Historical perspective and emergence of the GABAB receptor.

This chapter forms an introduction to the subsequent chapters in this volume which highlight the significance and potential therapeutic application of GABA(B) receptors. It is now 30 years since the GABA(B) site was first described in mammalian tissue. Since then much has emerged about its physiological role in the mammalian nervous system and its relationship to other neurotransmitter receptors. It appears to function at pre- and postsynaptic locations as both an auto- and a hetero-receptor where its activation modulates the membrane conductance of Ca(2+) and K(+). The receptor is G-protein coupled and was the first to be shown to exist, possibly in multiple forms, as a heterodimer. The primary agonist for the receptor is baclofen and this continues to be used therapeutically as a centrally active muscle relaxant. Other potential applications for agonists are suggested and positive allosteric modulators may provide an alternative and more effective approach. One application of an agonist, for which there are strong positive clinical data, is in gastroesophageal reflux disease where the receptor target is outside the brain. Antagonists of the GABA(B) receptor may also have therapeutic applications such as in cognitive deficits, affective disorders, and absence seizures but robust clinical evidence remains to be demonstrated. Each of these applications is also discussed in the chapters that follow.

GABA transporter type 1 (GAT-1) uptake inhibition reduces stimulated aspartate and glutamate release in the dorsal spinal cord in vivo via different GABAergic mechanisms.

Mechanisms through which the reported antinociceptive activity of GABA re-uptake inhibitors is mediated (and where on the sensory neuraxis) have not been defined. Here, microdialysis in the anaesthetised rat was used to examine the effect of selective GABA transporter type 1 (GAT-1) inhibition on basal and evoked amino acid release in the dorsal spinal cord. Reverse dialysis of the selective GAT-1 inhibitor NO-711 (10-300microM) induced a concentration-related increase in extracellular GABA (maximal approximately threefold of basal levels) without affecting other amino acids. Employing an S2/S1 paradigm, release evoked by brief high (45mM) K(+)-induced depolarisation of aspartate and glutamate, but not GABA or glycine, was found to be significantly reduced by reverse dialysis of NO-711 (300microM). Co-administration of selective antagonists for GABA(A) or GABA(B) receptors ((+)-bicuculline (100microM) or SCH 50911 (100microM), respectively) prevented the GAT-1 inhibition-induced reduction of evoked aspartate. In contrast, while (+)-bicuculline also antagonised the reduction of evoked glutamate, SCH 50911 (up to 1mM) was without effect. Inhibition of GAT-1 re-uptake was further found to play a permissive role in autoinhibitory effects on GABA release mediated through GABA(A) and GABA(B) receptors. These data demonstrate that augmentation of GABAergic neurotransmission by re-uptake inhibition activates pharmacologically distinguishable inhibitory influences on aspartate and glutamate release in the dorsal spinal cord. Thus, inhibition of spinal pro-nociceptive neurotransmitter release may contribute to the analgesic action of this drug class.

Activation by p-chloroamphetamine of the spinal ejaculatory pattern generator in anaesthetized male rats.

In urethane-anesthetized male rats, a branch of the hypogastric nerve was shown, anatomically and electrophysiologically, to supply the vas deferens. Recordings from this nerve revealed a low level of tonic activity, which was predominantly efferent motor activity. Administration of p-chloroamphetamine i.v., elicited a rhythmic burst of neuronal activity, coherent with rhythmic pressure increases in the vas deferens and contractions of the bulbospongiosus muscles, which together comprise ejaculation. This response to p-chloroamphetamine was still present after complete transection of the spinal cord at T8-T9. These data indicate that p-chloroamphetamine is capable of activating the spinal neuronal circuits that generate the pattern of autonomic and somatic responses similar to those of sexual climax. Furthermore based on the best documented action of p-chloroamphetamine, the results suggest that the excitability of the pattern generator is regulated by serotonergic, dopaminergic or noradrenergic receptors in the lumbosacral spinal cord. We conclude this animal model will enable robust studies of the pharmacology and physiology of central neural mechanisms involved in ejaculation and sexual climax.

