Waglerin-1 inhibits GABAA current of neurons in the nucleus accumbens of neonatal rats

The effect of Waglerin-1, a 22-amino acid peptide purified from the venom of Wagler's pit viper on the whole cell current response (I_GABA) to γ-aminobutyric acid (GABA) was examined for neurons freshly isolated from the nucleus accumbens of 3- to 7-day-old rats. Waglerin-1 depressed I_GABA induced by subsaturating concentrations of GABA; the IC₅₀ for I_GABA induced by 10 μM GABA was 2.5 μM Waglerin-1. This concentration of Waglerin-1 shifted the GABA concentration–response curve to the right in a parallel manner, increasing the GABA EC₅₀ from 12±3 to 27±5 μM. The depressant effect of Waglerin-1 was greater at negative holding potentials. Zn²⁺ also inhibited I_GABA with an IC₅₀ of 0.3 μM. Phosphorylation state appeared to modulate GABA_A receptor sensitivity to the inhibitory effect of Waglerin-1 since dialysis of neurons with N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide HCl (H-89), an inhibitor of protein kinase A, prevented inhibition. The data are discussed in terms of developmental influences on the subunit composition of GABA_A receptors in neurons of the nucleus accumbens.     

Cocaine and lidocaine have additive inhibitory effects on the GABAA current of acutely dissociated hippocampal pyramidal neurons

Inhibition mediated by γ-aminobutyric acid (GABA) is a major target for the central actions of cocaine and lidocaine, which can result in seizures, especially when these drugs are abused in combination. In the present study, we investigated how cocaine and lidocaine interact to depress GABA current (I_GABA), recorded by the whole-cell technique in freshly isolated rat hippocampal neurons. Cocaine depressed I_GABA in a concentration dependent manner, such that cocaine was more potent against lower than higher GABA concentrations: the cocaine IC₅₀ was 0.13, 0.62 and 1.2 mM for GABA at 2, 10 and 100 μM, respectively. Cocaine depressed I_GABA to the same extent in the absence and presence of 1 μM tetrodotoxin, indicating that cocaine inhibition of I_GABA is distinct from its Na⁺ channel blocking action. Lidocaine reversibly depressed I_GABA evoked by 10 μM GABA, with an IC₅₀ of 9.8 mM. In the presence of 3 mM lidocaine, 0.3 mM cocaine depressed I_GABA (10 μM GABA) to 30±7%. The significantly greater depression by the combined agents (p<0.05) indicates additive effects on the GABA receptor/channel complex, which are likely to contribute to the additive convulsant effects noted when these drugs are abused in combination.     

Glutamate selectively increases the high-threshold Ca channel current in sensory and hippocampal neurons

Previous studies resulted in conflicting conclusions that glutamate application either decreases or increases the activity of Ca²⁺ channels in hippocampal neurons. We studied whole-cell Ca²⁺ currents (I_Ca) in chick dorsal root ganglion neurons and rat hippocampal cells. For both cell types glutamate (1–30 μM) increased high-threshold Ca²⁺ current. It was independent of the charge carriers, Ca²⁺ or Ba²⁺. Low-threshold Ca²⁺ channel current and the fast sodium current were not changed with glutamate application. The effect developed within 1–2 min and then further facilitated after washout of the agonist. A second application of glutamate produced no additional increase in _ICa. No changes in the time-course of whole-cell currents were observed, suggesting that glutamate recruits ‘sleepy’ Ca²⁺ channels. Whatever its mechanism, overlasting increase of I_Ca by glutamate may be important in neuronal plasticity.     

