Different actions of gabapentin and baclofen in hippocampus from weaver mice

The pre- and postsynaptic effects of baclofen, a broad-spectrum gamma-aminobutyric acid (GABA)B receptor agonist, and gabapentin, a selective agonist at GABA(B) receptors composed of GABA(B)(1a,2) heterodimers, were examined in CA1 pyramidal cells using whole-cell patch-clamp recordings in hippocampal slices from different strains of mice. In slices from C57BL/6 mice, by means of GABA(B) receptors, gabapentin and baclofen activated outward K+ currents at resting membrane potential. In weaver mice with a Kir3.2 channel mutation, baclofen and gabapentin failed to activate postsynaptic K+ currents. However, in littermate controls of weaver mice, gabapentin failed to evoke K+ currents, whereas baclofen activated currents in the same cells. Thus, postsynaptic actions of gabapentin and baclofen on K+ currents are different in this mouse strain. Via presynaptic GABA(B) receptors, baclofen significantly reduced GABA(A) inhibitory postsynaptic currents (IPSCs) in slices from C57BL/6 mice, as well as weaver and control mice. In contrast, gabapentin did not affect IPSCs significantly in any group of mice. These results indicate that although baclofen and gabapentin are agonists at postsynaptic GABA(B) receptors positively coupled to K+ channels, their mechanism of action differs in certain strains of mice, including the weaver wild-type mice, suggesting a dissociation in their signaling mechanism and coupling to K+ channels.     

G-protein-coupled GABAB receptors inhibit Ca2+ channels and modulate transmitter release in descending turtle spinal cord terminal synapsing motoneurons

Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.     

GABA(B) receptor activation inhibits N- and P/Q-type calcium channels in cultured lamprey sensory neurons

In lamprey, sensory transmission from mechanosensory receptors (dorsal cells) to central neurons is presynaptically inhibited by GABA(B) receptor activation. The mechanisms underlying this effect were investigated using isolated dorsal cells, where voltage-dependent calcium currents were recorded in the whole-cell configuration. Activation of GABA(B) receptors by baclofen decreased the peak amplitude of high voltage-activated (HVA) calcium currents and slowed the activation phase. The role of G-proteins in mediating the effects of baclofen was examined. Intracellular dialysis of GTPgammaS occluded the effects of baclofen. Intracellular dialysis of GDPbetaS and preincubation in pertussis toxin both attenuated the effect of baclofen. Specific calcium channel blockers were used to study the types of HVA calcium channels involved in the GABA(B)-mediated modulation. The baclofen-induced inhibition was not affected by the L-type calcium channel antagonist nimodipine, but was partially blocked by the N-type blocker omega-conotoxin GVIA, and completely occluded by omega-conotoxin MVIIC, a blocker of both N- and P/Q-type channels. The pharmacology of dorsal cell GABA(B) receptors was studied using two agonists, baclofen and CGP 27492, and four antagonists, CGP 35348, CGP 55845, phaclofen and saclofen. The inhibition induced by either of the two agonists was blocked by CGP 55845, phaclofen and saclofen. The antagonist CGP 35348 completely blocked the inhibition of HVA calcium current induced by the agonist CGP 27492, but had no effect on baclofen-induced GABA(B) receptor activation. This study thus demonstrates that GABA(B) receptor activation in lamprey mechanosensory neurons inhibits N- and P/Q-type calcium channels in a voltage- and G-protein-dependent manner.     

Mechanism of GABA receptor-mediated inhibition of spontaneous GABA release onto cerebellar Purkinje cells

