Biomedical subjects
Publications and source records attributed to M Palkovits.
Several peptides are produced by central nervous system neurons, many of these are involved in the control of body fluid homeostasis. The presence of neuropeptides in the median eminence and circumventricular organs, in the neurosecretory hypothalamic cell groups and in the baroreceptor centres are briefly summarized.
The afferent connections of the retrochiasmatic area were examined in the rat after microintophoretic application of horseradish peroxidase. Labelled cells were found in amygdaloid nuclei, in the magnocellular preoptic nucleus, in the zona incerta and in all hypothalamic nuclei. In the brainstem reactive neurones were seen in the caudal part of the nucleus of the solitary tract (A2-C2 catecholaminergic cell groups), in and adjacent to the lateral reticular nucleus (A1-C1 catecholaminergic cell groups), in the nuclei reticularis pontis caudalis, pontis oralis, reticularis medullae oblongatae pars dorsalis and nucleus reticularis tegmenti pontis. The parabigeminal nucleus also contained a few labelled cells. A large number of labelled cells were seen around the superior cerebellar peduncle, in the caudal part of the periaqueductal grey and in the peripeduncular nucleus. Scattered cells appeared in the brainstem raphe nuclei, in the locus coeruleus and in the lateral part of the substantia nigra. Although it is not certain that all of these neurones terminate on retrochiasmatic cells, the extensive labelling suggests that this brain area is a critical station of many ascending and descending pathways. These may potentially affect a variety of neuro-endocrine mechanisms via the efferent projections of the retrochiasmatic area.
Using a specific radioimmunoassay, we have found that immunoreactive (ir) alpha-neo-endorphin has a widespread and unique distribution in the rat brain and spinal cord. The highest concentration in brain is in the substantia nigra (1692.1 fmol/mg of protein). Very high concentrations of ir-alpha- neo-endorphin (greater than 500 fmol/mg of protein) are also found in the lateral preoptic nucleus, dentate gyrus, parabrachial nuclei, nucleus accumbens, globus pallidus, median eminence, and anterior hypothalamic nucleus. Relatively high concentrations of ir-alpha-neo-endorphin (250 to 500 fmol/mg of protein) are present in the bed nucleus of the stria terminalis, paraventricular nucleus, nucleus of the solitary tract, dorsomedial nucleus, central amygdaloid nucleus, periaqueductal gray matter, suprachiasmatic nucleus, periventricular nucleus, hippocampus, prepositus hypoglossal nucleus, arcuate nucleus, ventromedial nucleus, medial preoptic nucleus, zona incerta, dorsal premamillary nucleus, medial forebrain bundle (hypothalamic and preoptic), nucleus of the diagonal band, locus ceruleus, lateral septal nucleus, and nucleus ambiguus . Moderate levels (100 to 250 fmol/mg of protein) are found in the posterior hypothalamic nucleus, ventral premamillary nucleus, dorsal raphe nucleus, motor hypoglossal nucleus, caudate-putamen, periventricular thalamic nucleus, subcommissural organ, sensory trigeminal nucleus, perifornical nucleus, area postrema, supraoptic nucleus, cuneate nucleus, medial amygdaloid nucleus, and organum vasculosum laminae terminalis. Low concentrations of ir-alpha-neo-endorphin (less than 100 fmol/mg of protein) are found in many cortical structures, claustrum, thalamic nuclei, habenular nuclei, lateral geniculate body, red nucleus, superior and inferior colliculi, paramedian reticular nucleus, pontine nuclei, superior olive, vestibular nuclei, motor facial nucleus, gigantocellular reticular nucleus, and subfornical organ.(ABSTRACT TRUNCATED AT 250 WORDS)
The distribution of immunoreactive (ir)-beta-neo-endorphin in 101 microdissected rat brain and spinal cord regions as well as in the neurointermediate lobe of pituitary gland was determined using a highly specific radioimmunoassay. The highest concentration of beta-neo-endorphin in brain was found in the median eminence (341.4 fmol/mg of protein). High concentrations of ir-beta-neo-endorphin (greater than 250 fmol/mg of protein) were found in 11 nuclei, including dorsomedial nucleus, substantia nigra, parabrachial nuclei, periaqueductal gray matter, anterior hypothalamic nucleus, and lateral preoptic areas. Moderate concentrations of the peptide (between 100 and 250 fmol/mg of protein) were found in 66 brain nuclei such as the amygdaloid and septal nuclei, most of the diencephalic structures (not including the hypothalamus), and the majority of the medulla oblongata nuclei and others. Low concentrations of ir-beta-neo-endorphin (less than 100 fmol/mg of protein) were found in 21 nuclei, e.g., cortical structures (frontal., cingulate, piriform, parietal, entorhinal, occipital), olfactory tubercle, and cerebellum (nuclei and cortex). The olfactory bulb has the lowest beta-neo-endorphin concentration (21.3 fmol/mg of protein). Spinal cord segments exhibit low peptide concentrations. The neurointermediate lobe of the pituitary gland is extremely rich in ir-beta-neo-endorphin.
