Following the cloning and sequencing of the A subunit of the 5‐HT3 receptor, two alternatively spliced isoforms, 5‐HT3‐AS and 5‐HT3‐AL, have been identified. In order to analyse the distribution of the receptor, a polyclonal antibody has been produced against the short form which is the most abundant in the central nervous system [Doucet et al. (2000) Neuroscience 95, 881–892]. As expected from the recognition of functional 5‐HT3 receptors, immunostaining by this anti‐5‐HT3‐R‐AS antibody matched the distribution of the high‐affinity 5‐HT3 binding sites in the rat brain and spinal cord. 5‐HT3‐AS‐like immunoreactivity was detected at low levels in the limbic system, particularly in the amygdala and the hippocampus, and in the frontal, piriform and entorhinal cortices. High levels of immunoreactivity were found in the brainstem, mainly in the nucleus tractus solitarius and the nucleus of the spinal tract of the trigeminal nerve, and in the dorsal horn of the spinal cord. At the ultrastructural level, immunostaining was generally found associated with axons and nerve terminals (70–80%) except in the hippocampus, where labelled dendrites were more abundant (56%). This preferential localization on nerve endings is consistent with the well‐documented physiological role of 5‐HT3 receptors in the control of neurotransmitter release. However, the different distribution in the hippocampus raises the question of whether differential addressing mechanisms exist for preferentially targeting 5‐HT3 receptors to postsynaptic dendritic sites as compared to presynaptic nerve endings, depending on the nature of the neurons bearing these receptors.

Serotonin-1A (5-HT1A) receptors in the CNS are a major target for psychotropic drugs. In nucleus raphe dorsalis (NRD) and hippocampus (CA3), the selective 5-HT1A agonist (+)-8-hydroxy-2-(di-N-propylamino) tetralin (8-OH-DPAT) reduces the firing activity of serotoninergic (5-HT) and pyramidal neurons, respectively. When located on 5-HT (autoreceptors), but not on non-5-HT (heteroreceptors) neurons, 5-HT1A receptors are known to be subject to desensitization. Using quantitative electron microscopy after pre-embedding immunogold labeling with specific antibodies, we examined the subcellular distribution of these receptors after acute administration of 8-OH-DPAT (0.5 mg/kg, i.v.). Silver-intensified immunogold particles associated with the plasma membrane or the cytoplasm were counted in somata and dendrites within the NRD, 15 min, 1 hr and 24 hr after 8-OH-DPAT injection, and in hippocampal dendrites 1 hr after the same treatment. Significant decrease in the density of membrane labeling and concomitant increase of cytoplasmic labeling were demonstrated in the NRD, 15 min and 1 hr after 8-OH-DPAT administration, with a return to baseline level at 24 hr. Internalization was blocked by previous administration of the 5-HT1A antagonistN-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexane-carboxamide (WAY 100635), which, by itself, was without apparent effect. In hippocampus (CA3), there were no apparent changes in the distribution of the receptor after 8-OH-DPAT administration. These findings are in line with earlier results showing a desensitization of 5-HT1A autoreceptors but not heteroreceptors after treatment with 5-HT1A receptor agonist. They suggest that this desensitization is the result of autoreceptor internalization.

