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.

GABA and its receptors in the spinal cord.

The importance of the inhibitory neurotransmitter, GABA, within higher centres of the mammalian brain is unquestionable. However, its role within the spinal cord is of equal significance. There have been numerous studies over the past two decades that have established GABA as a neurotransmitter at both post- and presynaptic sites in the cord. Here, Marzia Malcangio and Norman Bowery review the current status of GABA in relation to nociception and skeletal muscle tone, and indicate that its contribution to spinal cord function should not be overlooked.

Peptide autoreceptors: does an autoreceptor for substance P exist?

The presence of autoreceptors for simple neurotransmitters at synapses in the mammalian nervous system is well established. By contrast, the evidence for such receptors modifying neuropeptide transmission is less obvious. Probably the most well characterized of the neuropeptides is substance P (SP), which appears to play a major role as a primary afferent modulator. This article highlights evidence to support the existence of autoreceptors that might modulate the release of this neuropeptide and which, therefore, could be important in the design of drugs affecting SP function, not only in sensory processing, but also elsewhere in the brain.

Dextromethorphan and neuromodulation: old drug coughs up new activities.

Dextromethorphan is one of the most widely used non-opioid cough suppressants, representing the active ingredient in several over-the-counter antitussive formulations. It does not possess the CNS pharmacology of other opiates in humans (i.e. analgesia, respiratory depression, abuse liability or psychotomimetic properties), but since the discovery in 1981 of high affinity recognition sites in brain for dextromethorphan a unique neuropharmacological profile has emerged for this relatively innocuous drug. Anticonvulsant and neuroprotective properties have been demonstrated, and treatment with dextromethorphan has been shown to improve the cerebrovascular and functional consequences of global cerebral ischemia. Frank Tortella and colleagues review the CNS pharmacology of dextromethorphan, its possible involvement with NMDA or sigma-receptors, and the potential clinical importance of this old 'new' drug.

Calcitonin gene-related peptide content, basal outflow and electrically-evoked release from monoarthritic rat spinal cord in vitro.

In this study, Freund's adjuvant-induced monoarthritis in the rat hind paw was used to induce chronic pain and inflammation. In order to compare the basal outflow, electrically-evoked release and total content of calcitonin gene-related peptide like immunoreactivity (CGRP-LI) with previously reported changes in substance P (SP-LI), the lumbar enlargement of monoarthitic (complete Freund's adjuvant-treated, CFA rat) and control (incomplete Freund's adjuvant-treated, IFA rat) spinal cords were used. During the 4-wk period after injection, neither the basal nor the evoked release of CGRP-LI from CFA cords differed from controls. By contrast, we have previously reported that SP-LI release from CFA rat spinal cords was significantly higher than from controls, 21 days after inoculation with Freund's adjuvant. Electrically-evoked CGRP-LI release from 21-day CFA rat spinal cord slices was not modified by superfusion with a GABAB antagonist, CGP 36742 (100 microM) which could greatly increase SP-LI release. However, the release of both peptides was significantly increased to the same extent in IFA and normal tissue but to a lesser extent in CFA cords, by superfusion with the opioid antagonist naloxone (1 microM). In conclusion, CGRP-LI, unlike SP-LI, did not appear to be susceptible to any changes in the lumbar enlargement of the rat spinal cord during inflammation of the hind paw. In addition, CGRP-LI release was increased by antagonism of opiate but not GABAB receptors, suggesting that during chronic inflammation of one hind paw, the GABAB ergic system, unlike the opioid system, might be activated to selectively inhibit the enhanced SP-LI release but not CGRP-LI release which is not changed.

The 5-HT3 receptor ligand, [3H]BRL 43694, binds to presynaptic sites in the nucleus tractus solitarius of the rat.

This study has employed receptor autoradiography to localise the distribution of binding sites for the 5-HT3 receptor ligand [3H]BRL 43694 in sections of the brain of the rat (using a concentration of 10 nM [3H]BRL 43694 with 100 microM GR38032F to define non-specific binding). The highest density of binding sites for [3H]BRL 43694 was observed in the nucleus tractus solitarius and amounted to 652 fmol/mg tissue. The binding of [3H]BRL 43694 was also examined in sections prepared 10 days after unilateral nodose ganglionectomy, in an attempt to determine the neuronal location of these binding sites. Denervation reduced the binding of [3H]BRL 43694 by around 50% in the ipsilateral side of the nucleus tractus solitarius, relative to the contralateral side. This would indicate that the 5-HT3 binding sites may have a presynaptic location on vagal afferent terminals.

The pharmacology of adenylyl cyclase modulation by GABAB receptors in rat brain slices.

