Morphological evidence for direct interaction between gonadotrophin-releasing hormone neurones and astroglial cells in the human hypothalamus.

In rodents, there is compelling evidence indicating that dynamic cell-to-cell communications involving cross talk between astroglial cells (such as astrocytes and specialised ependymoglial cells known as tanycytes) and neurones are important in regulating the secretion of gonadotrophin-releasing hormone (GnRH), the neurohormone that controls both sexual maturation and adult reproductive function. However, whether such astroglial cell-GnRH neurone interactions occur in the human brain is not known. In the present study, we used immunofluorescence to examine the anatomical relationship between GnRH neurones and glial cells within the hypothalamus of five women. Double-staining experiments demonstrated the ensheathment of GnRH neurone perikarya by glial fibrillary acidic protein (GFAP)-immunoreactive astrocyte processes in the periventricular zone of the tuberal region of the hypothalamus. GFAP immunoreactivity did not overlap that of GnRH at the GnRH neurone's projection site (i.e. the median eminence of the hypothalamus). Rather, human GnRH neuroendocrine fibres were found to be closely associated with vimentin or nestin-immunopositive radial glial processes likely belonging to tanycytes. In line with these light microscopy data, ultrastructural examination of GnRH-immunoreactive neurones showed numerous glial cells in direct apposition to pre-embedding-labelled GnRH cell bodies and/or dendrites in the infundibular nucleus, whereas postembedding immunogold-labelled GnRH nerve terminals were often seen to be enwrapped by glial cell processes in the median eminence. GnRH nerve button were sometimes visualised in close proximity to fenestrated pituitary portal blood capillaries and/or evaginations of the basal lamina that delineate the pericapillary space. In summary, these data demonstrate that GnRH neurones morphologically interact with astrocytes and tanycytes in the human brain and provide evidence that glial cells may contribute physiologically to the process by which the neuroendocrine brain controls the function of GnRH neurones in humans.

Galanin modulates the activity of proopiomelanocortin neurons in the isolated mediobasal hypothalamus of the male rat.

It has become apparent that galanin as well as proopiomelanocortin-derived peptides, such as beta-endorphin, play an important role in the hypothalamic circuitry that regulates neuroendocrine functions and appetite behavior. We have recently shown that GalR1 and GalR2 galanin receptor mRNAs are expressed in proopiomelanocortin neurons of the arcuate nucleus, suggesting a direct modulatory action of galanin on the proopiomelanocortin neuronal system. In the present study, we investigated the effect of galanin on beta-endorphin release and proopiomelanocortin mRNA expression from male rat mediobasal hypothalamic fragments incubated ex vivo. Galanin induced a decrease of spontaneous beta-endorphin release within the first 30-60 min of incubation and this effect was blocked by the galanin receptor antagonist galantide. Co-incubation of galanin with FK-506 (tacrolimus), a calcineurin inhibitor, suppressed the inhibitory effect of galanin on beta-endorphin release, suggesting that calcineurin is involved in the galanin-evoked decrease in beta-endorphin release. Measurement of beta-endorphin levels in the tissues at the end of the incubation period (120 min) revealed that galanin caused a two-fold increase of beta-endorphin peptide concentration in the mediobasal hypothalamic tissues. Concurrently, galanin induced an increase in the mean density of silver grains overlying proopiomelanocortin neurons after 60 min of incubation, an effect antagonized by galantide. Finally, reverse transcription-polymerase chain reaction analysis revealed that the mRNAs for the three galanin receptor subtypes (i.e. GalR1, GalR2, and GalR3) were expressed in the incubated mediobasal hypothalamic fragments. Taken as a whole, our results indicate that galanin plays a modulatory role on proopiomelanocortin neurons and this interrelation contributes to the elucidation of the neural circuitry that controls, among others, gonadotropin-releasing hormone function.

Microwave strategy for improving the simultaneous detection of estrogen receptor and galanin receptor mRNA in the rat hypothalamus.