The 5-HT1A and 5-HT1B receptors of serotonin play important roles as auto- and heteroreceptors controlling the release of serotonin itself and of other neurotransmitters/modulators in the central nervous system (CNS). To determine the precise localization of these receptors, we examined their respective cellular and subcellular distributions in the nucleus raphe dorsalis and hippocampal formation (5-HT1A) and in the globus pallidus and substantia nigra (5-HT1B), using light and electron microscopic immunocytochemistry with specific antibodies. Both immunogold and immunoperoxidase preembedding labelings were achieved. In the nucleus raphe dorsalis, 5-HT1A immunoreactivity was found exclusively on neuronal cell bodies and dendrites, and mostly along extrasynaptic portions of their plasma membrane. After immunogold labeling, the density of membrane-associated 5-HT1A receptors could be estimated to be at least 30–40 times that in the cytoplasm. In the hippocampal formation, the somata as well as dendrites of pyramidal and granule cells displayed 5-HT1A immunoreactivity, which was also prominent on the dendritic spines of pyramidal cells. In both substantia nigra and globus pallidus, 5-HT1B receptors were preferentially associated with the membrane of fine, unmyelinated, preterminal axons, and were not found on axon terminals. A selective localization to the cytoplasm of endothelial cells of microvessels was also observed. Because the 5-HT1A receptors are somatodendritic, they are ideally situated to mediate serotonin effects on neuronal firing, both as auto- and as heteroreceptors. The localization of 5-HT1B receptors to the membrane of preterminal axons suggests that they control transmitter release from nonserotonin as well as serotonin neurons by mediating serotonin effects on axonal conduction. The fact that these two receptor subtypes predominate at extrasynaptic and nonsynaptic sites provides further evidence for diffuse serotonin transmission in the CNS. J. Comp. Neurol. 417:181–194, 2000. ©2000 Wiley-Liss, Inc.

Polyclonal antibodies were raised against a synthetic hexadecapeptide corresponding to the portion of the second intracytoplasmic loop of the short form of the mouse 5-hydroxytryptamine-3A receptor subunit (5-HT3A-S), which differs from the long form (5-HT3A-L) by the removal of six amino acids. Antibodies were detected by enzyme-linked immunosorbent assay as soon as two months after the first injection to rabbits of the peptide coupled to keyhole limpet hemocyanin. Immunoblot detection of fusion proteins comprising glutathione-S-transferase and the second intracellular loop of 5-HT3A-S or 5-HT3A-L, and immunoprecipitation of cloned receptors showed that antibodies exhibited some selectivity for the short variant. Affinity chromatography allowed the purification of selective anti-5-HT3A-S antibodies which yielded a strong positive labeling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing murine 5-HT3A-S. In contrast, COS-7 cells expressing similar levels of 5-HT3A-L exhibited only a very weak labeling. Selectivity was also observed on immunoblots of cloned receptors transiently expressed in COS-7 cells, or stably expressed in CHO cells, both systems showing an immunolabeled component at 53,000–54,000 mol. wt. Immunoautoradiographic labeling of central nervous system sections showed that 5-HT3A-S-like immunoreactivity was found mostly within the nucleus of the solitary tract, the nucleus of the spinal tract of the trigeminal nerve, and the dorsal horn of the the spinal cord in the rat. After unilateral ablation of the nodose ganglion, 5-HT3A-S-like immunoreactivity decreased markedly in the ipsilateral part of the nucleus of the solitary tract, as expected of the presynaptic localization of 5-HT3 receptors. Finally, immunohistochemistry at the light and electron microscope levels revealed that 5-HT3A-S-like immunoreactivity was associated essentially with terminals and axonal profiles. All these results demonstrate that the immunolabeling exhibited by these antibodies is consistent with a specific and partially selective recognition of the short isoform of the 5-HT3A subunit. Because the pattern of immunoautoradiographic labeling matches the distribution previously established with selective radioligands, it can be inferred that these antibodies probably recognized the same fully assembled form of the 5-HT3A-S receptor subunit.

The effects of long-term (7, 14 or 21 days) administration of the 5-HT1A receptor agonist alnespirone [5 mg/(kg day), i.p.] on the binding characteristics of 5-HT1A, 5-HT2A and 5-HT3 receptors, and the functional status of 5-HT1A autoreceptors were assessed using biochemical and electrophysiological approaches in rats. Whatever the treatment duration, the specific binding of [3H]8 hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT), [3H]trans,4-[(3Z)3-(2-dimethylaminoethyl) oxyimino-3(2-fluorophenyl) propen-1-yl] phenol hemifumarate ([3H]SR 46349B), and [3H]S-zacopride to 5-HT1A, 5-HT2A and 5-HT3 receptors, respectively, were unaltered in all the brain areas examined. In contrast, in vitro electrophysiological recordings performed 24 h after the last injection of alnespirone showed that the potency of the 5-HT1A receptor agonist, 8-OH-DPAT, to depress the firing of serotoninergic neurons in the dorsal raphe nucleus, was significantly reduced after a 21-day treatment with alnespirone. However, no changes were noted after a 7-day or 14-day treatment. These data indicate that desensitization of somatodendritic 5-HT1A autoreceptors is a selective but slowly developing adaptive phenomenon in response to their chronic stimulation in rats.

