M. Riad

Immunocytochemical localization of serotonin1A receptors in the rat central nervous system

Specific anti‐rat 5‐hydroxytryptamine1A (serotonin1A) receptor antibodies raised in a rabbit injected with a synthetic peptide corresponding to a highly selective portion of the third intracellular loop of the receptor protein (El Mestikawy et al. [1990] Neurosci. Lett. 118: 189–192) were used for immunohistochemical mapping of serotonin1A receptors in the brain and spinal cord of adult rats. The highest density of immunostaining was found in limbic areas (lateral septum, CA1 area of Ammons horn and dentate gyrus in the hippocampus, and frontal and entorhinal cortices), in the anterior raphe nuclei, and in the interpeduncular nucleus, in agreement with previous autoradiographic studies with selective radioligands showing the enrichment of these regions in serotonin1A receptor binding sites. Serotonin1A receptor‐like immunoreactivity was also present, but at a moderate level, in the neocortex, in some thalamic and hypothalamic nuclei, in the nucleus of the solitary tract, in the dorsal tegmentum, in the nucleus of the spinal tract of the trigeminal nerve, and in the superficial layers of the dorsal horn in the spinal cord. In contrast, extrapyramidal areas, including the caudate putamen, the globus pallidus, and the substantia nigra as well as the cerebellum, exhibited very low to no immunostaining by antiserotonin1A receptor antibodies.

Production of specific anti-rat 5-HT1A receptor antibodies in rabbits injected with a synthetic peptide

Polyclonal antibodies were raised by the repeated injection of rabbits with a synthetic peptide corresponding to a highly selective portion (amino acid residues 243 to 268) of the amino acid sequence of the rat 5-HT1A receptor. The anti-peptide antiserum allowed the immunoprecipitation of 5-HT1A receptors but not of other 5-HT1 sites solubilized from rat hippocampal membranes. Immunoautoradiographic labelling of rat brain sections with the anti-peptide antiserum was superimposed with the autoradiographic distribution of 5-HT1A sites labelled by the selective radioligand [3H]8-OH-DPAT.

Effect of the selective lesion of serotoninergic neurons on the regional distribution of 5-HT1A receptor mRNA in the rat brain.

The effects of the selective lesion of serotoninergic neurons by an intra-raphe administration of 5,7-dihydroxytryptamine on the 5-HT1A receptor protein and the 5-HT1A receptor mRNA were examined in various regions of the rat brain using specific antibodies and an antisense riboprobe, respectively. Twenty one days after the treatment, the 5-HT1A receptor protein was no longer detected within the dorsal raphe nucleus but was still present in the hippocampus and entorhinal cortex. Quantitative in situ hybridization showed an 85% decrease in the levels of 5-HT1A receptor mRNA within the dorsal raphe nucleus, but no significant change in the hippocampus, interpeduncular nucleus and entorhinal cortex of 5,7-dihydroxytryptamine-treated rats. These data demonstrate that 5-HT1A receptors are synthesized by serotoninergic neurons in the dorsal raphe nucleus, and by neurons located postsynaptically with regard to serotoninergic projections in other areas. The unchanged levels of 5-HT1A receptor mRNA in the hippocampus, interpeduncular nucleus and entorhinal cortex three weeks after the extensive lesion of serotoninergic neurons are consistent with the absence of 5-HT1A receptor up regulation already reported under this condition.