In a attempt to improve the sensitivity of the simultaneous use of immunohistochemistry (IHC) with estrogen receptor (ER) and in situ hybridization (ISH) with a neuropeptide receptor, we first applied an existing microwave (MW) irradiation protocol for immunohistochemical detection of the estrogen receptor in frozen brain sections. Regions of interest were the preoptic area and the arcuate nucleus of the hypothalamus. ER signal was effective only after MW heating of sections in the two regions. Control sections without pretreatment exhibited no staining for ER. Second, the MW protocol was applied in a novel procedure that consists of evaluation of the expression of the galanin receptor mRNA with a radioactive riboprobe after MW pretreatment. The galanin receptor mRNA signal intensity obtained after heating was quantitatively at least as good or significantly increased according to the region, with no discernible loss of tissue morphology. Finally, we describe a novel application of MW pretreatment on the same frozen section processed with ER antibody and a radioactive galanin receptor riboprobe. The stainings for estrogen and galanin receptors were intense in many cells of the preoptic area, with very low background. These results show that both IHC and ISH can be significantly improved by subjecting frozen sections to MW heating before the double labeling. This approach may provide a potential method to answer the important question of whether or not estrogen has a direct action on the expression of a peptide receptor. (J Histochem Cytochem 49:901-910, 2001)

Activities of the pituitary-adrenal and gonadal axes during the estrous cycle in adult female rats prenatally exposed to morphine.

The present investigation concerns 80-90-day-old female rats born from morphine-exposed mothers (2x10 mg/kg per day from day 11-18 of gestation) or saline-treated ones (controls). The former showed reduced size and activity of the adrenals at birth. At adult stage, they present: (1) higher increase of plasma adrenocorticotrophic hormone level on proestrus; (2) significant rise of plasma corticosterone level on diestrus morning and estrus evening; (3) adrenal atrophy which was significant only on diestrus and estrus morning; (4) more corticosterone binding sites of type I (mineralocorticoid receptors) on proestrus morning in the hippocampus; (5) more corticosterone binding sites of type II (glucocorticoid receptors) in the hippocampus on proestrus morning and in the hypothalamus on estrus morning. In both experimental groups, B(max) for hypothalamic mineralocorticoid receptors were drastically higher on estrus morning than on the other stages of the estrous cycle. The activity of the pituitary-gonadal axis is poorly affected by prenatal morphine-exposition. In both experimental groups drastic and comparable surges of both plasma progesterone and luteinizing hormone were observed during proestrus. Nevertheless morphine-exposed females showed higher levels of plasma estradiol on diestrus morning but lower levels on metestrus morning. In conclusion, prenatal exposition to morphine has long-term effects mainly on pituitary-adrenal axis as well as on binding sites for corticosterone in the hypothalamus and the hippocampus which are dependent on the estrous cycle stages in adult females.

Growth hormone secretagogues and hypothalamic networks.

Growth hormone secretagogues (GHSs) act at distinct levels to control growth hormone (GH) secretion. At the pituitary level they reinforce or extend a tonic GH-releasing-hormone (GHRH)-induced activated state by mobilizing intracellular Ca2+ store. At the hypothalamic level GHS actions are more complex than originally anticipated. Chronic treatments with GHS result in loss of responsiveness to the secretagogues, an effect probably accounted for by indirect negative feedback of GHS stimulated plasma GH levels over GHRH release. Moreover, intracerebroventricular treatments with GHS can have paradoxical, inhibitory effects on GH secretion. Several mechanisms can account for such dual effects. GHS receptors were found to extend far beyond the arcuate nucleus and are mainly coexpressed by GHRH, somatostatin, and neuropeptide Y (NPY) neurons. Activation of GHRH neurons by GHS can be direct or indirect. Indeed using antisense strategy we found that sstl are physiological activators of arcuate GHRH neurons and we propose that activation of SRIH arcuate interneurons by GHS can increase GHRH neuron activity. Moreover, GHS can stimulate distinct populations of NPY neurons having opposite effects on GH secretion: arcuate NPY interneurons, act as indirect facilitators of GHRH release, whereas, on the contrary, a different subset of NPY neurons projecting to the periventricular hypothalamus (those also involved in mediating leptin effects on GH) seems able to activate SRIH release.

Comparative distribution of mRNA encoding the growth hormone secretagogue-receptor (GHS-R) in Microcebus murinus (Primate, lemurian) and rat forebrain and pituitary.