GABAB receptor activation inhibits forskolin-stimulated adenylyl cyclase activity but augments noradrenaline-stimulated adenylyl cyclase activity. The present study investigated the pharmacology of these two GABAB receptor mediated responses. In a cross-chopped rat cortical slice preparation, it was confirmed that (-)baclofen inhibited forskolin-stimulated adenylyl cyclase activity and augmented noradrenaline-stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CGP44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin-stimulated adenylyl cyclase than as augmenters of noradrenaline-stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 = 6.07 +/- 0.29 and 5.04 +/- 0.17, respectively (p < 0.05)), and CGP47656 (pEC50 = 6.44 +/- 0.05 and 4.48 +/- 0.26, respectively (p < 0.05)). It is possible to explain this difference in potency by proposing that these compounds have low intrinsic efficacy, and the augmentation of noradrenaline-stimulated adenylyl cyclase has a low receptor reserve. In addition six antagonists (CGP49311A, CGP46381, CGP45024, CGP45397, CGP36742) were also tested for their ability to antagonize 10 microM (-)baclofen in these two assays. These antagonists ranged in potency as inhibitors of forskolin-stimulated adenylyl cyclase activity from CGP49311A (pEC50 = 5.45 +/- 0.30) to CGP36742 (pEC50 = 3.87 +/- 0.16). Each antagonist had similar potency in the two assays, suggesting that these two responses are mediated by pharmacologically similar receptors.

Benzodiazepine receptor autoradiography in corpus striatum of rat after large frontal cortex lesions and chronic treatment with diazepam.

It has previously been shown that diazepam impairs behavioural recovery from partial unilateral ablation of the cerebral cortex in rats. The present study confirmed this in rats with large unilateral lesions of the frontal cortex and showed that diazepam (5 mg/kg i.p. daily for 14 days immediately after surgery) prevented recovery from sensory asymmetry even after 120 days. In saline-treated rats greater than 80% recovery had occurred by this time. A study of binding to benzodiazepine receptors, using an in vitro autoradiographic technique, was performed to determine whether the lack of recovery after administration of diazepam was associated with any long-term receptor changes on the damaged side of the brain. Binding of [3H]Ro15-1788 was increased by up to 40% in the caudate putamen on the decorticated side at 14-120 days. This was not significantly altered by treatment with diazepam. Binding of [3H]Ro15-1788 in the nucleus accumbens was not altered by lesion of the frontal cortex alone or after treatment with diazepam. It is concluded that the lack of recovery from sensory asymmetry, produced by diazepam after lesion of the frontal cortex cannot be correlated with any change in binding to benzodiazepine receptors within the corpus striatum.

Effect of lamotrigine on the electrically-evoked release of endogenous amino acids from slices of dorsal horn of the rat spinal cord.

The novel anti-epileptic, lamotrigine (LTG) has been shown to exhibit antinociceptive effects in the rat. In the present study, the effect of LTG on the electrically-evoked release of endogenous amino acids from rat isolated spinal dorsal horn slices with intact dorsal roots has been examined and compared with those of morphine in the same preparation. LTG (0.1-300 microM) inhibited the release of aspartate, glutamate and GABA in a concentration-dependent manner. The lowest concentrations of morphine (0.001-0.01 microM) enhanced the stimulated release of aspartate and glutamate while the higher concentrations inhibited their release. Stimulated GABA release was reduced in a concentration-dependent manner. The anticonvulsant was more potent at inhibiting the release of glutamate (IC50 = 20 microM) than that of GABA (IC50 = 44 microM) supporting the previous suggestion that lamotrigine is a selective inhibitor of glutamate release. This suggests that the reduction in glutamate release could be one of the mechanisms by which lamotrigine exerts its antinociceptive effect.

Behavioural and neuropathological effects produced by tetanus toxin injected into the hippocampus of rats.

The behavioural effects of tetanus toxin, injected into the rostral hippocampus, have been studied in rats. A single dose (1000 mouse minimum lethal doses; n = 10) of the toxin produced tail rigidity, hunched back and sound- and touch-evoked stimuli, 48 hr after the injection in all rats treated and these culminated in generalized convulsions 5-7 days later. Seizures were also observed 4 days after the injection of 2000 MLDs (n = 10), whereas a dose of 500 MLDs (n = 10) was ineffective. Similarly, dose- and time-dependent lethal effects were observed. In comparison to the contralateral (untreated) hippocampus, tetanus toxin (1000 MLDs; n = 3) produced a statistically significantly reduction in the number of cells in the CA1 pyramidal cell layer of the injected side, 7 and 10 days after the injection. No changes were observed in other sectors (CA2 and CA3 areas) of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the hippocampus produced dose- and time-dependent behavioural stimulation and lethal effects in rats.

[3H](-)Baclofen: an improved ligand for GABAB sites.

