Michel Geffard

The dopaminergic innervation of the supraoptic and paraventricular nucleus. A light and electron microscopical study.

An antiserum that has been raised against glutaraldehyde-conjugated dopamine was used to demonstrate specifically dopamine in the rat hypothalamus. This dopamine antiserum permitted an optimal fixation with glutaraldehyde and therefore enabled the simultaneous light and electron microscopic immunocytochemical localization of dopamine. It was demonstrated that the paraventricular and supraoptic nuclei of the hypothalamus were innervated by thin dopaminergic fibers, in contrast to the suprachiasmatic nucleus, which hardly received any dopaminergic input. Ultrastructural observations revealed that the dopamine fibers terminated synaptically on the magnocellular neurons and their processes. It is concluded that the present results may explain the effect of centrally injected dopamine on vasopressin and oxytocin release. In the dopamine-containing terminals the reaction product was frequently observed in 90 nm dense core vesicles and around clear vesicles.

Serotonergic innervation of the dorsal horn of rat spinal cord: light and electron microscopic immunocytochemical study

SummaryThe ultrastructure of serotonergic projections to the dorsal horn of the rat spinal cord has been investigated, using a highly specific polyclonal antiserum. The highest concentrations of immunoreactive profiles were found in lamina I and the outer part of lamina II (IIo). Intermediate concentrations were found in laminae III and IV, while the inner part of lamina II (IIi) was almost devoid of immunoreactivity. Whereas 60% of the profiles show at least one varicosity studded with synaptic vesicles, only one-fifth of the latter contributes to classical synapses, the remaining profiles being devoid of a facing postsynaptic density. The results are compared with those in the literature and our own results relative to other regions of the cord. It is concluded that the pauci-synaptic projections to the dorsal horn could correspond to a diffuse influence of serotonin, the targets for which are determined by the corresponding serotonergic receptors.

Demonstration of GABAergic cell bodies in the suprachiasmatic nucleus: In situ hybridization of glutamic acid decarboxylase (GAD) mRNA and immunocytochemistry of GAD and GABA

The existence of GABAergic neurons in the rat suprachiasmatic nucleus (SCN) was demonstrated by three specific markers; mRNA coding for glutamic acid decarboxylase (GAD) and visualized by in situ hybridization using a 35S-labelled cDNA probe, and GAD protein and GABA were identified by immunocytochemistry using specific antisera. In situ hybridization demonstrated well labelled GAD mRNA positive cells throughout SCN, and GABA and GAD immunoreactive cells showed similar distributions. These results indicate that GABA is a transmitter of a large portion of the SCN neuronal population.

Immunocytochemical demonstration of octopamine-immunoreactive cells in the nervous system of Locusta migratoria and Schistocerca gregaria

SummaryThe distribution of octopamine in the metathoracic ganglion, brain and corpus cardiacum of Locusta migratoria and Schistocerca gregaria was investigated by means of immunocytochemistry with an antiserum against octopamine. The dorsal unpaired median (DUM) cells of the metathoracic ganglion were found to be strongly octopamine-immunoreactive. In the rostroventral part of the protocerebrum a group of seven immunopositive cells was demonstrated. Stained nerve fibres of these cells run into three directions: circumoesophageal connectives, midbrain, and optic lobes. As far as the protocerebrum is concerned, immunoreactive fibres were found in the central body, the protocerebral bridge, and in other neuropile areas. In the optic lobe a dense plexus of immunopositive fibres was found in the lobula and in the medulla. In the brain one other immunopositive cell was demonstrated, situated at the lateral border of the tritocerebrum. Octopamine could not be shown to occur either in the globuli cells of the mushroom bodies or in the dorsolateral part of the protocerebrum, where the perikarya of the secretomotor neurones are located that innervate the glandular cells of the corpus cardiacum. In the nervi corporis cardiaci II, which contain the axons of the neurones that extend into the glandular part of the corpus cardiacum, and in the corpus cardiacum proper no specific octopamine immunoreactivity could be found.

Neurons containing messenger RNA encoding glutamate decarboxylase in rat hypothalamus demonstrated by in situ hybridization, with special emphasis on cell groups in medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus

Previous deafferentation studies have suggested that most hypothalamic GABAergic innervation originates from neurons within the hypothalamus. We have investigated the distribution of GABAergic cell groups in the rat hypothalamus by means of the in situ hybridization technique, using a cDNA probe for messenger RNA encoding glutamate decarboxylase. Several major GABAergic cell groups were demonstrated, including cells of the tuberomammillary nucleus, arcuate nucleus, suprachiasmatic nucleus, medial preoptic area, anterior hypothalamic area, the dorsomedial hypothalamic nucleus, perifornical area, and lateral hypothalamic area. The most prominent glutamate decarboxylase mRNA-containing cell groups were located in the medial preoptic area, anterior hypothalamic area and dorsomedial hypothalamic nucleus, and were composed of small- to medium-sized neurons. Compared to previously well-characterized GABAergic cell groups in the tuberomammillary nucleus, reticular thalamic nucleus, and non-pyramidal cells of cerebral cortex, the cells of these GABAergic groups demonstrated only weak cDNA labelling, indicating that they contain lower levels of glutamate decarboxylase mRNA. Several types of control experiments supported the specificity of this cDNA labelling, and the GABAergic nature of these cell populations was further supported by detection of glutamate decarboxylase and GABA immunoreactivity. Abundance of GABAergic cells in many hypothalamic nuclei indicates that GABA represents quantitatively the most important transmitter of hypothalamic neurons, and may be involved in neuroendocrine and autonomic regulatory functions.