2,3-Butanedione monoxime protects mice against the convulsant effect of picrotoxin by facilitating GABA-activated currents

While adult mice receiving picrotoxin (PTX) alone responded with clonic and tonic-clonic seizures, this response was greatly suppressed for mice simultaneously injected with 2,3-butanedione monoxime (BDM). For example, 60% and 10% of the mice convulsed when injected (i.p.) with 3.0 mg/kg PTX alone or PTX plus 205 mg/kg of BDM, respectively. In contrast, a non-oxime analogue of BDM, 2,3-butanedione (BTD), did not have this anticonvulsant effect. In order to explore the basis for the anticonvulsant effect of BDM, we recorded GABA-activated currents (I_GABA) of frontal cortical as well as ventromedial hypothalamic neurons before, during and after exposure to this oxime. BDM had a biphasic effect on concentrations (100 μM-40 mM) decreased and lower concentrations (0.01 μM–0.001 μM) potentiatedI_GABA; these effects of BDM reversed upon washout of the oxime. In contrast, BTD had no effect onI_GABA. Finally, when 0.001 μM BDM, 10–30 μM PTX and GABA were co-applied the inhibitory effect of the toxin onI_GABA was markedly suppressed. These data suggest that the anticonvulsant effect of oximes involves facilitation of the inhibitory action of GABA.     

GABA-induced responses in electrophysiologically characterized neurons within the rat rostro-ventrolateral medulla in vitro

Rostro-ventrolateral medulla (RVL) neurons were recorded using conventional intracellular recording techniques in brain slices maintained in vitro at 32°C and classified into 3 major groups. The first group included neurons having endogenous pacemaker-like (PL) activity with regular firing frequency (mean = 8 Hz) and a linear current-voltage relationship (I–V). The second group of neurons were slowly and irregularly firing (IF) or quiescent, presenting membrane potential oscillations and their I–V usually displayed an inward rectification. These neurons had a relatively longer action potential duration. The third group included silent neurons (S) with no apparent membrane oscillations and they differed from the first two groups by having relatively shorter action potential duration and amplitude and lower cell input resistance. When recorded with KCl-filled electrodes, the majority of silent neurons displayed a time-dependent inward rectification. With KAc-filled electrodes, irregular slow hyperpolarizing and depolarizing spontaneous potentials could be recorded primarily on PL and IF neurons, respectively. Moreover, fast spontaneous inhibitory postsynaptic potentials (PSPs) were detected in about 15% of PL and S neurons. They generally exhibited a regular pattern and were depolarizing when KCl-filled electrodes were used for recording. The amplitude of these inhibitory PSPs was reversibly reduced by the GABAA antagonists bicuculline, SR 95531 and picrotoxin. With KAc-filled electrodes, pressure-applied GABA (20 mM) evoked complex responses. In PL neurons, it consisted of a fast hyperpolarization followed by a slower depolarization that were both sensitive to SR 95531 and picrotoxin. The response was terminated by a long-lasting hyperpolarization that was reduced, but not abolished, by the GABA_B antagonist CGP 35348. In IF and S neurons, GABA application usually produced a fast followed by a slow monophasic hyperpolarization and depolarization, respectively. The fast component of these responses was sensitive to the GABA_A antagonists. Pressure application of isoguvacine (10 mM) always induced monophasic responses in all types of neurons recorded. Baclofen (1–30 μM) reduced the firing frequency and hyperpolarized PL and IF neurons, an effect that was antagonized by CGP 35348 (50–100 μM); however, it had little effect on silent neurons. It is concluded that RVL neurons have heterogeneous electrophysiological characteristics. Their predominant synaptic input and GABA responsiveness might be additional criteria to identify the excitatory and inhibitory elements in the RVL circuitry. All neuronal types seem to have functional GABA_A and GABA_B receptors; however, only a subpopulation is under tonic inhibitory control in vitro, probably from local GABAergic pacemaker intemeurons. Our results further emphasize the role of GABA as an important neurotransmitter in the RVL network.     

