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.

The inhibitory effects of (gamma)-aminobutyric acid (GABA) on growth hormone secretion in the goldfish are modulated by sex steroids.

Double-labelling studies at the electron microscopic level demonstrated that gamma-aminobutyric acid (GABA)-immunoreactive nerve endings are associated with growth-hormone-secreting cells in the proximal pars distalis of the goldfish pituitary gland, suggesting that GABA may be important for the control of growth hormone release in this species. An in vitro assay for GABA-transaminase activity demonstrated that the pituitary is a site for the metabolism of GABA to succinic acid. In vitro, GABA or the GABA antagonists bicuculline and saclofen did not affect the rate of growth hormone release from dispersed pituitary cells in static incubation. In contrast, intracerebroventricular injection of GABA reduced serum growth hormone levels within 30 min. During the seasonal gonadal cycle, intraperitoneal injection of GABA was without effect in sexually regressed goldfish, but caused a significant decrease in serum growth hormone levels in sexually recrudescent animals. Intraperitoneal implantation of solid silastic pellets containing oestradiol increased serum GH levels fivefold in sexually regressed and recrudescent goldfish; in both groups, GABA suppressed the oestradiol-stimulated increase in circulating growth hormone levels. The effect of oestradiol on basal serum growth hormone levels was specific since progesterone and testosterone were without effect. However, in recrudescent animals treated with progesterone and testosterone, the inhibitory effects of GABA on serum growth hormone levels were absent, indicating a differential role for these steroids in growth hormone release. Taken together, these results demonstrate that GABA has an inhibitory effect on growth hormone release in goldfish.

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.

[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.

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.

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.

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.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

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.

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.

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.

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.

The reproductive brain in fish.

In fish as in other vertebrates, the brain is actively involved in the control of reproduction, first by participating, under the influence of external factors, in the establishment of an appropriate endocrine status, but also by allowing synchronization of the partners by the time of spawning. It is now well established that the pituitary gonadotropic function is controlled by multiple stimulatory and inhibitory factors, originating mainly from the preoptic region and the mediobasal hypothalamus, both target regions for sexual steroids. Little is known about the mechanisms involved in the mediation of external and internal factors, however there is indication that internal factors, such as androgens and melatonin, known to trigger particular behavioural and endocrine responses, act both at the level of neuroendocrine territories, but also on sensorial systems, which are the actual sites of action for external factors. This paper represents an attempt to summarize and integrate the recent literature devoted to the different aspects of the brain as a major participant in the complex endocrine and behavioural mechanisms of reproduction in fish.

Identification of neurotensin-immunoreactive cells in the anterior pituitary of normal and castrated rats. A double immunocytochemical investigation at the light- and electron-microscopic levels.

The localization of neurotensin (NT) has been studied in the rat pituitary by means of immunocytochemistry at the light- and electron-microscopic levels. Cells immunoreactive for NT (NT-IR) were observed in the anterior lobe of rats of both sexes. Following an elution-restaining procedure, it was found that most of these NT-IR cells correspond to cells also reacting to beta-LH or FSH antisera. However, a few NT-IR cells did not stain for beta-LH nor FSH, but for TSH. The subcellular localization of NT was studied at the electron-microscopic level by means of the immunogold procedure. The immunoreactivity was always localized over the secretory granules. By using two sizes of gold particles, it was shown that NT- and beta-LH-IR were colocalized in part of the cell granules. Castration caused a progressive decrease of the NT-IR in the beta-LH- or FSH-positive cells, while the number of NR-IR, TSH-IR cells increased. After 2 weeks, NT-IR was mainly observed in this latter cell type. The present work demonstrates that NT-IR is localized within the secretory granules of the gonadotrophs and a few thyrotrophs, indicating that NT could be released together with gonadotrophins and TSH. Moreover, since gonadectomy results in typical qualitative and quantitative changes of NT-IR gonadotrophs, the intervention of the pituitary NT in the regulation of the hypophysogonadal axis is strongly suggested.

Influence of GABA on gonadotrophin release in the goldfish.

The influence of GABA on pituitary gonadotrophin (GTH) release in the goldfish was studied by means of in vivo and in vitro techniques. It was found that GABA injected intraperitoneally caused an increase of serum GTH levels in regressed or early maturing fish, but not in late maturing animals. Moreover, injection of a GABA transaminase inhibitor caused a significant increase of GABA within the hypothalamus and pituitary, and a dose-dependent increase in serum GTH levels. To determine if this effect could be exerted directly at the level of the pituitary, dispersed pituitary cells in static incubation or in perifusion were exposed to increasing concentrations of GABA or its agonists muscimol and baclofen. None of these drugs was able to modify the spontaneous or GnRH-induced secretion of GTH, indicating that the in vivo effect of GABA was most likely mediated via another hypothalamic factor. Using in vitro incubation of pituitary slices, it was found that GABA caused a dose-related stimulation of GnRH release at the level of the pituitary, providing a possible explanation for the observed in vivo stimulatory effect of GABA on GTH release. Since the seasonal effect of GABA in vivo indicated a possible interaction of GABA with sexual steroids, GABA was given intraperitoneally to female goldfish implanted with either testosterone or estradiol. We found that the stimulatory effect of GABA on GTH release was abolished in estradiol-treated females but was still observed in testosterone-implanted fish. Moreover, estradiol but not testosterone caused a decrease of the GABA concentration within the telencephalon.(ABSTRACT TRUNCATED AT 250 WORDS)

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.