GABAB receptor: a site of therapeutic benefit.

Although the presence of functional GABAB receptors in mammalian brain has been known for more than 20 years, there is still only one therapeutic agent in use, baclofen, which mediates its effects directly via this receptor. However, activation of this receptor can produce numerous effects that might be amenable to drug development. Evidence from preclinical studies also suggests that antagonism of the GABAB receptor produces beneficial clinical effects.

Plasticity of GABA(B) receptor-mediated heterosynaptic interactions at mossy fibers after status epilepticus.

Several neurotransmitters, including GABA acting at presynaptic GABA(B) receptors, modulate glutamate release at synapses between hippocampal mossy fibers and CA3 pyramidal neurons. This phenomenon gates excitation of the hippocampus and may therefore prevent limbic seizure propagation. Here we report that status epilepticus, triggered by either perforant path stimulation or pilocarpine administration, was followed 24 hr later by a loss of GABA(B) receptor-mediated heterosynaptic depression among populations of mossy fibers. This was accompanied by a decrease in the sensitivity of mossy fiber transmission to the exogenous GABA(B) receptor agonist baclofen. Autoradiography revealed a reduction in GABA(B) receptor binding in the stratum lucidum after status epilepticus. Failure of GABA(B) receptor-mediated modulation of mossy fiber transmission at mossy fibers may contribute to the development of spontaneous seizures after status epilepticus.

Reflections on more than 30 years association with Hanns.

I first met Hanns in 1977 and soon learnt of his extraordinary ability as a researcher. He became a friend as well as a mentor providing enthusiasm for my own research. I watched closely over the years how his research uncovered details of the association of the benzodiazepines and GABA and delineated the structural composition of the GABAA receptor associated with the action of individual drugs such as antianxiety and antiepileptic agents. His work produced many important contributions to medicine notable of which was the discovery of the first benzodiazepine antagonists, which are now routinely used in clinical practice. But for me his most important contribution was the discovery of the benzodiazepine receptor. During this time, my group uncovered a novel receptor for GABA and my progress in this work was encouraged and enhanced by discussions with Hanns.

GABA(B) receptor alterations as indicators of physiological and pharmacological function.

Given the widespread distribution of GABA(B) receptors throughout the central nervous system, and within certain peripheral organs, it is likely their selective pharmacological manipulation could be of benefit in the treatment of a variety of disorders. Studies aimed at defining the clinical potential of GABA(B) receptor agonists and antagonists have included gene deletion experiments, examination of changes in receptor binding, subunit expression and function in diseased tissue, as well as after the chronic administration of drugs. The results indicate that a functional GABA(B) receptor requires the combination of GABA(B(1)) and GABA(B(2)) subunits, that receptor function does not always correlate with subunit expression and receptor binding, and that GABA(B) receptor modifications may be associated with the clinical response to antidepressants, mood stabilizers, and GABA(B) receptor agonists and antagonists. Moreover, changes in GABA(B) binding or expression suggest this receptor may be involved in mediating symptoms associated with chronic pain, epilepsy and schizophrenia. This, together with results from other types of studies, indicates the potential therapeutic value of developing drugs capable of selectively activating, inhibiting, or modulating GABA(B) receptor function.

Reduced G-protein coupling to the GABAB receptor in the nucleus accumbens and the medial prefrontal cortex of the rat after chronic treatment with nicotine.

The effect of repeated administration of nicotine (0.4 mg/kg, daily, s.c., for 14 days) on GABAB receptor density, affinity and G-protein coupling was investigated in the mesocorticolimbic system of the rat brain. Baclofen-stimulated [35S]GTPgammaS binding autoradiography revealed that the level of G-protein coupling to GABAB receptors was significantly reduced in the medial prefrontal cortex and the nucleus accumbens of nicotine-treated rats as compared to vehicle-injected controls. By contrast, GABAB receptor density and affinity, as revealed by [3H]GABA saturation binding autoradiography, were not altered by the nicotine exposure in any of the regions examined. Reduced G-protein coupling to the GABAB receptor may result in disinhibition of mesocorticolimbic dopaminergic neurones, which would contribute to the development of sensitised dopaminergic responses to repeated administration of nicotine.

Modification of GABA(B1) and GABA(B2) receptor subunits in the somatosensory cerebral cortex and thalamus of rats with absence seizures (GAERS).

In the present study, we have investigated GABA(B) receptor expression in somatosensory cortex (S1) and the ventrobasal (VB) and reticular (Rt) thalamic nuclei of Genetic Absence Epilepsy Rats from Strasbourg (GAERS), which represent an animal model for the human absence epilepsy. We focused our attention on the thalamocortical network because it has been demonstrated that absence seizures are generated in this specific circuit, which is under the control of several inhibitory, e.g. GABA, and excitatory systems. Autoradiography data obtained with the GABA(B) receptor antagonist [3H]CGP62349 did not show any differences in Kd or Bmax values between control rats and GAERS. In situ hybridisation (ISH) results showed a significant increase in messenger RNA for GABA(B1) in the S1 and a decrease in the VB thalamic nucleus but not in the Rt thalamic nucleus. By contrast the immunocytochemical data revealed an increased expression of both GABA(B1) and GABA(B2) receptor subunits in all the regions examined, somatosensory cerebral cortex, VB thalamus and Rt nucleus in GAERS compared to controls. The main finding was an up-regulation of GABA(B) receptor protein in the corticothalamic circuit in GAERS compared to controls.

