M. E. Stoeckel

Oxytocin receptors on oxytocin neurones: histoautoradiographic detection in the lactating rat.

1. The purpose of the present study was the detection at the cellular scale of the oxytocin (OT) receptors involved in the facilitatory effect of this neuropeptide on its own release during the milk ejection reflex. 2. OT binding sites were demonstrated in brain sections by using a highly selective 125I‐labelled OT antagonist detected by film‐ and histoautoradiography. 3. Film autoradiographs revealed the presence of OT binding sites in the hypothalamic magnocellular (supraoptic, paraventricular and anterior commissural) nuclei in lactating rats, suckled or not. This detection was only possible after acute i.c.v. injection of OT antagonist which probably induced an upregulation of the OT binding sites to autoradiographically detectable levels. 4. Combined application of histoautoradiographic and immunohistochemical techniques showed that the OT binding sites were concentrated on OT magnocellular neurones. Labelling concerned cell bodies and dendrites but not the axons and endings in the pituitary neural lobe. 5. The presently detected somatodendritic autoreceptors on OT neurones probably mediate the facilitatory effect of OT on its own release during the milk ejection reflex.

GABA- and peptide-immunoreactivities co-localize in the rat central extended amygdala

THE central amygdaloid nucleus and the lateral bed nucleus of the stria terminalis are two similar telencephalic structures belonging to the central extended amygdala. These regions contain numerous peptidergic and GABAergic neurones which maintain the neurones projecting to the brain stem under tight intrinsic control. Using immunocytochemistry in colchicinetreated rats, we showed that, in the lateral subdivision of the central amygdaloid nucleus and in the dorsal part of the lateral bed nucleus of the stria terminalis, a population of GABAergic neurones is able to co-synthesize either corticotropin-releasing factor or methionineenkephalin, but never both peptides. These results suggest that, in the GABAergic intrinsic circuits of the central extended amygdala, co-liberated peptides can have a modulatory role on GABAergic actions.

Localization of Oxytocin Binding Sites in the Thoracic and Upper Lumbar Spinal Cord of the Adult and Postnatal Rat: A Histoautoradiographic Study

Oxytocin binding sites were detected by autoradiography on films and emulsion‐coated sections in the spinal cord of adult and postnatal rats from C8 to L2, using a highly selective 125l‐labelled oxytocin antagonist. Oxytocin binding sites were detected on all transverse sections in the dorsal horn, where labelling was scattered over laminae I and II. The autonomic areas, i.e. the intermediolateral cell column, the central grey (lamina X) and the nucleus intercalatus were labelled. Binding in the intermediolateral cell column was most frequently observed on sections from T9 to T11 in adult and T7 to T8 in postnatal rats. In this location, oxytocin binding sites were highly concentrated on cell bodies of putative sympathetic preganglionic neurons; however, not all of these cells were labelled. Diffuse labelling occurred on the dorsal part of the central grey, mainly between T8 and L2. Isolated labelled cells belonging to the nucleus intercalatus were scattered between the central canal and the intermediolateral cell column. In addition, oxytocin binding sites were found on some motoneurons of the lateral group of T12‐T13, but only in postnatal rats. The distribution of oxytocin binding sites in the rat spinal cord coincides with that of the oxytocin innervation and strongly suggests a modulatory role of this peptide in sensory and autonomic functions.

An unusual familial cardiomyopathy characterized by aberrant accumulations of desmin-type intermediate filaments

Immunofluorescence microscopy using antibodies specific for different intermediate filament types has been used to study a rare familial cardiomyopathy characterized electron microscopically by massive accumulations of unordered intermediate filaments. The results show that the inclusions in cardiac muscle cells are composed of the desmin type of intermediate filament characteristic of muscle tissues, and draw attention to the importance of these filaments in maintaining normal cardiac ultrastructure and function.

Expression of the oxytocin and vasopressin genes in the rat hypothalamus during development: an in situ hybridization study

Oxytocin (OT)- and vasopressin (VP)-mRNAs were detected in the hypothalamus, during development, by in situ hybridization with synthetic oligonucleotide probes. The presence of VP- and OT-mRNAs was first detected in the supraoptic nucleus at E16 and E17 respectively, and simultaneously at E18 in the paraventricular nucleus. VP- (but not OT-) mRNA then appeared in the suprachiasmatic nucleus at E21, and OT- (but not VP-) mRNA, in the anterior commissural nucleus at time of birth. In the different nuclei, the relative distribution of cells containing OT- or VP-mRNA was comparable, from the earliest stages on, with that observed in the adult. These data suggest that the later appearance of mature OT (E20), versus VP (E16), reported in immunocytochemical studies, may not be due to a delayed transcription. Moreover, since the presence of both OT- and VP-prohormones has been reported at E16, the results support the idea of a rapid translation of both OT- and VP-mRNAs. In no location could OT- and VP-mRNAs be detected before final cell settlement; the possible role of environmental factors in final non-proliferative differentiation is discussed.