gamma-Aminobutyric acid (GABA)(B) receptor-mediated modulation of spontaneous GABA release onto Purkinje cells was investigated in cerebellar slices from 3- to 5-week-old mice. The GABA(B) receptor agonists baclofen and CGP 44533 each reduced the frequency of miniature inhibitory postsynaptic currents (mIPSCs), with no significant effect on mIPSC amplitude; together, consistent with a presynaptic site of action. The GABA(B) receptor antagonist CGP 55845 blocked baclofen-induced inhibition. The sulphydryl alkylating agent N-ethylmaleimide occluded baclofen effects, implicating G(i/o) subunits in mediating a GABA(B) G protein-coupled receptor pathway. Baclofen-induced inhibition persisted in the presence of Ba(2+), a blocker of K(+) channels, and Cd(2+), a blocker of Ca(2+) channel-mediated GABA release. Application of nominally Ca(2+)-free extracellular solutions reduced mIPSC frequency and amplitude; however, baclofen produced a significant inhibition in mIPSC frequency, further suggesting that this pathway was independent of Ca(2+) influx. Spontaneous GABA release was increased by the adenylate cyclase activator, forskolin, and the phorbol ester, phorbol 12,13-dibutyrate. However, baclofen-induced inhibition was not significantly changed in either condition. Baclofen action was also not affected by the adenylate cyclase inhibitor SQ 22536 or the protein kinase C inhibitor chelerythrine chloride. Baclofen still reduced mIPSC frequency in the presence of the polyvalent cation ruthenium red, which acts as a secretagogue here; however, baclofen-induced inhibition was reduced significantly. Furthermore, baclofen produced no clear inhibition during high-frequency mIPSCs bursts induced by the potent secretagogue alpha-Latrotoxin. Together, these results suggest that GABA(B) inhibition occurs downstream of Ca(2+) influx and may be mediated, in part, by an inhibition of the vesicular release mechanism.     

Inwardly rectifying and voltage-gated outward potassium channels exhibit low sensitivity to methylmercury

The concentration- and time-dependence of effects of methylmercury (MeHg) on voltage-gated outward K(+) (Kv) channels, inwardly rectifying K(+) (Kir) channels, voltage-gated Ca(2+) channels and GABA(A) receptor activated channels were compared in cerebellar granule cells in culture using whole cell patch clamp recording techniques. The objective was to determine if MeHg equally affects different types of ion channels. Under similar experimental conditions, these four ion channel types displayed markedly different sensitivity to MeHg. At 0.1-1 microM, MeHg caused apparent inhibition of Ca(2+)-channel and GABA(A) receptor-mediated currents, but did not cause any significant effect on Kv or Kir channels. Among the four channel types examined, GABA(A) receptors appeared to be the most sensitive to MeHg. The Kv channels, particularly the delayed rectifiers (DRs), appeared to be relatively resistant to MeHg compared with GABA(A) receptors and Ca(2+) channels. Kir channels were virtually unaffected by MeHg in the concentration range of 10-100 microM. The differential sensitivity of GABA(A) receptors and Kv channels to MeHg was also observed in granule and Purkinje cells in freshly isolated cerebellar slices of rat. The insensitivity of Kir channel to MeHg was also seen in Xenopus laevis oocytes expressing cloned Kir7.1 channels. Thus, these appear to be general properties of these channels as opposed to distinct effects associated with granule cells in culture. These results suggest that MeHg does preferentially affect certain types of ion channels. Hence, the effects of MeHg on membrane ion channels are not due simply to nonspecific actions on the membrane. Furthermore, at least certain types of Kir channels appear to be the most resistant type of ion channel reported to date to effects of MeHg.     

Gamma-aminobutyric acid type B receptors facilitate L-type and attenuate N-type Ca(2+) currents in isolated hippocampal neurons

Activation of presynaptic gamma-aminobutyric acid type B (GABA(B)) receptors inhibits neurotransmitter release at many synapses (both excitatory and inhibitory), and activation of postsynaptic GABA(B) receptors leads to a general inhibition of the postsynaptic cell in mature neurons. Although the action of GABA(B) receptors at the soma of excitatory hippocampal pyramidal cells has been resolved to be regulation of a potassium or calcium conductance, it is not clear that all neurons in the hippocampus demonstrate similar effects of GABA(B) receptor activation. In the current study, GABA(B) receptor-mediated effects on calcium currents in acute cultures composed of heterogeneous cells from the superior region of neonatal hippocampi were studied. In 54.5% of cells, the GABA(B) receptor agonist baclofen (10 microM) attenuated the whole-cell calcium current by 21.0% +/- 1.1%. In 29.9% of cells, baclofen facilitated the calcium current by 43.5% +/- 8.1%. The component of current attenuated by baclofen was blocked by the N-type calcium channel antagonist omega-conotoxin GVIA (3 microM). The component of current facilitated by baclofen was blocked by the L-type channel antagonist nimodipine (20 microM). For cells that showed calcium current facilitation, baclofen shifted the half-maximal activation by approximately -14 mV. The data indicate that activation of GABA(B) receptors in neurons of the superior hippocampus attenuates current through N-type channels and facilitates current through L-type channels. The two opposing effects of GABA(B) receptor activation may reflect the heterogeneity of the cultured cells or may be a developmentally regulated phenomenon.     