GABA and glycine as neurotransmitters: a brief history.

gamma-Aminobutyric acid (GABA) emerged as a potentially important brain chemical just over 50 years ago, but its significance as a neurotransmitter was not fully realized until over 16 years later. We now know that at least 40% of inhibitory synaptic processing in the mammalian brain uses GABA. Establishing its role as a transmitter was a lengthy process and it seems hard to believe with our current knowledge that there was ever any dispute about its role in the mammalian brain. The detailed information that we now have about the receptors for GABA together with the wealth of agents which facilitate or reduce GABA receptor mechanisms make the prospects for further research very exciting. The emergence of glycine as a transmitter seems relatively painless by comparison to GABA. Perhaps this is appropriate for the simplest of transmitter structures! Its discovery within the spinal cord and brainstem approximately 40 years ago was followed only 2 years later by the proposal that it be conferred with 'neurotransmitter' status. It was another 16 years before the receptor was biochemically isolated. Now it is readily accepted as a vital spinal and supraspinal inhibitory transmitter and we know many details regarding its molecular structure and trafficking around neurones. The pharmacology of these receptors has lagged behind that of GABA. There is not the rich variety of allosteric modulators that we have come to readily associate with GABA receptors and which has provided us with a virtual treasure trove of important drugs used in anxiety, insomnia, epilepsy, anaesthesia, and spasticity, all stemming from the actions of the simple neutral amino acid GABA. Nevertheless, the realization that glycine receptors are involved in motor reflexes and nociceptive pathways together with the more recent advent of drugs that exhibit some subtype selectivity make the goal of designing selective therapeutic ligands for the glycine receptor that much closer.

Positive N-methyl-D-aspartate receptor modulation by selective glycine transporter-1 inhibition in the rat dorsal spinal cord in vivo.

In this study we have employed the selective glycine transporter-1 (GlyT-1) and GlyT-2 transporter inhibitors R-(-)-N-methyl-N-[3-[(4-trifluoromethyl)phenoxy]-3-phenyl-propyl]glycine (1:1) lithium salt (Org 24598) and 4-benzyloxy-3,5-dimethoxy-N-[1-(dimethylaminocyclopently)methyl]benzamide (Org 25543), respectively, and microdialysis perfusion to determine the effect of GlyT transporter inhibition on extracellular amino acid concentrations in the lumbar dorsal spinal cord of the halothane-anaesthetised rat. Reverse dialysis of Org 24598 (0.1-10 microM) induced a concentration-related increase in extracellular glycine accompanied by a progressive increase in citrulline, but not aspartate, glutamate or GABA, efflux. Org 25543 (10 microM) by the same route induced a similar increase in glycine levels without affecting the efflux of other amino acids quantified. To test the hypothesis that the increase in citrulline efflux resulted from activation of the N-methyl-D-aspartate receptor (NMDA-R)/nitric oxide synthase (NOS) signalling cascade, the sensitivity was determined of GlyT-1 inhibition-induced effects to NMDA-R antagonism or NOS inhibition. Co-administration by reverse dialysis of the selective NMDA-R channel blocker MK-801 (0.5 mM) or the selective antagonist of the strychnine-insensitive glycine site, 7-chlorokynurenic acid (1 mM), with Org 24598 (10 microM) did not affect the uptake inhibition-induced increase in glycine efflux, but did significantly attenuate the increase in extracellular citrulline. Similarly, co-administration with Org 24598 of the isoform non-selective and selective neuronal NOS inhibitors Nomega-nitro-L-arginine methyl ester (1 mM) or 1-(2-trifluoromethylphenyl)imidazole (0.2 mM), respectively, prevented Org 24598-induced citrulline efflux with no effect on increased glycine efflux. These data provide evidence that the observed increased in extracellular citrulline is a consequence of positive modulation of NMDA-R, secondary to increased extracellular glycine and support a protective role for GlyT-1 against fluctuations in extracellular glycine uptake at glutamatergic synapses in the dorsal spinal cord. Such a mechanism could be important to NMDA-R-mediated synaptic plasticity in the spinal cord and be of relevance to the clinical usage of GlyT-1 inhibitors.

Interaction of gamma-aminobutyric acid receptor type B receptors and calcium channels in nociceptive transmission studied in the mouse hemisected spinal cord in vitro: withdrawal symptoms related to baclofen treatment.