Quantitative autoradiographic mapping of neurohypophysial hormone binding sites in the rat forebrain and pituitary gland—I. Characterization of different types of binding sites and their distribution in the long-evans strain

Oxytocin and vasopressin binding sites were localized and characterized by quantitative autoradiography on consecutive sections of Long-Evans rat forebrains and pituitary glands, incubated in the presence of 5 nM [3H]oxytocin or 5 nM [3H]vasopressin. In the forebrain, two types of neurohypophysial hormone binding sites were thus defined. (1) Oxytocin/vasopressin sites with similar nanomolar-range affinities for [3H]oxytocin and [3H]vasopressin; both tritiated peptides were displaced from these sites in the presence of 10 microM of either oxytocin or vasopressin. The main areas bearing such sites were the ventral subiculum, several nuclei of the amygdala, the ventromedial hypothalamic nucleus, the bed nucleus of the stria terminalis and the olfactory tubercle. (2) Selective vasopressin sites, binding [3H]vasopressin with nanomolar-range affinity and [3H]oxytocin with a much lower affinity; these sites were not labelled in the presence of 5 nM [3H]oxytocin, and 10 microM oxytocin displaced [3H]vasopressin binding by 80%. Such sites occurred in several thalamic nuclei, in the dopaminergic A13 cell group of the zona incerta, the suprachiasmatic nucleus, the fundus striati and the lateral septal nucleus. No selective oxytocin sites were detected. Different oxytocin and vasopressin binding characteristics were found in the hypothalamo-neurohypophysial system. In the paraventricular and supraoptic nuclei and in the pituitary neural lobe the [3H]vasopressin binding density was twice that of [3H]oxytocin; vasopressin was always more potent than oxytocin in displacing both [3H]vasopressin and [3H]oxytocin binding from those sites. Interaction of the tritiated peptides with neurophysins cannot be completely ruled out in these locations. The present data are discussed in correlation with the functional roles of the neurohypophysial peptides in the brain and the pharmacological characteristics of their receptors.

Cerebrospinal fluid‐contacting neurons in the rat spinal cord, a γ‐aminobutyric acidergic system expressing the P2X2 subunit of purinergic receptors, PSA‐NCAM, and GAP‐43 immunoreactivities: Light and electron microscopic study

Cerebrospinal fluid‐contacting neurons (CSFcNs) occur in various brain regions of lower vertebrates. In mammals, they are restricted to medullospinal areas, and little is known about their projection sites. In the present work, we investigated some morphofunctional characteristics of such neurons in the rat spinal cord by light and electron microscopic immunocytochemistry. CSFcNs expressing the P2X2 subunit of purinergic receptors were present throughout the spinal cord, though more numerous at lower thoracolumbar and sacral levels. These neurons coexpressed GAD and the polysialylated neural cell adhesion molecule (PSA‐NCAM), a marker of cellular plasticity. From low thoracic levels downward, tiny amyelinic axons (less than 200 nm in diameter) were tightly packed in bundles, which ran along the ependyma and extended ventrally, eventually concentrating against the walls of the ventral median fissure. In addition to P2X2, GAD, γ‐aminobutyric acid (GABA), and PSA, these axons expressed GAP‐43 immunoreactivity. Moreover, they were labelled along their entire lengths with antibodies against synaptotagmin and synaptophysin, but these failed to reveal intraspinal terminal fields. Taken together, our observations indicate the presence in the rat spinal cord of a highly plastic system of GABAergic CSFcNs that express the P2X2 subunit of purinergic receptors. The function of this original system remains open to question. In these neurons, the P2X2 receptors may confer a sensitivity to ATP either present in the CSF or released by nearby neurons of the central autonomic area. J. Comp. Neurol. 457:159–174, 2003.

Colchicine effects on neurosecretory neurons and other hypothalamic and hypophysial cells, with special reference to changes in the cytoplasmic membranes

SummaryThe morphological effects of colchicine on the entire neurosecretory (NS) tract and on various hypothalamic nuclei have been studied. The perturbation in axonal flow, indicated by the accumulation of NS material, coincide with fragmentation of the cytoplasmic membranes, i. e. the Golgi apparatus and the endoplasmic reticulum, whereas the neurotubules remain relatively well preserved. Autophagic destruction of NS material is observed along the entire length of the NS fibres. The rapid and systematic changes in the axoplasmic reticulum, known to store calcium, lead us to envisage a role for this system — similar to that of the sarcoplasmic reticulum — in controlling the transport of NS vesicles. The junctional zone between the stalk and the neural lobe seems to play a particular rôle in the transport of NS material to the posthypophysial terminals of the NS axons. Colchicine provokes an increase in dense-cored vesicles in most of the neurons of the other hypothalamic nuclei studied: arcuate, suprachiasmatic, periventricular and ventromedial. Membranous alterations are also observed in these sites. Colchicine administered to animals which were hypothyroid, castrated or adrenalectomized, reveals stimulated neurons, identified by their excessive content of dense-cored vesicles. These neurons display no specific localization, for they occur in all hypothalamic nuclei, irrespective of the stimulation. The frequency of stimulation of neurons of the periventricular nucleus is striking.

