GABAA antagonists

The GABA antagonist action of the convulsant alkaloid bicuculline was discovered 20 years ago. This action is now used to define GABA_A receptors. Other more potent GABA_A receptor antagonists with a range of molecular structures have been discovered subsequently. The existence of subclasses of GABA_A receptors, proposed on the basis of agonist structure-activity data and molecular modelling, has been supported by recent cDNA studies which indicate a substantial molecular heterogeneity of GABA_A receptor subunit proteins. Further studies on GABA_A antagonists with a range of GABA_A agonists acting on receptors of known molecular composition may provide a pharmacologically useful subclassification of GABA_A receptors and lead to the development of new therapeutic agents acting on specific subclasses of GABA_A receptors.     

A tyrosine kinase regulates propofol‐induced modulation of the β‐subunit of the GABAA receptor and release of intracellular calcium in cortical rat neurones

Propofol, an intravenous anaesthetic, has been shown to interact with the β‐subunit of the γ‐amino butyric acid_A (GABA_A) receptor and also to cause changes in [Ca²⁺]_i. The GABA_A receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre‐incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca²⁺]_i were investigated. Propofol (3 μg mL^?1, 16.8 μM) increased intracellular calcium levels by 122 ± 34% (mean ± SEM) when applied to neurones in calcium free medium. This rise in [Ca²⁺]_i was lowered by 68% when the cells were pre‐incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 ± 10%) in tyrosine phosphorylation, with maximum at 120 s, of the β‐subunit of the GABA_A‐receptor. This tyrosine phosphorylation was decreased after pre‐treatment with herbimycin A (44 ± 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the β‐subunit of the GABA_A‐receptor.     

Activation of α6-containing GABAA receptors by pentobarbital occurs through a different mechanism than activation by GABA

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

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1β3γ2L GABAA receptor account for their in vivo effects

Key Points

• Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood.
• We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABA_A receptors. In mice the S‐enantiomer is a convulsant, but the R‐enantiomer is an anticonvulsant.
• The convulsant S‐enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R‐enantiomer inhibits with equal affinity.
• In contrast, only the anticonvulsant R‐enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective.
• The in vivo actions of the convulsant S‐enantiomer are accounted for by its interactions with GABA_A receptors.

Abstract

Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABA_A receptors (GABA_ARs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD‐MPPB). In mice, S‐mTFD‐MPPB acted as a convulsant, whereas R‐mTFD‐MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α₁β₃γ_2L GABA_ARs expressed in HEK cells, we found that S‐mTFD‐MPPB inhibited GABA‐induced currents, whereas R‐mTFD‐MPPB enhanced them. S‐mTFD‐MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration–response curve to the right. R‐mTFD‐MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate‐limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration–response curve to the left. Under conditions when most GABA_ARs were open, an inhibitory action of R‐mTFD‐MPPB was revealed that had a similar IC₅₀ to that of S‐mTFD‐MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S‐mTFD‐MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABA_ARs underlie the enantiomers’ different pharmacological activities in mice.

Abbreviations

C

channel closed, resting state

CD₅₀

median clonic siezure dose

CI

confidence interval

G₂O

open channel state bound with two GABA molecules

GABA_AR

GABA receptor Type A

HEK

human embryonic kidney

I

peak current amplitude

I_max

maximal peak current amplitude

k₋₁

dissociation rate constant

k₊₁

binding rate constant

k_act

activation energy

LoRR

loss of righting reflex

MPPB

1‐methyl‐5‐phenyl‐5‐propyl‐barbituric acid

nAChR

nicotinic acetylcholine receptor

O

open channel state

O’

stabilized open channel state

pK

acid dissociation constant

PTZ

pentylenetetrazol

R‐mTFD‐MPAB

R‐5‐allyl‐1‐methyl‐5‐(m‐trifluoromethyl‐diazirynylphenyl) barbituric acid

R‐mTFD‐MPPB

R‐1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid

S‐mTFD‐MPAB

S‐5‐allyl‐1‐methyl‐5‐(m‐trifluoromethyl‐diazirynylphenyl) barbituric acid

S‐mTFD‐MPPB

S‐1‐methyl‐5‐propyly‐5‐(m‐trifluoromethyldiazirinyl) phenyl barbituric acid

TID

3‐(trifluoromethyl)‐3‐(m‐iodophenyl) diazirine

α

channel closing rate

β

channel opening rate

     