An in vitro mouse hemisected spinal cord was used to characterize the gamma-aminobutyric acid receptor type B (GABA(B)) modulation of the ventral root potential (VRP) in response to electrical stimulation of the dorsal root (DR). Low-intensity (LI) and high-intensity (HI) stimulation induced VRPs with progressively higher amplitude and duration. Repetitive HI-stimulation of the DR (1-10 Hz) produced windup. The selective GABA(B) receptor agonist, CGP35024, inhibited the VRPs in a dose-dependent manner. The inhibitory action of CGP35024 was blocked by CGP52432, a potent GABA(B) receptor antagonist. Following washout of the GABA(B) receptor agonist, VRPs and windup were significantly enhanced. The rebound increase of the VRP following removal of CGP35024 was also blocked by the GABA(B) receptor antagonist, CGP52432. This phenomenon is not linked to receptor desensitization, but rather due to GABA(B) receptor-induced hyperactivity of N-, P/Q-type Ca(2+) channels, as omega-CgTx GVIA and MVIIC abolished/prevented the increase. The 'rebound' enhancement of the spinal transmission after exposure to GABA(B) agonists sheds light on the possible mechanism of the severe withdrawal effects after abrupt termination of baclofen treatment in patients suffering from multiple sclerosis.

Cortical-area specific block of genetically determined absence seizures by ethosuximide.

Absence epilepsy is characterised by a paroxysmal loss of consciousness, of abrupt onset and termination, and is associated with a bilateral synchronous spike and wave discharge (SWD) on the electroencephalogram. Absence seizures involve an interplay between thalamic and cortical structures, although most research has so far focussed on sensory thalamic nuclei and the reticular thalamic nucleus (RTN). Thus, microinfusion of ethosuximide (ETX), a first choice anti-absence drug, into either the ventrobasal thalamus or RTN of the genetic absence epilepsy rat from Strasbourg (GAERS), a validated rat model of absence epilepsy, does not produce immediate cessation of seizure activity, as is seen following systemic administration. As recent evidence indicates a seizure initiation site within the peri-oral region of the primary somatosensory cortex (S1po), we have now applied ETX into S1po as well as the somatosensory cortex forelimb region (S1FL) and the motor cortex (M1) of freely moving GAERS. Microinfusion of 10 or 20 nmol/side of ETX into S1po produced an immediate cessation of seizure activity. A less marked response was produced when even a higher dose (200 nmol/side) was infused into S1FL. No reduction of SWD was seen when ETX was infused into M1. Microinfusion of CGP 36742 (5 nmol/side), a GABA(B) antagonist, produced immediate cessation of seizure activity in both S1po and M1 and a delayed effect in S1FL. These data suggest that the ability of ETX to abolish genetically determined absence seizures is cortical-area specific and support the involvement of S1po in the initiation of SWDs.

GABA(B1a), GABA(B1b) AND GABA(B2) mRNA variants expression in hippocampus resected from patients with temporal lobe epilepsy.

The aim of this study was to investigate the mRNA expression of the two GABA(B1) receptor isoforms and the GABA(B2) subunit, in human postmortem control hippocampal sections and in sections resected from epilepsy patients using quantitative in situ hybridisation autoradiography. Utilising human control hippocampal sections it was shown that the oligonucleotides employed were specific to the receptor. Hippocampal slices from surgical specimens obtained from patients with hippocampal sclerosis and temporal lobe epilepsy were compared with neurologically normal postmortem control subjects for neuropathology and GABA(B) mRNA expression. Neuronal loss was observed in most of the hippocampal subregions, but in the subiculum no significant difference was detected. The localisation of GABA(B1a) and GABA(B1b) isoform mRNAs in human control hippocampal sections supported and extended earlier studies using the GABA(B1) pan probe, which does not distinguish between the two GABA(B1) isoforms. Moreover, the GABA(B2) mRNA location confirmed the heterodimerisation of the receptor. Thus, although there was an apparent correlation between GABA(B1b) and GABA(B2), GABA(B1a) exhibited no such relationship. GABA(B1b) and GABA(B2) showed a similar intensity of expression whilst GABA(B1a) displayed a lower hybridisation signal. Comparison of the expression of the three mRNAs between control and epileptic subjects showed significant decreases or increases in different hippocampal subregions.GABA(B) isoforms and subunit mRNA expression per remaining neuron was significantly increased in the hilus and dentate gyrus. These results demonstrate that altered GABA(B) receptor mRNA expression occurs in human TLE; possibly the observed changes may also serve to counteract ongoing hyperexcitability.

