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Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [3H] serotonin or [3H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABAA receptor agonist muscimol (3 to 30 μM) and the GABAB receptor agonist baclofen (30 and 100 μM) inhibited [3H]serotonin and [3H]GABA release. These effects of muscimol were reversed by the GABAA antagonists bicuculline (100 μM). The GABAB antagonist phaclofen (100 μM) also antagonized the baclofen-induced inhibition of [3H]serotonin and [3H]GABA release. Phaclofen by itself increased [3H]serotonin release but it did not alter [3H]GABA overflow. Muscimol (10 μM) and baclofen (100 μM) also inhibited [3H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABAA and GABAB receptors located on serotonergic neurons. The 5-HT1A receptor agonist 8-OH-DPAT (0.01 to 1 μM) and the 5-HT1B receptor agonist CGS-12066A (0.01 to 1 μM) inhibited the electrically stimulated [3H]serotonin and [3H]GABA release. The 5-HT1A antagonist WAY-100135 (1 μM) was without effect on [3H]serotonin and [3H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 μM) did not alter the inhibitory effect of CGS-12066A (1 μM) on [3H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 μM) to reduce [3H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 μM) still inhibited [3H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 μM) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT1A/B and GABAA/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.

Slices from rat midbrain containing the raphe nuclei and from hippocampus were prepared, loaded with [3H]5-HT and superfused and the resting and the electrically stimulated [3H]5-HT release was measured. The 5-HT3 receptor agonist 2-methyl-5-HT (1 to 10 micromol/l) increased the resting tritium outflow in superfused raphe nuclei slices, EC50 5.3 micromol/l. The 2-methyl-5-HT-induced increase of tritium outflow was an external Ca2+-independent process and was not altered by reserpine pretreatment but it was reversed by addition of the 5-HT uptake inhibitor fluoxetine (1 micromol/l). The 5-HT3 receptor antagonists ondansetron and GYKI-46 903 (1 micromol/l) did not antagonize the stimulatory effect of 2-methyl-5-HT on resting tritium outflow. 2-Methyl-5-HT in lower concentration increased the electrically induced tritium overflow from raphe nuclei slices (EC50 0.56 micromol/l) and also from hippocampal slices preloaded with [3H]5-HT. These effects were reversed by 1 micromol/l of ondansetron and GYKI-46903. The 5-HT3 receptor antagonists (1 micromol/l) were without effects on depolarization-evoked [3H]5-HT release at 2 Hz stimulation, when 10 Hz stimulation was used, ondansetron and GYKI-46 903 reduced the tritium overflow from raphe nuclei slices. These data indicate that 5-HT3 receptors positively alter depolarization-induced somatodendritic 5-HT release in the raphe nuclei. They also show that 2-methyl-5-HT is able to evoke 5-HT release not only from vesicles but also from cytoplasmic stores via a transporter-dependent exchange process.

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [3H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium efflux from raphe nuclei slices preloaded with [3H]serotonin, and this inhibition was reversed by the 5-HT1A receptor antagonist (+)WAY-100135. The 5-HT1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [3H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubated with [3H]serotonin was completely external Ca(2+)-dependent, and omega-conotoxin GVIA and Cd2+, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced tritium release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT1A and 5-HT1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca2+ channels; (3) the 5-HT1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K+ channels.

Stroke or cerebrovascular accident reduces blood flow and decreases oxygen supply (ischemia) in brain tissue. This may be resulted from vascular obstruction when a blood vessel is blocked or by hemorrhage when bleeding occurs into the brain tissue. Decrease in oxygen supply shifts pH to acidosis and increases extracellular K+ concentration, which depolarizes neural cell membrane. Anoxic depolarization leads to excessive release of glutamate, which then activates various glutamate receptors in the synapse or the extrasynaptic space. Opening of ionotropic glutamate receptors (NMDA, AMPA and kainate receptors) causes influx of Na+ through the activated glutamate-gated ion channels. In response to anoxia, Ca2+ also enters the cells in excessive amounts via activated NMDA receptors and Ca2+-permeable AMPA receptors. This will lead to activation of several Ca2+-dependent intracellular signal transduction pathways (proteases, kinases, endonucleases, lipoxygeneses and nitric oxide synthase), which ultimately leads to neural death (Vizi et al., 1996; Parsons et al., 1998).