Herpes Simplex Virus Organizes Cytoplasmic Membranes To Form a Viral Assembly Center in Neuronal Cells.

Herpes simplex virus (HSV) is a neuroinvasive virus that has been used as a model organism for studying common properties of all herpesviruses. HSV induces host organelle rearrangement and forms multiple, dispersed assembly compartments in epithelial cells, which complicates the study of HSV assembly. In this study, we show that HSV forms a visually distinct unitary cytoplasmic viral assembly center (cVAC) in both cancerous and primary neuronal cells that concentrates viral structural proteins and is a major site of capsid envelopment. The HSV cVAC also concentrates host membranes that are important for viral assembly, such as Golgi- and recycling endosome-derived membranes. Finally, we show that HSV cVAC formation and/or maintenance depends on an intact microtubule network and a viral tegument protein, pUL51. Our observations suggest that the neuronal cVAC is a uniquely useful model to study common herpesvirus assembly pathways and cell-specific pathways for membrane reorganization.IMPORTANCE Herpesvirus particles are complex and contain many different proteins that must come together in an organized and coordinated fashion. Many viruses solve this coordination problem by creating a specialized assembly factory in the host cell, and the formation of such factories provides a promising target for interfering with virus production. Herpes simplex virus 1 (HSV-1) infects several types of cells, including neurons, but has not previously been shown to form such an organized factory in the nonneuronal cells in which its assembly has been best studied. Here, we show that HSV-1 forms an organized assembly factory in neuronal cells, and we identify some of the viral and host cell factors that are important for its formation.

Presynaptically silent synapses are modulated by the density of surrounding astrocytes.

Astrocytes, comprising the primary glial-cell type, are involved in the formation and maturation of synapses, and thus contribute to sustainable synaptic transmission between neurons. Given that the animals in higher phylogenetic tree have brains with a higher density of glial cells with respect to neurons, there is a possibility that the relative astrocytic density directly influences synaptic transmission. However, the notion has not been tested thoroughly. Here we addressed it, by using a primary culture preparation where single hippocampal neurons are surrounded by a variable but a countable number of cortical astrocytes in dot-patterned microislands, and recording synaptic transmission by patch-clamp electrophysiology. Neurons with a higher astrocytic density showed a higher amplitude of the evoked excitatory postsynaptic current than that of neurons with a lower astrocytic density. The size of the readily releasable pool of synaptic vesicles per neuron was significantly larger. The frequency of spontaneous synaptic transmission was higher, but the amplitude was unchanged. The number of morphologically identified glutamatergic synapses was comparable, but the percentage of functional ones was increased, indicating a lower ratio of presynaptically silent synapses. Taken together, the higher astrocytic density enhanced excitatory synaptic transmission by increasing the fraction of functional synapses through presynaptic un-silencing.

Disruption of Protein Processing in the Endoplasmic Reticulum of DYT1 Knock-in Mice Implicates Novel Pathways in Dystonia Pathogenesis.

Dystonia type 1 (DYT1) is a dominantly inherited neurological disease caused by mutations in TOR1A, the gene encoding the endoplasmic reticulum (ER)-resident protein torsinA. Previous work mostly completed in cell-based systems suggests that mutant torsinA alters protein processing in the secretory pathway. We hypothesized that inducing ER stress in the mammalian brain in vivo would trigger or exacerbate mutant torsinA-induced dysfunction. To test this hypothesis, we crossed DYT1 knock-in with p58(IPK)-null mice. The ER co-chaperone p58(IPK) interacts with BiP and assists in protein maturation by helping to fold ER cargo. Its deletion increases the cellular sensitivity to ER stress. We found a lower generation of DYT1 knock-in/p58 knock-out mice than expected from this cross, suggesting a developmental interaction that influences viability. However, surviving animals did not exhibit abnormal motor function. Analysis of brain tissue uncovered dysregulation of eiF2α and Akt/mTOR translational control pathways in the DYT1 brain, a finding confirmed in a second rodent model and in human brain. Finally, an unbiased proteomic analysis identified relevant changes in the neuronal protein landscape suggesting abnormal ER protein metabolism and calcium dysregulation. Functional studies confirmed the interaction between the DYT1 genotype and neuronal calcium dynamics. Overall, these findings advance our knowledge on dystonia, linking translational control pathways and calcium physiology to dystonia pathogenesis and identifying potential new pharmacological targets. SIGNIFICANCE STATEMENT: Dystonia type 1 (DYT1) is one of the different forms of inherited dystonia, a neurological disorder characterized by involuntary, disabling movements. DYT1 is caused by mutations in the gene that encodes the endoplasmic reticulum (ER)-resident protein torsinA. How mutant torsinA causes neuronal dysfunction remains unknown. Here, we show the behavioral and molecular consequences of stressing the ER in DYT1 mice by increasing the amount of misfolded proteins. This resulted in the generation of a reduced number of animals, evidence of abnormal ER protein processing and dysregulation of translational control pathways. The work described here proposes a shared mechanism for different forms of dystonia, links for the first time known biological pathways to dystonia pathogenesis, and uncovers potential pharmacological targets for its treatment.