Long-lasting enhancement of synaptic excitability of CA1/subiculum neurons of the rat ventral hippocampus by vasopressin and vasopressin(4–8)

Vasopressin (VP) is axonally distributed in many brain structures, including the ventral hippocampus. Picogram quantities of VP injected into the hippocampus improve the passive avoidance response of rats, presumably by enhancing memory processes. Vasopressin is metabolized by the brain tissue into shorter peptides, such as [pGlu⁴r,Cyt⁶]VP(4–9[ and [pGIu⁴,Cyt⁶,]VP(4–8), which preserve the behavioral activity but lose the peripheral activities of the parent hormone. Using brain slices, we investigated whether VP or VP(4–8) affects excitatory postsynaptic potentials (EPSPs) and/or membrane responses to depolarization in neurons of the CA 1 /subiculum of the ventral hippocampus. The EPSPs were evoked by stimulating the stratum radiatum of the CAI field; the membrane responses were elicited by current injections. Exposure of slices for 15 min to 0.1 nM solution of these peptides resulted in an increase in the amplitude and slope of the EPSPs in 21 neurons (67%) tested. No consistent change in either the resting membrane potential or the input resistance of the neurons was observed. The peptide-induced increase in EPSPs reached a maximum 30–45 min after peptide application. In 14 of these neurons (66%), the peptide-induced increase in EPSPs remained throughout the entire 60–120 min washout period. In the remaining 7 neurons (33%), the initial increase in EPSPs amplitude was followed by a gradual decline to the pre-administration level. The increase in EPSP amplitude was often. but not always, associated with a decrease in the threshold and increase in the number of action potentials in response to depolarizing current injection. Suppression of GABA_A receptor-mediated inhibition and N-methyl-d-aspartate (NMDA) receptor-mediated excitation did not prevent the effects of VP and VP(4–8[ on the EPSP amplitude or the threshold for action potentials. The results demonstrate that 0.1 nM concentrations of these neuropeptides can elicit a long-lasting enhancement of the excitability of CA1/subiculum neurons of the ventral hippocampus to excitatory, glutamatergic synaptic input. This novel action of VP and its metabolite in the ventral hippocampus may be the physiological action, mediating the memory-enhancing effect of these peptides.     

Virol A, a toxic trans-polyacetylenic alcohol of Cicuta virosa, selectively inhibits the GABA-induced Cl current in acutely dissociated rat hippocampal CA1 neurons

The effects of virol A (VA), a toxic component of Cicuta virosa (water hemlock), on the GABA-induced Cl⁻ current (I_GABA) in acutely dissociated rat hippocampal CA1 neurons were investigated using whole-cell patch-clamp techniques. VA reversibly reduced I_GABA and the muscimol (Mus)-induced current (I_Mus) in a concentration-dependent manner. The IC₅₀ values for VA against I_GABA and I_Mus were 9.6×10⁻⁷ and 9.8×10⁻⁷ M, respectively. VA shifted the EC₅₀ value of I_GABA from 6.5×10⁻⁶ to 2.1×10⁻⁵ M, whereas it had no effect on the maximum response, thereby suggesting that VA inhibited I_GABA in a competitive manner. VA had no apparent effect on current–voltage relationships for I_GABA, thus indicating the lack of voltage-dependency. On the other hand, application of VA (10⁻⁶ M) did not additionally reduce the I_GABA suppressed by >10⁻⁵ M picrotoxin. VA but not bicuculline accelerated the decay phase of I_GABA, as was seen with picrotoxin. Moreover, pre-application of 10⁻⁵ M VA reduced I_GABA. VA did not inhibit that induced by glycine (10⁻⁴ M). These results indicate that VA inhibits I_GABA by acting both on the GABA agonist site and on the Cl⁻ channel of the GABA_A receptor–channel complex. VA is a structurally novel type of compound that selectively inhibits the GABA_A receptor–Cl⁻ channel complexes in mammalian central nervous system neurons.     

Reactive oxygen species modulate neuronal excitability in rat intrinsic cardiac ganglia