The localization of 5-hydroxytryptamine1B receptors in the rat central nervous system was investigated using anti-peptide antibodies that recognize a selective portion of the third intracytoplasmic loop of the receptor protein. At the light microscope level the densest 5-hydroxytryptamine1B receptor-like immunoreactivity was observed in ventral pallidum, globus pallidus, substantia nigra and dorsal subiculum. In addition, moderate immunoreactivity was found in the entopeduncular nucleus, the superficial gray layer of the superior colliculus, the caudate–putamen and the deep nuclei of the cerebellum. This distribution matched perfectly that previously described from radioligand binding studies. At the ultrastructural level, 5-hydroxytryptamine1B receptor-like immunoreactivity was associated with axons and axon terminals in the three areas examined: substantia nigra, globus pallidus and superficial gray layer of the superior colliculus. In all cases, immunostaining was located on the plasma membrane of unmyelinated axon terminals and in the cytoplasm close to the plasmalemma. Synaptic differentiations were never labelled, but, in some cases, 5-hydroxytryptamine1B receptor-like immunoreactivity was found in their close vicinity. Injection of kainic acid into the neostriatum resulted in a marked decrease in receptor-like immunoreactivity in the globus pallidus and the substantia nigra, consistent with the location of 5-hydroxytryptamine1B receptors on terminals of striatopallidal and striatonigral fibres, respectively. A reduction in 5-hydroxytryptamine1B receptor-like immunoreactivity was also noted in the superficial gray layer of the superior colliculus after contralateral enucleation, as expected of the location of 5-hydroxytryptamine1B receptors on the terminals of retinocollicular fibres. In both lesion experiments, immunolabelled degenerating terminals were observed in the projection areas. Anterograde labelling experiments coupled with immunocytochemical detection further showed that 5-hydroxytryptamine1B receptors in the substantia nigra are located on axons of striatal neurons.These data provide anatomical supports to the idea that 5-hydroxytryptamine1B receptors act as terminal receptors involved in presynaptic regulation of the release of various neurotransmitters, including 5-hydroxytryptamine itself.

The mouse 5-hydroxytryptamine3 (5-HT3) type of serotonin receptors is expressed as two forms, 5-HT3R-AL and 5-HT3R-AS, generated by alternative splicing of its primary transcript, that differ by a stretch of six amino acids in the second intracellular loop domain. Because this six-amino acid region contains a putative phosphorylation site that may be important for the function and/or regulation of 5HT3R-AL receptor, specifically, we developed polyclonal antibodies as appropriate tools for studies relevant to this question. Antibodies against a 20-amino acid peptide corresponding to the sequence of 5-HT3R-AL at the level of this six-amino acid region were obtained as soon as one month after injection of this synthetic peptide to rabbits. Immunocytochemistry with these antibodies led to a strong positive labelling of plasma membrane, reticulum and Golgi apparatus of COS-7 cells expressing cloned murine 5-HT3R-AL, whereas COS-7 cells expressing similar levels of 5-HT3R-AS exhibited only a very weak labelling. Immunoblots of fusion proteins combining glutathion-S-transferase and the second cytoplasmic loop of 5-HT3R-AL or 5-HT3R-AS revealed a c. 20-fold selectivity of the antibodies for the first, long form, as evaluated by densitometric analysis of enhanced chemiluminescence detection. Similarly, immunoblots of COS-7 cells transfected with cloned 5-HT3 receptors showed that the anti-peptide antibodies detected a band at 53,000 mol. wt only in cells transfected with 5-HT3R-AL. Under optimal conditions, antibodies immunoprecipitated 52% of 5-HT3R-AL, but only 11% of 5-HT3R-AS, solubilized from COS-7 cells transfected with the respective encoding plasmids. In the rat, no immunoautoradiographic labelling by the anti-peptide antibodies could be detected in brain structures which had previously been described to express preferentially a short form of the 5-HT3 receptor. In contrast, a strong immunolabelling was found in the intestinal mucosa, especially in the rat fetus (at the 17th embryonic day), suggesting the possible participation of the 5-HT3R-AL isoform in the development of this tissue. These results show that specific antibodies are useful tools for the visualization of the least abundant 5-HT3 receptor isoform in rat tissue.