The forebrain and pituitary sites of synthesis of growth hormone secretagogue-receptor mRNA were identified in four adult lemurs (Microcebus murinus) by in situ hybridisation performed with a radiolabeled cRNA probe transcribed from human Growth Hormone Secretagogue-Receptor cDNA. The cRNA sense and antisense probes were hybridised to cryostat sections containing structures extending from the rostral hypothalamus to its caudal limit as defined by the mammillary bodies. The pituitary gland and areas adjacent to the hypothalamus were also analyzed. For comparative purposes, sections from five adult rats containing these structures were hybridised with the same probes. The results point to a widespread distribution of Growth Hormone Secretagogue-Receptor mRNA in the hypothalamus, hippocampal formation, and cerebellar cortex of both lemurs and rats. As in the rat, specific hybridisation was particularly dense in the arcuate nucleus. Significant species differences were observed in the periventricular nucleus, the ventromedial nucleus, the lateral hypothalamic area, and the pituitary gland. In contrast to the rat, the lemur exhibited marked labelling in the infundibular nucleus, the periventricular nucleus and the pars tuberalis of the pituitary gland, whereas no labeling was detectable in the ventromedial nucleus and the lateral hypothalamic area. These results are discussed in terms of difference between the control of growth hormone secretion, feeding behaviour and seasonal rhythmicity among murine species and primates.

Evidence that members of the TGFbeta superfamily play a role in regulation of the GnRH neuroendocrine axis: expression of a type I serine-threonine kinase receptor for TGRbeta and activin in GnRH neurones and hypothalamic areas of the female rat.

The present study was designed to determine whether transforming growth factor (TGF)beta and/or activin participate in the regulation of the gonadotropin releasing hormone (GnRH) neuroendocrine axis in vivo. Single-label in situ hybridization histochemistry was used to determine the anatomical distribution of a TGFbeta and activin type I receptor (B1) mRNA, in the adult female rat hypothalamic areas that are known to be important sites for the regulation of reproduction. Dual-label in situ hybridization histochemistry was performed to determine whether B1 mRNA was expressed in GnRH neurones. The results of these studies revealed an extensive distribution of B1 mRNA in the hypothalamic regions, including diagonal bands of Broca, preoptic area, arcuate nucleus and median eminence. In the median eminence, B1 mRNA was detected in tanycytes and in the endothelial cells of the pituitary portal blood capillaries. Dual-label in situ hybridization histochemistry showed that 31+/-5% of GnRH neurones expressed B1 mRNA, thus providing evidence that TGFbeta and/or activin can act directly on GnRH neurones to modulate their activity. Taken together, these data provide morphological arguments in favour of a participation of TGFbeta and/or activin in the regulation of reproduction at the hypothalamic level.

Expression of the galanin receptor subtype Gal-R2 mRNA in the rat hypothalamus.

The distribution of galanin receptor subtype 2 (Gal-R2) mRNA-expressing cells was examined by in situ hybridization in the rat hypothalamus using a full-length rat 35S-riboprobe. Gal-R2 receptor mRNA-expressing cells were found at moderate to high levels of expression in most nuclei and regions of hypothalamus. The labeling was observed within well-defined anatomical nuclei: preoptic, suprachiasmatic, periventricular, paraventricular, arcuate, dorsomedial, mammillary nuclei. The supraoptic and ventromedial nuclei were almost devoid of labeling. Some scattered labeled cells were also observed in the pituitary. This distribution of Gal-R2 mRNA-expressing cells corresponds well with that of galanin binding sites studies. As compared to the distribution of the galanin receptor subtype 1 (Gal-R1), our results indicate that the Gal-R2 type is differentially distributed, although a significant overlap exists in some regions such the preoptic area, arcuate and dorsomedial nuclei. The functional implications of these results are discussed in light of the role of galanin receptors plays in neuroendocrine regulation and feeding behavior.

Hypothalamic-pituitary-adrenocortical and gonadal axes and sympathoadrenal activity of adult male rats prenatally exposed to morphine.

