Jean Thibault

Ultrastructural morphology of dopaminergic nerve terminals and synapses in the striatum of the rat using tyrosine hydroxylase immunocytochemistry: A topographical study

Structures immunoreactive for TH were examined in the rat striatum (including caudate-putamen, nucleus accumbens and globus pallidus) by electron microscopy using the indirect peroxidase-labeled antibody method. Axon profiles and nerve terminals were the only structures stained by DAB precipitates in the axoplasm. The reactive boutons frequently contained a population of large pleomorphic vesicles (40-60 nm in diameter) but their interiors remained free of reactions. The synaptic contacts formed belonged principally to the symmetric type 2 of Gray while asymmetric Grays type 1 synapses were rarely observed. The former were mostly apposed to dendritic trunks (rarely to perikarya) and the latter to dendritic spines. In addition, numerous immunoreactive nerve terminals were often found in close contact with small structures identified as the neck of dendritic spines. The active zone of these presumed synapses was characterized by a prominent thickening of the presynaptic membrane but the post-synaptic thickening was lacking. For similar reasons, it was difficult to assert the existence of one axo-axonic synapse when a positive nerve terminal was closely apposed to another one (generally unreactive). The exact morphology of dopaminergic synapses, or even their existence, have not been firmly established owing to large discrepancies between previous reports. No synapses, or synaptic contacts of either asymmetric or symmetric type were described according to the technique used. Our work was undertaken to elucidate further this problem, and in particular, we thought that regional differences in the synaptic organization might explain the divergent data. However, regional quantitative analysis performed in this study did not show significant differences in the percentage of either kind of synapses in the various striatal regions.

Neuroanatomical substrate for the inhibition of gonadotrophin secretion in goldfish: existence of a dopaminergic preoptico-hypophyseal pathway.

To investigate the existence of a dopaminergic preoptico-hypophyseal pathway in the goldfish, electrolytic lesions were placed in the rostral preoptic area and their effects on gonadotrophin levels and pituitary innervation examined. In a first experiment, the fish were sacrificed 2 days after surgery and the pituitary studied by electron microscopy. Numerous exocytosis profiles were observed in the gonadotrophs, confirming the large increase in serum gonadotrophin levels measured in the animals. In addition, type A and B degenerating fibers were detected in the neurohypophysis and the pars distalis, in particular at the level of the gonadotrophs. In the second experiment, the distribution of tyrosine hydroxylase-immunopositive fibers was studied in the pituitary of controls and lesioned animals. It was found that lesioning the anterior ventral preoptic region resulted in the disappearance of all positive fibers in the pars distalis, while those in the neurointermediate lobe appeared unaffected. The presence of a large group of catecholaminergic perikarya in the destroyed area was confirmed in control animals. These results and other data strongly support the existence of a dopaminergic preoptico-hypophyseal pathway, providing a morphological support for the inhibitory effect of dopamine on the release of anterior pituitary hormones in teleosts, in particular gonadotrophin.

In vitro translation of mRNA from rat pheochromocytoma tumors, characterization of tyrosine hydroxylase.

Studies are presented which demonstrate that rat pheochromocytoma tumors are a convenient material for the preparation of tyrosine hydroxylase mRNA. Total pheochromocytoma poly(A)+mRNA has been extracted from tumors, then translated in a reticulocyte lysate cell-free system. Neo-synthesized tyrosine hydroxylase has been identified by direct immunoprecipitation followed by sodium dodecyl sulfate acrylamide gel electrophoresis. The proportion of this specific mRNA has been calculated; it represents 0.15 per cent of the total poly(A)+mRNA. The molecular weight of the invitro synthesized tyrosine hydroxylase is 62,000.

Topographic immunocytochemical mapping of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase in human post mortem brain stem