Proton inhibition of GABA-activated current in rat primary sensory neurons

 The modulation of the Cl^– current activated by γ-aminobutyric acid (GABA) by changes in extracellular pH in freshly isolated rat dorsal root ganglia (DRG) neurons was studied using the whole-cell patch-clamp technique. In the pH range of 5.0–9.0, increased extracellular pH enhanced, and decreased extracellular pH suppressed, current activated by 10 μM GABA in a reversible and concentration-dependent manner with an IC₅₀ of pH 7.1 in these neurons. Acidification to pH 6.5 inhibited currents activated by the GABA_A-selective agonist muscimol in all neurons tested. The antagonism of GABA-activated current by lowering the pH was equivalent at holding potentials between –80 and +40 mV and did not involve a significant alteration in reversal potential. Acidification shifted the GABA concentration/response curve to the right, significantly increasing the EC₅₀ for GABA without appreciably changing the slope or maximal value of the curve. Inhibition of the GABA-activated current by protons was not significantly different when the patch-pipette solution was buffered at pH 7.4 or pH 6.5. These results suggest that extracellular protons inhibit GABA_A receptor channels in primary sensory neurons by decreasing the apparent affinity of the receptor for GABA. This represents a novel mechanism of inhibition by protons of a neurotransmitter-gated ion channel. Proton inhibition of GABA_A receptor channels may account in part for the modulation by protons of sensory information transmission under certain pathophysiological conditions. Received: 1 July 1997 / Received after revision: 29 September 1997 / Accepted: 15 October 1997     

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

 In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca²⁺ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca²⁺ channel current was inhibited by 28.4±6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca²⁺ channel currents (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca²⁺ channel inhibition was dose dependent with an IC₅₀ of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 μM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 μM) prevented Ca²⁺ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca²⁺ imaging showed that no substantial rise in intracellular Ca²⁺ levels occurred during ET-1 application excluding a Ca²⁺-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca²⁺ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger. Received: 13 March 1998 / Received after revision and accepted: 14 May 1998     

Investigation of ethanol-induced impairment of spatial memory in γ2 heterozygous knockout mice

GABA_A receptors, the major inhibitory receptors in the mammalian central nervous system, are affected by a number of drug compounds, including ethanol. The pharmacological effects of certain drugs have been shown to be dependent upon specific GABA_A receptor subunits. Because benzodiazepines and ethanol have similar effect signatures, it has been hypothesized that these drugs share the γ2-containing GABA_A receptors as a mechanism of action. To probe the involvement of the γ2 subunit in ethanol's actions, spatial memory for the Morris water maze task was tested in γ2 heterozygous knockout mice and wild type littermate controls following ethanol administration at the following doses: 0.0, 1.25, 1.75, and 2.25 g/kg. While baseline learning and memory were unaffected by reduction of γ2 containing GABA_A receptors, ethanol dose-dependently impaired spatial memory equally in γ2 heterozygous knockouts and wild type littermate controls.     

Trifluoperazine: a rynodine receptor agonist

Trifluoperazine (TFP), a phenothiazine, is a commonly used antipsychotic drug whose therapeutic effects are attributed to its central anti-adrenergic and anti-dopaminergic actions. However, TFP is also a calmodulin (CaM) antagonist and alters the Ca²⁺ binding properties of calsequestrin (CSQ). The CaM and CSQ proteins are known modulators of sarcoplasmic reticulum (SR) Ca²⁺ release in ventricular myocytes. We explored TFP actions on cardiac SR Ca²⁺ release in cells and single type-2 ryanodine receptor (RyR2) channel activity in bilayers. In intact and permeabilized ventricular myocytes, TFP produced an initial activation of RyR2-mediated SR Ca²⁺ release and over time depleted SR Ca²⁺ content. At the single channel level, TFP or nortryptiline (NRT; a tricyclic antidepressant also known to modify CSQ Ca²⁺ binding) increased the open probability (Po) of CSQ-free channels with an EC₅₀ of 5.2 μM or 8.9 μM (respectively). This Po increase was due to elevated open event frequency at low drug concentrations while longer mean open events sustained Po at higher drug concentrations. Activation of RyR2 by TFP occurred in the presence or absence of CaM. TFP may also inhibit SR Ca uptake as well as increase RyR2 opening. Our results suggest TFP and NRT can alter RyR2 function by interacting with the channel protein directly, independent of its actions on CSQ or CaM. This direct action may contribute to the clinical adverse cardiac side effects associated with these drugs.     