A widespread distribution of immunoreactive dynorphin (ir-Dyn) in rat brain and spinal cord was demonstrated by means of a highly specific radioimmunoassay. The highest concentrations of ir-Dyn (greater than 399 pg/mg protein) were found in hypothalamic nuclei, i.e. the premamillary, anterior hypothalamic and dorsomedial nuclei and median eminence. Relatively high concentrations of ir-Dyn (between 320 and 399 pg/mg protein) were found in other hypothalamic nuclei such as the medial and lateral preoptic, perifornical, suprachiasmatic, ventromedial nuclei and in the medulla oblongata in the area postrema and in the nucleus of the solitary tract (commissural part). Moderate levels of ir-Dyn (between 140 and 320 pg/mg protein) were found in most diencephalic areas other than the hypothalamic nuclei and further nuclei in the medulla oblongata, in the mesencephalon, pons and spinal cord. Low to moderate levels of ir-Dyn were found in the telencephalon, with lowest levels (less than 140 pg/mg protein) found in the cerebral cortex, olfactory bulb, dorsal septal nucleus, medial amygdaloid nucleus, caudate-putamen, superior collicle, cerebellum and certain areas of the reticular formation.
Concentrations of dynorphin and alpha-neo-endorphin were measured in rat supraoptic and paraventricular nuclei, median eminence and posterior pituitary. Paraventricular lesions did not alter the level of either peptide in the median eminence or posterior pituitary. Knife cuts in the lateral retrochiasmatic area that severed the supraoptico-hypophyseal tract reduced dynorphin and alpha-neo-endorphin levels by 64-80% in the posterior pituitary and by somewhat less in the median eminence. Knife cuts in the vicinity of the supraoptic nucleus that did not interrupt the supraoptico-hypophyseal tract did not affect peptide concentrations in the posterior pituitary. Our data suggest that dynorphin and alpha-neo-endorphin in the posterior pituitary are in processes of supraoptic nucleus neurons. These neurons also project to the median eminence which may receive fibers from other dynorphin and alpha-neo-endorphin containing cells too.
The distribution of thyrotropin releasing hormone (TRH) in individual nuclei of the rat brainstem was examined by radioimmunoassay. TRH was detectable in 36 of 40 brainstem nuclei investigated and the localization of TRH in the brainstem was unlike other known brainstem neuropeptides. By far the highest concentration of TRH in the brainstem (1.2 ng/mg protein) is present in the nucleus of the solitary tract. The concentration of TRH was relatively high in the motor nuclei of the IIIrd, Vth, VIIth, Xth and XIIth cranial nerves, and less high in the area postrema, nucleus gracilis, locus coeruleus, lateral reticular nucleus (A1-catecholamine cell group), dorsal raphe and central gray matter. Cerebellum and pontine nuclei contained very low levels (less than 0.03 ng/mg protein) of TRH.
The total number and the numerical ratio of extrinsic and intrinsic (local) innervations of magnocellular neurons in the paraventricular nucleus (mPVN) were determined after surgical isolation of the nucleus in rats. Various lesions and transections of fibers running to the mPVN were performed to determine the number and possible sources of septal, hippocampal and caudal periventricular fibers to the mPVN. The relatively high proportion of possibly intrinsic connections (43%) suggests a local, integrative function of neuronal activity in the PVN. On the average, 57% of the total number of presynaptic boutons have been found to originate from outside the nucleus (extrinsic afferentation). Only 7% of these fibers ascend from caudal through the periventricular area. mPVN afferents originating from the ventral subiculum and from the lateral septal nucleus comprise about 3 and 5% of the extrinsic afferentation, respectively.