Synaptic vesicle recycling is enhanced by torsinA that harbors the DYT1 dystonia mutation.

Early-onset generalized dystonia, DYT1, is caused by a mutation in the gene encoding the evolutionarily conserved AAA+ ATPase torsinA. Synaptic abnormalities have been implicated in DYT1 dystonia, but the details of the synaptic pathophysiology are only partially understood. Here, we demonstrate a novel role for torsinA in synaptic vesicle recycling, using cultured hippocampal neurons from a knock-in mouse model of DYT1 dystonia (ΔE-torsinA) and live-cell imaging with styryl FM dyes. Neurons from heterozygous ΔE-torsinA mice released a larger fraction of the total recycling pool (TRP) during a single round of electrical stimulation than did wild-type neurons. Moreover, when the neurons were subjected to prior high activity, the time course of release was shortened. In neurons from homozygous mice, these enhanced exocytosis phenotypes were similar, but in addition the size of the TRP was reduced. Notably, when release was triggered by applying a calcium ionophore rather than electrical stimuli, neither a single nor two ΔE-torsinA alleles affected the time course of release. Thus, the site of action of ΔE-torsinA is at or upstream of the rise in calcium concentration in nerve terminals. Our results suggest that torsinA regulates synaptic vesicle recycling in central neurons. They also indicate that this regulation is influenced by neuronal activity, further supporting the idea that synaptic abnormalities contribute to the pathophysiology of DYT1 dystonia.

Miniature release events of glutamate from hippocampal neurons are influenced by the dystonia-associated protein torsinA.

TorsinA is an evolutionarily conserved AAA+ ATPase, and human patients with an in-frame deletion of a single glutamate (ΔE) codon from the encoding gene suffer from autosomal-dominant, early-onset generalized DYT1 dystonia. Although only 30-40% of carriers of the mutation show overt motor symptoms, most experience enhanced excitability of the central nervous system. The cellular mechanism responsible for this change in excitability is not well understood. Here we show the effects of the ΔE-torsinA mutation on miniature neurotransmitter release from neurons. Neurotransmitter release was characterized in cultured hippocampal neurons obtained from wild-type, heterozygous, and homozygous ΔE-torsinA knock-in mice using two approaches. In the first approach, patch-clamp electrophysiology was used to record glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in the presence of the Na⁺ channel blocker tetrodotoxin (TTX) and absence of GABA(A) receptor antagonists. The intervals between mEPSC events were significantly shorter in neurons obtained from the mutant mice than in those obtained from wild-type mice. In the second approach, the miniature exocytosis of synaptic vesicles was detected by imaging the unstimulated release of FM dye from the nerve terminals in the presence of TTX. Cumulative FM dye release was higher in neurons obtained from the mutant mice than in those obtained from wild-type mice. The number of glutamatergic nerve terminals was also assessed, and we found that this number was unchanged in heterozygous relative to wild-type neurons, but slightly increased in homozygous neurons. Notably, in both heterozygous and homozygous neurons, the unitary synaptic charge during each mEPSC event was unchanged. Overall, our results suggest more frequent miniature glutamate release in neurons with ΔE-torsinA mutations. This change may be one of the underlying mechanisms by which the excitability of the central nervous system is enhanced in the context of DYT1 dystonia. Moreover, qualitative differences between heterozygous and homozygous neurons with respect to certain synaptic properties indicate that the abnormalities observed in homozygotes may reflect more than a simple gene dosage effect.