Targeting thalamic nuclei is not sufficient for the full anti-absence action of ethosuximide in a rat model of absence epilepsy.

Absence epilepsy is characterised by recurrent periods of physical and mental inactivity coupled to bilateral, synchronous spike and wave discharges (SWDs) on the electroencephalogram. The mechanism of action of ethosuximide (ETX), a drug specific for absence seizures, is believed to involve a reduction in the low threshold T-type Ca(2+) current in thalamocortical and nucleus reticularis thalami (NRT) neurones, although other electrophysiological data have questioned this. Here, we employed a genetic rat model of absence seizures to investigate the effects of directly administering ETX to the thalamus.SWDs were immediately and substantially reduced (approximately 90%) by systemic administration of ETX (177-709 micromol/kg), or by bilateral microinfusion into the thalamus of the GABA(B) antagonist, CGP 36742 (5-27 nmol per side). However, infusion of ETX (1-200 nmol per side) into the ventrobasal complex or the NRT resulted in a reduction of SWDs that was delayed (30-60 min) and less marked (approximately 50%). Administration of ETX (0.2 mM to 1M) to a greater volume of thalamus by reverse microdialysis also produced significant but delayed reduction of SWDs at concentrations >1mM. Only at 5mM were seizures significantly reduced (approximately 70%) within 30 min of administration. These results suggest that targeting of the thalamus alone may be insufficient for an immediate and full anti-absence action for ETX. Concomitant or exclusive actions in the cortex remain a possibility.

Reduced inhibitory action of a GABAB receptor agonist on [3H]-dopamine release from rat ventral tegmental area in vitro after chronic nicotine administration.

BACKGROUND: The activation of GABAB receptors in the ventral tegmental area (VTA) has been suggested to attenuate the rewarding properties of psychostimulants, including nicotine. However, the neurochemical mechanism that underlie this effect remains unknown. Since GABAB receptors modulate the release of several neurotransmitters in the mammalian brain, we have characterised the effect of the GABAB receptor agonist baclofen on the release of [3H]-dopamine ([3H]-DA) from VTA slices of naive rats and of rats pre-treated with nicotine. RESULTS: In naive rats, baclofen concentration-dependently inhibited the electrically evoked release of [3H]-DA from the isolated VTA (EC50 = 0.103 microM, 95% CI = 0.043-0.249), without affecting the basal [3H]-monoamine overflow. This effect was mediated by activation of GABAB receptors as it was blocked by the selective receptor antagonist CGP55845A. Chronic administration of nicotine (0.4 mg kg(-1), s.c., for 14 days) affected neither the basal nor the electrically evoked release of [3H]-DA from VTA slices. However, the inhibitory effect of baclofen (10 microM) on the stimulated [3H]-monoamine overflow was abolished in rats pre-treated with nicotine as compared to saline-injected controls. CONCLUSIONS: Our results demonstrate that GABAB receptor activation reduces the release of DA from the rat VTA. In addition, a reduced sensitivity of VTA GABAB receptors appears to develop after chronic exposure to nicotine. The resulting disinhibition of VTA DA neurones might therefore contribute to the sensitised dopaminergic responses observed in the rat mesocorticolimbic system following repeated administration of nicotine.

Reversal of GABA-mediated inhibition of the electrically and potassium chloride evoked [3H]-GABA release from rat substantia nigra slices by DL-3-hydroxy-3-phenyl pentanamide.

