Direct and indirect enkephalinergic synaptic inputs to the rat arcuate nucleus studied by combination of retrograde tracing and immunocytochemistry.

The origin of both direct and indirect enkephalinergic innervation potentially able to influence neurons of the rat arcuate nucleus has been investigated by combining enkephalin immunocytochemistry and retrograde axonal transport of a wheatgerm agglutinin-Apo horseradish peroxidase-gold complex. Twenty four hours after tissue injections of small volumes (20 nl) of the tracer into the arcuate nucleus, rats were treated with colchicine and killed. In order to localize the enkephalinergic cells which directly innervate the arcuate nucleus, Vibratome sections were first silver-stained for detection of the wheatgerm agglutinin-Apohorseradish peroxidase-gold complex and then processed for enkephalin immunohistochemistry. To study the indirect enkephalinergic input to the arcuate nucleus, an electron microscope detection of immunoreactive synapses was carried out in areas rich in retrogradely labeled perikarya. Perikarya both immunoreactive and retrogradely labeled were observed ipsilaterally to the injection site in telencephalic structures such as the bed nucleus of the stria terminalis, medial preoptic and adjacent periventricular areas. Hypothalamic ipsilateral doubly labeled cells were localized principally in the dorsomedial nucleus and rostral arcuate nucleus. The major direct inputs arising from brainstem structures concerns the dorsal and ventral parabrachial nuclei. Moreover, at the ultrastructural level, numerous enkephalinergic terminals were demonstrated to synapse with retrogradely labeled perikarya and dendrites localized in the medial preoptic area, the hypothalamic paraventricular nucleus and the parabrachial nuclei providing evidence for an important enkephalinergic input on neurons projecting to the arcuate nucleus. Taken together, our light and electron microscope studies strongly suggest that the arcuate nucleus is the target of an enkephalinergic control originating from several regions and acting either directly or indirectly on neurons projecting to the arcuate nucleus.

Immune challenge-stimulated hypophysiotropic corticotropin-releasing hormone messenger RNA expression is associated with an induction of neurotensin messenger RNAs without alteration of vasopressin messenger RNAs.

The corticotropin-releasing hormone neurons of the hypothalamic paraventricular nucleus are the final common pathway of the neuroendocrine adaptative response to a variety of stressors. To meet varied homeostatic needs, corticotropin-releasing hormone neurons exhibit a marked phenotypical plasticity, enabling them to rapidly modify their neuroendocrine output. In particular, they synthesize the neuropeptides vasopressin and neurotensin. Under many experimental circumstances, it is observed that corticotropin-releasing hormone and vasopressin are regulated in parallel, whereas the expression of neurotensin seems dissociated, in these neurons, evoking different transcriptional control over the co-existing neuropeptides depending on the adaptative response required. Using radioactive and dual-label in situ hybridization techniques, we have studied the respective expression of paraventricular corticotropin-releasing hormone, vasopressin and neurotensin messenger RNAs in the context of an immune challenge. A single intraperitoneal injection of the endotoxin lipopolysaccharide was administered to adult male rats that were killed 8 h later. Compared to control animals, lipopolysaccharide-injected rats showed elevated plasma corticosterone (614+/-65 vs 185+/-40 ng/ml in control) and increased expression of paraventricular corticotropin-releasing hormone messenger RNA (+200%); expression of neurotensin messenger RNA was induced in about one-third of corticotropin-releasing hormone neurons, whereas vasopressin messenger RNA expression remained unchanged. Therefore, in this experimental context and at the time-point examined, co-existing corticotropin-releasing hormone and vasopressin appeared differentially expressed, and an additional stimulus (inflammation) is demonstrated to result in neurotensin expression in neuroendocrine corticotropin-releasing hormone neurons. Neurotensin may be released in the pituitary portal blood to trigger pituitary response associated with mobilization of the immune system.

Ultrastructural colocalization of vesicular cholecystokinin and corticoliberin in the periportal nerve terminals of the rat median eminence.

