Vasopressin-deficient paraventricular magnocellular neurons of homozygous Brattleboro rats synthesize neuropeptide Y

Immunohistochemical study was carried out to determine whether neuropeptide Y (NPY), which was only found in certain experimental procedures in arginine vasopressin (AVP)-containing neurons of the magnocellular paraventricular nucleus, might also be synthesized in AVP-deficient homozygous Brattleboro (BB) rats. After an intraventricular colchicine administration, NPY was found in many AVP-deficient non-oxytocinergic magnocellular neurons of the paraventricular and supraoptic nuclei in BB rats, but not in suprachiasmatic nucleus neurons. The results suggest that the NPY synthesis is a phenotype of magnocellular non-oxytocinergic neurosecretory neurons and occurs independently from the synthesis of AVP.     

Protein synthetic machinery in the dendrites of the magnocellular neurosecretory neurons of wild-type Long-Evans and homozygous Brattleboro rats

There is growing evidence of local protein synthesis in neuronal dendrites, especially in relation to synaptic activity. The hypothalamic magnocellular system is a robust model for peptidergic neurons, especially for the study of dendrites. Quantitative electron microscopy, immunocytochemistry and non-radioactive in situ hybridization (with tyramide signal amplification) were used to compare dendrites of magnocellular neurons in the supraoptic nucleus of wild-type rats and of homozygous Brattleboro (BB) rats which are subject to long-term hyper-osmotic stimulation because they cannot secrete vasopressin. The dendrites contained free polyribosomes, cisterns of rough endoplasmic reticulum (ER) and small Golgi-like elements. These were clustered in the dendrites, mostly near the plasma membrane. All were increased in amount in the enlarged dendrites of the BB rats. The presence of polyribosomes and cisterns of rER implies that both cytosolic and membrane-inserting proteins are synthesized in the dendrites. The ER marker protein disulfide isomerase extended far into dendrites, but Golgi element markers (mid-Golgi and trans-Golgi network) were distributed mainly in their proximal parts. In BB rats, all the labeling was stronger. 28S rRNA, initiator tRNA(Met), and poly(A) mRNA were revealed extending into proximal and middle parts of dendrites where intensely reactive punctate structures were common. 28S rRNA could be detected in the distal parts of the dendrites. The length of positively stained dendrites was increased significantly for all these RNAs in BB rats. The results provide morphological evidence that magnocellular dendrites have the capacity for local protein syntheses and that this is increased in chronic hyperosmotic stress.     

Dye-coupled magnocellular peptidergic neurons of the rat paraventricular nucleus show homotypic immunoreactivity

Magnocellular neurons in rat hypothalamic slices are known to exhibit dye coupling: the transfer of the fluorescent dye, Lucifer Yellow, from an intracellularly-injected neuron to one or more nearby neurons. The question of the hormonal identity of coupled cells and the possibility of dye coupling as an artefact led us to determine the immunoreactivity of dye-coupled magnocellular neurons in the paraventricular nucleus of the rat hypothalamus using antisera to oxytocin- and vasopressin-associated neurophysins. In 23 pairs, one triplet, and one quadruplet, immunoreactivity to one or the other antiserum was always exclusive, and dye coupling was always homotypic, that is, coupled neurons in each instance were reactive to the same antiserum. The quadruplet, triplet and 17 pairs were immunoreactive to vasopressin-associated neurophysin, and oxytoxin-associated neurophysin immunoreactivity was observed in the remaining pairs. Immunoreactivity to each antiserum was found for somasomatic and non somasomatic modes of coupling and for coupled neurons in the three magnocellular areas of the nucleus. A relationship between mode of coupling and hormone content was not detected. The data support the hypothesis that coupling is a real, functionally significant mechanism for coordinating neuronal activity in this nucleus, particularly under conditions of high hormone demand. They do not support the idea that coupling is artefact. The possibility of a relationship between hormone content and mode of coupling, and the projection pathway(s) of the coupled neurons of each type require further study.     

Local cerebral glucose utilization in the homozygous Brattleboro rat

The [14C]deoxyglucose technique was used to measure brain glucose utilization in homozygous male Brattleboro and age-matched Long-Evans control rats. Brattleboro homozygotes had significantly higher daily water intakes and plasma osmolalities and significantly lower body weights than controls. Glucose utilization for the brain as a whole and for 46 discrete brain structures was not significantly different for the two strains. Our results indicate that vasopressin is not essential for the maintenance of overall brain glucose utilization in resting, awake rats.     

Delayed ontogenesis of histamine in the hypothalamus of the homozygous Brattleboro rat

The ontogenetic development of histamine was studied in the diabetes insipidus rat to clarify the possible interference between the lack of vasopressin and the development of histaminergic systems in the hypothalamus. Rat pups were decapitated at different ages between the 2nd and 38th postnatal days. In addition to homozygous Brattleboro (diabetes insipidus) rats, Long Evans controls and heterozygous animals were studied. In all three genotypes hypothalamic histamine was almost equal during the first 6 postnatal days. In homozygous Brattleboro rats the period of most rapid increase occurred between days 14 to 26, which was significantly later than in Long Evans rats. In the remainder of the brain no such difference was seen. On the contrary, histamine values were highest in the youngest animals. It remains to be elucidated whether the delayed ontogenesis is causally related to vasopressin deficiency and what is the underlying mechanism.     