The present investigation concerns 80-90 day-old male rats born from morphine-exposed mothers (2 x 10 mg/kg per day from days 11 to 18 of gestation which showed at birth reduced size and activity of the adrenals). This prenatal treatment did not significantly disturb under resting conditions: (1) the postnatal body growth up to week 10 after birth, (2) the activity of the pituitary gonadal axis (circulating luteinizing hormone (LH) and testosterone (T), weight of the testicles and seminal vesicles), (3) the activity of the hypothalamo-pituitary-adrenal axis (HPA) (hypothalamic corticoliberin (CRF) content, plasma adrenocorticotrophic hormone (ACTH) level, adrenal weight and corticosterone (B) content, plasma B level) as well as Bmax and Kd of mineralocorticoid (type I) and glucocorticoid (type II) receptors to B in both the hippocampus and the hypothalamus. In contrast these rats showed reduced content of adrenals in noradrenaline (NA) and adrenaline (A) but increased circulating levels of A.

Semiquantitative distribution of galanin-receptor (GAL-R1) mRNA-containing cells in the male rat hypothalamus.

The semiquantitative distribution of mRNA encoding for rat galanin receptor (GAL-R1) was examined by in situ hybridization in the rat hypothalamus using a 35S-riboprobe. Most hypothalamic nuclei expressed GAL-R1 mRNA. In the anterior hypothalamus, high levels of expression were found in the medial preoptic area, paraventricular and supraoptic nuclei. Numerous cells also expressed the GAL-R1 mRNA with a moderate level of expression in the periventricular region. Very few GAL-R1-expressing cells were present in the suprachiasmatic nucleus. In the medial hypothalamus, numerous expressing cells were detected in the dorsomedial and ventromedial nuclei. The arcuate nucleus was moderately labeled throughout its rostrocaudal extent; labeled cell bodies were visible in the ventromedial and ventrolateral subdivisions as well. These results indicate that the GAL-R1 mRNA is not only expressed in anterior hypothalamic nuclei but also in the mediobasal hypothalamus and periventricular region. This hypothalamic distribution correlates well with that of 125I-GAL-binding sites and GAL-immunoreactive fibers. This distribution represents the morphological substrate for GAL roles in the hypothalamic regulation of neuroendocrine, behavioral and autonomic functions.

The fetal expression of proenkephalin mRNAs and Met-enkephalin immunoreactivity in the hypothalamoseptal tract and adjacent hypothalamic areas of the guinea pig brain.

The development of the enkephalinergic hypothalamoseptal tract in the guinea pig brain was studied from embryonic day 30 until birth. Proenkephalin (PE) mRNAs were detected in the hypothalamic magnocellular dorsal nucleus (MDN) by in situ hybridization with a synthetic 35S-labeled oligonucleotide. The Met-enkephalin-like immunoreactivity (Met-enk-LI) in the MDN and the lateral septum (LS) was detected with antibodies against Met-enkephalin, on adjacent cryostat sections. At the same time, an immunohistochemical study of the arrangement of enkephalinergic axon terminals in the LS at birth was performed at the electron microscopic level. PE mRNAs were first found to be expressed in the MDN at embryonic day 32 (E32) and increased to reach a maximal level at E48. Met-enk-LI was consistently detectable from E38 in numerous perikarya of the MDN as well as in nerve terminals of the LS. The number of Met-enk-LI cells of the MDN decreased after this stage until birth, whereas positive nerve endings in the LS increased. At the electron microscopic level, numerous cell bodies of the LS at birth were consistently surrounded by Met-enk immunoreactive nerve terminals. Cells expressing the PE gene and Met-enk-LI were also observed from E38 to E44 in the periventricular area. Some of these cells were found double-labeled with Met-enkephalin and Somatostatin antisera. The enkephalinergic system of the hypothalamoseptal tract appears at early embryonic stages and may be essential in regulating septal neuronal functions early in gestation. Differing ontogenic onsets of the enkephalinergic hypothalamoseptal and periventricular-median eminence tracts suggest possible developmental and functional differences.

Immunocytochemical and in vitro autoradiographic evidence for a direct somatostatinergic modulation of the enkephalinergic hypothalamoseptal tract of the guinea-pig.

The present study was undertaken to determine whether the enkephalinergic hypothalamoseptal tract originating in the magnocellular dorsal nucleus in the guinea-pig brain is under the influence of somatostatin. In the first step, double immunocytochemical labeling of enkephalinergic cells and somatostatinergic fibers was combined at the light and electron microscopic levels in the magnocellular dorsal nucleus. As a second step, an in vitro radioautography was used to determine whether somatostatin receptors are present in the same area. A close relationship between somatostatin nerve endings and enkephalin perikarya was observed at both the light and electron microscopic levels. Contracts were more numerous in the ventral part of the magnocellular dorsal nucleus. Whenever synaptic images were clearly observable, they appeared symmetrical. In the same area, a moderate concentration of G-protein-coupled somatostatin binding sites was also visualized. These results suggest that somatostatin has a regulator role on the enkephalinergic hypothalamoseptal tract, directly at the level of the magnocellular dorsal nucleus.