The regional distribution of the mRNA encoding the serotonin 5-HT1B receptor was studied in the central nervous system of the rat by in situ hybridization histochemistry and Northern blot analysis. A 180 base pair probe, corresponding to a highly selective portion of the third intracellular loop of the rat 5-HT1B receptor, was used. In most regions, a single 5 kb message was found by Northern blot analysis. However, two additional bands (2.5 and 4 kb) were detected in the striatum. The rank order of 5-HT1B mRNA abundance was striatum >> septum = ventral tegmentum greater than or equal to colliculi = hypothalamus = hippocampus > brain stem greater than or equal to cerebellum greater than or equal to dorsal horn of the spinal cord > cerebral cortex greater than or equal to ventral horn of the spinal cord > olfactory tubercle. This distribution was confirmed by in situ hybridization, which further revealed that the 5-HT1B mRNA was present in dorsal root ganglia, the layer IV of the cerebral cortex, the Purkinje cell layer of the cerebellum, the pyramidal neurons in the CA1 area of the hippocampus, and the dorsal and median raphe nuclei. In situ hybridization was also performed in nomifensine (10 mg/kg/i.p.)-pretreated rats whose serotoninergic neurons were extensively and selectively lesioned by microinjection of 5,7-dihydrox-ytryptamine (8 mu g/1 mu l) directly into the anteroventral vicinity of anterior raphe nuclei 3 weeks before sacrifice. In lesioned rats, 5-HT1B mRNA was present in the same areas and at the same levels as in control rats, except in the dorsal and median raphe nuclei, where a marked decrease (-75%) in its local concentration was observed. These data provide the first demonstration of the synthesis of 5-HT1B receptor within serotoninergic neurons, as expected of their presynaptic autoreceptor function at the level of serotoninergic terminals. (C) 1995 Wiley-Liss, Inc.

The localization of the rat brain 5-HTIA receptor mRNA was analyzed by RNAse mapping and in situ hybridization during postnatal development, particularly in the cerebellum. The regional distribution of 5-HTIA mRNA during the first 2 postnatal weeks was different from that found in adults. In some areas of the immature brain (hippocampus, cerebral cortex), 5-HTIA mRNA was found in lower density than in the adult brain. In contrast, high concentrations of the transcript were present in other brain structures only during the first days after birth. Thus, in the cerebellum, the density of 5-HTIA mRNA decreased markedly from day 2 to day 9 after birth and could hardly be detected in the adult animal. The localization of the mRNA in the molecular/Purkinje cell layer of the immature cerebellum agreed with that of the 5-HTIA receptor protein visualized by immmunocytochemistry and was consistent with the hypothesis that Purkinje cells express this receptor.

