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A comparison has been made of the effects of carbon monoxide (CO) or nitrogen (N2) exposure on synaptic transmission in the hippocampal slice. CA1 field potentials, evoked by Schaffer collateral stimulation, were unaffected by superfusion of slices with artificial cerebral spinal fluid (ACSF) equilibrated with either 15% CO or 15% N2 for 120 min. However, superfusion with hypoxic ACSF equilibrated with either 85% CO or 85% N2 caused a rapid depression of synaptic transmission. Reperfusion with control ACSF following 30 min hypoxia led to recovery of evoked responses and a slight hyperexcitability. In the hippocampal slice, synaptic transmission, as assessed by input/output curves, was not different during or following hypoxia induced by exposure to CO or N2. In the short term, CO is not toxic.

The actions of 2-hydroxy-saclofen (2-OH-S), a recently developed analog of baclofen, were studied at presynaptic GABAB receptors in the rat hippocampal slice. Baclofen (0.5-20 microM) reduces the amplitude of excitatory postsynaptic potentials (EPSPs) recorded from hippocampal CA1 pyramidal neurons. In the presence of 200-500 microM 2-OH-S, the synaptic depressant action of baclofen is significantly reduced. These data show that 2-OH-S is an effective antagonist at presynaptic GABAB receptors on excitatory terminals in the hippocampus.

The inhibition of the binding of the GABAB agonist [3H](-)-baclofen to rat cerebellar membranes by some sulfonic and phosphonic acid analogues of GABA has been studied. These analogues have been shown to act as GABAB antagonists in the rat cortical wedge and the guinea-pig isolated ileum preparations. The order of potency of phaclofen (IC50 118 microM), 2-hydroxysaclofen (IC50 5.1 microM) and saclofen (IC50 7.8 microM) as inhibitors of [3H](-)-baclofen binding was similar to the order of potency of these compounds as GABAB antagonists, whereas 3-aminopropylphosphonic acid (IC50 1.5 microM) and 4-aminobutyl-phosphonic acid (IC50 3.9 microM) were much more potent than anticipated from their relatively weak GABAB antagonist actions. These results indicate that inhibition of [3H](-)-baclofen binding to rat cerebellar membranes does not reflect antagonist activity at GABAB receptors seen in the rat cortical wedge preparation or the guinea-pig isolated ileum preparation. This may indicate a heterogeneity of GABAB binding and receptor sites.

Baclofen reduced the frequency and aborted the bursts of spontaneous paroxysmal discharges in rat neocortical slices maintained in magnesium-free medium. This action was prevented by pretreatment with barium or caesium, which each increased the ictaform burst frequency, amplitude and duration. 4-Amino-pyridine also increased the burst frequency but reduced the amplitude and did not completely prevent the action of baclofen. Evidently baclofen suppresses such discharges by opening potassium channels normally involved in limiting the burst activity.

Rat neocortical slices maintained in Mg2(+)-free Krebs medium developed spontaneous paroxysmal discharges which were attenuated or suppressed by the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen, occasionally accompanied by a slight hyperpolarisation, and antagonised by the specific GABAB-receptor antagonist, 2-OH-saclofen. Over the same dose range, the GABA-analogue 3-amino-propylphosphinic acid (3-APA) caused a marked, prompt hyperpolarisation with little or no effect on the frequency of the discharges, although their amplitude was attenuated. In the presence of 2-OH-saclofen, 3-APA still induced a hyperpolarisation but the amplitude of the discharges was no longer affected. This marked difference in action between baclofen and 3-APA in the rat neocortical slices suggests there may be a heterogeneity of GABAB-receptors.

Localization of [3H]GABA in the guinea-pig myenteric plexus has been studied using light microscopic autoradiography. In the presence of beta-alanine, 10(-3) M, an inhibitor of glial cell high affinity GABA transport, [3H]GABA was transported by a high affinity uptake system into neuronal elements of the plexus. Scattered neurones accumulating [3H]GABA showed localization of silver grains over the soma and axonal processes. In addition a large population of uptake sites for [3H]GABA was found within the secondary and tertiary meshworks of the plexus so that dense accumulations of silver grains were observed localized over distinct 'tracts' within all three meshworks of the plexus. These results are considered to provide strong evidence for GABAergic neurons in the enteric nervous system.

When administered microelectrophoretically, a sulphonic acid derivative of baclofen, 3-amino-2-(4-chlorophenyl)-2-hydroxy-propylsulphonic acid, reversibly reduced the presynaptic reduction by (-)-baclofen of the monosynaptic excitation of spinal interneurones by impulses in low threshold primary afferent fibres of the cat as well as the postsynaptic depression by (-)-baclofen of the firing of these neurones. This compound, 2-hydroxy-saclofen, may be useful in assessing the physiological significance of central baclofen receptors.

In the isolated ileum of the guinea pig, neurally mediated rhythmic longitudinal mechanical activity was induced by avermectin, a macrolide anthelmintic that releases gamma-aminobutyric acid (GABA) and modulates the GABAA-receptor-ionophore complex. This avermectin-induced activity was dependent on GABA, being reduced or abolished by bicuculline, a GABAA-receptor antagonist, and by 3-mercaptopropionic acid which prevents neural GABA release. These results provide additional direct evidence that GABA is a functional neurotransmitter in the myenteric plexus of the guinea pig intestine, evidently involved in the regulation of intestinal motility.

