Two structurally different T-type Ca 2+ channel inhibitors, mibefradil and pimozide, protect CA1 neurons from delayed death after global ischemia in rats.

Recent in vitro evidence suggests that T-type Ca(2+) channels are implicated in the mechanisms of ischemia-induced delayed neuronal cell death. The aim of this work was to study the neuroprotective potential of mibefradil and pimozide, both T-type Ca(2+) channel inhibitors, in an in vivo rat model of global ischemia. We performed blinded and randomized placebo vs. treatment experiments using 57 animals to test mibefradil and fourteen animals to test pimozide. Each treated animal received a single stereotactic intraventricular injection of mibefradil or intraperitoneal injection of pimozide prior to transient global cerebral ischemia. The primary endpoint was the number of neurons surviving in the CA1 region 72 h after insult as evaluated by NeuN-labeled cell counts. All physiological variables monitored immediately before and after ischemic insult were equivalent between all groups. Surviving neurons in the CA1 region were significantly more frequent in the treated groups compared to the placebo group (mibefradil: 36.8 ± 2.8 cells in a 200 × 100 µm counting area vs. placebo: 25.2 ± 3.2 [P < 0.01]; pimozide: 39.4 ± 1.12 vs. placebo: 27.8 ± 0.7 [P < 0.0001]). Thus, administration of mibefradil or pimozide effectively prevents neuronal death after ischemia in a rat model of global ischemia. This study provides further support for a neuroprotective effect of T-type Ca(2+) current inhibition during ischemia.

Inhibition of T-type calcium channels protects neurons from delayed ischemia-induced damage.

Intracellular calcium increase is an early key event triggering ischemic neuronal cell damage. The role of T-type voltage-gated calcium channels in the neuronal response to ischemia, however, has never been studied. Using an in vitro model of ischemia-induced delayed cell death in rat organotypic hippocampal slice cultures, we show that T-type calcium channels inhibitors drastically reduce ischemic cell damage. Immunostaining studies reveal the existence of Ca(V)3.1 and Ca(V)3.2 types of low-voltage-activated calcium channels in rat organotypic hippocampal cultures. Low extracellular calcium (100 nM) or increase of intracellular calcium buffering ability by BAPTA-acetoxymethyl ester significantly reduced ischemia-induced neuronal damage. Pharmacological inhibition of the T-type calcium current by mibefradil, kurtoxin, nickel, zinc, and pimozide during the oxygen-glucose deprivation episode provided a significant protection against delayed neuronal death. Mibefradil and nickel exerted neuroprotective effects, not only if administrated during the oxygen-glucose deprivation episode but also in conditions of postischemic treatment. These data point to a role of T-type calcium currents in ischemia-induced, calcium-mediated neuronal cell damage and suggest a possible new pharmacological approach to stroke treatment.

Evidence for a direct projection from the paraventricular nucleus of the hypothalamus to putative serotoninergic neurons of the nucleus paragigantocellularis involved in the control of erection in rats.

In the male rat, serotoninergic neurons of the ventrolateral medulla send direct projections onto spinal preganglionic neurons that innervate the penis. The role of the paraventricular nucleus of the hypothalamus in the control of penile erection is well recognized. Our aim was to demonstrate anatomical relation between paraventricular neurons and medullary serotoninergic neurons innervating the penis. In adult male rats, stereotaxic iontophoretic injections of Phaseolus vulgaris leuco-agglutinin were performed in the paraventricular nucleus. Neurons in the ventrolateral medulla were retrogradely labelled using transneuronal retrograde transport of pseudorabies virus injected in the corpus cavernosum. Sections of the ventro-lateral medulla were processed for double immunofluorescence to reveal both Phaseolus vulgaris leuco-agglutinin and pseudorabies virus using specific antibodies. Sections were also processed for the simultaneous detection of pseudorabies virus and serotonin. Pseudorabies virus-infected neurons in the ventrolateral medulla were present in the nucleus paragigantocellularis, reticular formation of the medulla, raphe pallidus and raphe magnus. In the nucleus paragigantocellularis, all pseudorabies virus-infected-neurons were immunoreactive for serotonin. Some of them received Phaseolus vulgaris leuco-agglutinin-labelled varicose fibres that ran along the soma of pseudorabies virus-infected neurons. Confocal microscopy suggested the presence of several close appositions between them, which were demonstrated using three-dimensional reconstruction of serial optical sections. Our results show that paraventricular neurons send direct projections in the nucleus paragigantocellularis onto neurons that innervate the penis. They suggest a possible role of the paraventricular nucleus in penile erection through the control of descending serotoninergic raphe-spinal neurons. The neurotransmitter used in this pathway remains to be determined.

Relationships of 5-hydroxytryptamine immunoreactive terminal-like varicosities to 5-hydroxytryptamine-2A receptor-immunoreactive neuronal processes in the rat forebrain.