Studies of GABA(B) receptors labelled with [(3)H]-CGP62349 in hippocampus resected from patients with temporal lobe epilepsy.

1 The aim of this study was to investigate the binding of a novel GABA(B) receptor radioligand, [(3)H]-CGP62349, to human post-mortem control and epileptic hippocampal sections using quantitative receptor autoradiography. Utilizing human control hippocampal sections it was shown that [(3)H]-CGP62349 bound with high affinity (K(D) 0.5 nM) to this tissue. 2 Hippocampal slices from surgical specimens obtained from patients with hippocampal sclerosis (HS) and temporal lobe epilepsy (TLE) were compared with neurologically normal post-mortem control subjects for neuropathology and GABA(B) receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, but in the subiculum no significant difference was detected. 3 The localization of GABA(B) receptors with the antagonist [(3)H]-CGP62349 in human control hippocampal sections supported and extended earlier studies using the agonist ligand [(3)H]-GABA. 4 The kinetics of binding to the GABA(B) receptor in human hippocampus using this novel compound was comparable to previous data obtained in rat hippocampal membranes. 5 GABA(B) receptor density (B(max)) was significantly reduced in CA3, hilus, and dentate gyrus (DG); the affinity was increased exclusively in DG. The trend is identical in all the hippocampal subregions with the agonist and the antagonist, although significant differences with the antagonist where recorded in CA3 and hilus, whereas with the agonist a significant reduction was reported in all of the hippocampal subfields. 6 GABA(B) receptor expression per remaining neuron appeared significantly increased in CA3 and hilus. These results suggest altered GABA(B) receptor function may occur in human TLE, possibly as a result of synaptic reorganization, and may contribute to epileptogenesis.

International Union of Pharmacology. XXXIII. Mammalian gamma-aminobutyric acid(B) receptors: structure and function.

The gamma-aminobutyric acid(B) (GABA(B)) receptor was first demonstrated on presynaptic terminals where it serves as an autoreceptor and also as a heteroreceptor to influence transmitter release by suppressing neuronal Ca(2+) conductance. Subsequent studies showed the presence of the receptor on postsynaptic neurones where activation produces an increase in membrane K(+) conductance and associated neuronal hyperpolarization. (-)-Baclofen is a highly selective agonist for GABA(B) receptors, whereas the established GABA(A) receptor antagonists, bicuculline and picrotoxin, do not block GABA(B) receptors. The receptor is G(i)/G(o) protein-coupled with mixed effects on adenylate cyclase activity. The receptor comprises a heterodimer with similar subunits currently designated 1 and 2. These subunits are coupled via coiled-coil domains at their C termini. The evidence for splice variants is critically reviewed. Thus far, no unique pharmacological or functional properties have been assigned to either subunit or the variants. The emergence of high-affinity antagonists for GABA(B) receptors has enabled a synaptic role to be established. However, the antagonists have generally failed to establish the existence of pharmacologically distinct receptor types within the GABA(B) receptor class. The advent of GABA(B1) knockout mice has also failed to provide support for multiple receptor types.

Interaction of group I mGlu and NMDA receptor agonists within the dorsal horn of the spinal cord of the juvenile rat.