Reactive oxygen species (ROS) are produced as by-products of oxidative metabolism and occur in the heart during ischemia and coronary artery reperfusion. The effects of ROS on the electrophysiological properties of intracardiac neurons were investigated in the intracardiac ganglion (ICG) plexus in situ and in dissociated neurons from neonatal and adult rat hearts using the whole-cell patch clamp recording configuration. Bath application of ROS donors, hydrogen peroxide (H₂O₂) and tert-butyl hydroperoxide (t-BHP) hyperpolarized, and increased the action potential duration of both neonatal and adult ICG neurons. This action was also recorded in ICG neurons in an adult in situ ganglion preparation. H₂O₂ and t-BHP also inhibited voltage-gated calcium channel (VGCC) currents and shifted the current–voltage (I–V) relationship to more hyperpolarized potentials. In contrast, H₂O₂ increased the amplitude of the delayed rectifier K⁺ current in neonatal ICG neurons. In neonatal ICG neurons, bath application of either superoxide dismutase (SOD) or catalase, scavengers of ROS, prior to H₂O₂ attenuated the hyperpolarizing shift but not the inhibition of VGCC by H₂O₂. In contrast, in adult ICG neurons, application of SOD alone had no effect upon either VGCC current amplitude or the I–V relationship, whereas application of SOD prior to H₂O₂ exposure abolished both the H₂O₂-mediated hyperpolarizing shift and inhibition. These data indicate that ROS alter depolarization-activated Ca²⁺ and K⁺ conductances which underlie neuronal excitability of ICG neurons. This affects action potential duration and therefore probably modifies autonomic control of the heart during ischemia/reperfusion.     

Forskolin attenuates the evoked release of [H]GABA from striatal neurons in primary culture

The actions of the diterpene forskolin, and cyclic AMP analogues, on the evoked release of [³H]GABA (γ-aminobutyric acid) was examined in intact striatal neurons in primary culture, generated from the fetal mouse brain. Exposure of striatal neurons to forskolin (100 μM) resulted in a 40–55% attenuation of [³H]GABA release evoked by either KCl (30 mM) or veratrine (2 μg/ml), while baseline levels of release were unaffected. The dose-dependence for forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable identical to the dose-dependent elevation of cyclic AMP levels by forskolin in striatal neurons. Exposure of striatal neurons to membrane-permeable analogues of cyclic AMP, such as p-chlorophenylthio cyclic AMP (0.5 mM) and dibutyryl cyclic AMP (1 mM), resulted in a 25 and 26% attenuation of [³H]GABA release, respectively; dibutyryl cyclic GMP (1 mM) was without effect. The similarity between the actions of forskolin and the cyclic AMP analogues suggests that, in striatal neurons in primary culture, the elevation of cyclic AMP levels results in the attenuation of the evoked release of [³H]GABA. The greater effectiveness of forskolin, compared to the cyclic AMP analogues, may be related to the recently reported, additional direct actions of forskolin on neuronal membrane ion channels.     

Postnatal ethanol exposure blunts upregulation of GABAA receptor currents in Purkinje neurons

Recently, we found that early postnatal ethanol exposure inhibits the maturation of GABA_A receptors (GABA_ARs) in developing medial septum/diagonal band (MS/DB) neurons, suggesting that these receptors may represent a target for ethanol related to fetal alcohol syndrome (FAS). To determine whether GABA_ARs on other neurons are also sensitive to a postnatal ethanol insult, postnatal day (PD) 4–9, rat pups were artificially reared and exposed to ethanol (4.5 g kg⁻¹ day⁻¹, 10.2% v/v). The pharmacological profile of acutely dissociated cerebellar Purkinje cell GABA_ARs from untreated, artificially reared controls and ethanol-treated animals was examined with conventional whole-cell patch clamp recordings during PD 12–16 (juveniles) and PD 25–35 (young adults). For untreated animals, GABA (0.3–100 μM) consistently induced inward Cl⁻ currents in a concentration-dependent manner showing an age-related increase in maximum response without change in EC₅₀ or slope value. Acute ethanol (100 mM) consistently inhibited 3 μM GABA currents (10–20%); positive modulators, pentobarbital (10 μM), midazolam (1 μM) and loreclezole (10 μM), consistently potentiated; the negative modulator, Zn²⁺ (30 μM), inhibited GABA currents across both juvenile and young adult groups. Loreclezole potentiation increased while Zn²⁺ inhibition decreased with age in untreated Purkinje neurons. Postnatal ethanol exposure (PD 4–9) decreased GABA_AR maximum current density in young adult Purkinje cells but not in juvenile neurons. However, sensitivity to allosteric modulators did not change after ethanol. These data are consistent with the hypothesis that postnatal ethanol exposure during the brain growth spurt can disturb GABA_AR development across the brain, although the mechanism(s) underlying this action remains to be determined.     