Cholecystokinin (CCK) is present in axon terminals distributed around the fenestrated capillary loops of the hypothalamo-hypophysial portal system. In the hypothalamic paraventricular nucleus, CCK has been shown to coexist with corticoliberin (CRH). However, in the median eminence (ME) nothing is known about the chemical phenotype of the CCK immunoreactive terminals. This study, carried out in the male rat, was designed to examine the possibility of coexistence of CCK immunoreactivity (CCK-IR) and CRH-IR in fibres of the ME and to describe, at the electron microscopic level, the vesicular pattern of distribution of CCK-IR in the pericapillary endings of the ME. The use of the elution-restaining procedure showed notable similarities between stainings directed against CCK or CRH, respectively, suggesting a colocalization of both peptides in the same terminals. This result was confirmed using a simultaneous double-staining procedure. At the electron microscope level, double immunogold staining procedure enabled us to observe a consistent localization of CCK-IR and CRH-IR over dense-cored vesicles. Most of the terminals were seen to contain both immunoreactivities which, in addition, were often present together in the same vesicles. However, some rare endings remained exclusively stained either for CCK or for CRH. Our results provide evidence for a concomitant release of CCK and CRH into the portal blood.

Synaptic inputs of tachykinin-containing nerve terminals to target tyrosine-hydroxylase-, beta-endorphin- and neuropeptide Y-producing neurons of the arcuate nucleus. Double pre-embedding immunocytochemical study in the rat.

Anatomical connections between tachykinin-containing terminals and three neuronal populations of the arcuate nucleus, chemically defined respectively by beta-endorphin (beta-END), tyrosine-hydroxylase or neuropeptide Y (NPY) and well represented in the arcuate nucleus, were studied using electron microscope double pre-embedding immunocytochemistry involving a combination of two sensitive chromogens: diaminobenzidine and tetramethylbenzidine. Following tachykinin immunodetection by diaminobenzidine, and tyrosine-hydroxylase, beta-END or NPY immunolabelling by tetramethylbenzidine, tachykinin-immunoreactive terminals were seen presynaptic to tyrosine-hydroxylase immunopositive cells and dendrites principally in the dorsomedial portion of the arcuate nucleus. Tachykinin-immunoreactive processes were also seen in synaptic contact with ventrolaterally located beta-END immunopositive perikarya. Tachykinin-immunopositive terminals also contacted NPY-immunoreactive cells and dendritic processes ventromedially. These results demonstrate the existence of a direct tachykinergic input onto three neuronal populations expected to play a role in the control of reproductive events. Consequently, they suggest, at least, an indirect action for tachykinins in the regulation of reproduction. Especially, tachykinins may indirectly control the luteinizing hormone-releasing hormone neurons via dopamine, beta-END and NPY cells and thereby influence luteinizing hormone secretion.

Ultrastructural evidence for synaptic inputs of enkephalinergic nerve terminals to target neurons in the rat arcuate nucleus.

The morphological support of interactions between enkephalins and three systems--beta-endorphin (beta-END), tyrosine hydroxylase (TH), or neuropeptide Y (NPY)--well represented in the arcuate nucleus, was examined by using an electron microscopic double immunostaining combining two sensitive chromogens, diaminobenzidine (DAB) and tetramethylbenzidine (TMB). The first step consisted of visualizing Metenkephalinergic terminals with DAB reaction product, and the second one involved detecting the antigens TH, beta-END, and NPY in their respective neurons with TMB reaction product. Ultrastructural analysis revealed enkephalinergic terminals presynaptic to TH-immunopositive cells and dendrites, principally in the dorsal portion of the arcuate nucleus. Enkephalinergic nerve terminals also contacted synaptically ventrolaterally located beta-END-immunoreactive cells. In the ventromedial arcuate nucleus, few synaptic contacts were observed between enkephalinergic boutons and NPY neurons, which were principally in close apposition with glial processes. Enkephalin-immunoreactive synapses were more frequently seen on TH-immunopositive neurons. This TH neuronal group is known to correspond to the dopaminergic tuberoinfundibular neurons implicated in the control of reproductive functions. The pattern of distribution of the different synapses within the arcuate nucleus (TH dorsal, beta-END ventrolaterally; NPY ventromedially) suggests that enkephalins may play a role in the neuroendocrine regulation of gonadotropin and prolactin secretion. The results provide evidence that enkephalins, in the arcuate nucleus, exert a postsynaptic action on the beta-END cells in addition to the presynaptic regulation previously demonstrated in the mediobasal hypothalamus, related to beta-END release. Moreover, the arcuate nucleus is a site of intercellular relationships between enkephalins and dopamine and between enkephalins and other peptides such as NPY.

Preparation and characterization of a specific antibody for the immunohistochemical detection of L-dopa in paraformaldehyde-fixed rodent brains.

