Prostaglandins mediate lipopolysaccharide effects upon cholecystokinin and neurotensin phenotypes in neuroendocrine corticotropin-releasing hormone neurons: in situ hybridization evidence in the rat.

Intraperitoneal injection of the endotoxin lipopolysaccharide produces an inflammation accompanied by immune system activation and secretion of cytokines that stimulate the hypothalamo-pituitary-adrenal (HPA) axis to release the anti-inflammatory corticosterone. Upstream in HPA axis are neuroendocrine corticotropin-releasing hormone neurons in the paraventricular nucleus whose multipeptidergic phenotype changes during inflammation: coexisting corticotropin-releasing hormone and cholecystokinin mRNAs are up-regulated whereas neurotensin mRNA expression is induced de novo. These changes may be mediated by prostaglandins released from perivascular and microglial cells in response to circulating cytokines. We examined by quantitative in situ hybridization histochemistry whether blockade of prostaglandin synthesis by indomethacin alters phenotypic expression in paraventricular nucleus neurons after lipopolysaccharide. Because indomethacin also elevated circulating corticosterone, animals were adrenalectomized and corticosterone replaced. Results showed that i.p. indomethacin administration suppressed lipopolysaccharide effects in a phenotype non-specific manner: one injection was sufficient to prevent both the increase in corticotropin-releasing hormone and cholecystokinin mRNAs expression and the induction of neurotensin mRNA expression. Therefore, neuroendocrine corticotropin-releasing hormone neurons with different peptidergic phenotypes appear to respond as a whole in the acute phase response to systemic infection.

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.

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.

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.

Involvement of AMPA receptors in posterior locomotor activity in the rabbit: an in vivo study.

Although AMPA receptors are known to be widely involved in excitatory synaptic neurotransmission at the spinal level, very little is known about their role in modulating motor activity in mammals. In curarized decerebrate or spinalized rabbit preparations, fictive locomotion was monitored on hindlimb nerves after either activation or blockade of AMPA receptors. In decerebrate preparations, the administration of the antagonist, NBQX (3.5 mg/kg i.p.) or the agonist, AMPA (0.5 mg/kg i.v.) produced, in both cases, a depression of locomotor activities induced by stimulation of cutaneous afferents (evoked locomotor activity). This potent effect was transient with AMPA (recovery after 20 min) and followed by the occurrence of spontaneous locomotor sequences, while no recovery was observed with NBQX treatment. In spinal preparations where a continuous 'spontaneous' locomotor activity resulted from the pharmacological activation of noradrenergic descending pathways (nialamide-DOPA pretreatment), the same drugs injected at higher doses (5 mg/kg NBQX i.p. and 1 mg/kg AMPA i.v.) only weakly affected the frequency of 'spontaneous' and evoked locomotor bursts while they exerted inhibitory and facilitatory effects on the burst amplitude respectively. The results suggest that AMPA receptors are involved at spinal level: 1) in direct mediation of cutaneous afferent excitatory effects on the posterior locomotor generators (pLG); 2) in indirect mediation of a supraspinal descending inhibition controlling, likely presynaptically, the cutaneous afferent activation; and 3) in transmission to motoneurons of the output signals from the pLG. Finally, tight spinal interactions between potent descending noradrenergic pathways and spinal AMPA neurotransmission were disclosed.

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.

Immunocytochemical distribution of ionotropic glutamate receptor subunits in the spinal cord of the rabbit.

Several histochemical and physiological studies in the literature suggest that ionotropic glutamate receptors are involved in various sensory and motor control mechanisms at the spinal level. The present immunocytochemical study used three specific antibodies to GluR2,4, GluR5,6,7 and to NMDAR1 to differentiate between the regional distribution of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate and N-methyl-D-aspartate (NMDA) subtypes of glutamate receptors throughout the rabbit spinal cord. All of these immunoreactivities were prominent in the superficial dorsal horn and motor column. Each antibody gave rise to regionally specific immunostaining patterns but which were similar at all spinal levels. Numerous small neurons in superficial laminae were immunostained with GluR2,4 antibody while only neuropilar elements were immunostained with the two other antibodies. Cell bodies of the intermediate zone and fibres in the motor column were particularly densely immunostained with GluR5-7. Such an immunostaining pattern, which was particularly abundant with the GluR5-7 antibody, suggests the presence, at the spinal level, of an extensive population of neurons exhibiting a high density of kainate receptors. Immunostaining with NMDAR1 antibody was less dense in comparison with the two others and especially in the motoneuron area. The present results provide the first immunohistochemical comparison between the respective regional distributions of the three types of ionotropic glutamate receptors in the spinal cord. Their parallel distributions throughout the spinal cord support the concept of a tight functional cooperation between NMDA and non-NMDA receptors which has been extensively described for spinal events.