Glutamate increases the [Ca]i but stimulates Ca-independent release of [H]GABA in cultured chick retina cells

The effect of glutamate of [Ca²⁺]_i and on [³H]γ-aminobutyric acid (GABA) release was studied on cultured chick embryonic retina cells. It was observed that glutamate (100 μM) increases the [Ca²⁺]_i by Ca²⁺ influx through Ca²⁺ channels sensitive to nitrendipine, but not to ω-conotoxin GVIA (ω-Cg Tx) (50%), and by other channels insensitive to either Ca²⁺ channel blocker. Mobilization of Ca²⁺ by glutamate required the presence of external Na⁺, suggesting that Na⁺ mobilization through the ionotropic glutamate receptors is necessary for the Ca²⁺ channels to open. The increase in [Ca²⁺]_i was not related to the release of [³H]GABA induced by glutamate, suggesting that the pathway for the entry of Ca²⁺ triggered by glutamate does not lead to exocytosis. In fact, the glutamate-induced release of [³H]GABA was significantly depressed by Ca_o²⁺, but it was dependent on Na_o⁺, just as was observed for the [³H]GABA release induced by veratridine (50 μM). The veratridine-induced release could be fully inhibited by TTX, but this toxin had no effect on the glutamate-induced [³H]GABA release. Both veratridine- and glutamate-induced [³H]GABA release were inhibited by 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid (NNC-711), a blocker of the GABA carrier. Blockade of the NMDA and non-NMDA glutamate receptors with MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively, almost completely blocked the release of [³H]GABA evoked by glutamate. Continuous depolarization with 50 mM K⁺ induced maximal release of [³H]GABA of about 1.5%, which is much smaller than the release evoked by glutamate under the same conditions (6.0–6.5%). Glycine (3 μM) stimulated [³H]GABA release induced by 50 mM K⁺, and this effect was blocked by MK-801, suggesting that the effect of K⁺ on [³H]GABA release was partially mediated through the NMDA receptor which probably was stimulated by glutamate released by K⁺ depolarization. We conclude that glutamate induces Ca²⁺-independent release of [³H]GABA through reversal of the GABA carrier due to Na⁺ entry through the NMDA and non-NMDA, TTX-insensitive, channels. Furthermore the GABA carrier seems to be inhibited by Ca²⁺ entering by the pathways open by glutamate. This Ca²⁺ does not lead to exocytosis, probably because the Ca²⁺ channels used are located at sites far from the active zones.     

Synthetic ω-conotoxin: a potent calcium channel blocking neurotoxin

The Ca²⁺ channel blocking action of synthetic ω-conotoxin (ωCTX) was studied on isolated frog dorsal root ganglion neurons using a ‘concentration clamp’ technique which enabled internal perfusion and rapid external solution change. At 100 nM, ωCTX showed a time-dependent depression of Ca²⁺ current (I_Ca). At higher concentrations, ωCTX exhibited a dose-dependent depression of I_Ca amplitude without changing the current-voltage relationship. Increases in external Ca²⁺ concentration partly overcame the inhibitory action of ωCTX on the I_Ca amplitude. At 10 μM ωCTX totally blocked I_Ca without effect on the Na⁺ current. It was likely that ωCTX had high selectivity for the Ca²⁺ channel.     

Attenuation of a voltage-dependent sodium current by GABA (identified neurons, buccal ganglia, Helix pomatia

The action of systemically applied GABA on voltage-dependent currents of the identified neurons B1–B4 in the buccal ganglia of Helix pomatia were investigated by conventional voltage clamp techniques. In the B4 neuron, superfusion with sodium-free solution or addition of tetrodotoxin to the bath medium abolished the voltage dependent inward current. This voltage dependent sodium current was reduced with GABA application. Muscimol exerted the same effect as GABA whereas administration of baclofen had no effect. Voltage-dependent sodium and calcium currents of the neurons B1–B3 remained unchanged with GABA application. It is concluded that GABA is capable of reducing voltage-dependent sodium currents of distinct neuronal individuals via a GABA_A receptor-like structure.     