The phenyl alcohol amides, DL-2-hydroxy-2-phenyl butyramide (CAS 52839-87-9), DL-3-hydroxy-3-phenyl pentanamide (CAS 131802-69-2, DL-HEPP) and DL-4-hydroxy-4-phenyl hexanamide (CAS 67880-30-2) and their fluorine and chlorine analogs, at a concentration of 100 micromol/L, did not displace [3H]-gamma-aminobutyric acid ([3H]-GABA, CAS 108158-36-7) from GABAA receptors and only weakly displaced [3H]-GABA and [3H]-CGP62349 (CAS 186986-97-0), a GABAB receptor antagonist, from GABAB receptors in rat brain crude synaptic membranes. The electrically and potassium chloride (15 mmol/L) evoked [3H]-GABA release in the presence of DL-HEPP, GABA and GABAB receptor ligands from rat brain substantia nigra (SN) slices was studied. R-Baclofen (CAS 69308-37-8) (10 micromol/L), a GABAB receptor agonist, produced an inhibition of the electrically evoked [3H]-GABA release and this inhibition was blocked by CGP 55845A (CAS 149184-22-5) (10 micromol/L), a GABAB receptor antagonist, but was not affected by DL-HEPP (100 micromol/L). CGP 55845A (10 micromol/L) did not alter the electrically evoked [3H]-GABA release in the absence of baclofen. The addition of DL-HEPP (100 micromol/L) alone did not affect the electrically-evoked release of [3H]-GABA release control, but it was able to significantly reduce the inhibitory effect of GABA (CAS 56-12-2) (10 micromol/L) on [3H]-GABA release evoked both by electrical and potassium chloride stimulation, in the presence of tiagabine (CAS 115103-54-3) (10 micromol/L), a GABA uptake blocker. In three preliminary experiments, bicuculline (CAS 485-49-4) (10 micromol/L) and picrotoxinin (CAS 17617-45-7) (10 micromol/L), two GABAA antagonists, inhibited the electrically evoked release of [3H]-GABA from rat SN slices, and DL-HEPP (100 micromol/L) reversed this inhibition. The mechanism of action of DL-HEPP is not known but, it might act as a negative GABA modulator in rat brain slices.

Extracellular glutamine to glutamate ratio may predict outcome in the injured brain: a clinical microdialysis study in children.

The amino acid content of hourly microdialysis samples from nine severely head-injured children was examined. Of particular interest was the measurement of the excitatory amino acid glutamate, as high levels of this substance, which are associated with the excitotoxicity cascade, have been linked to high intracranial pressure and poor outcome in a similar study in adults. Interpretation of these data is complicated by many clinical and methodological factors and these are discussed in relation to the findings. Our findings from this pilot study in children confirm the associations between glutamate levels, intracranial pressure and outcome, but fail to corroborate the correlation between excitatory and structural amino acid levels seen in adult patients, that was interpreted as evidence of non-specific leakage of amino acids through damaged cell membranes. In addition, we have shown that the role of glutamine in glutamate homeostasis is an important consideration and that estimation of the extracellular glutamine/glutamate ratio may have some prognostic value in head trauma cases as there is evidence of links to clinical outcome.

Extracellular glutamate in the brains of children with severe head injuries: a pilot microdialysis study.

OBJECTIVE: To determine, using a microdialysis technique, the extracellular levels of glutamate and non-transmitter amino acids in the brain of children with severe head injuries. METHODS: A microdialysis probe was inserted along side the intracranial pressure (ICP) bolt in 9 children (age range 2-14 years) with severe head injuries (Glasgow coma scale<8). Dialysate samples were collected hourly and analysed, using High Performance Liquid Chromatography (HPLC), for glutamate, glutamine and various structural amino acids. Clinical monitoring parameters were also correlated with amino acid concentrations. CONCLUSIONS: Mean glutamate concentrations in the dialysate varied from very low (<5 micro M) to very high (>30 micro M). No correlation with outcome at discharge was demonstrated. Structural amino acid levels did not follow the glutamate fluctuations. Low glutamine/glutamate ratio was associated with increased morbidity, while a high ratio was associated with clinical improvement. Glutamate metabolism may have a more significant role in the pathophysiology of paediatric head injury than has already been recognised.

5-Hydroxytryptamine drives apoptosis in biopsylike Burkitt lymphoma cells: reversal by selective serotonin reuptake inhibitors.

Serotonin (5-HT), a well-known neurotransmitter of the central nervous system, has been implicated in diverse aspects of immune regulation. Here we show that 5-HT can efficiently drive programmed cell death in established Burkitt lymphoma (BL) lines that remain faithful to the original biopsy phenotype (group 1). Group 1 BL cells cultured in the presence of 5-HT exhibited marked suppression of DNA synthesis that was accompanied by extensive apoptosis-serotonin-driven apoptosis was complete within 24 hours, was preceded by early caspase activation, and was accompanied by a decline in mitochondrial membrane potential. BL cells that had drifted to a lymphoblastic group 3 phenotype were relatively resistant to these actions of serotonin, and the forced ectopic expression of either bcl-2 or bcl-x(L) provided substantial protection from 5-HT-induced apoptosis. 5-HT receptor antagonists (SDZ205-557, granisetron, methysergide) failed to inhibit serotonin-induced apoptosis, whereas the selective serotonin reuptake inhibitors (SSRI)-fluoxetine (Prozac), paroxetine (Paxil), and citalopram (Celexa)-substantially blocked the monoamine actions. Western blot analysis showed that BL cells expressed protein for the 5-HT transporter, and transport assays confirmed active uptake of serotonin by the cells. Unlike what was suggested for neuronal cells, there was no evidence that intracellular oxidative metabolites were responsible for the 5-HT-induced programmed death of BL cells. These data indicate that serotonin drives apoptosis in biopsylike BL cells after its entry through an active transport mechanism, and they suggest a novel therapeutic modality for Burkitt lymphoma.