CB1 cannabinoid receptor-dependent and -independent inhibition of depolarization-induced calcium influx in oligodendrocytes

Regulation of Ca(2+) homeostasis plays a critical role in oligodendrocyte function and survival. Cannabinoid CB(1) and CB(2) receptors have been shown to regulate Ca(2+) levels and/or K(+) currents in a variety of cell types. In this study we investigated the effect of cannabinoid compounds on the Ca(2+) influx elicited in cultured oligodendrocytes by transient membrane depolarization with an elevated extracellular K(+) concentration (50 mM). The CB(1) receptor agonist arachidonoyl-chloro-ethanolamide (ACEA) elicited a concentration-dependent inhibition of depolarization-evoked Ca(2+) transients in oligodendroglial somata with a maximal effect (94+/-3)% and an EC(50) of 1.3+/-0.03 microM. This activity was mimicked by the CB(1)/CB(2) agonist CP55,940, as well as by the endocannabinoids N-arachidonoyl-ethanolamine (anandamide, AEA) and 2-arachidonoylglycerol (2-AG), whereas the CB(2) receptor selective agonist JWH133 was ineffective. The CB(1) receptor antagonist AM251 (1 microM) also reduced the Ca(2+) response evoked by high extracellular K(+) and did not prevent the inhibition elicited by ACEA (3 microM). Nevertheless, the ability of ACEA and AEA to reduce depolarization-evoked Ca(2+) transients was significantly reduced in oligodendrocytes from CB(1) receptor knockout mice, as well as by pretreatment with pertussis toxin. Bath application of the inwardly rectifying K(+) channels (Kir channels) blockers BaCl(2) (300 microM) and CsCl(2) (1 mM) reduced the size of voltage-induced Ca(2+) influx and partially prevented the inhibitory effect of ACEA. Our results indicate that cannabinoids inhibit depolarization-evoked Ca(2+) transients in oligodendrocytes via CB(1) receptor-independent and -dependent mechanisms that involve the activation of PTX-sensitive G(i/o) proteins and the blockade of Kir channels.     

K(+)-Evoked [(3)H]D-aspartate release in rat spinal cord synaptosomes: modulation by neuropeptide Y and calcium channel antagonists

This study was conducted to investigate mechanisms regulating the release of [(3)H]D-aspartate (or endogenous glutamate) in the rat spinal cord. Presynaptic modulation of glutamate release was studied in superfused synaptosomes depolarized with 20 mM KCl. Calcium-channel antagonists, omega-conotoxin GVIA (omega-CgTx GVIA; N-type), nifedipine (L-type), and omega-conotoxin MVIIC (omega-CmTx MVIIC; P/Q type), were used to characterize the voltage-operated Ca(2+) channels (VOCCs) involved in this release. Nifedipine had no significant effect on the K(+)-evoked release of [(3)H]D-aspartate, but the omega-conotoxins GVIA and MVIIC produced dose-dependent inhibitory effects that were additive. The most substantial reduction (54.30% +/- 4.40%) was seen with omega-CgTx GVIA, indicating that N-type channels play a major role in the release of glutamate in this tissue. We investigated the effects of neuropeptide Y (NPY), NPY(13-36), and [Leu(31)][Pro(34)]NPY on Ca(2+)-dependent, K(+)-evoked [(3)H]D-aspartate release. NPY and NPY(13-36) equipotently inhibited the release of glutamate in a concentration-dependent manner. The half-maximal response was observed at about 12 nM; maximal inhibition of 44.22% +/- 4.60% was achieved with 0.3 microM. The selective GABA(B) agonist (-)baclofen inhibited K(+)-evoked [(3)H]D-aspartate release from superfused spinal cord synaptosomes by 50.00% +/- 4.80% at 10 microM. When NPY(13-36) and (-)baclofen were used together at maximal doses, their release-inhibiting effects were not additive. In addition, neither of the agonists was able to enhance the inhibition produced by pretreating the synaptosomes with the selective inhibitor of N-type VOCCs omega-CgTx GVIA. These results are consistent with the hypothesis that presynaptic Y(2)-like and GABA(B) receptors regulate glutamate release by blocking Ca(2+) currents through N-type VOCCs. Characterization of the receptors that can inhibit the release of glutamate may provide useful information for treatment of conditions characterized by excessive glutamatergic transmission in the spinal cord.     