Calcium-induced calcium release in noradrenergic neurons of the locus coeruleus.

The locus coeruleus (LC) is a nucleus within the brainstem that consists of norepinephrine-releasing neurons. It is involved in broad processes including cognitive and emotional functions. Understanding the mechanisms that control the excitability of LC neurons is important because they innervate widespread brain regions. One of the key regulators is cytosolic calcium concentration ([Ca2+]c), the increases in which can be amplified by calcium-induced calcium release (CICR) from intracellular calcium stores. Although the electrical activities of LC neurons are regulated by changes in [Ca2+]c, the extent of CICR involvement in this regulation has remained unclear. Here we show that CICR hyperpolarizes acutely dissociated LC neurons of the rat and demonstrate the underlying pathway. When CICR was activated by extracellular application of 10 mM caffeine, LC neurons were hyperpolarized in the current-clamp mode of patch-clamp recording, and the majority of neurons showed an outward current in the voltage-clamp mode. This outward current was accompanied by increased membrane conductance, and its reversal potential was close to the K+ equilibrium potential, indicating that it is mediated by opening of K+ channels. The outward current was generated in the absence of extracellular calcium and was blocked when the calcium stores were inhibited by applying ryanodine. Pharmacological blockers indicated that it was mediated by Ca2+-activated K+ channels of the non-small conductance type. The application of caffeine increased [Ca2+]c, as visualized by fluorescence microscopy. These findings show CICR suppresses LC neuronal activity, and indicate its dynamic role in modulating the LC-mediated noradrenergic tone in the brain.

Structure of the Golgi apparatus is not influenced by a GAG deletion mutation in the dystonia-associated gene Tor1a.

Autosomal-dominant, early-onset DYT1 dystonia is associated with an in-frame deletion of a glutamic acid codon (ΔE) in the TOR1A gene. The gene product, torsinA, is an evolutionarily conserved AAA+ ATPase. The fact that constitutive secretion from patient fibroblasts is suppressed indicates that the ΔE-torsinA protein influences the cellular secretory machinery. However, which component is affected remains unclear. Prompted by recent reports that abnormal protein trafficking through the Golgi apparatus, the major protein-sorting center of the secretory pathway, is sometimes associated with a morphological change in the Golgi, we evaluated the influence of ΔE-torsinA on this organelle. Specifically, we examined its structure by confocal microscopy, in cultures of striatal, cerebral cortical and hippocampal neurons obtained from wild-type, heterozygous and homozygous ΔE-torsinA knock-in mice. In live neurons, the Golgi was assessed following uptake of a fluorescent ceramide analog, and in fixed neurons it was analyzed by immuno-fluorescence staining for the Golgi-marker GM130. Neither staining method indicated genotype-specific differences in the size, staining intensity, shape or localization of the Golgi. Moreover, no genotype-specific difference was observed as the neurons matured in vitro. These results were supported by a lack of genotype-specific differences in GM130 expression levels, as assessed by Western blotting. The Golgi was also disrupted by treatment with brefeldin A, but no genotype-specific differences were found in the immuno-fluorescence staining intensity of GM130. Overall, our results demonstrate that the ΔE-torsinA protein does not drastically influence Golgi morphology in neurons, irrespective of genotype, brain region (among those tested), or maturation stage in culture. While it remains possible that functional changes in the Golgi exist, our findings imply that any such changes are not severe enough to influence its morphology to a degree detectable by light microscopy.

Therapeutic treatment of New Zealand mouse disease by a limited number of anti-idiotypic antibodies conjugated with neocarzinostatin.