[3H](-)Baclofen, the radiolabelled form of the active isomer of baclofen, has been used as a ligand for GABAB (gamma-aminobutyric acid) receptors on synaptic membranes from whole brain of rat. The pharmacological profile for displacement of this ligand was observed to be identical with that for the racemic ligand [3H](+/-)baclofen and [3H]GABA under conditions where GABAB, but not GABAA sites, were labelled. The displaceable (specific) portion of membrane-bound [3H](-)baclofen was 47.5 +/- 2.3% of the total which was twice that obtained previously with [3H](+/-)baclofen. Two binding components were observed with affinities of 19 and 304 nM and binding capacities of 0.37 and 1.58 pmol/mg protein respectively. It is suggested that [3H](-)baclofen is an improvement over the labelled racemic form and binds to the same sites. It should provide a more reliable tool for studying GABAB receptors.

High vulnerability of dentate granule cells to the neuropathological effects induced by intrahippocampal injection of tetanus toxin.

The behavioural and neuropathological effects of tetanus toxin, injected into the dentate gyrus, were studied in rats. The monolateral injection of a single dose (1000 mouse minimum lethal doses, MLDs; n = 14 rats) of tetanus toxin produced time-dependent behavioural stimulation. Wet-dog shakes and facial stereotypy were observed 3-4 days after the injection, culminating 4-5 days after treatment, in "limbic motor seizures". Ten days after injection, histological examination revealed death of dentate granule cells in the tetanus toxin-treated side but not in the contralateral, control side (treated with neutralized toxin). This effect was observed in all rats (n = 5) receiving tetanus toxin in the dentate gyrus and no damage was reported in other sectors (e.g. CA1 and CA3 pyramidal cell layers) of the hippocampus. Quantification of the neuronal damage yielded an approximately 70% reduction (P less than 0.01) in the number of granule cells in the toxin-injected dentate gyrus, compared with the control side. This was greater than that previously reported (30% reduction) in the CA1 pyramidal cell layer of rats receiving the same dose of toxin, into the regio superior of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the dentate gyrus produced behavioural excitation and selective death of dentate granule cells.

Comparative receptor autoradiography of ex vivo and in vitro [3H]dizocilpine binding in mouse brain after middle cerebral artery occlusion.

In the present study the in vitro and ex vivo distributions of [3H]dizocilpine binding sites in mouse brain after middle cerebral artery occlusion (MCA-O) were compared using receptor autoradiography. The distribution patterns of [3H]dizocilpine binding sites obtained in vitro and ex vivo in normal mouse brain were the same with the highest densities occurring in the hippocampus and cerebral cortex. MCA-O had little or no effect on the in vitro binding density for at least 24 hr post-ischaemia. However after 2-3 days binding densities in the region of infarct were significantly reduced compared to the contralateral cerebral cortex. Further reductions occurred after 5-7 days. By contrast ex vivo [3H]dizocilpine binding was reduced in the infarcted area by 78.7 +/- 4% within 2 hr of the ischaemic insult and at all subsequent times binding was reduced by more than 75%. Ex vivo binding after ischaemia was always less than 30% of in vitro binding and this decrease was apparent within 2 hr of the ischaemic insult whereas in vitro binding was maintained at control levels for at least 24 hr. The neuroprotective activity of the NMDA antagonists dizocilpine and CGP 37849 in this model at different times after MCA-O was assessed. The time scale for receptor access following MCA-O is discussed and it is suggested that although the population of NMDA receptors is maintained in the infarct region for some days access to them in vivo may be sufficiently impaired within 2 or 4 hr of ischaemic insult to reduce the neuroprotective activity of NMDA antagonists after this time.

Hippocampal extracellular amino acids and EEG spectral analysis in a genetic rat model of absence epilepsy.

Absence seizures have a clearly defined thalamocortical origin. However, there is evidence from a genetic rat model of absence epilepsy, GAERS, that the underlying cellular and molecular abnormalities may also manifest themselves in other brain regions. As enhanced learning has previously been associated with this rat model, we have studied extracellular amino acid levels and EEG spectra in the hippocampus of these rats, this being a brain region associated with memory and learning. We report significantly higher levels of basal extracellular glutamate within the hippocampus of GAERS, together with transient increases in citrulline and glycine following aggravation of the absence seizures with the GABA(B) agonist, (-)baclofen. Furthermore, there is a reduction in the relative power of the EEG theta frequencies in GAERS, and a slowing of the EEG following administration of (-)baclofen which is not evident in control animals. Administration of a GABA(B) antagonist, CGP 56999, at a dose which blocks absence seizures in GAERS, caused a shift to faster frequencies of the EEG in both GAERS and control rats. It is speculated that the mechanisms underlying absence seizures in GAERS may manifest themselves in other functions modulated by thalamocortical oscillations such as cognitive processing.

Evidence that SL75102 is an agonist at GABAb as well as GABAa receptors.