Fine-structural localization of proenkephalin mRNAs in the hypothalamic magnocellular dorsal nucleus of the guinea pig: a comparison of radioisotopic and enzymatic in situ hybridization methods at the light- and electron-microscopic levels.

With the aim of localizing proenkephalin mRNAs in neurons of the hypothalamic magnocellular dorsal nucleus of the guinea pig, we compared the in situ hybridization signals obtained on Vibratome sections with a method employing either a biotinylated or a digoxigenin-labeled oligonucleotide detected by means of the alkaline phosphatase reaction. Since the hybridization approach using the biotinylated probe was more sensitive than the digoxigenin method, the ultrastructural localization of hybrids in neurons of the magnocellular dorsal nucleus was studied by the use of the former procedure, and was further compared with results of in situ hybridization using a 35S-labeled probe. Biotin was detected via an amplified avidin-biotin-peroxidase complex. Radioactive hybrids were localized over extended cytoplasmic compartments rich in rough endoplasmic reticulum and also in nuclear indentations. The method based on biotinylated probe proved to be sensitive and provided high-resolution labeling in well-preserved specimens. Proenkephalin mRNAs were clearly localized within circumscribed cytoplasmic compartments. The immunoprecipitates were mainly observed within the rough endoplasmic reticulum, especially at the periphery of the cell. The reticulum was dominated by elongated parallel cisternae. The labeling also appeared in a paranuclear position, mainly in nuclear indentations. The labeling was found on the outer surface of the endoplasmic lamellae. The remainder of the reticulum was unlabeled. Neuronal processes were free of labeling.

Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.

We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.

Effects of inhibition of GABA catabolism by aminooxyacetic acid on enkephalinergic neurons--immunocytochemical and ultrastructural study in the enkephalinergic hypothalamoseptal tract of the guinea-pig.

The immunocytochemical and ultrastructural features within [Met]enkephalin neurons of the guinea-pig hypothalamoseptal tract were investigated under chronic inhibition of GABAergic catabolism. This was achieved by raising the brain GABA concentration with aminooxyacetic acid which inhibits GABA-transaminase, the enzyme responsible for the catabolism of GABA. Guinea-pigs were injected intraperitoneally with 10 or 20 mg/kg per day aminooxyacetic acid for two, four or eight days and killed 16 h post-dose. Repeated injections of aminooxyacetic acid produced a great increase in immunoreactivity for GABA in nerve endings surrounding enkephalinergic perikarya in the magnocellular dorsal nucleus of the guinea-pig. Extensive immunocytochemical studies stressed the increase and redistribution of the immunoreaction for [Met]enkephalin in the perikarya of the magnocellular dorsal nucleus under such GABAergic activation. Quantitative and statistical analyses showed that administration of aminooxyacetic acid for eight days significantly increased the intensity of labelling within stimulated perikarya (P less than 0.001). A concomitant accumulation of immunopositive large granules in the enkephalinergic boutons of the lateral septum was observed. In the same way, ultrastructural changes in enkephalinergic cell bodies were analysed and reflected disturbances in the biosynthetic and digestive activities of enkephalinergic perikarya. We postulate that chronic inhibition of the GABAergic catabolism leads to modification in the metabolism of enkephalinergic neurons and to an inhibitory action of GABA on the [Met]enkephalin release from nerve endings. This study give morphological support to the complex functional interactions between GABA and opioid peptide transmitter system.

Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.

GABA axon terminals in synaptic contact with enkephalin neurons in the hypothalamus of the guinea pig. Demonstration by double immunocytochemistry.

By using a method combining pre-embedding immunoperoxidase staining for enkephalin and postembedding immunocolloidal gold labeling for gamma-aminobutyric acid (GABA) it has been demonstrate that many GABAergic boutons made synapses on enkephalin-reacting soma in the magnocellular dorsal nucleus of the guinea pig hypothalamus. The gold particles revealing the presence of GABA were essentially located over the small clear vesicles and mitochondria present in these GABAergic nerve endings. All the synapses observed were symmetrical. Taking into account the great number of these nerve endings, we conclude for a strong regulatory role of GABA on enkephalin-containing cells of the magnocellular dorsal nucleus.