The portion of the complementary DNA encoding the third intracellular loop of the rat 5-hydroxytryptamine1A (serotonin) receptor was subcloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein coupled with the glutathione S-transferase of Schistosoma japonicum. The fusion protein was purified on a glutathione-agarose affinity column and used to immunize rabbits for the production of polyclonal anti-5-hydroxytryptamine1A receptor antibodies. Enzyme-linked immunosorbent assay revealed that antibodies were produced as early as one month after the first injection of the fusion protein, and immune response plateaued at a maximum after the third (monthly) booster injection. These antibodies only marginally affected the specific binding of [3H]8-hydroxy-2-(di-n-propyl-amino) tetralin to solubilized and membrane bound 5-hydroxytryptamine1A receptors, and did not interfere with serotonin-induced inhibition of forskolin-stimulated adenylate cyclase negatively coupled to 5-hydroxytryptamine1A receptors in rat hippocampal membranes. However, antibodies were able to immunoprecipitate 5-hydroxytryptamine1A receptor binding sites solubilized from rat hippocampal membranes. The distribution of immunoautoradiographic labelling and immunohistochemical staining of rat brain sections exposed to the antibodies raised against the fusion protein superimposed to that of 5-hydroxytryptamine1A receptor binding sites labelled by specific radioligands, with marked enrichment in the limbic areas (dentate gyrus and CA1 area in the hippocampus, lateral septum, entorhinal cortex) and the anterior raphe nuclei. The differential cellular location of immunoreactivity within the hippocampus (where dendritic fields but not pyramidal cell somas were immunostained) and the median raphe nucleus (where the plasmic membrane of somas was strongly immunoreactive) suggests that the addressing of 5-hydroxytryptamine1A receptors might differ from one neuronal cell type to another.

Anti-5-HT receptor antibodies were raised by injecting rabbits with either a synthetic peptide corresponding to the aa 243-268 portion of the third intracellular loop of the rat 5-HTAreceptor, or a fusion protein made of the complete loop (aa 216-350) coupled to glutathione-S-transferase. Immunohistochemical staining with these antibodies allowed the visualization of 5-HTAreceptors mainly in the limbic areas and anterior raphe nuclei, as expected from previous autoradiographic data with selective 5-HTAreceptor radioligands. Double immunostainingwith anti-5-HT,, receptor antibodies and anti-5-HT or anti-choline acetyl-transferase antibodies demonstrated that these receptors are located in the plasma membrane of practically all serotoninergic neurones in anterior raphe nuclei and a small fraction of cholinergic neurones in the median septum.

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.

The regional distribution of the mRNA encoding the 5-HT1A serotonin receptor (whose selective agonists are potential anxiolytic and antidepressant drugs) was investigated in rat brain sections by in situ hybridization histochemistry using two sets of [32P]labelled nucleoprobes, a riboprobe of 156 bases and oligoprobes of 30 bases corresponding to highly selective portions within the third intracellular loop and the N terminus domain of the amino acid sequence. These probes allowed the visualization of the 5-HT1A mRNA mainly in the limbic regions: dentate gyrus and area CA1 of the hippocampus, amygdala, entorhinal cortex, lateral septum and the dorsal raphe nucleus. These structures were also those which could be labelled by the specific 5-HT1A radioligand [125I]BH-8-MeO-N-PAT and antibodies raised against a synthetic 26 amino acid peptide whose sequence was taken from the most selective portion of the rat 5-HT1A receptor protein. These data suggest that the 5-HT1A receptors are not transported to a long distance from their site of synthesis, as it has been already reported for the somato-dendritic 5-HT1A autoreceptors in the dorsal raphe nucleus. Combined autoradiographic quantification of the 5-HT1A binding sites (labelled by a selective radioligand such as [125I]BH-8-MeO-N-PAT, the 5-HT1A receptor binding subunit (by radioimmunohistochemistry) and the 5-HT1A mRNA on adjacent brain sections should be a relevant approach for assessing the molecular mechanisms responsible for the functional alterations of these receptors under various pathological and pharmacological conditions.

SUMMARY A population of plant-parasitic nematode Nacobbus aberrans (Thorne, 1935) Thorne & Allen, 1944 from Rio Cuarto, Province of Cordoba, Argentina, was found parasitizing roots of the weed Sisyn~brium irio L. The root galls contained mature females with egg masses. The histological changes induced by the nematode - a syncytium formed by a mass of cells with partially dissolved walls - is commonly observed in plants parasitized by Nacobbus spp. We suggest that S. irio represents an efficient host for N. aberrans and describe this association for the first time.