Quantitative histological analysis of serial sections of the adult male rat brain gives mean total estimates of the numbers of cells in the magnocellular and parvicellular divisions of the paraventricular hypothalamic nuclei (PVN) of 6600 and 11,500, respectively. The numbers and densities (number/mm3) of presynaptic bouton profiles have been measured and calculated on electron micrographs of the magnocellular paraventricular nucleus (mPVN). There are approximately 18 x 10(6) presynaptic boutons in the magnocellular subdivision: 76% of the synaptic boutons are axodendritic, 17% axosomatic and 7% are unidentified but include a few axo-axonic contacts. One third of the boutons contain dense-core vesicles although their postsynaptic contacts do not differ from other boutons. The estimated ratio of the number of boutons per neuron in the magnocellular paraventricular nucleus is 2820:1.
Using both the 'punch' microdissection and a radioimmunological technique, circadian variations in beta-endorphin concentrations can be observed in the pituitary and in some discrete brain regions of the male rat (Wistar CFY). Animals were synchronized with light from 06.00 to 18.00 h, then darkness. Water and food were available ad libitum. Very well marked circadian rhythms were in evidence in the anterior lobe of the pituitary, the septum, the pons, the medulla oblongata and the cerebellum. There crest time locations were situated between 20.00 and 24.00 h. No significant circadian rhythms but biphasic variations were observed in the intermediate lobe of the pituitary, the POA, the thalamus, the central gray and the caudatus. There crest time locations were synchronized around 08.00 and 20.00 h. The most striking finding was that, regardless of the brain area investigated so far, maximal values were observed a short time after the beginning of the activity period of rats. This fact is identical with the one which has been observed for substance P and LH-RH contents in brain areas where these peptides are mostly present in nerve terminals in high concentrations.
Ovine corticotropin releasing factor-like (oCRF-LI) immunoreactivity was detected in the rat hypothalamus by immunocytochemistry. The unlabeled antibody peroxidase-antiperoxidase method was applied in 40 microM vibratome sections before embedding for examination under the electron microscope. Immunoreactivity was found in axons of the median eminence and the neural lobe, as well as in cell bodies and dendrites of parvocellular neurons the in paraventricular nucleus. Axon terminals in the external zone of the median eminence and in the neural lobe frequently abutted on the pericapillary space, suggesting the possible release of oCRF-LI into the fenestrated capillaries. Labeled cells in the paraventricular nucleus synapsed with unlabeled nerve terminals and were found in synaptic-like contact with protrusions of magnocellular neurons. The latter finding might represent the morphological basis for orthodromic interactions between parvocellular and magnocellular neurons of the paraventricular nucleus, which have been previously demonstrated by electrophysiological methods.
Serotonin-immunoreactive nerve fibers were demonstrated to form a plexus in the intermediate lobe of rat pituitary. Such fibers were sporadic in the posterior lobe where they occurred exclusively in the zone adjacent to the intermediate lobe, and in the anterior lobe. With the aid of electron-microscopic immunocytochemistry, labeled fibers were shown to constitute the majority of neuronal processes found in the intercellular region of the intermediate lobe. They were frequently seen to establish synaptoid contacts with endocrine cells.
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Previous work in our laboratory has shown that stimulation of beta 2-adrenergic receptors on mouse anterior pituitary tumor cells causes the secretion of immunoreactive adrenocorticotropin (ACTH). The present study was designed to test the hypothesis that catecholamines can cause the release of ACTH in vivo by the direct stimulation of beta 2-adrenergic receptors in the rat anterior pituitary. Systemic administration of a beta-adrenergic receptor agonist (-)-isoproterenol resulted in an increase in plasma ACTH levels in intact animals and in rats with transected pituitary stalks. This effect could be blocked by the beta-adrenergic receptor antagonist, propranolol, but not by the specific beta 1-adrenergic receptor antagonist, practolol. Salmefamol, a beta 2-adrenergic receptor agonist also elevated plasma ACTH levels in stalk-sectioned animals. Dexamethasone, a glucocorticoid that inhibits the synthesis and release of ACTH from the anterior pituitary but not the intermediate lobe, prevented the elevation of ACTH secretion by (-)-isoproterenol in stalk-transected rats. These data indicate that beta 2-adrenergic receptors are present on anterior pituitary cells and suggest that catecholamines can directly stimulate ACTH secretion.