Na influx through Ca channels can promote striatal GABA efflux in Ca-deficient conditions in response to electrical field depolarization

Electrical field depolarization releases γ-aminobutyric acid (GABA) in rat striatal slices in the absence of external Ca²⁺. ω-Conotoxin GVIA (ω-CgTx; 1–50 nM), a neuronal Ca²⁺ channel blocker, inhibits electrically evoked efflux of newly taken up [³H]GABA in a concentration-dependent manner in either normal or Ca²⁺-free medium. This suggests that ion influx occurs through Ca²⁺ channels in the absence of external Ca²⁺ and contributes to the efflux of GABA. Reducing external Na⁺ concentration to 27.25 mM (low [Na⁺]₀ medium) by equimolarly substituting choline chloride for sodium chloride has differential effects on electrically evoked GABA efflux depending on the external Ca²⁺ concentrations. In normal Ca²⁺ medium, electrically evoked GABA efflux increases whereas, in Ca²⁺-free medium, it is greatly inhibited when [Na⁺]₀ is reduced to 27.25 mM. In low [Na⁺]₀ medium, GABA efflux is largely tetrodotoxin (TTX)-sensitive, however, spike firing evoked by antidromic stimulation of striatal cells is inhibited. In Na⁺-free medium, resting GABA efflux increases 17-fold whereas evoked GABA efflux diminishes. In Ca²⁺-free medium, 70 min of incubation with 1–2-bis-(2-aminophenoxy)ethane-N,N,N′,N′ tetraacetoxy methyl ester (BAPTA-AM, 1 μM), an intracellular calcium chelator, increases both resting GABA efflux and electrically evoked GABA overflow by 100%. These results suggest that: (1) in Ca²⁺-free conditions, Na⁺ permeability of cells increases via Ca²⁺ channels and this profoundly affects GABA efflux. (2) Electrical field depolarization is likely to release GABA by directly depolarizing axon terminals. (3) Ca²⁺-independent GABA efflux is not promoted by an increase in intracellular free Ca²⁺ concentration via Na⁺/Ca²⁺ exchange processes from internal pools.     

Australian funnel-web spider toxin, versutoxin, enhances spontaneous synaptic activity in single brain neurons in vitro

Neurotoxins isolated from the venoms of Australian funnel-web spiders increase spontaneous action potential activity in a variety of excitable cells. In the present study intracellular recordings were made with microelectrodes (30–60 MΩ, 2 M KCl) from locus coeruleus, mesencephalic nucleus of the trigeminal nerve and laterodorsal tegmental neurons in brain slices. Versutoxin, a polypeptide toxin isolated from the venom ofHadronyche versutus produced a profound increase in spontaneous synaptic activity impinging on neurons, which did not fully recover for up to 3 h after washout. The threshold concentration was 1.5 nM in locus coeruleus neurons, with increasing concentrations (up to 50 nM) producing larger effects. A modest increase in synaptic activity was observed in mesencephalic nucleus of the trigeminal nerve neurons during superfusion with 50 nM versutoxin. The increase in spontaneous synaptic activity was reversed by agents which block synaptic potentials impinging on locus coeruleus neurons, i.e., tetrodotoxin (100 nM), Co²⁺ (3 nM) or the combination of 6-cyano-7-nitroquinoxaline-2,3-dione (10 μM) and bicuculline (30 μM). Threshold, peak amplitude, maximum rate of rise, duration, amplitude of afterhyperpolarisations and interspike intervals of action potentials in each type of neuron were unaffected by versutoxin. Voltage-current relationships were also unaffected. Calcium-dependent action potentials evoked in locus coeruleus neurons in the presence of tetrodotoxin were unaffected by versutoxin, as were depolarisations produced by exogenously applied glutamate. These results suggest that versutoxin increases spontaneous synaptic activity, but has no effect on the membrane properties of the soma of several types of rat brain neurons.     