-81 and NE-1 idiotypes (Id) of human nephritogenic anti-DNA antibodies are interspecies Id expressed also in NZB/W F1 mice. We tried to manipulate the synthesis of spontaneously occurring anti-DNA antibody using monoclonal anti-Id antibodies (D1E2 and 1F5) conjugated with a cytotoxic agent, neocarzinostatin (NCS). In vivo administration of anti-Id antibodies conjugated with NCS brought about an improvement in the survival rate of female NZB/W F1 mice. It also caused a retardation of development of lupus nephritis and decreased the numbers of anti-DNA-producing cells. The suppression of anti-DNA antibody synthesis was specific and Id-mediated. The results indicate that the use of a limited number of anti-Id antibodies in combination with a cytotoxic agent may be applicable therapeutically to autoimmune diseases.

Limited numbers of recycling vesicles in small CNS nerve terminals: implications for neural signaling and vesicular cycling.

The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo-endocytotic recycling following stimulated Ca(2+) entry. This recycling pool typically contained 30-45 vesicles, only a minority fraction (15-20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.

Increased expression of alpha 1A Ca2+ channel currents arising from expanded trinucleotide repeats in spinocerebellar ataxia type 6.

The expansion of polyglutamine tracts encoded by CAG trinucleotide repeats is a common mutational mechanism in inherited neurodegenerative diseases. Spinocerebellar ataxia type 6 (SCA6), an autosomal dominant, progressive disease, arises from trinucleotide repeat expansions present in the coding region of CACNA1A (chromosome 19p13). This gene encodes alpha(1A), the principal subunit of P/Q-type Ca(2+) channels, which are abundant in the CNS, particularly in cerebellar Purkinje and granule neurons. We assayed ion channel function by introduction of human alpha(1A) cDNAs in human embryonic kidney 293 cells that stably coexpressed beta(1) and alpha(2)delta subunits. Immunocytochemical analysis showed a rise in intracellular and surface expression of alpha(1A) protein when CAG repeat lengths reached or exceeded the pathogenic range for SCA6. This gain at the protein level was not a consequence of changes in RNA stability, as indicated by Northern blot analysis. The electrophysiological behavior of alpha(1A) subunits containing expanded (EXP) numbers of CAG repeats (23, 27, and 72) was compared against that of wild-type subunits (WT) (4 and 11 repeats) using standard whole-cell patch-clamp recording conditions. The EXP alpha(1A) subunits yielded functional ion channels that supported inward Ca(2+) channel currents, with a sharp increase in P/Q Ca(2+) channel current density relative to WT. Our results showed that Ca(2+) channels from SCA6 patients display near-normal biophysical properties but increased current density attributable to elevated protein expression at the cell surface.

Rapid genotyping of animals followed by establishing primary cultures of brain neurons.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (ΔE-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

Imaging single synaptic vesicles undergoing repeated fusion events: kissing, running, and kissing again.

At synapses of the mammalian central nervous system, release of neurotransmitter occurs at rates transiently as high as 100 Hz, putting extreme demands on nerve terminals with only tens of functional vesicles at their disposal. Thus, the presynaptic vesicle cycle is particularly critical to maintain neurotransmission. To understand vesicle cycling at the most fundamental level, we studied single vesicles undergoing exo/endocytosis and tracked the fate of newly retrieved vesicles. This was accomplished by minimally stimulating boutons in the presence of the membrane-fluorescent styryl dye FM1-43, then selecting for terminals that contained only one dye-filled vesicle. We then observed the kinetics of dye release during single action potential stimulation. We found that most vesicles lost only a portion of their total dye during a single fusion event, but were able to fuse again soon thereafter. We interpret this as direct evidence of "kiss-and-run" followed by rapid reuse. Other interpretations such as "partial loading" and "endosomal splitting" were largely excluded on the basis of multiple lines of evidence. Our data placed an upper bound of <1.4 s on the lifetime of the kiss-and-run fusion event, based on the assumption that aqueous departitioning is rate limiting. The repeated use of individual vesicles held over a range of stimulus frequencies up to 30 Hz and was associated with neurotransmitter release. A small percentage of fusion events did release a whole vesicle's worth of dye in one action potential, consistent with a classical picture of exocytosis as fusion followed by complete collapse or at least very slow retrieval.