Ovine corticotropin-releasing hormone (CRF)-like immunoreactivity has been examined in the rat hypothalamus by light microscopy. Immunoreactivity was found in nerve fibers of the median eminence, mainly in the external zone around the portal vessels. In rats pretreated with colchicine or with hypothalamic knife cuts, small to moderate sized cells with two (bipolar) or rarely more (multipolar) dendrites, showing CRF-like immunoreactivity were present in the anterior and medial parvocellular subdivisions of the paraventricular nucleus. Scattered CRF-like immunopositive cells were found in the periventricular and medial preoptic nuclei. CRF-like immunoreactivity was clearly enhanced in the median eminence and paraventricular nucleus 8-10 days after bilateral adrenalectomy. A variety of hypothalamic transections had to be performed to determine reliably the topography of CRF-like nerve fibers projecting to the stalk-median eminence. Axons left the paraventricular nucleus in a lateral direction, turned ventrally in the lateral hypothalamus then medially as they approached the base of the hypothalamus above and behind the optic chiasm (lateral retrochiasmatic area). Fibers reached the median eminence by traveling caudally and medially from the rostral half of the lateral retrochiasmatic area. Scattered fibers were present in the retroinfundibular (posterior) portion of the median eminence. No immunoreactive fibers remained in the stalk-median eminence 1 or 4 weeks after transection of that loop-like pathway of CRF-containing fibers in the lateral retrochiasmatic area.
The course of the hypothalamo-infundibular somatostatin containing pathway was visualized by immunocytochemistry (the unlabeled antibody peroxidase-antiperoxidase complex method) combined with various knife cuts in the hypothalamus. Somatostatin immunoreactive fibers left the perikarya in the anterior hypothalamic-preoptic periventricular cell mass in a lateral direction. After a loop-like path through the lateral hypothalamus, fibers enter the medial basal hypothalamus mainly in the lateral retrochiasmatic area near the ventral surface and project to the ipsilateral half of the median eminence and the pituitary stalk. Somatostatin immunoreactivity almost completely disappeared from the stalk-median eminence 3-7 days following a complete anterolateral transection while transections dorsal or posterior to the lateral retrochiasmatic area failed to influence it. 7 weeks after anterolateral deafferentation an increased density of somatostatin containing terminals and immunopositive perikarya were seen in the arcuate and ventromedial nuclei but these neurones failed to reinnervate the stalk-median eminence. Thus there appears to be a neuron system containing somatostatin-like immunoreactivity within the medial basal hypothalamus, cell bodies being most abundant in the posterior divisions of the ventromedial and arcuate nuclei. However, most if not all of the somatostatin immunoreactivity in the stalk median eminence originates from outside of the medial-basal hypothalamus. Further, we have demonstrated the importance of the careful histological evaluation of each individual operation if functional studies are to be carried out after hypothalamic deafferentation.
In sheep, 26 brain areas of 62 investigated contained corticotropin releasing factor (CRF)-like immunoreactivity in concentrations higher than 0.5 ng CRF/mg protein. Most of the CRF in the brain was in the hypothalamus. The highest level of CRF in the sheep brain was in the median eminence, almost two orders of magnitude higher than anywhere else. All of the hypothalamic nuclei had measureable amounts of CRF. A number of extrahypothalamic regions such as amygdala, hippocampus, claustrum, cingulate cortex, habenula and certain lower brainstem nuclei had CRF but generally in very low concentrations.
Serotonin (5-HT)-containing nerve fibers and terminals, but not cell bodies, have been demonstrated by light and electron microscopic immunocytochemistry in the intermediate lobe of the rat pituitary. Seven days after pituitary stalk transection, 5-HT immunoreactive fibers disappeared almost entirely from the intermediate lobe. Intermediate lobe 5-HT and 5-hydroxyindoleacetic acid concentrations, measured by high pressure liquid chromatography coupled to electrochemical detection, fell to 56% and 62% of control, respectively. There were no changes in 5-HT concentrations in anterior or posterior lobes of the pituitary or in the median eminence. These findings, and the failure of sympathectomy to cause a drop in pars intermedia 5-HT, indicate that in the intermediate lobe of the pituitary neuronal 5-HT originates in cells situated in the central nervous system.