Kindling increases the K -evoked Ca -dependent release of endogenous GABA in area CA1 of rat hippocampus

The release of endogenous amino acids from hippocampal CA1 subslices under basal conditions and the release evoked by high potassium (50 mM K⁺) depolarization was studied during kindling epileptogenesis. Emphasis was put on the release of the amino acid neurotransmitters γ-aminobutyric acid (GABA) and glutamate. Kindling was induced by tetanic stimulation of the Schaffer-collaterals/commissural fibers of the dorsal hippocampus of the rat. The calcium-dependent GABA release in the presence of high K⁺ was significantly increased (40–46%) in fully kindled animals, 24 h after the last seizure, in comparison to controls. At long-term, 28 days after the last seizure, the calcium-dependent GABA release was still significantly increased (45–49%). An increased release of GABA in kindled animals was still found when GABA uptake was blocked by nipecotic acid. In contrast, no significant alterations were encountered in the basal or high potassium induced release of the excitatory amino acids aspartate and glutamate. These results suggest that kindling epileptogenesis is accompanied by a specific and long-lasting enhancement of GABA exocytosis which may lead to a desensitization of the GABA receptor, and thus determine the increase of seizure sensitivity.     

AMPA and metabotropic glutamate receptors cooperatively generate inspiratory-like depolarization in mouse respiratory neurons in vitro

Excitatory transmission mediated by AMPA receptors is critical for respiratory rhythm generation. However, the role of AMPA receptors has not been fully explored. Here we tested the functional role of AMPA receptors in inspiratory neurons of the neonatal mouse preBötzinger complex (preBötC) using an in vitro slice model that retains active respiratory function. Immediately before and during inspiration, preBötC neurons displayed envelopes of depolarization, dubbed inspiratory drive potentials, that required AMPA receptors but largely depended on the Ca²⁺-activated non-specific cation current (I_CAN). We showed that AMPA receptor-mediated depolarization opened voltage-gated Ca²⁺ channels to directly evoke I_CAN. Inositol 1,4,5-trisphosphate receptor-mediated intracellular Ca²⁺ release also evoked I_CAN. Inositol 1,4,5-trisphosphate receptors acted downstream of group I metabotropic glutamate receptor activity but, here too, AMPA receptor-mediated Ca²⁺ influx was essential to trigger the metabotropic glutamate receptor contribution to inspiratory drive potential generation. This study helps to elucidate the role of excitatory transmission in respiratory rhythm generation in vitro. AMPA receptors in preBötC neurons initiate convergent signaling pathways that evoke post-synaptic I_CAN, which underlies inspiratory drive potentials. The coupling of AMPA receptors with I_CAN suggests that latent burst-generating intrinsic conductances are recruited by excitatory synaptic interactions among preBötC neurons in the context of respiratory network activity in vitro, exemplifying a rhythmogenic mechanism based on emergent properties of the network.     

GABA and glutamate receptor development of cultured neurons from rat hippocampus, septal region, and neocortex

The early development of functionally active GABA and glutamate receptors on neurons from hippocampus, septal region, and neocortex of embryonic rats were studied using primary dissociated serum-free cell cultures. The responses to GABA and glutamate, applied to individual neurons by pressure ejection, were tested at different developmental stages, starting at 1 day in vitro (DIV) until 3 weeks. In all three types of neuronal cultures, the GABAA-receptor developed prior to the glutamate receptors, and after 9 DIV most of the neurons were sensitive to both GABA and glutamate. N-methyl-D-aspartate (NMDA) and non-NMDA receptor subtypes of the glutamate receptors could be distinguished in hippocampal cultures. The development of GABA and glutamate receptors on septal region neurons appeared to be delayed as compared to hippocampal neurons. In neocortical cultures the majority of neurons was sensitive to GABA just after plating, whereas the sensitivity to glutamate was retarded. The differences in GABA and glutamate receptor development among these three neuronal cultures provide evidence that the appearance of transmitter receptors on cultured neurons is predominantly determined by intrinsic mechanisms rather than by environmental conditions. The proportion of spontaneously active networks in these cultures increased with a time course very similar to the rise in glutamate-sensitive neurons suggesting that functional active glutamate receptors may be involved in the generation of spontaneous activity.     

