M. Geffard

Anatomical distribution and ultrastructural organization of the gabaergic system in the rat spinal cord. An immunocytochemical study using anti-GABA antibodies

gamma-Aminobutyric acid (GABA)-containing elements have been studied by light and electron microscopy in the rat spinal cord, using immunocytochemistry with anti-GABA antibodies. Light microscopy showed immunoreactive somata localized principally in laminae I-III, and occasionally in the deeper laminae of the dorsal horn and in the ventral horn. Small somata were also observed around the central canal. Punctate GABA-immunoreactive profiles were particularly concentrated in laminae I-III, and moderately abundant in the deeper laminae and in the ventral horn where they were observed surrounding the unlabelled motoneurons. At the ultrastructural level, the punctate profiles corresponded to GABA-containing axonal varicosities or small dendrites. GABA-immunoreactive varicosities were presynaptic to labelled or unlabelled dendrites and cell bodies. Some unlabelled terminals presynaptic to unlabelled dendrites received symmetrical synaptic contacts from GABA-immunoreactive terminals. These results confirm data obtained with L-glutamate decarboxylase immunocytochemistry, and support the role of GABA in pre- and postsynaptic inhibition in the spinal cord, respectively via axoaxonal and axosomatic or axodendritic synapses.

Immunocytochemical localization of dopamine in the prefrontal cortex of the rat at the light and electron microscopical level

In the present study the dopaminergic innervation of the prefrontal cortex was studied by means of a recently developed anti-dopamine serum. This method can demonstrate endogenous dopamine in a specific way, and offers the opportunity to study the distribution of dopaminergic fibres in the cortex in detail in counterstained sections. Furthermore, dopaminergic nerve endings can be visualized at the electron microscopic level. Light microscopic observations demonstrated that the highest density of dopaminergic fibres in the frontal cortex is found in the prefrontal cortex and the infralimbic cortex. Within the prefrontal cortex, a good correlation is found between regional differences in distribution of dopaminergic fibres and the cytoarchitectonic parcellation of this part of the cortex. Outside the prefrontal cortex dopaminergic fibres were observed in adjacent frontal areas, the cortex surrounding the entire rhinal sulcus and the retrosplenial cortex. Electron microscopic observations demonstrated dopaminergic terminals through all cortical layers. The majority of dopaminergic terminals in the prefrontal cortex from synaptic contacts with dendritic processes. The synaptic profiles were usually symmetric and were characterized by the presence of many clear vesicles and an occasional dense-core vesicle.

First demonstration of highly specific and sensitive antibodies against dopamine

Dopamine was coupled to bovine serum albumin (BSA) with glutaraldehyde, precautions were taken in order to preserve the catechol ring. After injection of this immunogen into rabbits, anti-dopamine antibodies were obtained and tested using radioimmunochemical binding studies and adsorption to catecholamine covered sepharose beads. A good correlation was found between the results of the different test systems, allowing us to visualize dopamine specifically in glutaraldehyde-fixed rat brains.

Gamma-aminobutyric acid-immunoreactivity in the rat hippocampus. A light and electron microscopic study with anti-GABA antibodies

The distribution of GABA-immunoreactive neurons and axonal varicosities was investigated in the hippocampal region of the rat brain by means of an indirect peroxidase immunocytochemical method with recently developed anti-GABA antibodies. The immunolabeling was found to be restricted to nervous structures: neuronal cell bodies, dendrites and axon terminals. Myelinated axons showing GABA-immunoreactivity were also observed. GABA-immunoreactive neurons were found in great number in the stratum pyramidale, the superficial part of the stratum oriens and the deep part of the stratum radiatum in the Ammons horn. Less were found in the other regions; rare labeled cells were observed in the superficial part of the stratum radiatum and the middle part of the stratum oriens. The dentate gyrus exhibited numerous labeled cells in the granular layer, few in the hilus, rare in the molecular layer. A high density of GABA-immunoreactive terminals was found at the limit of the stratum oriens with the alveus, in the stratum pyramidale and in the stratum lacunosum. A lower density of labeled fibers was observed in the other areas. The somata and proximal dendrites of pyramidal and granular cells were encompassed by characteristic pericellular arrangements of GABA-immunoreactive varicosities. Ultrastructural observations revealed a diffuse immunoreaction product spread over the cytoplasm and the nucleus without specific relationship with the organelles, and immunoreactive aggregates in the cytoplasm. Labeled dendrites often showed enlargements displaying the immunoreaction whereas thinner segments were devoid of it. They received numerous asymmetrical synapses from unlabeled axon terminals. GABA-immunoreactive terminals were filled with small clear vesicles with immunopositive membranes and were observed in symmetrical contact with somata and dendrites.

Antisera against small neurotransmitter-like molecules.

Antisera were produced against three types of small neurotransmitter-like molecules: indolealkylamines, catecholamines and amino acid derivatives such as GABA. The specificity of the antisera were evaluated using radioimmunological or immunoenzymatic competition tests between a radiolabelled ligand or conjugated hapten, and analog molecules from the same metabolic pathway. The antibody site was characterized by the ratios of cross-reactivity and the affinity constants. On the basis of these in vitro studies, each immune response was found to be specific for the target molecule.