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.

Estrogen receptors are expressed in a subset of tyrosine hydroxylase-positive neurons of the anterior preoptic region in the rainbow trout.

A double immunocytochemical procedure, with two different chromogens, was used to compare the respective distribution of estrogen receptor-immunoreactive cells and tyrosine hydroxylase-immunoreactive neurons on the same sections of the preoptic region of adult female rainbow trout (Oncorhynchus mykiss). Estrogen receptor-immunoreactive cells were observed in the anterior preoptic region surrounding the preoptic recess and its large lateral extensions. Tyrosine hydroxylase-immunoreactive cells were consistently detected in the ventral and ventrolateral walls of the preoptic recess, in an area that was named nucleus preopticus pars anteroventralis. Dopamine immunohistochemistry and Dil retrograde transport studies indicated that part of these catecholaminergic neurons are dopaminergic and could project to the pituitary. Double staining studies showed consistently that most estrogen receptor-positive cells located ventral to the large extensions of the preoptic recess are also tyrosine hydroxylase-positive, indicating that this region is a major target for estradiol feedback. The results are discussed in relation to the role of the nucleus preopticus pars anteroventralis in mediating the negative feedback actions of estradiol on the secretion of gonadotrophin (GTH2) secretion. A hypothesis is drawn in order to explain the synchronizing role of estradiol at the time of ovulation in rainbow trout.

Neurotensin and tyrosine hydroxylase in the tuberoinfundibular system of the guinea pig hypothalamus with special emphasis to lactation status.

Immunohistochemical methods allowed us to study the distribution of neurotensin immunoreactivity (NT-IR) in the infundibular area of the guinea pig and to obtain evidence of a clear increase of such immunoreactivity in females during lactation. By use of the double immunolabeling method we demonstrated the presence of NT in the tuberoinfundibular dopaminergic (TIDA) neuronal system, in the nerve terminals of the median eminence as well as in the arcuate perikarya. In addition, NT-IR was detected in anterior pituitary cells especially stained in the rostral part of the gland of lactating females. These cells were identified exclusively as gonadotrophs by use of the elution-restaining procedure. These results suggests that NT is able to participate in the modulation by TIDA of prolactin secretion. Moreover, NT production by gonadotrophs could be responsible for other unknown pituitary actions.

Origin of the central entrainment of respiration by locomotion facilitated by MK 801 in the decerebrate rabbit.

In order to establish the origin of the central coupling between locomotion and respiration which operates in freely moving mammals during galloping, we sought experimental conditions that readily lead to such a coupling in decerebrate and curarised rabbit preparations. In such preparations, stimulation of the mesencephalic locomotor region (MLR) evokes locomotor activities, recorded from hindlimb muscle nerves, that are rarely totally coordinated with phrenic inspiratory activity. However, low doses (0.2 mg/kg i.v.) of MK 801, a non-competitive NMDA antagonist which has been shown to increase the activity of the spinal locomotion generators (Fenaux et al. 1991), dramatically enhanced this coupling during MLR stimulation in most experiments: 1/1 coupling was dominant but 2/1 and 3/1 couplings (i.e. two or three locomotor cycles per respiratory cycle) were also obtained. Compared with spontaneous respiratory activity, which was apneustic under these conditions, the respiratory period was drastically decreased during coupling. However, a further transection of the spinal cord at the C6 or C7 level, which isolated the spinal locomotion generators from the supraspinal levels, totally suppressed this reduction of the inspiratory period during MLR stimulation in the presence of MK 801. In experiments where locomotor activity was simultaneously recorded at forelimb and hindlimb levels, the 1/1 evoked locomotor-respiratory coupling remained after the lumbar cord had been isolated by L1 spinal transection. The present data do show that intact spinal mechanisms are required for entrainment to occur. They suggest either tha a common supraspinal drive cannot entrain locomotion and respiration when being depressed, or that respiration is entrained at the locomotor rate by the spinal locomotion generators.(ABSTRACT TRUNCATED AT 250 WORDS)

Distribution of catecholaminergic and serotoninergic systems in forebrain and midbrain of the newt, Triturus alpestris (Urodela).