The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na⁺ channel currents. Single Na⁺ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na⁺ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na⁺ channel is directly modulated by phosphorylation process through A-kinase.     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

GABAC receptors are functionally expressed in the intermediate zone and regulate radial migration in the embryonic mouse neocortex

Radial neuronal migration in the cerebral cortex depends on trophic factors and the activation of different voltage- and ligand-gated channels. To examine the functional role of GABA_C receptors in radial migration we analyzed the effects of specific GABA_A and GABA_C receptor antagonists on the migration of BrdU-labeled neurons in vitro using organotypic neocortical slice cultures. These experiments revealed that the GABA_A specific inhibitor bicuculline methiodide facilitated neuronal migration, while the GABA_C specific inhibitor (1,2,5,6-tetrahydropyridine-4-yl) methylphosphinic-acid (TPMPA) impeded migration. Co-application of TPMPA and bicuculline methiodide or the unspecific ionotropic GABA receptor antagonist picrotoxin both impeded migration, suggesting that the GABA_C receptor mediated effects dominate. Addition of the specific GABA_C receptor agonist cis-4-aminocrotonic acid (CACA) also hampered migration, indicating that a physiological GABAergic stimulation is required for appropriate function. RT-PCR experiments using specific probes for GABA_C receptor mRNA and Western blot assays using an antibody directed against rho subunits revealed the expression of GABA_C receptor mRNA and translated GABA_C receptor protein in the immature cortex. Microfluorimetric Ca²⁺ imaging in neurons of identified cortical layers using Calcium Green revealed the functional expression of GABA_A and GABA_C receptors in the intermediate zone, while only GABA_A receptor mediated responses were observed in the upper cortical plate. In summary, these results demonstrate that activation of GABA_C receptors is a prerequisite for accurate migration and that GABA_C receptors are functionally expressed in the intermediate zone.     

Mechanisms of kindling induced by norbornan intoxication

Single injection of norbornan induced kindling by disordering postsynaptic GABAergic structures. Modulation of GABA_A receptor chlorine ionic channels is most crucial, while disturbances in postsynaptic low-affinity GABA_A receptors are less important for this phenomenon. It is unlikely that GABA_B, dopamine, and muscarinic neurotransmitter structures are involved into this process. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 6, pp. 653–656, June, 1998     

Norbornan,a new irreversible ligand of the GABAA-receptor chloride channels

In vitro experiments demonstrate the high affinity of the toxic convulsant norbornan [2,2-di-(trifluoromethyl)-3,3-dicyano-5,6-dichloronorbornane] for the GABA_A-receptor chloride channels of membranes from rat brain. Repeated washing and centrifugation do not restore the ionophore density on norbornan-treated membranes, suggesting that norbornan may be counted among the agents acylating the chloride channels. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 121, No. 4, pp. 444–446, April, 1996     

The effect of a chemical phosphatase on single calcium channels and the inactivation of whole-cell calcium current from isolated guinea-pig ventricular myocytes

A chemical phosphatase, butanedione monoxime (BDM, at 12–20 mM), reduced open probability (P ₀) of single cardiac L-type Ca²⁺ channels in cellattached patches from guinea-pig ventricular myocytes, without effect on the amplitude of single-channel current, the mean open time or the mean shorter closed time, but it increased mean longer closed time and caused a fall in channel availability. A decrease in the mean time between first channel opening and last closing within a trace was principally due to an inhibition of the longer periods of activity. As a result, the time course of the mean currents, which resolved into an exponentially declining and a sustained component, was changed by an increase in the rate of the exponential phase and a profound reduction of the sustained current. Essentially similar results were obtained when studying whole-cell Ba²⁺ currents. The inactivation of the whole-cell Ca²⁺ currents was composed of two exponentially declining components with the slower showing a significantly greater sensitivity to BDM, an effect that was much more pronounced in myocytes exposed to isoprenaline with adenosine 5-O-(3-thiotriphosphate) (ATP[S]) in the pipette solution. The actions of BDM, which are the opposite of those produced by isoprenaline, suggest that the level of phosphorylation affects processes involved in the slow regulation of channel activity under basal conditions and that several sites (and probably several kinases) are involved. Channels with an inherently slow inactivation would seem to be converted into channels with a rapid inactivation by a dephosphorylation process.     