Three distinct thyrotropin-releasing hormone-immunoreactive axonal systems project in the median eminence-pituitary complex of the frog ran a ridibunda. Immunocytochemical evidence for co-localization of thyrotropinreleasing hormone and mesotocin in fibers innervating pars intermedia cells

The localization of thyrotropin-releasing hormone-immunoreactive structures was investigated in the hypothalamo-hypophyseal complex of the frog, Rana ridibunda, by light and electron microscopy using the conventional indirect immunoperoxidase technique and the immuno-gold technique, respectively. The localization of mesotocin-, vasotocin- and neurophysin-immunoreactive elements was compared to that of thyrotropin-releasing hormone either by comparing homologous fields on serial sections or by staining the same section with two different antibodies. Thyrotropin-releasing hormone-immunoreactive perikarya occurred mainly in the anterobasal periventricular area and dorsal extension of the preoptic nucleus, and in the lateral zone of the infundibular nucleus. In the anterobasal preoptic nucleus, the distribution of thyrotropin-releasing hormone-immunoreactive perikarya partially overlapped that of vasotocin- and mesotocin-containing neurons; however, co-localization of thyrotropin-releasing hormone with either nonapeptide could not be detected there. In contrast, in the caudal extension of the preoptic nucleus, thyrotropin-releasing hormone- and mesotocin-like immunoreactivities were frequently co-localized in the same neurons. In the external zone of the median eminence, abundant networks of thyrotropin-releasing hormone- and vasotocin-immunoreactive nerve fibers were found in the vicinity of portal capillaries, while mesotocin-immunoreactive axons were only found in the internal zone. Using the immuno-gold technique at the electron microscopic level, three distinct thyrotropin-releasing hormone-immunoreactive systems were identified in the median eminence-neurointermediate lobe complex. (1) In the external zone of the median eminence, a conspicuous population of pericapillary endings contained 100-nm dense core vesicles immunoreactive solely for thyrotropin-releasing hormone. (2) In the neural lobe of the pituitary, thyrotropin-releasing hormone immunoreactivity occurred on secretory vesicles in a subpopulation of the mesotocinergic axons containing 160-nm secretory granules; co-localization with vasotocin was never seen. (3) In the intermediate lobe, thyrotropin-releasing hormone- and mesotocin (or neurophysin I)-immunoreactivities were systematically found in the same 120-nm dense core vesicles; these thyrotropin-releasing hormone-/mesotocin-immunoreactive axon terminals frequently made synaptic contacts with melanotropic cells. The possible modulatory effect of mesotocin on thyrotropin-releasing hormone-induced alpha-melanocyte-stimulating hormone secretion was investigated using perifused frog neurointermediate lobes. Administration of graded doses of mesotocin (from 10(-10) to 10(-5) M) did not affect the spontaneous release of alpha-melanocyte-stimulating hormone. In addition, mesotocin (10(-7) and 10(-6) M) did not modify thyrotropin-releasing hormone-evoked alpha-melanocyte-stimulating hormone release.(ABSTRACT TRUNCATED AT 400 WORDS)

Systematic presence of GABA-immunoreactivity in the tubero-infundibular and tubero-hypophyseal dopaminergic axonal systems: an ultrastructural immunogold study on several mammals

SummaryImmunoreactivities for tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA) and, in some cases, glutamic acid decarboxylase (GAD) were detected by light and electron microscopy in axons projecting into the median eminence and pituitary gland of various mammals (rats, mice, guinea pigs, cats, rabbits and hares). Light microscope immunoperoxidase reactions were performed on adjacent semithin sections of plasticembedded samples. In the median eminence external zone, the distributions of the TH- and GAD- or GABA-immunoreactive endings were very similar in the anterior and lateral areas, while medially the GABA-labelled endings predominated. Comparable distribution patterns were found in the various species examined. In the pituitary gland, the distributions of GABA- and TH- immunoreactivities were superimposable in the intermediate lobes of all species examined, except in the rabbit and hare in which both types of innervation were lacking. For electron microscopy, the immunogold procedure was applied to sections of lowicryl-embedded samples; simultaneous detection of GABA- and TH-immunoreactivities was enabled by recto-verso double labelling with gold particles of distinct diameters. In the median eminence, GABA-immunoreactivity occurred systematically in the TH-positive endings, while distinct GABA-positive/TH-negative axons were also detected. In the intermediate lobe, the colocalization of TH- and GABA-immunoreactivities was a constant feature of the axons innervating the melanotrophic cells in all the species examined, except in the Leporidae. The functional significance of this colocalization remains to be determined.