Mapping of monoaminergic systems in the brain of the newt Triturus alpestris was achieved with antisera against (1) thyrosine hydroxylase (TH), (2) formaldehyde-conjugated dopamine (DA), and (3) formaldehyde-conjugated serotonin (5-HT). In the telencephalon, the striatum was densely innervated by a large number of 5-HT-, DA- and TH-immunoreactive (IR) fibers; IR fibers were more scattered in the amygdala, the medial and lateral forebrain bundles, and the anterior commissure. In the anterior and medial diencephalon, TH-IR perikarya contacting the cerebrospinal fluid (CSF-C perikarya) were located in the preoptic recess organ (PRO), the organum vasculosum laminae terminalis and the suprachiasmatic nucleus. Numerous TH-IR perikarya, not contacting the CSF, were present in the posterior preoptic nucleus and the ventral thalamus. At this level, DA-IR CSF-C neurons were only located in the PRO. In the posterior diencephalon, large populations of 5-HT-IR and DA-IR CSF-C perikarya were found in the paraventricular organ (PVO) and the nucleus infundibularis dorsalis (NID); the dorsal part of the NID additionally presented TH-IR CSF-C perikarya. Most regions of the diencephalon showed an intense monoaminergic innervation. In addition, numerous TH-IR, DA-IR and 5-HT-IR fibers, originating from the anterior and posterior hypothalamic nuclei, extended ventrally and reached the median eminence and the pars intermedia of the pituitary gland. In the midbrain, TH-IR perikarya were located dorsally in the pretectal area. Ventrally, a large group of TH-IR cell bodies and some weakly stained DA-IR and 5-HT-IR neurons were observed in the posterior tuberculum. No dopaminergic system equivalent to the substantia nigra was revealed. The possible significance of the differences in the distribution of TH-IR and DA-IR neurons is discussed, with special reference to the CSF-C neurons.

Distribution of serotonin- and dopamine-immunoreactivity in the brain of the teleost Clarias gariepinus.

The distribution of serotonergic and dopaminergic cell bodies and varicose fibres in the brain of the teleost Clarias gariepinus was studied immunohistochemically using antisera against formaldehyde-conjugated serotonin and dopamine. Many serotonergic and dopaminergic fibres innervated the areas dorsalis telencephali pars medialis and pars lateralis dorsalis, as well as the area ventralis telencephali pars ventralis. In the diencephalon, a large number of serotonergic and some dopaminergic fibres were found in the preoptic nucleus, innervating the cells of this nucleus. In addition, serotonergic and dopaminergic fibres were observed in the pituitary stalk and in all regions of the pituitary gland. Moreover, the diencephalon contained the highest number of serotonin- or dopamine-immunoreactive cell bodies. These cells were confined to the same periventricular nuclei as the nucleus ventromedialis thalami, the nucleus posterior periventricularis, the nucleus lateralis tuberis, the nuclei recessus lateralis and recessus posterioris. Most cells of these nuclei were in contact with the cerebrospinal fluid of the third ventricle. The brainstem contained serotonergic cell bodies in the raphe nuclei and a few serotonergic and dopaminergic fibres. The torus semicircularis was densely innervated by serotonergic fibres and, to a lesser extent, dopaminergic fibres. In the midbrain of Clarias gariepinus, no dopaminergic homologue of the substantia nigra was observed. The results are discussed both in a comparative and a physiological context. In this regard, special attention has been paid to the contribution of hypothalamic monoamines in the regulation of gonadotropin secretion as an essential step in the neuro-endocrine control of reproduction.

Localization of the lumbar pools of motoneurones which provide hindlimb muscles in the rabbit.

The localization of the pools of motoneurones (Mns) to the main hindlimb muscles was performed in the rabbit, using the retrograde transport of HRP from motor end plates. After 48 h survival time, the large alpha Mns were labeled. All the pools were met inside L6-S2 limits and a functional organization was observed: the pools to proximal muscles formed a ventral group of Mns and the pools to distal muscles a dorsal group, with flexor and extensor pools apart. The relative disposition of the different pools fits with that described in the cat and rat.

Effects of an NMDA-receptor antagonist, MK-801, on central locomotor programming in the rabbit.