A rat polyclonal antiserum has been obtained after coupling of L-3,4-dihydroxyphenylalanine (L-DOPA) to larger proteins using a low concentration of glutaraldehyde. The antiserum was tested for its affinity and specificity using an enzyme-linked-immunosorbent-assay (ELISA). From competition experiments, the most immunoreactive compound was found to be the non-reduced L-DOPA conjugate. Our specific L-DOPA antiserum enables us to visualize L-DOPA molecule on brain of guinea pigs and rats. We examined the immunohistochemical distribution of the polyclonal L-DOPA antiserum after the fixation of brains with a mixture of paraformaldehyde and picric acid. The presence of L-DOPA-immunoreactive (IR) neurons and fibers was described in the posterior, dorsal and periventricular hypothalamic areas and in the arcuate nucleus. Finally, the distribution of L-DOPA-IR cells was compared to that of tyrosine hydroxylase (TH)-IR cells, by means of a double staining procedure. The presence of two populations of TH-IR cells (TH-positive/L-DOPA-negative and TH-positive/L-DOPA-positive cells) was described in the dorsal part of the hypothalamus.

Reciprocal synaptic connections between neurotensin- and tyrosine hydroxylase-immunoreactive neurons in the mediobasal hypothalamus of the guinea pig.

Neurotensin (NT) and dopamine are two neurotransmitters which are present in the hypothalamus of mammals and are often distributed in identical areas. In particular, in the periventricular anterior hypothalamus and in the arcuate nucleus, images of apposition between perikarya and fibers containing dopamine or neurotensin have frequently been observed at the light microscope level. The aim of this study was to answer, at the ultrastructural level in the A12 and A14 catecholaminergic cell groups, the question as to the existence of the possible synaptic nature of such contacts. To this end, NT and tyrosine hydroxylase (TH) were simultaneously visualized using double pre-embedding immunocytochemical methods. In the A12 arcuate area, synaptic contacts were demonstrated between TH-immunoreactive terminals and NT-labeled perikarya and dendrites. The opposite pattern, i.e., NT-stained terminals synapsing onto TH-positive neurons, was also observed. In contrast, only NT synaptic inputs onto TH-stained cell bodies could be demonstrated in the hypothalamic periventricular nucleus. In addition, immunoreactive terminals stained for NT or TH were observed to make synaptic contacts with perikaryal profiles stained for the same antigen. These results demonstrate a strong synaptic NT input onto the dopaminergic neurons of the mediobasal hypothalamus and suggest a reciprocal influence, at least in part, of catecholaminergic terminals on arcuate NT-containing neurons.

CCK mRNA expression in neuroendocrine CRH neurons is increased in rats subjected to an immune challenge.

The expression of cholecystokinin (CCK) mRNA in neuroendocrine corticotropin-releasing hormone (CRH) neurons of the hypothalamic paraventricular nucleus (PVN) of male rats was examined 8 h following an acute immune challenge by intraperitoneal lipopolysaccharide (LPS, 250 microg/kg). Both quantitative, macroautoradiographic, single-label radioactive in situ hybridization histochemistry (ISHH) and qualitative dual-label ISHH were performed. Compared to controls, LPS-injected rats displayed increased (185%) parvicellular CCK mRNA expression levels, occurring in a majority (70%) of CRH neurons as revealed by dual-label ISHH.

Tachykinergic synaptic inputs to neurons of the medial preoptic region which project to the rat arcuate nucleus.

Anatomical relationships between tachykinin-containing terminals and neurons of the medial preoptic area that innervate the arcuate nucleus were studied using silver staining of the retrograde tracer wheat germ agglutinin-apoperoxidase-gold (WGA-ApoHRP-gold) complex injected in the arcuate nucleus and pre-embedding immunocytochemistry for neurokinin A (NKA). At the histological level, retrogradely labeled cells not stained for NKA were seen to be surrounded by numerous NKA-immunopositive punctate profiles, in particular in the dorsal part of the medial preoptic area. At the ultrastructural level, retrogradely labeled cell bodies and dendritic profiles displayed highly electron-dense silver particle accumulations over the cytoplasm. The were seen in synaptic contact with one or several NKA-immunoreactive axon terminals containing small clear vesicles and dense-cored vesicles. Such synapses were either symmetrical or asymmetrical. The occurrence of synaptic contacts between tachykinin terminals and cells innervating the arcuate nucleus in the medial preoptic region provides a morphological support for a tachykinergic regulation of preoptic afferences to the arcuate nucleus. These results suggest that tachykinins are implicated in the indirect control of neuronal activity in the arcuate nucleus notably via the preoptic area. Consequently, tachykinins are potentially able to regulate indirectly numerous neuroendocrine events involving the tuberoinfundibular system.