Altered GABAA receptor subunit expression and pharmacology in human Angelman syndrome cortex

The neurodevelopmental disorder Angelman syndrome is most frequently caused by deletion of the maternally derived chromosome 15q11-q13 region, which includes not only the causative UBE3A gene, but also the β₃-α₅-γ₃ GABA_A receptor subunit gene cluster. GABAergic dysfunction has been hypothesized to contribute to the occurrence of epilepsy and cognitive and behavioral impairments in this condition. In the present study, analysis of GABA_A receptor subunit expression and pharmacology was performed in cerebral cortex from four subjects with Angelman syndrome and compared to that from control tissue. The membrane fraction of frozen postmortem neocortical tissue was isolated and subjected to quantitative Western blot analysis. The ratios of β₃/β₂ and α₅/α₁ subunit protein expression in Angelman syndrome cortex were significantly decreased when compared with controls. An additional membrane fraction was injected into Xenopus oocytes, resulting in incorporation of the brain membrane vesicles with their associated receptors into the oocyte cellular membrane. Two-electrode voltage-clamp analysis of GABA_A receptor currents was then performed. Studies of GABA_A receptor pharmacology in Angelman syndrome cortex revealed increased current enhancement by the α₁-selective benzodiazepine-site agonist zolpidem and by the barbiturate phenobarbital, while sensitivity to current inhibition by zinc was decreased. GABA_A receptor affinity and modulation by neurosteroids were unchanged. This shift in GABA_A receptor subunit expression and pharmacology in Angelman syndrome is consistent with impaired extrasynaptic but intact to augmented synaptic cortical GABAergic inhibition, which could contribute to the epileptic, behavioral, and cognitive phenotypes of the disorder.     

Differential vasomotor adjustments in the evaporative tissues of the tongue and nose in the dog under heat load

1. Small sinoatrial-node preparations were voltage clamped by two micropipettes. Both the amplitude (mean current) and the fluctuation of acetylcholine-induced potassium current were recorded. The fluctuations were analyzed by both calculating their variance and establishing their power density spectrum.
2. It is assumed that acetylcholine receptors, when occupied, open momentarily ionic channels. Each channel is described by its conductance γ and the average open time τ.
3. The variance did not increase linearly with the mean current, but reached a saturation and even decayed with large mean currents, indicating that more than 50% of the total number of channels are open.
4. The average maximum mean current (all channels open) for a preparation approximately 300 μm ×200 μm×50 μm was 89±22 nA (n=7).
5. The average single channel conductance, calculated from the variance, was 3,71±0.48 pS (n=7).
6. The power density spectra of the fluctuations were fitted by single Lorentzian curves. The single channel conductance was thus determined to be γ= 3.79±1.25 pS and the corner frequencyf _c=0.96±0.25 Hz (n=21). This corresponds to an average open time τ=166 ms.
7. TheQ ₁₀ of the average open time (determined by the corner frequency (f _c) was 2.83±0.38 (n=4).
8. The density of channels was approximately 1 per 1 μm² of cell surface or roughly 2000 channels per cell.
9. The muscarinic acetylcholine receptor of S-A node cells may thus be characterized by a relatively long open time (160 ms), a low density of channels and a small elementary conductance of about 4 pS.

     

The behavioural and neuronal effects of the chronic administration of benzodiazepine anxiolytic and hypnotic drugs

Benzodiazepine anxiolytic and hypnotic drugs are some of the most widely prescribed drugs in the Western world. Despite this fact, the mechanisms that underlie the development of tolerance to, and dependence upon, benzodiazepines are poorly understood. The aim of this review is to summarize and critically evaluate the experimental evidence relating to the chronic behavioural and neuronal effects of benzodiazepines. Behavioural studies in animals generally indicate that tolerance gradually develops to the muscle relaxant, ataxic, locomotor and anticonvulsant effects of benzodiazepines. The evidence relating to the development of tolerance to the anxiolytic effects of benzodiazepines is less clear. The literature on the possible mechanisms of benzodiazepine tolerance and dependence is large, highly complex and difficult to interpret. The effect of chronic benzodiazepine treatment varies enormously as a function of the benzodiazepine used and the treatment schedule employed. Many studies have demonstrated a down-regulation of benzodiazepine binding sites, although affinity is usually unchanged. The evidence relating to the number and affinity of GABA_A binding sites is unclear. Some studies suggest that chronic benzodiazepine administration results in a reduction in the number of C⁻ channels associated with the GABA_A receptor complex, although it is not clear that the efficacy of the GABA binding site in operating the Cl⁻ channel necessarily changes. There is, however, substantial evidence to support the hypothesis that chronic benzodiazepine treatment results in a reduction in the coupling between the GABA_A and benzodiazepine binding sites (the “functional uncoupling hypothesis”). Although some electrophysiological studies suggest that chronic benzodiazepine treatment results in a subsensitivity to GABA, this effect seems to be highly area-specific.     