Immunocytochemical demonstration of monoamine oxidase-A, monoamine oxidase-B and tyrosine hydroxylase was performed in the human brain stem using monoclonal antibodies to monoamine oxidase-A and monoamine oxidase-B and polyclonal antibodies to tyrosine hydroxylase. In most of the brain areas examined, except the serotonergic dorsal nucleus of raphe, the noradrenergic locus coeruleus and the dorsal efferent nucleus of vagus, tyrosine hydroxylase-positive neurons were in greater number than monoamine oxidase-A-stained or monoamine oxidase-B-stained neurons. The dorsal nucleus of raphe showed no tyrosine hydroxylase immunoreactivity, but reacted positively to serotonin- and monoamine oxidase-B antibodies, while monoamine oxidase-A staining was moderate. In none of the investigated brain areas did neurons exclusively react with monoamine oxidase-B antibodies without expressing monoamine oxidase-A in a few neurons, while in some areas neurons expressed both monoamine oxidase-A and tyrosine hydroxylase (locus coeruleus; dorsal efferent nucleus of vagus). The oculomotor nucleus stained only with monoamine oxidase-A antibodies, substantia nigra neurons reacted only with tyrosine hydroxylase antibodies. Glial staining in most of the brain areas examined seemed, with slight differences, to have the same intensity with monoamine oxidase-A and monoamine oxidase-B antibodies used. No glial staining was obtained with tyrosine hydroxylase antibodies.

Immunohistochemical study of catecholaminergic cell bodies in the rat spinal cord

Immunohistochemistry of three specific synthesizing catecholamine enzymes was used in the rat spinal cord to determine precisely the distribution of catecholaminergic perikarya and the nature of the neurotransmitter they contain. Single and double labeling experiments were performed on cryostat sections from perfused rats. The peroxidase anti-peroxidase (PAP) and the indirect fluorescence techniques were used for labeling spinal catecholaminergic somata and separated into two completely different populations. The first is located in the upper cervical cord and includes three apparently distinct groups: a lateral cluster, of probably a noradrenergic nature, and two central subgroups where noradrenergic and dopaminergic neurons are intermingled. It is likely that these cervical cells represent caudal extensions of the medullary catecholaminergic cell groups. In the remaining cord, only tyrosine hydroxylase immunoreactive cell bodies have been found. Accordingly, this second population is probably dopaminergic. It is present almost exclusively in the first sacral segments, where it is located in the commissural (mostly lateral) grey matter and in the marginal dorsal horn.

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.

Tyrosine hydroxylase‐expressing and/or aromatic L‐amino acid decarboxylase‐expressing neurons in the mediobasal hypothalamus of perinatal rats: Differentiation and sexual dimorphism

In this quantitative and semiquantitative immunocytochemical study, the authors evaluated the differentiation of neurons expressing tyrosine hydroxylase (TH) and/or aromatic L‐amino acid decarboxylase (AADC) in the mediobasal hypothalamus (MBH) of male and female rats on embryonic day 18 ( E18 ), E20, and postnatal day 9 ( P9 ). Four neuronal populations were distinguished according to either enzyme expression or neuron location. The earliest and most prominent first population was represented by TH‐immunoreactive (IR)/AADC‐immunonegative (IN) neurons that were detected initially at E18 and always were located in the ventrolateral region of the MBH. The second population of TH‐IN/AADC‐IR neurons was observed first at E20 and, after that time, was distributed dorsomedially. The third minor population of TH‐IR/AADC‐IR neurons initially was detected at E20 and was located dorsomedially. The fourth population was represented by TH‐IR/AADC‐IN neurons that were distributed in the dorsomedial region at any studied age. The numbers of TH‐IR and AADC‐IR neurons increased from their initial detection at E18 and E20 until P9. The area of TH‐IR and AADC‐IR neurons also increased from E18 to E20 and from E20 to P9, respectively. Both TH‐IR and AADC‐IR neurons showed sex differences in the neuron number, size, and optic density (OD). The numbers of TH‐IR neurons in males exceeded those of females at E20 and at P9, although, at P9, sexual dimorphism was a characteristic only of the ventrolateral population. The area and OD of TH‐IR neurons from females exceeded those from males in the entire mediobasal hypothalamus (MBH) at E18 and E20 but only in its dorsomedial region at P9. Sexual dimorphism also was an attribute of AADC‐IR neurons at E20 and P9. Their number, size, and OD were significantly higher in females than in males. Thus, the MBH of perinatal rats contained two major populations of TH‐IR/AADC‐IN or TH‐IN‐AADC‐IR neurons and a minor population of TH‐IR/AADC‐IR neurons. The differentiating neurons expressing either enzyme showed sexual dimorphism. J. Comp. Neurol. 425:167–176, 2000.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. An atlas of neuronal cell bodies

