cultured Kenyon cells of the honeybee, Apis mellifera. Kenyon cells build up the mushroom body neuropils, which are involved in higher order neuronal processes such as olfactory learning. We measured whole-cell currents through nicotinic and γ-aminobutyric acid (GABA) receptors using patch-clamp techniques. Pressure applications of imidacloprid elicited inward currents, which were irreversibly blocked by α-bungarotoxin. Imidacloprid was a partial nicotinic agonist, since it elicited only 36% of ACh-induced currents and competitively blocked 64% of the peak ACh-induced currents. GABA-induced currents were partially blocked when imidacloprid was coapplied and this block was independent upon activation of nAChRs. Our results identify the honeybee nAChR as a target of imidacloprid and an imidacloprid-induced inhibition of the insect GABA receptor. [Copyright &y& Elsevier]

We have investigated the modulation of a cloned rat brain α-subunit of large conductance Ca2+-activated K+ channels (rSlo K+ channels) by glutathione (GSH), a physiological sulfhydryl-specific reducing reagent. The application of GSH to the intracellular side of excised inside-out macroscopic patches of rslo-transfected HEK293 cells reversibly activated the currents. The activation rate constants of the current were increased while the deactivation rate constants were decreased by GSH at all voltages tested without any change in the voltage dependence of the rate constants. GSH induced a leftward shift of the steady state conductance–voltage relationship curve of the current with no change in the slope of the curve. These results suggest that modulation by GSH may constitute an important regulatory mechanism of neuronal large conductance Ca2+-activated K+ channels. [Copyright &y& Elsevier]