Single-channel analysis of fast transient outward K+ currents in frog skeletal muscle

 The patch-clamp method was employed to examine the voltage-dependent gating mechanism of A-type K⁺ channels, which generate the transient outward K⁺ currents described previously in a study of vesicles derived from the sarcolemma of frog skeletal muscle. Channels were activated by depolarizing pulses. There is evidence for non-random grouping of records with channel openings and blank records when depolarizations were repeated at brief intervals, suggesting a slow process similar to slow inactivation. Binomial analysis was consistent with independent behaviour of the channels. Ensemble average currents obtained from multichannel patches had kinetics similar to those of the macroscopic A-type K⁺ current, I _A. The rate of activation, fitted to n ⁴ kinetics, was fast and voltage dependent. The rate of inactivation had an exponential time course with a voltage-independent time constant. The mean open time and the probability of a channel being open increased with depolarization. The histograms of latency to first opening revealed the presence of more than two voltage-dependent closed states. Channel openings occurred in bursts and the closed-time histograms could be fitted by the sum of two or three exponentials. These results suggest a gating scheme with at least three closed states, probably two open states, and two inactivated states. Received: 4 November 1997 / Received after revision: 9 January 1998 / Accepted: 12 January 1998     

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit of the GABAA receptor

A line of mice was recently created in which the gabrb3 gene, which encodes the β₃ subunit of the GABA_A receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA_A receptors in the CNS enabled an investigation of the role of GABA and GABA_A receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA_A and GABA_B receptor agonists. Homozygous null (β₃^−/−) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (β₃^+/+) and heterozygous (β₃^+/−) littermates. The β₃^−/− mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their β₃^+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in β₃^−/− mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA_A receptors in the spinal cord. As expected, subcutaneous administration of the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in β₃^−/− mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, β₃^+/+ and β₃^+/− mice. However, the antinociceptive effect of the GABA_B receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in β₃^−/− mice compared to the progenitor strains, β₃^+/+ or β₃^+/− mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA_A receptors can affect the production of antinociception by other analgesic drugs as well.     

Differing effects of intracortical circuits on plasticity

Practice of a motor task leads to an increase in amplitude of motor-evoked potentials (MEP) in the exercised muscle. This is termed practice-dependent plasticity, and is abolished by the NMDA antagonist dextromethorphan and the GABA_A agonist lorazepam. Here, we sought to determine whether specific subtypes of GABA_A circuits are responsible for this effect by comparing the action of the non-selective agonist, lorazepam with that of the selective GABA_A-alpha₁ receptor agonist, zolpidem. In seven healthy subjects, transcranial magnetic stimulation (TMS) was used to quantify changes in amplitude of MEP after practice of a ballistic motor task. In addition we measured how the same drugs affected MEP amplitudes and the excitability of a number of cortical inhibitory circuits [short-interval intracortical inhibition (SICI), short-interval afferent inhibition (SAI) and long-interval intracortical inhibition]. This allowed us to explore correlations between drugs effects in measures of cortical excitability and practice-dependent plasticity of MEP amplitudes. As previously reported, lorazepam increased SICI and decreased SAI, while zolpidem only decreased SAI. The new findings were that practice-dependent plasticity of MEPs was impaired by lorazepam but not zolpidem, and that this was negatively correlated with lorazepam-induced changes in SICI but not SAI. This suggests that the intracortical circuits involved in SICI (and not neurons expressing GABA_A-alpha₁ receptor subunits that are implicated in SAI) may be involved in controlling the amount of practice-dependent MEP plasticity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.