NMDA has been shown to disclose spinal fictive locomotor activity in various in vitro preparations. In the present work the NMDA-mediated effects of endogenously released excitatory aminoacids (EAA) on fictive locomotion in the adult rabbit preparation were assessed in vivo using systemic injections of a non competitive NMDA-antagonist, MK-801. In acute low spinal and curarized preparations, the amplitude of the "spontaneous" fictive locomotor activities recorded from hindlimb muscle nerves after nialamide-DOPA pretreatment was much decreased in flexor and extensor nerves after MK-801 administration (0.25 mg/kg i.v.) whereas the locomotor period increased slightly. The more potent locomotor bursts, evoked by repetitive sural nerve stimulation at 10 Hz during 10 s, were differently affected after MK-801: the main effect was a lengthening of the locomotor period and a less drastic drop in the burst amplitude. These changes in the burst period were maximal for activities evoked by A fibre group stimulation (+100%) and less when C fibres were recruited (+70%). In decerebrate curarized preparations where the locomotor sequences were evoked either by sural nerve stimulation or by stimulation of the mesencephalic locomotor region, MK-801 (0.25 mg/kg i.v.) caused the same drop in burst amplitude (by at least 50%) as in the spinal preparation but, in contrast, it reinforced rhythmic bursting: this was revealed by a clear shortening (up to -65%) of the locomotor period and by the prolongation of rhythmic bursting after stimulation. All these effects obtained in decerebrate preparations were maximal 20-30 min after MK-801 injection.(ABSTRACT TRUNCATED AT 250 WORDS)

Topographical relationships between catecholamine- and neuropeptide-containing fibers in the median eminence of the newt, Triturus alpestris. An ultrastructural immunocytochemical study.

Dopaminergic and peptidergic nerve fibers were simultaneously demonstrated with a double-labeling technique at the ultrastructural level. The first antibody, raised against tyrosine hydroxylase, was applied during the preembedding phase and visualized with the peroxidase method. The second antibody, raised against one of the peptides met-enkephalin, somatostatin or gonadotropin-releasing hormone (GnRH), was applied to the ultrathin sections and visualized with gold-labeled goat anti-rabbit IgG. The fibers of both categories were present in the zona externa of the median eminence, frequently contacting the basal lamina of the portal vessels. In addition, topographical relationships between different types of nerve fibers were observed in the perivascular areas, although there were no morphological signs of synaptic specializations. Using serial sections, it could be established that one GnRH-fiber contacted both a dopaminergic fiber and a fiber immunoreactive for met-enkephalin. The observations support earlier physiological data concerning the regulation of the hypothalamo-hypophyseal axis, with special emphasis on the release of neurohormones in the median eminence of the newt.

Further experimental evidence for the involvement of ecdysone in the control of meiotic reinitiation in oocytes of Locusta migratoria (Insecta, Orthoptera).

Ecdysone has recently been shown to be able to trigger meiotic reinitiation in vitro in submature oocytes of Locusta. In the present study we have experimentally depressed (by 60-70%) ecdysone biosynthesis in the ovaries of adult females by rearing them on a diet with a modified sterol profile. Mature oocytes from such females fail to undergo normal reinitiation, but when incubated in vitro, can be induced to break their meiotic arrest by the addition of exogenous ecdysone. These results lend further support to the hypothesis that in Locusta, ovarian ecdysone is involved in the control of meiotic reinitiation.

The monoaminergic system in the diencephalon of the newt tadpole, Triturus alpestris (Mert). A histofluorescence study.

The distribution of monoamines in the brain of Triturus alpestris tadpoles was investigated with the Falck-Hillarp histofluorescence method. This study concerns especially the diencephalon where is observed the major part of the monoaminergic (MA) neurons. MA fiber tracts are found in the striatum, the septal nucleus and the medial and lateral forebrain bundles of the telencephalon. In the anterior diencephalon cerebrospinal fluid (CSF)--contacting catecholaminergic cells are localized within the preoptic recess organ (PRO) and fibers in the preoptic nucleus. More caudally both CSF-contacting catecholaminergic and serotonergic cells can be demonstrated in the paraventricular organ (PVO) and in the nucleus infundibularis dorsalis (NID). In the hypothalamo-hypophysial tract, numerous catecholaminergic (CA) fibers occur in the median eminence and in the pars intermedia. The origin of these fibers is not elucidated but a participation of the PRO, PVO and NID seems possible. CA cells without contact with the CSF are localized laterally to the PVO, PVO-accompanying cells, and in the nucleus reticularis mesencephali (NRM). The distribution of MA neurons in the brain of Triturus alpestris appears more similar to that of anurans than that of the primitive urodeles.