The distributions of 5-hydroxytryptamine (5-HT)-immunoreactive (IR) varicosities and 5-hydroxytryptamine-2A receptor (5-HT2A)-IR neuronal structures in the rat brain have previously been described individually. Using double labeling immunocytochemistry, the relationships between 5-HT2A-IR and 5-HT-IR elements in the forebrain of male rats has been studied at the light microscopic level. In neocortical regions (frontal, parietal and retrosplenial cortex), the strongest 5-HT2A-IR was found in the apical dendrites of pyramidal cells in layers III-V, while 5-HT-IR terminal-like varicosities were present in all layers but most prominently in the outer layers. In other forebrain regions, the olfactory bulb, the hippocampal formation, and the islands of Calleja and Calleja magna, localized discrepancies were present between the 5-HT2A-IR neuronal profiles and the 5-HT-IR terminal-like varicosities. Hardly any additional juxtapositions between the 5-HT2A-IR neuronal profiles and 5-HT-IR terminal-like varicosities were revealed when the intraneuronal level of 5-HT was increased by monoamine oxidase inhibitor pretreatment (nialamide, 250 mg/kg, 3 h). Thus, in most forebrain regions, there were overall few juxtapositions between 5-HT terminal-like varicosities and 5-HT2A-IR neuronal structures. This observation suggests that 5-HT2A receptor mediated 5-HT transmission in the rat forebrain is mainly a volume transmission process mediated via short distance diffusion in the extra-cellular space.

Localization of 5-HT(6) receptors at the plasma membrane of neuronal cilia in the rat brain.

5-HT(6) receptor-like immunoreactivity has been previously found in association with both neuronal dendrites and cilia in the striatum, nucleus accumbens, olfactory tubercle and islands of Calleja of the rat brain. Using immunogold pre-embedding immunocytochemical techniques to investigate the subcellular localization of 5-HT(6) receptor-like immunoreactivity in cilia, we showed that immunogold particles were associated with their plasma membrane, and not with microtubules. This membrane localization is consistent with a possible physiological role, which is still unknown, of these receptors.

Plasticity of 5-hydroxytryptamine(1B) receptors during postnatal development in the rat visual cortex.

The distribution of 5-hydroxytryptamine1A and 5-hydroxytryptamine1B receptors in the visual cortex was studied by quantitative autoradiography during postnatal development. Overall, receptor densities increased throughout development, but exhibited regional rearrangements, particularly in the case of 5-hydroxytryptamine1B receptors. Neonatal treatment with 5,7-dihydroxytryptamine, which causes selective degeneration of serotoninergic neurons, had no effect on the density of 5-hydroxytryptamine1A receptors in the visual cortex. However, a transient increase in 5-hydroxytryptamine1B at postnatal days 10-12 was observed after this treatment, suggesting a regulation of postsynaptic receptors. Neonatal enucleation resulted in a marked increase in 5-hydroxytryptamine1B binding sites in all layers of the visual cortex by P16, whereas it had no effect upon 5-hydroxytryptamine1A binding sites. These results show that both receptor subtypes do not exhibit striking transient features in the visual cortex during postnatal development, but rather undergo discrete reorganizations. 5-Hydroxytryptamine1B receptors show changes in density after either neonatal degeneration of serotoninergic neurons or enucleation, indicating that the serotoninergic system involving this receptor subtype can exhibit some postnatal plasticity in the visual cortex.

5-Hydroxytryptamine2C receptors on spinal neurons controlling penile erection in the rat.

The localization of 5-hydroxytryptamine2C receptors in the lumbosacral spinal cord of the rat was investigated using selective antibodies raised against the carboxyl-terminal part of the rat receptor. The distribution of immunoperoxidase labelling at the light microscope level revealed numerous labelled neurons in the gray matter, with a higher intensity in the sacral parasympathetic nucleus, the dorsal gray commissure and particularly the motoneurons of the ventral horn. Confocal microscope analysis showed that immunostaining was mainly intracellular (motoneurons), but could also be associated with the membrane of cell bodies and dendrites. Actually, electron microscope immunogold experiments demonstrated an exclusive staining of the cis-Golgi apparatus. Following pseudo-rabies virus transsynaptic retrograde labelling from the corpus cavernosum, labelled neurons were found in the sacral parasympathetic nucleus and the dorsal gray commissure of the L6-S1 segments. All virus-labelled neurons exhibited 5-hydroxytryptamine2C receptor immunoreactivity. These results indicate that all parasympathetic preganglionic neurons and their related interneurons which contribute to the innervation of cavernosal tissue bear 5-hydroxytryptamine2C receptors. In the sacral parasympathetic nucleus, most neurons which were retrogradely-labelled from the pelvic ganglion with Fast Blue also showed 5-hydroxytryptamine2C receptor immunoreactivity. In the ventral horn, motoneurons retrogradely labelled from the ischiocavernosus muscle and the bulbospongiosus muscle, both of which are involved in erection and ejaculation, were also 5-hydroxytryptamine2C receptor-immunopositive. The supraspinal serotoninergic control of erection at the lumbosacral level therefore appears to be strongly associated with the activation of 5-hydroxytryptamine2C receptors, consistent with the proerectile properties of 5-hydroxytryptamine2C agonists.

Light and electron microscopic immunocytochemical visualization of 5-HT1B receptors in the rat brain.

Specific antipeptide antibodies were used for the immunohistochemical visualization of 5-HT1B receptors in the rat brain. A dense, specific 5-HT1B receptor-like immunoreactivity was found in the globus pallidus, the dorsal subiculum and the substantia nigra. At the light microscope level, immunostaining was diffuse within the neuropil but absent from cell bodies. Observations at the electron microscope level in the substantia nigra showed immunoperoxidase staining in fine unmyelinated axons and nerve terminals.