Cortisol had a biphasic action on GABAA-receptor-mediated contractile responses, enhancing at picomolar concentrations (1-10 pM) and inhibiting at higher concentrations (10-1000 nM). There was a sinistral shift of the GABA dose-response curve in the presence of 10 pM cortisol, with a significant potentiation of the GABA-induced contractions over the lower dose range of GABA (3-30 microM), whereas 100 nM cortisol caused a non-parallel dextral shift of the GABA dose-response curve, with a depression of the maximum GABA response indicative of non-competitive antagonism. Cortisol at various concentrations did not affect GABAB-receptor-mediated ileal relaxations, or the baclofen-induced depression of twitch contractions to transmural stimulation. Such concentrations of cortisol also did not affect ileal responses to exogenously applied acetylcholine or cholinergic twitch contractions themselves. These results suggest that cortisol is a specific, and very potent modulator at GABAA-receptor complexes in the guinea pig ileum.

In the guinea-pig isolated ileum, both gamma-aminobutyric acid (GABA) and baclofen induced a dose-dependent depression of cholinergic twitch contractions to transmural stimulation, sensitive to delta-aminovaleric acid (DAVA) and phosphonobaclofen (phaclofen). beta-Phenyl-GABA (BPG) antagonised this depressant action of baclofen and GABA, whilst itself weakly depressing ileal twitch contractions, an effect insensitive to DAVA or phaclofen, and thus unrelated to any GABAB-receptor-mediated effects. These results suggest that the baclofen receptors in the ileum that are antagonised by BPG differ from either of baclofen receptors in the spinal cord, where the presynaptic receptors are blocked by phaclofen and the postsynaptic receptors are insensitive to phaclofen, with BPG having baclofen-like actions at both sites. Interaction of BPG and baclofen with different receptor populations may explain the differing therapeutic actions of these compounds.

The effects of the GABAB receptor antagonist 2-hydroxy-saclofen were studied using intracellular recording of synaptic potential from CA1 hippocampal neurons. 2-Hydroxy-saclofen (50-200 microM) reversibly blocked the late, GABAB receptor-mediated inhibitory postsynaptic potential (IPSP) but not the early, GABAA receptor-mediated IPSP. In addition, the hyperpolarizing response to baclofen was reduced by similar concentrations of 2-hydroxy-saclofen. This suggests that 2-hydroxy-saclofen is a potent antagonist at postsynaptic GABAB receptors on hippocampal neurons.

Saclofen (the direct sulphonic analogue of baclofen) is a competitive antagonist of baclofen at GABAB receptors in guinea pig ileum and rat cortical slices (estimated pA2 = 5.3), at least twice as potent as 2-hydroxy-saclofen (pA2 = 5). A series of related sulphonic analogues also antagonised baclofen in the guinea pig ileum, including 2-hydroxy-saclofen amide (pA2 = 3.3), 3-amino-2-hydroxy-2-phenyl-propylsulphonic acid (pA2 = 3.5), 3-amino-2-(benzo-(b)-furan-2-yl)-propylsulphonic acid (pA2 = 4.3), and 3-amino-2-(4-chlorophenyl)-prop-1-enesulphonic acid (pA2 = 2.5-3), but none were more active than saclofen which is the most potent specific GABAB antagonist yet found.

2-hydroxy-saclofen (2-OH-S), a sulphonic analogue of baclofen, slightly increased the twitch height and reversibly antagonised the GABA- and baclofen-induced depression of twitch contractions in the guinea pig vas deferens and isolated ileum, causing a parallel dextral shift in the baclofen dose-response curve in a competitive manner (pA2 = 5.0) in the latter tissue. 2-OH-S (10-50 microM) reversibly elevated the spike height and antagonised the baclofen (8-20 microM)-induced suppression of ictal discharges in rat cortical slices superfused in Mg2+-free Krebs solution, the spike height declining to control level within 15 min of washout. The antagonism by 2-OH-S on GABAB receptor-mediated actions is selective, as 2-OH-S did not affect depressive responses to adenosine or morphine, or contractile responses to GABA (GABAA receptor-mediated), acetylcholine and carbachol in the ileum. Compared to phaclofen, 2-OH-S is a more potent competitive antagonist of GABAB receptor-mediated actions in the central and peripheral nervous system.

Rhythmic neurally mediated spontaneous relaxations of the longitudinal muscle in the isolated ileum of the guinea pig are modified by GABA antagonism. Bicuculline methochloride, a GABAA-receptor antagonist, and picrotoxinin, a Cl--ionophore blocker, as well as GABA desensitization reduced or most often abolished these naturally occurring periodic spontaneous relaxations, indicating a physiological involvement of GABA in the control of intestinal motility. The possible mechanism whereby GABA may induce such relaxations is discussed in relation to GABA actions in the enteric nervous system where GABA is a neurotransmitter.

Rhythmic neurally mediated spontaneous contractions of the longitudinal muscle in the isolated ileum of the guinea pig, sensitive to tetrodotoxin and atropine, were depressed and most often abolished by the GABAA receptor antagonists, bicuculline methiodide, RU 5135, and picrotoxin, a Cl- -ionophore blocker, as well as by GABA desensitization. 3-Mercaptopropionic acid, known to prevent GABA release, also reduced these naturally occurring spontaneous contractions. All these strongly indicate a physiological involvement of endogenous GABA in the control of spontaneous rhythmic activity in the intestine.