The daily pattern of Fos synthesis by hypothalamic pro-opiomelanocortin neurons is unaffected by adrenalectomy in the rat.

At the onset of dark, a large population of rat mediobasal hypothalamic (MBH) pro-opiomelanocortin (POMC) neurons starts spontaneously expressing Fos-immunoreactivity (Fos-IR). Here we studied the effect of adrenalectomy upon this expression since circulating corticosteroids, which increase in the rat with the onset of behavioural wakening, are thought to modulate the basal expression of MBH POMC mRNA. Hence, groups of intact, adrenalectomised and sham-operated rats were sacrificed at times when Fos synthesis by POMC neurons is known to show either nadir (at light-offset) or peak (6 h after light-offset) values. Brains were processed for Fos- and/or POMC immunohistochemistry. This allowed us to show that, in all experimental groups, Fos-IR is hardly expressed in MBH POMC neurons at the onset of dark, whereas it is strongly induced 6 h later. We concluded that such an induction is not triggered through the known evening rise of plasma corticosteroid levels.

Double immunocytochemistry in pre-embedding electron microscopy for the detection of neurotensin and tyrosine hydroxylase in the guinea pig, using two primary antisera raised in the same species.

In this study we identified for electron microscopy two different antigens (neurotensin and tyrosine hydroxylase) in the same pre-embedding section of nervous tissue, using two antibodies obtained in the same species. Optimal ultrastructural results were obtained without adding to the fixative either glutaraldehyde or acrolein (normally used for electron microscopy techniques). The different developing methods used in this study (DAB in combination with either 1 nm silver-enhanced colloidal gold or benzidine dihydrochloride) are perfectly distinguishable at the ultrastructural level, and show some advantages over other previously described developing procedures. For instance, the use of small gold particles (1 nm) reduces the severity of membrane damage caused by tissue penetration of the bigger gold particles (5 nm). In addition, the reaction products are stable, so there is no need to stabilize them before osmication, as is necessary in other developing methods such as the TMB procedure. The immunolabeling results obtained in this study were similar in both developing methods, although synaptic profiles were more readily visible when the DAB/colloidal gold procedure was used. Using electron microscopy, we have detected TH immunoreactivity in dendrites and perikarya receiving synaptic contacts from NT-positive terminals, as well as TH-immunoreactive inputs on NT-positive neurons, at both the somatic and dendritic levels.

[Polyradicular lesion revealing ankylosing spondylarthritis]. / Atteinte pluriradiculaire révélatrice d'une spondylarthrite ankylosante.

INTRODUCTION: Radicular manifestations of ankylosing spondylitis are rare and observed in the course of long-term ankylosing spondylitis. EXEGESIS: The case of a young man who presented with bilateral and multiple radicular involvement is reported. Neurological symptoms occur a few weeks before ankylosing spondylitis was diagnosed. CONCLUSION: This suggests that nerve root lesions might take place during initial stages of the disease. The role of inflammatory changes in the region of the intervertebral foramina is discussed. Disease evolution is marked by relief of neurological disorders in response to anti-inflammatory treatment.

[Ultrastructure of prolactin cells of three Mugilidae during adaptation to fresh-water (author's transl)]. / Etude ultrastructurale des cellules à prolactine chez trois espèces de mugilides au cours de l'adaptation à l'eau douce.

The behaviour of three species of Mugilidae (Mugil ramada, Crenimugil labrosus and Mugil auratus) during adaptation to fresh water is very different. Those differences may be explain by variants of their prolactin cells activity. The adaptation of Mugil ramada and Crenimugil labrosus to fresh water is easy and last over a long time; the prolactin cells, with a slow activity in sea water, become during adaptation more active. In Mugil auratus in sea water, prolactin cells present already a sign of strong activity. After transfert in fresh water, they appear to be unable to increase their activity. The adaptation of this species is difficult and short-lasting.

[Ultrastructure of neurons of nucleus preopticus and pars lateralis of the nucleus lateralis tuberis in Gambusia (Teleosta poeciliidae)]. / Ultrastructure des neurones du noyau préoptique et de la pars lateralis du noyau latéral du tuber chez Gambusia (Téléostéen poeciliidae).