Suppression of epileptiform activity by GABA(B) receptors in wild type and weaver hippocampus 'in vitro'

Inhibition by GABA(B) receptors comprises activation of K(+) conductance and inhibition of Ca(2+) conductance, thereby reducing action potential dependent transmitter release and silencing neuronal activity. We compared epileptiform activity and its inhibition by the activation of GABA(B) receptors in homozygous weaver (wv/wv) and wild type (+/+) CA3 neurons disinhibited by GABA(A) receptor blockade. In wv/wv mice GABA(B) receptors have lost their ability to activate K(+) conductance (J. Neurosci. 18 (1998) 4001). Spontaneous synchronous burst discharges in elevated [K(+)](o) displayed only subtle differences in +/+ and wv/wv slices, except that the GABA(B) receptor agonist R-baclofen in low concentration (0.1 microM) strongly reduced the frequency of synchronous bursts in +/+ CA3 neurons, but not in wv/wv CA3 neurons. A high affinity GABA(B) antagonist, CGP55845A (0.5 microM) promoted the incidence of bursts in low [K(+)](o). Concentration dependence of the reduction of evoked EPSCs was identical in wv/wv and +/+ neurons (IC(50)=0.3 microM). Amplitudes of evoked IPSCs were reduced by 0.01 microM R-baclofen in +/+, but not in wv/wv CA3 neurons. The effect of the low concentration was abolished by Ba(2+), which is known to block Kir conductance. The data suggest that activation of Kir conductance is important for the control of GABA release by GABA(B) autoreceptors in the CA3 network. We conclude that the loss of a contribution of Kir conductance to GABA(B) receptor-mediated autoinhibition reduces the inclination towards spontaneous bursts of wv/wv CA3 pyramidal neurons.     

Possible modulatory role of voltage-activated Ca(2+) currents determining the membrane properties of isolated pyramidal neurones of the rat dorsal cochlear nucleus.

Voltage-activated Ca(2+) currents have been studied in pyramidal cells isolated enzymatically from the dorsal cochlear nuclei of 6-11-day-old Wistar rats, using whole-cell voltage-clamp. From hyperpolarized membrane potentials, the neurones exhibited a T-type Ca(2+) current on depolarizations positive to -90 mV (the maximum occurred at about -40 mV). The magnitude of the T-current varied considerably from cell to cell (-56 to -852 pA) while its steady-state inactivation was consistent (E(50)=-88.2+/-1.7 mV, s=-6. 0+/-0.4 mV). The maximum of high-voltage activated (HVA) Ca(2+) currents was observed at about -15 mV. At a membrane potential of -10 mV the L-type Ca(2+) channel blocker nifedipine (10 microM) inhibited approximately 60% of the HVA current, the N-type channel inhibitor omega-Conotoxin GVIA (2 microM) reduced the current by 25% while the P/Q-type channel blocker omega-Agatoxin IVA (200 nM) blocked a further 10%. The presence of the N- and P/Q-type Ca(2+) channels was confirmed by immunochemical methods. The metabotropic glutamate receptor agonist (+/-)-1-aminocyclopentane-trans-1, 3-dicarboxylic acid (200 microM) depressed the HVA current in every cell studied (a block of approximately 7% on an average). The GABA(B) receptor agonist baclofen (100 microM) reversibly inhibited 25% of the HVA current. Simultaneous application of omega-Conotoxin GVIA and baclofen suggested that this inhibition could be attributed to the nearly complete blockade of the N-type channels. Possible physiological functions of the voltage-activated Ca(2+) currents reported in this work are discussed.     

Intracellular signalling pathways modulate K(ATP) channels in inspiratory brainstem neurones and their hypoxic activation: involvement of metabotropic receptors, G-proteins and cytoskeleton