[The ultrastructural level of different peptidergic nerve endings in the median eminence, dorsal hypothalamus and septum]. / Etude à l'échelle ultrastructurale de différentes terminaisons nerveuses peptidergiques dans l'éminence médiane, l'hypothalamus dorsal et le septum.

Comparison at the ultrastructural level of nerve endings containing 6 different peptides (LH-RH, C terminal ACTH, somatostatin, enkephalin, CRF, hp GRF) using immunocytochemical means has been performed. The results show that the granules are in each case very similar in size and appearance. This aspect can therefore be used to individualize the neuropeptide terminals but it does not permit to determine the nature of the peptide. In the central nervous system many different neuropeptide terminals have been observed at the ultrastructural level but in the majority of cases neither the role nor the pericarya of origin could be precised. On the contrary for the enkephalin containing nerve endings observed in the lateral septum it has been possible to prove that pericarya of origin were located in the hypothalamus and more precisely in the magnocellular dorsal nucleus.

Study of the efferent connections of the enkephalinergic magnocellular dorsal nucleus in the guinea-pig hypothalamus using lesions, retrograde tracing and immunohistochemistry: evidence for a projection to the lateral septum.

The efferents of enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus of the guinea-pig were studied using different neuroanatomical methods and indirect immunocytochemical technique. Following unilateral implantation of the fluorescent dye 4',6-diamidino-2-phenylindole in the lateral septal nucleus, retrogradely-labeled perikarya were found in the magnocellular dorsal nucleus. These labeled perikarya reacted with antiserum against enkephalin, demonstrating that enkephalin-immunoreactive neurons in the magnocellular dorsal nucleus project to the lateral septal nucleus. In other experiments, complete bilateral lesions were produced in the magnocellular dorsal nucleus by electrocoagulation. Enkephalin-immunoreactive nerve fibers and terminals were totally depleted in the lateral septal nucleus. This confirms that septal enkephalin-immunoreactive terminals originate in the magnocellular dorsal nucleus and further suggests that this nucleus is the source of all the enkephalin-immunoreactive material found in the septum. Experiments utilizing two different fluorescent dyes, 4',6-diamidino-2-phenylindole and propidium iodide, injected in each side of the lateral septal nucleus, respectively, demonstrated that the magnocellular dorsal nucleus gives off axon collaterals to both sides of the septum, since double-labeling of individual cell bodies was detected in the nucleus. By relating this finding to the results obtained after unilateral destruction of the nucleus, which caused an incomplete loss of enkephalin- immunoreactive material in the lateral septal nucleus ipsilaterally, it is suggested that the enkephalinergic hypothalamo-septal pathway contains unbranching neurons projecting ipsilaterally and branching neurons distributing fibers ipsilaterally and contralaterally. Lesion experiments, and experiments based on the retrograde axonal transport of horseradish peroxidase after intravenous injections, demonstrated that the magnocellular dorsal nucleus contributes neither to the tubero-infundibular nor to the hypothalamo-neurohypophyseal tracts. The lateral septal nucleus receives numerous aminergic and peptidergic projections, indicating the potential importance of this region in physiological and behavioral events. In the guinea-pig, the well-demarcated enkephalinergic pathway demonstrated in this study provides a convenient model for the experimental study of the enkephalinergic innervation of the lateral septal nucleus.

[Immunohistochemical arguments in favor of co-localization of neuropeptides in hypothalamo-infundibular neuron systems]. / Arguments immunohistochimiques en faveur de la colocalisation de neuropeptides dans des systèmes de neurones hypothalamo-infundibulaires.

Light and electron microscope methods which can be used to examine the possibilities of co-occurrence of peptides are reviewed. The results obtained using some of them are exposed and discussed, concerning i/the light microscope demonstration of the CRF/vasopressin coexistence in rat and guinea-pig. ii/the electron microscope demonstration of the granular colocalization of LH-RH/C-terminal ACTH and somatostatin/enkephalin in the guinea-pig median eminence.