1. The modulatory effects of mGlu receptors on NMDA-induced potential changes in spinal motoneurones were studied in vitro. 2. Selective activation of mGlu5 receptors by 10 microM (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG; EC(50)=280 +/- 24 microM) did not produce any change in the ventral root potential. However, the same concentration of CHPG (10 min perfusion) significantly attenuated the NMDA-induced ventral root depolarization (VRD). The effect persisted for 10 min after washout. NMDA-induced responses returned to control in 30 min. Brief co-application of CHPG and NMDA did not alter the NMDA-induced response indicating lack of direct receptor interaction. 3. The attenuating effect of CHPG on the NMDA-induced VRD was inhibited by the mGluR5 receptor antagonist, 2-methyl-6-phenyl-ethynylpyridine (MPEP). 4. In the presence of CGP56433A, a GABA(B) receptor antagonist, the NMDA-induced VRD was unchanged. However, NMDA-induced responses were potentiated after 10 min co-application of CHPG and CGP56433A. 5. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate ((2R,4R)-APDC), a group II mGlu receptor agonist did not attenuate the NMDA-induced response. 6. Under normal physiological conditions group I mGlu receptor agonists activate at least two populations of neurones: (1) GABA-ergic cells, which could release GABA and inhibit dorsal horn neurones, and (2) deep dorsal horn neurones/motoneurones which express NMDA receptors. Therefore, activation of mGlu5 receptors located on GABA-ergic interneurones could influence any direct potentiating interaction between mGlu5 and NMDA receptors in spinal cord and result in depression of the VRD. In the presence of a GABA(B) receptor antagonist, the direct synergistic interaction is unmasked. These data suggest that group I mGlu receptors provide a complex modulation of spinal synaptic processes.

Effects of high-affinity GABAB receptor antagonists on active and passive avoidance responding in rodents with gamma-hydroxybutyrolactone-induced absence syndrome.

RATIONALE: Absence seizures in man are behaviourally manifested as arrest and mild jerks mainly of facial muscles, associated in the electroencephalogram with synchronous spike and wave discharges. Gamma-hydroxybutyrolactone (GHBL) administration is currently used as an experimental model of absence seizures in rats and mice. OBJECTIVE: The aim of the present study was to examine the effects of three potent gamma-aminobutyric acid (GABA)B receptor antagonists CGP55845A, CGP62349 and CGP71982 (0.01 mg/kg) on the development of GHBL-induced absence epilepsy and in learning paradigms of active and passive avoidance tests in GHBL-treated mice and rats. METHODS: After 4 weeks of development of the absence syndrome, active and passive avoidance tests with negative reinforcement were performed. In both animal species, the absence syndrome was observed after 3 weeks of treatment in the saline group. RESULTS: The GABAB receptor antagonists CGP55845A and CGP62349 appeared to suppress the development of the absence syndrome to a greater degree in mice than in rats. CGP71982 suppressed it later than the other two antagonists (fifth week). In an active avoidance test in GHBL-treated mice, the GABAB antagonists had different effects - CGP62349 improved learning and memory retention to a greater extent than CGP55845A, whilst CGP71982 had no influence on it. In a passive avoidance test in GHBL-treated mice, the GABAB antagonists also had different effects - CGP71982 improved both learning and memory retrieval, whereas CGP55845A and CGP62349 had no effect. In the active avoidance test in GHBL-treated rats, the GABAB antagonist CGP55845A improved learning, whereas the other two, CGP62349 and CGP71982, had no effect. In the passive avoidance test the GHBL-treated rats showed an improvement in short memory retrieval. CGP55845A and CGP71982 improved this further, whilst CGP62349 had no effect. CONCLUSIONS: GHBL appeared to influence mice and rats in a different manner - rats learned the active avoidance task better than the GHBL-treated mice. The present study confirms previous data that GABAB antagonists suppress absence behaviour.

Determination of the extracellular concentration of glycine in the rat spinal cord dorsal horn by quantitative microdialysis.

Two quantitative microdialysis methods were used to determine the extracellular concentration of glycine in the dorsal spinal cord of halothane-anaesthetised rats. Extracellular glycine determined by zero net flux was 2.6+/-0.3 microM and by the zero flow method was 3.3+/-0.3 microM. For comparison the glycine content of cerebrospinal fluid was determined to be 6.4+/-1.1 microM. There was no correlation between the extracellular and the cerebrospinal fluid concentrations.

Allosteric modulation of [3H]-CGP39653 binding through the glycine site of the NMDA receptor: further studies in rat and human brain.