Sodium and chloride-dependent high and low-affinity uptakes of GABA by brain capillary endothelial cells

The mechanisms of carrier-mediated transport of γ-aminobutyric acid (GABA) at the blood–brain barrier (BBB) were examined by investigating [ ]GABA uptake by isolated bovine brain capillaries, monolayers of primary cultured brain capillary endothelial cells (BCECs) attached to plates or suspended BCECs. The uptake of [ ]GABA was concentration-dependent and saturable. Nonlinear regression analysis of the original data indicated the existence of two distinct high and low-affinity GABA transporters on isolated brain capillaries or suspended BCECs, with K_m1, K_m2, V_m1 and V_m2 equal to 25.3 μM, 485.2 μM, 3.6 and 8.4 nmol/5 min/mg protein, respectively, for the capillaries, and 21.3 μM, 322.0 μM, 6.1 and 15.7 nmol/5 min/mg protein, respectively, for the suspended BCECs. In contrast, a single low-affinity transporter was found for monolayers of BCECs attached to plates with K_m and V_m equal to 338.7 μM and 18.8 nmol/5 min/mg protein, respectively. Subcellular location of the two distinct transporters on BCECs is discussed, suggesting that the low-affinity GABA transporter is probably localized to the luminal membrane of BCECs, and the high-affinity GABA transporter is probably localized to the antiluminal membrane. Low temperature (4°C) and metabolic inhibitors markedly diminished both high and low-affinity uptakes of [ ]GABA by isolated brain capillaries. The substitution of Na⁺ with choline⁺, K⁺ or Li⁺ with the counter anion Cl⁻ almost completely abolished both uptakes. Substitution of Cl⁻ with Br⁻, I⁻, F⁻ or NO₃⁻ in the presence of Na⁺ significantly reduced both uptakes to different extents. Alanine, leucine, phenylalanine, arginine, glutamate and pyruvate had no obvious effect on either uptake. Probenecid, amino-oxyacetic acid, β-alanine, taurine, betaine, and nipecotic acid significantly reduced both uptakes. These data suggested that both the GABA transporters at the BBB were temperature, metabolic energy, Na⁺ and Cl⁻-dependent, and may be specific and different from the known monocarboxylic acid, GABA and other amino acid transporters, which may play a role in the disposition of GABA in the brain.     

Serotonin potentiates the response of neurons of the superficial laminae of the rat spinal dorsal horn to γ-aminobutyric acid

Employing the Nystatin-perforated whole-cell patch-clamp recording technique, the modulatory effects of serotonin (5-HT) on γ-aminobutyric acid (GABA)-activated whole-cell currents were investigated in neurons acutely dissociated from the superficial laminae (laminae I and II) of the rat spinal dorsal horn. The results showed: (1) GABA acted on GABA_A receptors and elicited inward Cl⁻ currents (I_GABA) at a holding potential (V_H) of −40 mV; (2) 5-HT potentiated GABA-induced Cl⁻ current without affecting the reversal potential of I_GABA and the apparent affinity of GABA to its receptor; (3) α-methyl-5-HT, a selective agonist of 5-HT₂ receptor, mimicked the potentiation effect of 5-HT on I_GABA, whereas ketanserine, an antagonist of 5-HT₂ receptor, blocked the potentiation effect of 5-HT; (4) Chelerythrine, an inhibitor of protein kinase C, reduced the potentiation effect of 5-HT on I_GABA. The present results indicate: (1) The potentiation of 5-HT on I_GABA is mediated by 5-HT₂ receptor and through a protein kinase-dependent transduction pathway; (2) The interactions between 5-HT and GABA might play an important role in the modulation of nociceptive information transmission at spinal cord level.