The development of the catecholaminergic system in the hypothalamus and in the septal region was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic structures were visualized with pre-embedding immunocytochemistry using antiserum to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to make the immunocytochemical technique more sensitive. In this paper only the data on the appearance and distribution of the tyrosine hydroxylase-immunopositive neurons (cell bodies) are presented, whereas the catecholaminergic innervation of the hypothalamus with the tyrosine hydroxylase-immunopositive fibers is the topic of an accompanying paper. Sparse tyrosine hydroxylase-immunopositive neurons were first observed in the anlage of the hypothalamus and septal region on the 13th fetal day. Their number increased progressively with age and by the 15th fetal day they already gave rise to a large dorsal accumulation. From the 18th fetal day on, tyrosine hydroxylase immunopositive neurons began to occupy their definitive positions, mainly concentrating within the hypothalamus: in the zona incerta, periventricular and arcuate nuclei. To a lesser extent, they were concentrated in the medial preoptic area, suprachiasmatic, supraoptic, paraventricular, dorsomedial, and anterior hypothalamic nuclei. The data on the distribution of the tyrosine hydroxylase-immunopositive neurons both in the hypothalamus and in the septal region during ontogenesis are summarized in the precise atlas. Primarily small bi- and unipolar catecholaminergic neurons first observed in the youngest fetuses undergo cytodifferentiation during ontogenesis, giving rise to at least two different populations localized ventrally, mainly in the arcuate nucleus, and dorsally, in the zona incerta. The neurons of the former population remain similar to those of the youngest fetuses, whereas the neurons of the latter increase significantly in size, forming several long, highly ramified processes.

Quantitative Autoradiography of Tyrosine Hydroxylase Immunoreactivity in the Rat Brain

Levels of tyrosine hydroxylase (TH) were quantified in discrete areas of unfixed rat brain tissue sections using a rapid and sensitive radioimmunohistochemical method. The immunological reaction with the TH monoclonal antibody was revealed by a 35S‐labelled secondary antibody and thus permitted autoradiographic detection of the enzyme. Autoradiograms were generated by apposition of tissue sections to high‐sensitivity films or by dipping into autoradiographic emulsion. A detailed analysis of antibody concentration, incubation time, tissue section thickness, and exposure time of the film was undertaken to determine optimal conditions to produce a linear radiolabelling intensity with respect to the amount of antigen. Quantification of the antigen at regional levels was assessed by computer‐assisted image analysis. Autoradiographic optical density of radiolabelling in brain areas was converted to enzyme concentrations by interpolation with a constructed TH calibration curve processed in parallel with tissue sections. The specificity of the labelling and the validity and reproducibility of the quantification were investigated. The distribution of TH radiolabelling was comparable to that described using immunofluorescence histochemistry or measuring TH enzymatic activity on homogenates. Using a 35S‐labelled antibody, the detection of TH could be performed at the cellular level.

Ontogenesis of tyrosine hydroxylase-immunopositive structures in the rat hypothalamus. Fiber pathways and terminal fields.

The innervation of the hypothalamus and septal region by catecholaminergic fibers was studied in rats from the 12th fetal day until the 9th postnatal day. Catecholaminergic fibers were visualized with preembedding immunocytochemistry using antibodies to tyrosine hydroxylase. An intensification of diaminobenzidine product with silver and gold was additionally applied to increase the sensitivity and resolution power of the routine immunocytochemical technique. It has been demonstrated that, from the 13th fetal day, the hypothalamus and the septal region receive catecholaminergic fibers either belonging to the hypothalamic neurons or coming with the medial forebrain bundle from the outside of the hypothalamus. As the development of the hypothalamus proceeds, these fibers form the extensive networks within some neurosecretory centers either containing (the zona incerta, periventricular nucleus, etc.) or almost lacking (suprachiasmatic and paraventricular nuclei) the catecholaminergic neurons. In the former case, they terminate on the processes or perikarya of catecholaminergic neurons, while in the latter case their varicosities surround the immunonegative presumptive neurons in a basket-like manner. Moreover, from the 18th fetal day catecholaminergic fibers penetrate between the ependymal cells towards the 3rd ventricle and the primary capillary plexus of the hypophysial portal circulation, apparently providing the release of catecholamines to the cerebrospinal fluid and portal blood, respectively. The data obtained in this study are considered as the morphological basis for the involvement of the hypothalamic catecholamines in neuroendocrine regulations during ontogenesis.