Binding of D,L-(E)-2-amino-4-[(3)H]-propyl-5-phosphono-3-pentenoic acid ([(3)H]-CGP39653), a selective antagonist at the glutamate site of the NMDA receptor, is modulated by glycine in rat brain tissue. We have further investigated this phenomenon in rodent and human brain by means of receptor binding and quantitative autoradiography techniques. In rat cerebral cortical membranes the glycine antagonist 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt (GV150526A) did not change basal [(3)H]-CGP39653 binding, but competitively reversed the high affinity component of [(3)H]-CGP39653 binding inhibition by glycine, with a pK(B) value of 8.38, in line with its affinity for the glycine site (pK(i)=8.49 vs. [(3)H]-glycine). Glycine (10 microM) significantly decreased [(3)H]-CGP39653 affinity for the NMDA receptor (with no change in the B(max)), whereas enhanced L-glutamate affinity (P<0.05, paired-samples Student's t-test). In rat brain sections the addition of GV150526A (30 microM) to the incubation medium increased [(3)H]-CGP39653 binding to 208% of control (average between areas), indicating the presence of endogenous glycine. The enhancement presented significant regional differences (P<0.05, two-way ANOVA), with striatum higher than cerebral cortex (282 and 187% of control, respectively; P<0.05, Fisher's LSD). On the contrary, there was not any significant variation in affinity values of [(3)H]-CGP39653, L-glutamate, glycine and GV150526A in striatal and cortical membranes. These results confirmed the existence of regionally distinct NMDA receptors subtypes with different glycine/glutamate allosteric modulation. Whole brain autoradiography revealed an uneven distribution of [(3)H]-CGP39653 binding sites in human brain. High levels of binding were determined in hippocampus and in cingulate, frontoparietal and insular cortex. Intermediate to low levels of binding were found in diencephalic nuclei and basal ganglia. [(3)H]-CGP39653 binding was increased to 216% of control (mean between areas) by 30 microM GV150526A. The enhancement, however, did not present significant regional differences. These results introduce GV150526A as a useful tool to identify NMDA receptor subtypes by means of receptor autoradiography; moreover, they demonstrate that the allosteric inhibition of [(3)H]-CGP39653 binding by glycine parallels an increase in receptor affinity to the endogenous ligand L-glutamate. Finally, this study provides the first detailed anatomical description of the regional distribution of [(3)H]-CGP39653 binding sites in human brain.

Distribution of GABA(B(1a)), GABA(B(1b)) and GABA(B2) receptor protein in cerebral cortex and thalamus of adult rats.

The distribution of GABA(B) receptor subunits GABA(B(1a)), GABA(B(1b)) and GABA(B2), has been examined in the cerebral cortex and thalamus of adult rats using an immunocytochemical technique. GABA(B(1a)) and GABA(B(1b)) subunits co-localized with GABA(B2) in the cortex, where afferent thalamic GABAergic axons project to pyramidal neurones. The expression patterns of GABA(B(1a)), GABA(B(1b)) and GABA(B2) were similar throughout the thalamus. The data suggest that the GABA(B(1b)) subunit might be the presynaptic isoform in the thalamo-cortical pathway with the GABA(B(1a)) subunit possibly present at postsynaptic sites on cell bodies. This contrasts with our previous data, obtained in cerebellum and spinal cord which indicate opposite locations. Thus, it seems unlikely that functional role along with cellular location can be assigned in a general manner to specific GABA(B) receptor subunit splice variants.

GABA(B) receptor autoradiography in hippocampal sclerosis associated with human temporal lobe epilepsy.