K(ATP) channels regulate the neuronal excitability and their activation during hypoxia/ischemia protect neurons. The activation of K(ATP) channels during hypoxia is assumed to occur mainly due to the fall in intracellular ATP levels, but other intracellular signalling pathways can be also involved. We measured single K(ATP) channel currents in inspiratory brainstem neurones of neonatal mice. The activity of K(ATP) channels was enhanced in hypoosmotic bath solutions, or after applying negative pressure to the recording pipette. Cytochalasin B activated K(ATP) channels and prevented the effects of osmo-mechanical stress, indicating that cytoskeleton rearrangements, which occur during hypoxia, contribute to the activation of K(ATP) channels. During hypoxia, extracellular levels of many neurotransmitters increase, leading to activation of corresponding metabotropic receptors that can modulate K(ATP) channels. K(ATP) channels were activated by GABA(B) agonist, baclofen, by mGLUR2/3 agonists and were inhibited by mGLUR1/5 agonists. K(ATP) channels were activated by phorbol esters and were inhibited by staurosporine. These treatments did not occlude the modulating actions of mGLUR agonists, indicating that they are not mediated by protein kinase C. Activator of alpha-subunits of G-proteins Mas 7 increased and their inhibitor GPant-2 decreased the activity of K(ATP) channels. In the presence of either agent, the modulatory actions of baclofen and mGLUR agonists were not observed. We conclude that K(ATP) channels are modulated by G-proteins that are activated by metabotropic receptors for GABA and glutamate and their release during hypoxia complements activation of channels by osmo-mechanical stress and [ATP](i) depletion.     

Dopamine modulates a voltage-gated calcium channel in rat olfactory receptor neurons

Dopamine D2 receptors exist in the soma of rat olfactory receptor neurons. Actions of dopamine on the voltage-gated Ca(2+) channels in the neurons were investigated using the perforated whole-cell voltage-clamp. In 10 mM Ba(2+) solution, rat olfactory receptor neurons displayed the inward currents elicited by the voltage ramp (167 mV/s) and depolarizing step pulses from a holding potential of -91 mV. The inward Ba(2+) currents were greatly reduced by 10 microM nifedipine (L-type Ca(2+) channel blocker). The Ba(2+) currents were inhibited by the external application of dopamine. The IC(50) for the inhibition was about 1 microM. Quinpirole (10 microM, a D2 dopamine agonist) also inhibited the Ba(2+) currents. Quinpirole did not affect the activation and inactivation kinetics of the Ba(2+) currents. The results suggest that dopamine modulates the L-type Ca(2+) channels in rat olfactory receptor neurons via the mechanism independent of voltage.     

5-HT inhibits N-type but not L-type Ca(2+) channels via 5-HT1A receptors in lamprey spinal neurons

5-HT is a potent modulator of locomotor activity in vertebrates. In the lamprey, 5-HT dramatically slows fictive swimming. At the neuronal level it reduces the postspike slow afterhyperpolarization (sAHP), which is due to apamin-sensitive Ca(2+)-dependent K+ channels (KCa). Indirect evidence in early experiments suggested that the sAHP reduction results from a direct action of 5-HT on KCa channels rather than an effect on the Ca(2+) entry during the action potential. In view of the characterization of different subtypes of Ca(2+) channels with very different properties, we now reinvestigate if there is a selective action of 5-HT on a Ca(2+) channel subtype in dissociated spinal neurons in culture. 5-HT reduced Ca(2+) currents from high voltage activated channels. N-type, but not L-type, Ca(2+) channel blockers abolished this 5-HT-induced reduction. It was also confirmed that 5-HT depresses Ca(2+) currents in neurons, including motoneurons, in the intact spinal cord. 8-OH-DPAT, a 5-HT1A receptor agonist, also inhibited Ca(2+) currents in dissociated neurons. After incubation in pertussis toxin, to block Gi/o proteins, 5-HT did not reduce Ca(2+) currents, further indicating that the effect is caused by an activation of 5-HT1A receptors. As N-type, but not L-type, Ca(2+) channels are known to mediate the activation of KCa channels and presynaptic transmitter release at lamprey synapses, the effects of 5-HT reported here can contribute to a reduction in both actions.     