Reinnervation of the nucleus accumbens and frontal cortex of the rat by dopaminergic grafts and effects on hoarding behavior

Embryonic dopaminergic neurons were implanted in the form of a cellular suspension in the nucleus accumbens previously deprived of its dopaminergic innervation by a local injection of 6-hydroxydopamine. The graft provided a dopaminergic reinnervation to the nucleus accumbens, the anteromedial striatum, the anteromedial frontal cortex and also, in some cases, of the septum. The pattern of reinnervation was specific for each structure and similar to the innervation provided by mesocorticolimbic dopaminergic neurons to these same structures in the normal animal. The graft restored the locomotor stimulatory action of amphetamine which was abolished in the lesioned controls. Hoarding behavior, which was disrupted following the lesion, was not reinstated by the graft alone. However, if the grafted neurons were stimulated by a small dose (0.2 mg/kg, i.p.) of (+)-amphetamine, hoarding reappeared in the grafted animals, while the same dose of amphetamine had no effect in the lesioned controls.

Antisera Against the Indolealkylamines: Tryptophan, 5-Hydroxytryptophan, 5-Hydroxytryptamine, 5-Methoxytryptophan, and 5-Methoxytryptamine Tested by an Enzyme-Linked Immunosorbent Assay Method

Abstract: Antisera were raised against tryptophan, 5‐hydroxytryptophan, 5‐hydroxytryptamine, 5‐methoxytryptophan, and 5‐methoxytryptamine, by conjugating each molecule to bovine serum albumin and to human serum albumin via glutaraldehyde, in such a way as to preserve the original part. Antibody specificity was tested with the enzyme‐linked immunosorbent assay method. The specificity of each anti‐indolealkylamine‐glutaraldehyde antibody was established with competition experiments by using an adsorbed immunogenic conjugate and indolealkylamines either free or conjugated with poly‐L‐lysine. The nonconjugated compounds were poorly recognized. In the same way, the nonreduced conjugates always appeared less immunoreactive than the reduced ones. Calculated from the specificity study of each antiserum, the cross‐reactivity ratios were found to be smallest for the most immunoreactive conjugates. Thus, a specific immune response was defined for each compound belonging to the same metabolic pathway.

Existence of dopaminergic neurons in the preoptic region of the goldfish

Three morphofunctional techniques for the detection of biogenic monoamines have been used in order to find evidence for the presence of dopaminergic neurons in the preoptic region of the goldfish. The formaldehyde-induced fluorescence technique and the immunohistochemical demonstration of tyrosine hydroxylase allowed the detection of cell bodies containing catecholamines in the ventral and lateral walls of the preoptic recess of the goldfish. Specific antibodies indicated that at least part of these perikarya contain dopamine. Evidence for the projection of these neurons to the pituitary are given. These results support the assumption that dopamine, originating from the preoptic region, may act as a gonadotrophin release-inhibiting factor in goldfish.

Ultrastructural immunocytochemical study of the dopaminergic innervation of the rat lateral septum with anti-dopamine antibodies

The dopaminergic innervation of the rat lateral septum has been investigated at ultrastructural level by immunocytochemistry using the unlabelled peroxidase-anti-peroxidase method with anti-dopamine antibodies. The specificity of the reaction has been carefully checked by immunological and histochemical controls. A strong immunoreaction was observed in fibres of the lateral septum as well as in their cells of origin in the ventral tegmental area. In the lateral septum, dopamine-immunoreactive fibres were localized in two distinct areas. A first area, located ventrally in the anterior part of the septum was characterized by a high density of immunoreactive varicosities with barely visible intervaricose segments. A more dorsal area, extending throughout the anteroposterior region of the septum, was characterized by immunoreactive fibres in pericellular arrangements. Electron microscopic observations revealed no difference in the ultrastructure of dopamine-immunoreactive profiles in the different areas. Reaction product was found in vesicles, linked to microtubules and in the cytoplasm. Three types of vesicles were seen: (i) small vesicles (30-50 nm) with varying intensity of immunoreaction, filling up the varicosities; (ii) rare large clear vesicles (50-80 nm) with no internal immunoreaction; (iii) very rare large dense vesicles (50-100 nm) with a strong dopamine immunoreactivity. Labelled profiles were observed in clearly defined asymmetrical synaptic contacts with somata and dendrites. Due to the lack of previous work dealing with the use of anti-dopamine antibodies for electron microscope immunocytochemistry, our observations are compared to previous data obtained by more indirect labelling techniques.

Antibodies to dopamine: radioimmunological study of specificity in relation to immunocytochemistry

Abstract: Two classes of anti‐3,4‐dihydroxyphenylethylamine (dopamine) antibodies were raised in rabbits using dopamine conjugated to albumin either via formaldehyde or via glutaraldehyde. Each was usable for immunohistochemical detection of dopamine neurons provided that the tissue was fixed by the homologous cross‐linking agent. However, anti‐dopamine‐glutaraldehyde antibodies turned out to be of more general use because of the better fixative properties of glutaraldehyde which fixed dopamine in rat and in teleost, whereas formaldehyde only worked in lower vertebrates (such as goldfish) and not in rat brain. The specificity of antidopamine‐glutaraldehyde antibodies was firmly established by competition experiments in equilibrium dialysis, using an immunoreactive tritiated derivative synthesized by coupling dopamine to N‐α‐acetyl‐L‐lysine N‐methylamide via glutaraldehyde. Specificity studies in vitro and immunohistological results demonstrating the specific staining of dopaminergic neurons were found to correlate well.