The nucleus preopticus (NPO) and the pars lateralis of the nucleus lateralis tuberis (NLT) have been examined at the ultrastructural level. In the NPO two types of large neurons occur, both containing 140-150 nm secretory granules. The pars lateralis of the NLT contains only one cell type with similar granules. Both of these nuclei may be the source of type A fibres innervating the gonadotropic cells of Gambusia.

Ultrastructural changes in the pars intermedia of the goldfish kept in calcium-free environments.

The PAS-positive-calcium-sensitive (Ca-s) cells of the pars intermedia (PI) were studied in goldfish kept in fresh water (FW), deionized water (DW), 1/3 sea water (SW) and 1/3 Ca-free SW. Ultrastructural studies show that Ca-s cells of control goldfish kept in FW have a low activity with elongated or deeply indented nuclei. This activity is slightly reduced after 19 days in 1/3 SW. A considerable stimulation of most Ca-s cells is noted in goldfish kept in DW for 20 or 40 days. The stimulation is similar in 1/3 Ca-free SW, but it affects sometimes a smaller percentage of cells and may be less marked in peripheral areas of the PI. Exocytotic figures are more numerous in Ca-s cells of goldfish in 1/3 Ca-free SW than in DW. A basal lamina is rarely present and direct contacts between PI cells and nervous tissue are frequent, although a single synaptic contact with a type B fiber was observed. MSH cells are not affected in goldfish kept in DW. They are stimulated in 1/3 Ca-free SW: the physiological significance of this response remains unclear. Few agranular (Agr) cells are scattered in the PI. Evident changes are not observed in the different environments. The present ultrastructural data support the hypothesis that the Ca-s cells of the PI secrete a factor involved in calcium regulation in some teleosts.

Ultrastructural changes in the calcium-sensitive (PAS-positive) cells of the pars intermedia of eels kept in deionized water and in normal and concentrated sea water.

The ultrastructure of the calcium-sensitive (Ca-s) (PAS-positive) cells of the pars intermedia was investigated in eels kept in hypo- and hyperosmotic environments. Although the cells were moderately active in fresh water (FW), they were highly stimulated in deionized water (DW) and displayed an enlarged Golgi apparatus, a distinct rough endoplasmic reticulum, few secretory granules, some microtubules and an extended area of contact with the basal lamina that separates nervous and glandular tissues. Some mitosing cells were seen. A similar picture was observed in eels kept in sea water (SW) for 45 days, returned to FW and subsequently to DW for 21 days. In SW (20 and 33%), and particularly in concentrated SW (50, 60 and 63%), the Ca-s cells were inactive. Their granules were significantly smaller than in eels kept in FW, and the area of contact with the basal lamina was greatly reduced. However, signs of granule-release were seen in eels adapted to 50 and 60% SW. Nerve fibers rarely contacted the Ca-s cells and did not synapse with them. The ultrastructural data support the hypothesis that the Ca-s cells of Anguilla, like those of Carassius, are involved in ionic regulation. MSH cells were not greatly affected by the present experiments.

Cytological and ultrastructural changes in the pituitary pars distalis of the goldfish kept in calcium-free environments.

The cytology and ultrastructure of the pars distalis, mainly that of prolactin (PRL) cells, were investigated in goldfish adapted to fresh water (FW) or deionized water (DW) for 20 and 40 days, or gradually adapted to 1/3 artificial sea water (ASW) or 1/3 Ca-free sea water. When compared to PRL cells of goldfish kept in FW, those of goldfish adapted to DW did not show signs of increased activity. The lack of exocytotic activity and the low development of various organelles suggested that cell activity was slightly reduced. In 1/3 ASW, PRL cells were smaller and less active. In 1/3 Ca-free ASW, PRL cells appeared slightly stimulated compared with those of fish in 1/3 ASW. The Golgi area was more developed and a few lamellae of endoplasmic reticulum were observed in some cell islets. However, there was no significant difference between PRL cells of goldfish kept in 1/3 Ca-free ASW and in FW. In 1/3 ASW, which is isosmotic to the blood, thyrotrophs (TSH cells) corticotrophs (ACTH cells) and somatotrophs (STH cells) were not clearly affected. In DW, these cells and their nuclei were significantly enlarged. Their stimulation was also evident in 1/3 Ca-free ASW; values for cellular and nuclear areas were maximal in this environment and significantly higher than those of fish in FW and 1/3 ASW. These data suggest that in addition to the PAS-positive cells of the pars intermedia, highly stimulated in Ca-free environments, other cell types of the pars distalis may be involved in osmoregulation, and that the role of PRL cells is not primordial in the goldfish.