Inwardly rectifying potassium channels in rat retinal ganglion cells

Inwardly rectifying potassium channels (Kir channels) are important for neuronal signalling and membrane excitability. In the present work we characterized, for the first time, Kir channels in rat retinal ganglion cells (RGCs), the output neurons in the retina, using immunocytochemical and patch-clamp techniques. Various subunits of Kir channels (Kir1.1, 2.1, 2.3, 3.1, 3.2 and 3.3) were expressed in RGCs, but with distinct subcellular localization. Kir1.1 was mainly expressed in axons of RGCs. Kir2.1 and Kir2.3 were both present in somata of RGCs. Whereas staining for Kir3.1 was profoundly present in an endoplasmic reticulum-like structure and Kir3.2 was strongly expressed in the cytoplasm and the cytomembrane of somata, dendrites and axons of RGCs, faint, sparse labelling for Kir3.3 was seen in the cytomembrane. Immunoreactivity for Kir4.1 and Kir4.2 was not detectable in RGCs. Whole-cell currents mediated by Kir channels were recorded in isolated RGCs and they differed from hyperpolarization-activated currents (I(h)) by showing full activation in < 10 ms, no inactivation, and being significantly suppressed by 300 microM Ba2+. Unlike in retinal horizontal cells and bipolar cells, these currents were mainly mediated by G-protein-coupled Kir3 (GIRK) channels, as demonstrated by the fact that GDP(beta)S and GTP(gamma)S included in the pipette solution markedly decreased and increased the currents, respectively. Furthermore, the GIRK channels were probably coupled to GABA(B) receptors, because baclofen considerably increased the Kir currents and the increased currents were suppressed by Ba2+. These characteristics of the Kir currents provide more versatility for signalling of RGCs.     

Inwardly rectifying K(+) (Kir) channels antagonize ictal-like epileptiform activity in area CA1 of the rat hippocampus

Reactive glial cells, for example, from patients with temporal lope epilepsy have a reduced density of inward rectifying K(+) (Kir) channels and thus a reduced K(+) buffering capacity. Evidence is accumulating that this downregulation of Kir channels could be implicated in epileptogenesis. In rat hippocampal brain slices, prolonged exposure to the nonselective Kir channel antagonist, Cs(+) (5 mM), gives rise to an epileptiform field potential (Cs-FP) in area CA1 composed of an initial positive (interictal-like) phase followed by a prolonged negative (ictal-like) phase. We have previously shown that the interictal-like phase depends on synaptic activation. The present study extends these findings by showing that the ictal-like phase of the Cs-FP is (i) sensitive to osmotic expansion of the extracellular space, (ii) reversed very quickly during wash out of Cs(+), and (iii) re-established in the presence of Ba(2+) (30-200 microM) or isosmotic low extracellular concentration of Na(+) ([Na(+)](o), 51.25 mM). The interictal-like phase showed less or no sensitivity to these treatments. In the complete absence of Cs(+), the Cs-FP could be fully reconstructed by the combined application of 4-aminopyridine (0.5 mM), an isosmotic high extracellular concentration of K(+) ([K(+)](o), 7 mM), and low [Na(+)](o) (51.25 mM). These results suggest that the interictal-like phase is initiated through synaptic activation and results from an unspecific increase in neuronal excitability, whereas the ictal-like phase is entirely dependent on blockade of Kir channels in CA1. We propose that glial dysfunction-related loss of Kir channels may not alone be sufficient for starting the induction process, but will likely increase the tendency of an epileptogenic process to proceed into seizure activity.     

The reduction in paired-pulse inhibition in the rat hippocampus by gabapentin is independent of GABA(B) receptor receptor activation

Previously we have shown that gabapentin causes a reduction of paired-pulse inhibition in the dentate gyrus of the urethane-anesthetized rat, which looks very much like the effect of baclofen on paired-pulse inhibition. In addition, it has been proposed that gabapentin increases release of GABA from non-vesicular stores and may, therefore, interact with GABA(B) mechanisms. Here we tested the ability of a GABA(B) agonist, baclofen, and a GABA(B) antagonist, CGP35348, to block the effect of gabapentin on paired-pulse inhibition in the dentate gyrus in urethane-anesthetized adult Sprague-Dawley rats. Both baclofen (6 mg/kg) and gabapentin (100 mg/kg) caused a long-lasting reduction of paired-pulse inhibition in the dentate gyrus when given alone or in combination. CGP35348 (45 mg/kg) blocked the effect of baclofen on paired-pulse inhibition, but did not alter the effect of gabapentin. Gabapentin also caused a reduction of inhibition in the CA1 region, indicating that its effect is not specific for the dentate gyrus. These results suggest that gabapentin produces its effect on paired-pulse inhibition independent from the effect of baclofen and not through non-vesicular release of GABA interacting with the GABA(B) receptor system.     