Excitatory amino acid responses in relay neurons of the rat lateral geniculate nucleus.

Responses to glutamate receptor agonists were recorded from identified relay neurons in the dorsal lateral geniculate nucleus of the rat, using the nystatin-perforated patch-clamp technique. Rapid application of glutamate, N-methyl-D-aspartate, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate induced inward currents at a holding potential of -44 mV. The responses to low concentrations of each agonist were composed only of steady-state currents, but the responses to high concentrations were additionally composed of a rapid transient peak component except in the kainate-induced current. The currents induced by 10(-3)M N-methyl-D-aspartate in the external solution containing 0 mM Mg2+ and 10(-6)M glycine were reduced in amplitude when the external solution contained 1 mM Mg2+, and were abolished when the solution contained no glycine. The currents induced by a neurotransmitter candidate at retinogeniculate synapses, N-acetyl-aspartyl-glutamate, were markedly reduced in amplitude when the solution contained 1 mM Mg2+ or 10(-4)M DL-2-amino-5-phosphonovaleric acid. The current abolished in the Mg2+-containing, glycine-free solution (N-methyl-D-aspartate component) and the current remaining in the same solution (non-N-methyl-D-aspartate component) of the N-acetyl-aspartyl-glutamate response were both increased in a concentration-dependent manner, as the N-acetyl-aspartyl-glutamate concentration was increased. The current-voltage relationship of the currents induced by N-methyl-D-aspartate and N-acetyl-aspartyl-glutamate was characterized by Mg2+-dependent block at hyperpolarized potentials. The inward currents induced by 3 x 10(-4)M AMPA and 3 x 10(-4)M glutamate were markedly potentiated by 10(-4)M cyclothiazide, but the currents induced by 3 x 10(-4)M kainate and 10(-3)M N-acetyl-aspartyl-glutamate (non-N-methyl-D-aspartate component) were little affected. The currents induced by any agonist were not affected by 3 x 10(-4)g/ml concanavalin A. The current induced by 10(-4)M kainate was markedly suppressed by pretreatment with 10(-4)M AMPA or 10(-4)M glutamate, but only weakly by 10(-3)M N-acetyl-aspartylglutamate. The Ca2+ permeability (PCa/PCs) of the N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors was 9.57 and 0.16, respectively. These results suggest that dorsal lateral geniculate nucleus relay neurons of the rat possessed both Ca2+-permeable N-methyl-D-aspartate receptors and less permeable non-N-methyl-D-aspartate (presumably AMPA) receptors, and that N-acetyl-aspartyl-glutamate mainly acts at N-methyl-D-aspartate receptors with a weak kainate-like action on non-N-methyl-D-aspartate receptors.

Kinetics of sevoflurane action on GABA- and glycine-induced currents in acutely dissociated rat hippocampal neurons.

Effects of a new kind of volatile anaesthetics, sevoflurane, on GABA- and glycine-gated chloride current (ICl) were examined in single pyramidal neurons acutely dissociated from the rat hippocampal CA1 region, using the voltage-clamp mode of the nystatin-perforated patch-clamp technique. Rapid application of sevoflurane-induced ICl by itself, with the time to peak reduced as the sevoflurane concentration was increased from 10(-3) to 3 x 10(-3) M. Although a pretreatment with 10(-3) M sevoflurane enhanced the peak amplitude of GABA (3 x 10(-6) M)-induced ICl and suppressed the peak amplitude when the GABA concentration was increased to 10(-4) M, the pretreatment decreased the time to peak of the ICl induced by any concentration of GABA (from 3 x 10(-6) to 10(-4) M). The treatment also accelerated the decay phase of the GABA-induced ICl. On the other hand, sevoflurane suppressed the peak ICl induced by 3 x 10(-5) M glycine in a concentration-dependent manner. In the presence of 3 x 10(-4) M sevoflurane, the peak amplitude of the glycine-induced ICl was decreased without changes in EC50 or Hill coefficients. Pretreatment with 10(-3) M sevoflurane did not affect the time to peak of the ICl induced by any concentration of glycine (from 3 x 10(-5) to 10(-3) M). Pretreatment with 3 x 10(-8) M strychnine markedly prolonged the time to peak of the glycine-induced ICl. These results suggest that sevoflurane modulated the amplitude of the GABA responses, depending on the balance of the accelerated activation and decay phases, and that sevoflurane suppressed the glycine-induced ICl in a non-competitive manner without noticeable effect on the kinetics. The reversible and differential modulation of GABA(A) and glycine receptors might underlie a part of the anaesthetic actions and less adverse clinical effects of sevoflurane.