Trophic Effects of Neurotransmitters during Brain Maturation

Besides their neurotransmitter and/or neuromodulatory roles, many neuroactive substances synthesized and released during brain development can also directly influence neuronal differentiation. Transitory expression of neurotransmitters, their metabolic enzymes and their receptors is only one aspect of this trophic role. The most considerable progress in neurotrophic factor research has been made with the use of primary cultures of neuronal cells, and numerous studies have focused on the effects of neurotransmitters on the differentiation of cells at various stages of development. Thus, several neuropeptides like VIP, substance P, enkephalins, somatostatin, and monoamines, can modulate neuronal differentiation, but only during a limited period of fetal life. Among the monoamines, it was shown that, depending on the target, 5-HT stimulates the development of the neuropile, the myelinization of axons, the differentiation of the synaptic contacts, induces markers of monoaminergic neuron differentiation, inhibits the development of the growth cone, decreases the branching of neurites, and influences the survival, cell body size, and neurite outgrowth in several neuronal cultures. 5-HT can also indirectly influence the differentiation of serotonergic neurons by the intermediate of astrocytes, and it was shown in our laboratory that 5-HT1A agonists can stimulate the cholinergic parameters of primary cultures of rat fetal septal neurons. At the molecular level, the events triggered by neurotransmitters that underlie their neurotrophic action probably involve the transmembrane influx of calcium. To date, calcium regulation of cellular processes is one of the most rapidly expanding areas of research in developmental neurobiology.

Neurotrophic effects of ipsapirone and other 5-HT1A receptor agonists on septal cholinergic neurons in culture

Repeated treatment of primary cultures of fetal rat septal neurons with 5-HT1A receptor agonists (8-OH-DPAT, ipsapirone, gepirone and buspirone) increased choline acetyltransferase activity after 6-7 days in culture. This effect was optimal with ipsapirone (+ 50-80% at 1 microM of the agonist), and could be prevented by potent 5-HT1A receptor antagonists such as (-)-tertatolol and (+)-WAY 100135. Under conditions where they completely suppressed the stimulatory effect of NGF on choline acetyltransferase in these cultures, specific anti-NGF antibodies did not alter the stimulatory effect of ipsapirone, suggesting that a possible release of NGF from some septal cells did not account for the effect of 5-HT1A receptor stimulation. Autoradiographic investigations with [3H]8-OH-DPAT as radioligand and immunocytochemistry with specific anti-choline acetyltransferase antibodies and anti-rat 5-HT1A receptor antibodies showed that 5-HT1A receptors were expressed on septal neurons in culture, notably on the cholinergic neurons identified by their positive staining with anti-choline acetyltransferase antibodies. Detailed morphometrical analysis by computer-assisted imaging revealed that repeated exposure to ipsapirone (1 microM for 7 days) did not influence the survival of cholinergic as well as non-cholinergic neurons, but specifically altered the neuritic tree (i.e. the total length of neurites and the number of branching points) of cholinergic neurons only. These data suggest that under in vitro conditions ipsapirone and other 5-HT1A receptor agonists may exert a direct trophic action on septal cholinergic neurons.

Characteristics of [14C]guanidinium accumulation in NG 108-15 cells exposed to serotonin 5-HT3 receptor ligands and substance P.

Abstract: In the presence of substance P (SP; 10 μM), serotonin (5‐HT; 1 μM) triggered a cation permeability in cells of the hybridoma (mouse neuroblastoma X rat glioma) clone NG 108‐15 that could be assessed by measuring the cell capacity to accumulate [14C]guanidinium for 10‐15 min at 37°C. In addition to 5‐HT (EC50, 0.33 μM), the potent 5‐HT3 receptor agonists 2‐methyl‐serotonin, phenylbiguanide, and m‐chlorophenylbiguanide, and quipazine, markedly increased [14C]guanidinium uptake in NG 108‐15 cells exposed to 10 μM SP. In contrast, 5‐HT3 receptor antagonists prevented the effect of 5‐HT. The correlation (r= 0.97) between the potencies of 16 different ligands to mimic or prevent the effects of 5‐HT on [14C]guanidinium uptake, on the one hand, and to displace [3H]zacopride specifically bound to 5‐HT3 receptors on NG 108‐15 cells, on the other hand, clearly demonstrated that [14C]guanidinium uptake was directly controlled by 5‐HT3 receptors. Various compounds such as inorganic cations (La3+, Mn2+, Ba2+, Ni2+, and Zn2+), D‐tubocurarine, and memantine inhibited [14C]guanidinium uptake in NG 108‐15 cells exposed to 5‐HT and SP, as expected from their noncompetitive antagonistic properties at 5‐HT3 receptors. However, ethanol (100 mM), which has been reported to potentiate the electrophysiological response to 5‐HT3 receptor stimulation, prevented the effects of 5‐HT plus SP on [14C]guanidinium uptake. The cooperative effect of SP on this 5‐HT3‐evoked response resulted neither from an interaction of the peptide with the 5‐HT3 receptor binding site nor from a possible direct activation of G proteins in NG 108‐15 cells. Among SP derivatives, [D‐Pro9]SP, a compound inactive at the various neurokinin receptor classes, was the most potent to mimic the stimulatory effect of SP on [14C]guanidinium uptake in NG 108‐15 cells exposed to 5‐HT. Although the cellular mechanisms involved deserve further investigations, the 5‐HT‐evoked [14C]guanidinium uptake appears to be a rapid and reliable response for assessing the functional state of 5‐HT3 receptors in NG 108‐15 cells.