l-DOPA-immunoreactive neurons in the rat hypothalamic tuberal region

The presence of L-DOPA-immunoreactivity is reported for the first time in the rat hypothalamic tuberal region. L-DOPA-immunoreactive neurons were demonstrated to be present in the ventrolateral part of the arcuate nucleus and periarcuate region just dorsal to the ventral surface of the brain (VLAR/PA). Weakly L-DOPA-immunostained neurons were found in the dorsomedial part of the arcuate nucleus and its neighboring periventricular nucleus (DMAR/PV). In contrast, dopamine (DA)-immunoreactive neurons were detected only in the DMAR/PV. These findings suggest that L-DOPA exists not only as a precursor of DA in neurons of the DMAR/PV, but also as an end-product in cells of the VLAR/PA.

Specific antisera against the catecholamines: L-3,4-dihydroxyphenylalanine, dopamine, noradrenaline, and octopamine tested by an enzyme-linked immunosorbent assay.

Abstract: Antisera were raised against L‐3,4‐dihydroxyphe‐nylalanine (L‐DOPA), dopamine (DA), noradrenaline (NA), and octopamine (OA). This was achieved by coupling each molecule to bovine serum albumin or human serum albumin using glutaraldehyde. The conjugated aromatic amines were kept in a reducing medium containing sodium metabisulfite. Antiserum specificity was tested using an enzyme‐linked immunosorbent assay method for catecholamines. Competition experiments were done between the immunogen coated on the well plates and each catechol‐amine, either in the free state or in conjugated form, previously incubated with an antiserum. In each case, the non‐conjugated compound was poorly recognized. The nonre‐duced conjugates of L‐DOPA and DA were well recognized, whereas those of NA and OA were poorly immunoreactive. The cross‐reactivity ratios established in the competition experiments allowed the specificity of the immune response to be defined. In each case, it was found to be high. The results suggest that the antibodies of L‐DOPA and DA antisera recognize preferentially the catechol moiety, whereas for the anti‐NA and anti‐OA antibodies, the lateral chain is important.

Immunological approach to the detection of taurine and immunocytochemical results

Abstract: An immunological approach to the detection of taurine resulted in antibodies specific enough to be used for immunocytochemical studies. The experimental conditions were similar to those previously described for raising antibodies against some small‐sized neurotrans‐mitter molecules: antisera were obtained from rabbits immunized with taurine conjugated to carrier proteins via glutaraldehyde and purified by adsorption on the glutar‐aldehyde‐treated protein carriers. Antibody affinity and specificity were determined in competition experiments between conjugated taurine and other conjugated amino acids or derivatives by enzyme‐linked immunosorbent assay. The resulting cross‐reactivity ratios, calculated at half‐displacement, showed conjugated taurine to be the best recognized compound. Given the molecular structure of taurine and the method used to prepare the conjugate, it seemed necessary to perform an oxidation step. However, adsorption of antisera on reoxidized or non‐reoxidized taurine conjugates suggested that reoxidation did not make a significant difference. Immunocytochemical application of the sera revealed populations of strongly immunopositive nerve cells in the cerebellum, striatum, and septum. The results confirmed that anti‐taurine antibodies can be used as specific tools for a better understanding of the role of taurine in the central nervous system.

Glycine neurons in the brain and spinal cord: antibody production and immunocytochemical localization

Antibodies were raised against glycine and they were specific for immunocytochemistry. Obtained from rabbits immunized with glycine conjugated to glutaryled protein-carriers, antisera were then purified by adsorption on the various glutaraldehyde-conjugated protein-carriers. Using a modified ELISA method, their specificity was determined in competition experiments between conjugated glycine and either non-conjugated glycine or other conjugated amino acids or derivatives, preincubated with anti-glycine antibodies. Calculated at half-displacement, the resulting cross-reactivity ratios showed conjugated glycine to be the best recognized compound. By revealing the presence of the majority of the glycine-containing cell bodies in the brainstem and spinal cord, immunocytochemical applications of glycine antibodies confirmed their use as specific tools for a better understanding of the role of glycine in the central nervous system.

Comparative study of dopamine- and noradrenaline-immunoreactive terminals in the paraventricular and supraoptic nuclei of the rat

The distribution of dopaminergic and noradrenergic terminal fields of the paraventricular (PVN) and supraoptic (SON) nuclei of the rat was investigated at the optic and electron microscopical level using antibodies directed against dopamine (DA) and noradrenaline (NA). The DA innervation was uniform among these nuclei, although more important in the PVN than in the SON. NA fibers were preferentially distributed in the parvocellular parts of the PVN and in areas of the magnocellular nuclei where vasopressinergic neurons were mainly located. Both DA and NA terminals synaptically contacted magnocellular neurons on their cell body or dendrites. This study thus provides morphological evidence for a double and independent catecholaminergic control, by DA and NA, on neuroendocrine mechanisms at the hypothalamic level.