Caffeine and related compounds block inhibitory amino acid-gated Cl- currents in freshly dissociated rat hippocampal neurones.

1. The effects of caffeine and related compounds on responses mediated by inhibitory amino acids were investigated in freshly dissociated rat hippocampal pyramidal neurones by conventional and nystatin perforated patch-clamp techniques. 2. Glycine and gamma-aminobutyric acid (GABA) evoked Cl- currents in hippocampal neurones. The half-maximum effective concentrations (EC50) of glycine and GABA were 8.5 x 10(-5) and 5 x 10(-6) M, respectively. 3. Caffeine reversibly inhibited both 10(-4) M glycine- and 10(-5) M GABA-induced Cl-currents in a concentration-dependent manner. The half-maximum inhibitory concentrations (IC50) of caffeine were 4.5 x 10(-4) M for the glycine response and 3.6 x 10(-3) M for the GABA response. 4. Caffeine shifted the concentration-response curve of IGly to the right without affecting the maximum response. 5. The inhibitory action of caffeine did not show voltage-dependency. 6. The blocking action of caffeine was not affected by intracellular perfusion with 5 mM BAPTA or by pretreatment with the protein kinase A inhibitor, H-8. This excludes the participation of Ca2+ or cyclic AMP in the inhibitory action of caffeine. 7. Caffeine failed to inhibit the augmentations of aspartate- and N-methyl-D-aspartate (NMDA) -gated current by glycine, suggesting that caffeine has no effect on the allosteric glycine binding site on the NMDA receptor. 8. The inhibitory effects of some xanthine derivatives on IGly were compared. The inhibitory potency of those compounds on IGly was in the order of pentoxifylline > theophylline > or = caffeine > paraxanthine > IBMX > or = theobromine > dyphylline. Xanthine had no effect. 9. The results indicate that methylxanthines including caffeine may act directly on the glycine receptor Cl- channel complex in rat hippocampal pyramidal neurones. The blockade of the inhibitory amino acid response by methylxanthines may be involved in the excitatory side effects of methylxanthines in the mammalian central nervous system.

Potentiation by sevoflurane of the gamma-aminobutyric acid-induced chloride current in acutely dissociated CA1 pyramidal neurones from rat hippocampus.