Endothelial expression of the 5-hydroxytryptamine1B antimigraine drug receptor in rat and human brain microvessels

In addition to triggering vasoconstriction of peripheral blood vessels, which led to its discovery as a circulating neurohormone 50 years ago, serotonin (5-hydroxytryptamine) acts as a neurotransmitter/ modulator in the central nervous system and regulates local cerebral blood flow and vascular permeability through direct and indirect effects on intraparenchymal microvessels. Among the various 5-hydroxytryptamine receptors which mediate these effects, particular attention has been paid to the 5-hydroxytryptamine1B and 5-hydroxytryptamine1D subtypes, as the preferred targets of modern antimigraine agents. Immunoelectron microscopic labeling of the 5-hydroxytryptamine1B receptor in rat brain parenchyma has revealed a distinct localization to the endothelium of microvessels, which was predominantly cytoplasmic as opposed to membrane-bound, contrary to that on preterminal unmyelinated axons [Riad et al. (1997) Soc. Neurosci. Abstr. 23, 1214]. Similar observations have now been made in human cortical tissue, in which the expected localization of the vascular 5-hydroxytryptamine1B receptor to periarteriolar myocytes was also confirmed. Such a dual localization in human brain microvessels suggests that the 5-hydroxytryptamine1B receptor might mediate opposite effects, vasodilatory and contractile, depending upon its activation by circulating or centrally released 5-hydroxytryptamine. It raises new possibilities as regards 5-hydroxytryptamine effects on human brain microvessels in health and disease, and notably the triggering of migraine headache.

Unexpected localization of the Na+/Cl–-dependent-like orphan transporter, Rxt1, on synaptic vesicles in the rat central nervous system

Numerous features of its primary structure demonstrate that the orphan transporter Rxt1 belongs to the Na+/Cl–‐dependent neurotransmitter plasma membrane transporter superfamily, which includes the dopamine, norepinephrine, serotonin and γ‐aminobutyric acid (GABA) transporters. Initial immunocytochemical investigations with affinity‐purified antibodies have established that Rxt1 is localized, almost exclusively, in axon terminals of glutamatergic neurons and subsets of GABAergic neurons in the CNS. Further studies were carried out to determine its subcellular distribution. In a first series of experiments, PC‐12 cells were transfected with plasmids encoding either the dopamine transporter or Rxt1. Immunofluorescence experiments showed that the dopamine transporter was expressed in these cells, and, as expected, addressed to their plasma membrane. Surprisingly, this was never the case with Rxt1, which was targeted to the same subcellular compartment as synaptophysin, a vesicular protein. In a second set of experiments, subcellular fractionation of rat striatum showed that Rxt1, but not the dopamine transporter, was relatively abundant in the purified synaptic vesicle fraction. Finally, electron microscopic immunocytochemistry with anti‐Rxt1 antibodies showed peroxidase as well as pre‐ and post‐embedding immunogold labelling confined to the intracellular compartment in various brain regions. Moreover, quantitative analysis of post‐embedding experiments demonstrated that the immunogold particles corresponding to Rxt1 immunoreactivity were mostly localized to small synaptic vesicles. These data indicate that, in contrast with the other members of the Na+/Cl–‐dependent neurotransmitter transporter superfamily, which are targeted to the plasma membrane, Rxt1 is distributed as a vesicular protein in the CNS.