The compound SL75102 ([alpha(4-chlorophenyl)5-fluoro 2-hydroxy benzilidene-amino]-4-butanoate sodium) is a GABA-mimetic at bicuculline-sensitive and -insensitive receptors. It depolarized rat isolated superior cervical ganglia in a dose-dependent manner (relative potency = 0.101 +/- 0.013; GABA = 1). Bicuculline methobromide (13 microM) antagonized this action of SL75102 and shifted the log dose-response curve to the same extent as the GABA curve. The evoked release of [3H]noradrenaline from rat isolated atria was also reduced by SL75102 in a GABA-like manner. SL75102 also displaced [3H]GABA and [3H]baclofen specifically bound to divalent cation dependent GABAB sites on rat synaptic membranes (relative potency approximately 0.1 GABA throughout). The butyramide from which SL75102 can be formed within the body (SL76002) was much less active at GABAB sites (less than 0.02 atria, 0.001 binding, GABA = 1). It is suggested that in addition to any direct action of SL76002 itself the products of SL76002 metabolism, SL75102 and GABA may exert effects via baclofen-sensitive GABAB as well as GABAA sites in mammalian brain.

Effect of paroxetine, a selective 5-hydroxytryptamine uptake inhibitor, on beta-adrenoceptors in rat brain: autoradiographic and functional studies.

Quantitative receptor autoradiography was used to investigate the effects of paroxetine (8.3 mg/kg), amitriptyline (26 mg/kg) and desipramine (17 mg/kg), administered daily in the drinking water for 21 days, on the number of beta 1- and beta 2-adrenoceptors in the cortex of the rat. In addition, the effect of these drugs on the function of beta-adrenoceptors was examined by measuring noradrenaline- and isoprenaline-stimulated production of cyclic AMP in slices of cortex. Paroxetine did not alter the number of cortical beta 1 or beta 2-adrenoceptors nor did it induce any functional changes in beta-adrenoceptor-linked adenylyl cyclase. In contrast, desipramine caused a significant reduction in the density of beta 1-adrenoceptors and in the sensitivity of both noradrenaline and isoprenaline-stimulated adenylyl cyclase. Although amitriptyline significantly reduced the number of beta 1-adrenoceptors in cortical membranes, no such changes could be detected by autoradiography. It is apparent from these and other studies, that the ability of antidepressants to down-regulate central beta-adrenoceptors is not a property shared by all antidepressants. In particular, the more potent and selective inhibitors of the uptake of 5-HT, such as paroxetine, appear to be devoid of effects on this receptor system.

NMDA NR1 subunit mRNA and glutamate NMDA-sensitive binding are differentially affected in the striatum and pre-frontal cortex of Parkinson's disease patients.

Changes in the levels of mRNA for the NR1 subunit of the glutamate NMDA receptor and in NMDA-sensitive glutamate binding were investigated in consecutive sections of the prefrontal cortex and striatum of control and Parkinson's disease (PD) post-mortem brain using in-situ hybridisation and receptor autoradiography. Both markers of NMDA receptors were found to be relatively unaffected when measured by microdensitometry in the prefrontal cortex of control and PD brains. At a cellular level, a subpopulation of small and medium neurons in the superficial layers of the prefrontal cortex of the PD group showed a decreased expression of NMDA NR1 mRNA, with the maximal decrease in cortical layer IV. In the striatum, levels of glutamate binding to the NMDA receptor detected by receptor autoradiodgraphy were significantly reduced in the PD group, while no change could be detected at a macroscopical level in NMDA NR1 mRNA expression. Consequently, we suggest that the important decrease in agonist binding to the NMDA receptor observed in this study in the caudate and putamen of PD brains, in the absence of any major change in NMDA NR1 mRNA levels might reflect the degeneration of pre-synaptic NMDA receptors located on nigro-striatal projections particularly affected by the disease. Small changes observed at a cellular level in subsets of neurons of both prefrontal cortex and striatum will be discussed at the light of neurochemical changes characteristics of PD.

Distribution of GABA(B) binding sites in the thalamus and basal ganglia of the rhesus monkey (Macaca mulatta).

The regional distribution of GABA(B) receptor binding sites in the thalamus and basal ganglia of rhesus monkey has been determined by receptor autoradiography using the agonist ligand, [3H]-GABA. Whilst binding sites were evident throughout the thalamus, the internuclear differences in the Bmax were up to 10-fold. In the basal ganglia the binding density was on average lower than in the thalamus. The highest number of binding sites was in striatum followed closely by substantia nigra. In both the thalamus and basal ganglia, the binding density was higher than previously described in the rat. Although our results do not allow us to differentiate between presynaptic and postsynaptic locations of GABA(B) sites we conclude that with a few exceptions the distribution pattern of GABA(B) binding sites in the monkey thalamus appears to correlate with the known innervation from the NRT.