1. The effects of a new kind of volatile anaesthetic, sevoflurane (Sev), on gamma-aminobutyric acid (GABA)-gated chloride current (Icl) in single neurones dissociated from the rat hippocampal CA1 area were examined using the nystatin perforated patch recording configuration under the voltage-clamp condition. All drugs were applied with a rapid perfusion system, termed the "Y-tube' method. 2. When the concentrations were higher than 3 x 10(-4) M, Sev, itself, induced an inward current (ISev) at a holding potential (VH) of -40 mV. The concentration-response curve of ISev was bell-shaped, with a suppressed peak and plateau currents at high concentrations (above 2 x 10(-3) M). The reversal potential of ISev (ESev) was close to the theoretical Cl- equilibrium potential, indicating that ISev was carried mainly by Cl-. 3. ISev was reversibly blocked by bicuculline (Bic), an antagonist of the GABAA receptor, in a concentration-dependent manner with a half-inhibitory concentration (IC50) of 7.2 x 10(-7) M. But ISev was insensitive to strychnine (Str), an antagonist of the glycine receptor. 4. At low concentrations (between 3 x 10(-4) and 10(-3) M), Sev markedly enhanced the 10(-6) M GABA induced current (IGABA) but reduced the IGABA with accelerating desensitization accompanied by a "hump' current after washout at high concentrations (higher than 2 x 10(-3) M). 5. Sev, 10(-3) M potentiated the current induced by low concentrations of GABA (between 10(-7) and 3 x 10(-6) M) but reduced the current induced by high concentrations (higher than 10(-5) M) of GABA with a clear acceleration of IGABA desensitization. 6. Sev, like pentobarbitone (PB), pregnanolone (PGN) or diazepam (DZP), potentiated the 10(-6) M GABA-induced response without shifting the reversal potential of IGABA. 7. ISev was augmented by PB, PGN, or DZP at concentrations that maximally potentiated IGABA, suggesting that Sev enhanced IGABA at a binding site distinct from that for PB, PGN, or DZP. 8. It is concluded that Sev acts on the GABAA receptor complex mimicking the GABA-induced chloride current at high concentrations. At low concentrations, Sev enhances GABA-gated chloride current at a binding site independent of the allosteric modulator sites of barbiturates, benzodiazepines or neurosteroids. The reversible potentiation of the inhibitory GABAA receptor-mediated Cl- current may result in the depressing of postsynaptic excitability and may, at least in part, underlie the anaesthetic action of Sev.

Sevoflurane-induced ionic current in acutely dissociated CA1 pyramidal neurons of the rat hippocampus.

The electrophysiological properties of sevoflurane (Sev)-induced current (ISev) were investigated in CA1 pyramidal neurons freshly dissociated from the rat hippocampus by using the nystatin perforated patch recording configuration under voltage-clamp condition. Within the range of Sev concentrations from 3 x 10(-4) to 2 x 10(-3) M, ISev was an inward current which consisted of an initial transient peak component and a successive steady-state plateau component. The peak current component increased in a concentration-dependent manner with a conductance increase. The application of Sev over 2 x 10(-3) M, however, suppressed the peak and steady-state current components with a concomitant decrease in conductance and elicited a transient inward current ('hump' current) immediately after wash out. The current-voltage relationship for ISev showed some outward rectification suggesting a slight voltage-dependency of the ISev. The reversal potential of ISev (ESev) was close to the ECl and shifted by 52 mV for a 10-fold change in extracellular Cl- concentrations, indicating that ISev is passing through Cl- channels. The single channel conductance obtained from the analysis of the variance of ISev fluctuations was 15.3 +/- 1.3 pS.

Delayed potassium current and non-specific outward current in pyramidal neurones acutely dissociated from rat hippocampus.

The electrical property of delayed K+ currents (IKD) was studied in pyramidal neurones freshly isolated from the rat hippocampal CA1 region. The IKD was separated pharmacologically from other membrane currents. Activation and inactivation processes of the IKD were highly voltage-dependent in the potential range between -30 and +20 mV. The steady-state inactivation of IKD was observed at -100 mV or more positive potentials. The potential for half steady-state inactivation was -65 mV. The IKD was fully inactivated around -20 mV. Reactivation of IKD consisted of two exponential components. After pharmacological suppression of IKD, the small amount of residual voltage-dependent outward current (one-fifteenth to one-twentieth of IKD amplitude) was observed. The current kinetics was similar to that of IKD and greatly reduced by substitution of internal K+ with N-methyl-D-glucamine+. It was concluded that the properties of IKD was basically similar to those of IKD in other excitable tissues and that the residual current might be non-specific outward current.

Quinolones do not interact with NMDA receptor in dissociated rat hippocampal neurons.

Interaction of quinolone antimicrobials and fenbufen, anti-inflammatory agents, with the N-methyl-D-aspartate (NMDA) receptor was investigated in rat hippocampal neurons with the conventional patch-clamp technique. Simultaneous applications of quinolones and a metabolite of fenbufen had no effects on the glutamate- and NMDA-induced currents. The blocking effects of Mg2+ and MK-801 on NMDA responses were also unaffected. Therefore, the convulsive action induced by quinolones and fenbufen does not seem to involve the function change of NMDA receptor.