Visualization and quantification of central 5-HT1A receptors with specific antibodies

Abstract Polyclonal antibodies were raised by the repeated injection of rabbits with synthetic peptides corresponding to selective portions (peptide 1: aminoacid residues 12–23, and peptide 2: aminoacid residues 243–268) of the aminoacid sequence of the rat 5-HT1A receptor. Both antisera allowed the immunoprecipitation of 5-HT1A receptors but not of other 5-HT receptor types and adrenergic receptors solubilized from rat hippocampal membranes. Immunoblots demonstrated that a single protein of ∼63 kDa, corresponding to the molecular weight of the rat 5-HT1A receptor binding subunit, was recognized by each antiserum. Immunoautoradiographic labelling of rat brain sections with the anti-peptide 2-antiserum exhibited the same regional distribution as 5-HT1A sites labelled by selective radioligands such as [3H]8-OH-DPAT and [125I]BH-8-MeO-N-PAT. However regional differences apparently existed between the respective intensity of labelling by the agonist radioligands and the antiserum, which might be explained by variations in the degree of coupling of 5-HT1A receptor binding subunits with G proteins from one brain area to another.

Pharmacological Evidence for the Involvement of Calciuml Calmodulin in Serotonin 5‐HT3 Receptor‐Mediated Cation Permeability in NG 108‐15 Cells

Abstract: In NG 108–15 clonal cells, extracellular application of micromolar concentrations of serotonin [5‐hydroxy‐tryptamine (5‐HT)] and substance P induces the opening of a cation permeability monitored by the influx of [14C]‐guanidinium. The serotoninergic component of this cation permeability Is linked to 5‐HT3 receptor activation, whereas the substance P component probably involves an “N‐terminal‐dependent substance P receptor.” In this study, [14C]guanidinium influx triggered by 1 μM 5‐HT plus 10 μM substance P was shown to be insensitive to tetrodotoxin, verapamil, diltiazem, nimodipine, and ω‐conotoxin, as expected from a process independent of voltage‐sensitive sodium and calcium channels. In contrast, [14C]guanidinium influx was inhibited by millimolar concentrations of extracellular calcium and by the chelation of intracellular calcium by bis‐O‐aminophenoxyethanetetraacetic acid. The inhibition by extracellular calcium apparently involved a competition between the divalent cation and [14C]guanidinium for the same channel. When NG 108–15 cells were exposed to X537A, an ionophore that specifically induces release of calcium from intracellular stores, [14C]guanidinium uptake was markedly increased even in the absence of 5‐HT and/or substance P. Conversely, [14C]guanidinium influx due to the latter substances could be reversibly and dose‐dependently blocked by various drugs that possess calmodulin‐antagonizing properties. These results strongly suggest that the cation permeability opened by 5‐HT and substance P in NG 108–15 cells involves a calcium/calmodulin‐dependent process. However, as the phosphodiesterase inhibitor isobutylmethylxanthine, the nitric oxide synthase inhibitor A/monomethylarginine, the protein kinase C inhibitor staurosporine, and the protein kinase C activator 12‐O‐tetradeca‐noylphorbol 13‐acetate did not alter [14C]guanidinium influx in NG 108–15 cells exposed to 5‐HT and substance P., this process probably does not involve the calcium‐dependent nitric oxide pathway and protein kinase C activation.