1. Metabotropic gamma-aminobutyric acid receptors (GABA(B)) exist both pre- and postsynaptically throughout the brain, mediating the suppression of neurotransmitter release and late inhibitory postsynaptic potentials. Investigation of GABA(B) receptors in rodent models of temporal lobe epilepsy (TLE) suggests that expression or function of these receptors may be altered in the disorder. 2. The aim of the present study was to investigate the expression of GABA(B) receptors in samples of hippocampus surgically resected from patients with hippocampal sclerosis (HS) related intractable TLE, and compare this expression with samples of neurologically normal post-mortem (PM) control hippocampal tissue. Appropriate measures of neuronal loss associated with HS were investigated for comparison with receptor binding data. 3. Receptor autoradiography with [(3)H]-GABA in the presence of isoguvacine, and quantitative densitometric analysis were used to investigate GABA(B) receptor expression (B(max)) and affinity (K(D)) in 11 HS samples and eight controls. A three-dimensional cell counting technique was used to assess neuronal density in both groups. 4. GABA(B) receptor density was significantly reduced in CA1, CA2, CA3, hilus and dentate gyrus, and increased in the subiculum, of HS cases as compared with PM controls. Neuronal loss was significant in all regions measured. When adjusted for neuronal loss, CA1 GABA(B) receptor expression appeared significantly upregulated (P:<0.05). 5. In HS/TLE, GABA(B) receptor expression per remaining neurone appears increased in CA1. This finding, and increased [(3)H]-GABA affinity at CA3 and hilar GABA(B) receptors, suggests altered GABA(B) receptor function may occur in human HS/TLE, possibly as a result of synaptic reorganization.

GABA(B(1)) mRNA expression in hippocampal sclerosis associated with human temporal lobe epilepsy.

GABA(B) receptors act to inhibit neurotransmitter release from presynaptic terminals, and mediate the late inhibitory postsynaptic potential. Studies of GABA(B) receptor function in rodent models of temporal lobe epilepsy (TLE) suggest that GABA(B) receptor expression and/or function may be perturbed. GABA(B(1)) mRNA levels were investigated in 10 hippocampal resection samples obtained at surgery from intractable hippocampal sclerosis (HS) associated TLE patients and five neurologically normal post-mortem (PM) control samples. In situ hybridisation with a 35S-dATP-labelled oligonucleotide was carried out to measure mRNA levels, along with three-dimensional cell counting, for assessment of neuronal density in hippocampal subregions. GABA(B(1)) mRNA was significantly up-regulated in the subiculum of HS samples as compared with PM controls. When adjusted for the characteristic neuronal density changes observed in HS, GABA(B(1)) mRNA was significantly up-regulated in CA1, hilus and dentate gyrus granule cell layer of HS samples as compared with PM controls. The possibility of increased GABA(B(1)) expression suggests that changes in GABA(B) receptor mechanisms may be involved in the pathogenesis of human HS-associated TLE.

GABA(B(1)) splice variant mRNAs are differentially affected by electroshock induced seizure in rats.

Receptor autoradiography with the high affinity antagonist radioligand [3H]CGP62349 and in situ hybridization with radiolabelled oligonucleotides were used to investigate GABA(B) receptor protein expression, and GABA(B(1)) mRNA splice variant (GABA(B(1a)) and GABA(B(1b)) levels, in brain sections from rats 4 h following a single electroshock-induced generalized seizure. Densitometric analysis indicated that GABA(B(1a)) mRNA levels were not significantly altered by an acute electroshock seizure, but that GABA(B(1b)) mRNA levels were significantly increased throughout the brain. GABA(B) receptor expression at this time point was unaffected by the seizure. The observed up-regulation of GABA(B(1b)) mRNA levels may imply increased importance of this splice variant in the regulation of further seizure activity.

Effects of alpha-dendrotoxin and dendrotoxin K on extracellular excitatory amino acids and on electroencephalograph spectral power in the hippocampus of anaesthetised rats.

Dendrotoxins, important pharmacological tools for studying K(+) channels, are potently convulsant in the central nervous system and evidence suggests that different members of the dendrotoxin family may act at pre- or post-synaptic sites. Using a combination of intrahippocampal infusion, microdialysis and electroencephalograph (EEG) recording, we have compared the effects of alpha-dendrotoxin and dendrotoxin K on extracellular levels of excitatory amino acids in anaesthetised rats. Our findings show that although infusion of 35 pmol of both peptides was associated with elevated extracellular aspartate and glutamate, these increased levels were more sustained with dendrotoxin K. Furthermore, there was EEG evidence of an associated transient functional change consistent with an action on pre-synaptic K(+) channels. In contrast, infusion of alpha-dendrotoxin produced only a brief effect on amino acid levels and no evidence of a functional consequence.

GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia.

The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, approximately 90% was of the GABAB(1a) form and approximately 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.