Stimulatory effects of chlorzoxazone,a centrally acting muscle relaxant,on large conductance calcium-activated potassium channels in pituitary GH3 cells

Chlorzoxazone, a centrally acting muscle relaxant, has been used as a marker for hepatic CYP2E1 activity. However, little is known about the mechanism of chlorzoxazone actions on ion currents in neurons or neuroendocrine cells. We thus investigated its effects on ion currents in GH(3) lactotrophs. Chlorzoxazone reversibly increased Ca(2+)-activated K(+) current (I(K(Ca))) in a concentration-dependent manner with an EC(50) value of 30 microM. The chlorzoxazone-stimulated I(K(Ca)) was inhibited by iberitoxin (200 nM) or clotrimazole (10 microM), but not by glibenclamide (10 microM) or apamin (200 nM). Chlorzoxazone (30 microM) suppressed voltage-dependent L-type Ca(2+) current. In the inside-out configuration, chlorzoxazone applied to the intracellular side of the patch did not modify single-channel conductance of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, but did increase channel activity by increasing mean open time and decreasing mean closed time. Chlorzoxazone also caused a left shift in the activation curve of BK(Ca) channels. However, Ca(2+)-sensitivity of these channels was unaffected by chlorzoxazone. 1-Ethyl-2-benzimidazolinone (30 microM), 2-amino-5-chlorobenzoxazole (30 microM) or chlormezanone (30 microM) enhanced BK(Ca) channel activity, while 6-hydroxychlorzoxazone (30 microM) slightly increased it; however, chlorphenesin carbamate (30 microM) had no effect on it. Under the current-clamp condition, chlorzoxazone (10 microM) reduced the firing rate of action potentials. In neuroblastoma IMR-32 cells, chlorzoxazone (30 microM) also stimulated BK(Ca) channel activity. The stimulatory effects of chlorzoxazone on these channels may be responsible for the underlying mechanism of chlorzoxazone actions on neurons and neuroendocrine cells.     

Potassium channels underlie postsynaptic but not presynaptic GABA(B) receptor-mediated inhibition on ventrolateral periaqueductal gray neurons

γ-Aminobutyric acid (GABA) is the principle inhibitory neurotransmitter in adult mammalian brain. GABA receptors B subtype (GABA(B)Rs) are abundantly expressed at presynaptic and postsynaptic neuronal structures in the rat ventrolateral periaqueductal gray (PAG), an area related to pain regulation. Activation of GABA(B)Rs by baclofen, a selective agonist, induces presynaptic inhibition by decreasing presynaptic glutamate release. At the same time, baclofen induces a postsynaptic inhibitory membrane current or potential. We here report that in the ventrolateral PAG, the postsynaptic inhibition is mediated by activation of G protein-coupled inwardly rectifying K(+) (GIRK) channels. Blockade of K(+) channels largely prevents postsynaptic action of baclofen. In contrast, presynaptic inhibition of baclofen is insensitive to K(+) channel blockade. The data indicate that potassium channels play different roles in GABA(B)R-mediated presynaptic and postsynaptic inhibition on PAG neurons.     

Kv3 K+ channels enable burst output in rat cerebellar Purkinje cells

The ability of cells to generate an appropriate spike output depends on a balance between membrane depolarizations and the repolarizing actions of K(+) currents. The high-voltage-activated Kv3 class of K(+) channels repolarizes Na(+) spikes to maintain high frequencies of discharge. However, little is known of the ability for these K(+) channels to shape Ca(2+) spike discharge or their ability to regulate Ca(2+) spike-dependent burst output. Here we identify the role of Kv3 K(+) channels in the regulation of Na(+) and Ca(2+) spike discharge, as well as burst output, using somatic and dendritic recordings in rat cerebellar Purkinje cells. Kv3 currents pharmacologically isolated in outside-out somatic membrane patches accounted for approximately 40% of the total K(+) current, were very fast and high voltage activating, and required more than 1 s to fully inactivate. Kv3 currents were differentiated from other tetraethylammonium-sensitive currents to establish their role in Purkinje cells under physiological conditions with current-clamp recordings. Dual somatic-dendritic recordings indicated that Kv3 channels repolarize Na(+) and Ca(2+) spikes, enabling high-frequency discharge for both types of cell output. We further show that during burst output Kv3 channels act together with large-conductance Ca(2+)-activated K(+) channels to ensure an effective coupling between Ca(2+) and Na(+) spike discharge by preventing Na(+) spike inactivation. By contributing significantly to the repolarization of Na(+) and especially Ca(2+) spikes, our data reveal a novel function for Kv3 K(+) channels in the maintenance of high-frequency burst output for cerebellar Purkinje cells.