Endogenous vasopressin and the weaning period in Brattleboro rats

Vasopressin, which is important for behavior and brain development, begins to influence osmoregulation with the onset of weaning. We studied the role of vasopressin in the development of feeding behavior since its mechanisms might be essential for the age determination of the suckling and weaning periods. Radionuclide methods were employed to follow maternal milk, solid food and water consumption in developing Brattleboro rats. The appearance of solid food intake and the spontaneous extinction of maternal milk intake indicated the onset and the end of weaning. The absence of endogenous vasopressin did not influence the onset and/or the duration of the weaning period. Both vasopressin-deficient homozygous Brattleboro rats and their heterozygous littermates (with preserved vasopressin synthesis) began to consume solid food and water at the age of 16 days and their intake of maternal milk was terminated about the 27th day of age. Thus, the maturation of feeding behavior in the suckling and weaning periods is vasopressin-independent.     

Nitric oxide synthase-containing magnocellular neurons of the rat hypothalamus synthesize oxytocin and vasopressin and express Fos following stress stimuli

We investigated the chemical and anatomical features of nitric oxide synthase (NOS)-containing neurons in the paraventricular and supraoptic nuclei in the rat hypothalamus using combinations of enzyme histochemistry, in situ hybridization and immuno-histochemistry. Neurons expressing NOS mRNA completely overlapped with NADPH-diaphorase-positive neurons. Topographical distribution of NOS was segregated from that of CRF-containing parvicellular neurons in the posterior paraventricular nucleus but overlapped with that of magnocellular neurons. In the paraventricular nucleus, 70% of oxytocin neurons contained NOS, which corresponded to one half of NOS neurons. About one third of vasopressin-immunoreactive neurons were NADPH-diaphorase-positive and the same proportion of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive. In the supraoptic nucleus, 50% of oxytocin neurons were NADPH-diaphorase-positive, which corresponded to 40% of NOS neurons. About 25% of vasopressin neurons were NADPH-diaphorase-positive, and 30% of NADPH-diaphorase-positive neurons were vasopressin-immunoreactive. When NADPH-diaphorase histochemistry was performed first, subsequent immunostaining was markedly perturbed. Using fluoro-gold as a retrograde tracer, 4% of NADPH-diaphorase-positive neurons were shown to contribute to the descending projection to the spinal cord. About 40%–50% of NADPH-diaphorase-positive neurons exhibited Fos immunoreactivity after injection of lipopolysaccharide or hypertonic saline, while only 10%–15% of these neurons expressed Fos in response to immobilization or pain. Endogenous NO may be involved in the regulation of magnocellular functions, especially when the internal environment is disturbed.     

The kappa opioid receptor and dynorphin co-localize in vasopressin magnocellular neurosecretory neurons in guinea-pig hypothalamus

The relationship between the cloned kappa opioid receptor, dynorphin, and the neurohypophysial hormones vasopressin and oxytocin was analysed in the guinea-pig hypothalamic magnocellular neurosecretory neurons. This analysis was performed in order to understand better which population of neuroendocrine neurons in the guinea-pig is modulated by kappa opioid receptors and its endogenous ligand dynorphin. Extensive co-localization was observed between kappa opioid receptor immunoreactivity and preprodynorphin immunoreactivity in neuronal cell bodies in the paraventricular and supraoptic nuclei. Cells positive for either the kappa opioid receptor or both the kappa opioid receptor and preprodynorphin were restricted to the vasopressin expressing neuronal population and not found in the oxytocin expressing neuronal population. The kappa opioid receptor and dynorphin were examined in the posterior pituitary and both were found to be extensively distributed. Staining for the kappa opioid receptor and dynorphin B co-localized in posterior pituitary. In addition, immunogold electron microscopy confirmed that kappa opioid receptor and dynorphin B immunoreactivity were found in the same nerve terminals. Ultrastructural analysis also revealed that kappa opioid receptor immunoreactivity was associated with both nerve terminals and pituicytes. Within nerve terminals, kappa opioid receptor immunoreactivity was often associated with large secretory vesicles and rarely associated with the plasma membrane.Our data suggest that the cloned kappa opioid receptor may directly modulate the release of vasopressin but not oxytocin in guinea-pig hypothalamic magnocellular neurosecretory neurons and posterior pituitary. Furthermore, we propose that this receptor is an autoreceptor in this system because our results demonstrate a high degree of co-localization between kappa opioid receptor and dynorphin peptide immunoreactivity in magnocellular nerve terminals.     

The single-prolonged stress paradigm alters both the morphology and stress response of magnocellular vasopressin neurons

Vasopressin (AVP) plays an important role in anxiety-related and social behaviors. Single-prolonged stress (SPS) has been established as an animal acute severe stress model and has been shown to induce a lower adrenocorticotropic hormone (ACTH) response upon cortisol challenge. Here, we show results from immunoassays for AVP, ACTH, and corticosterone (CORT), and in situ hybridizations for AVP mRNA performed 7 days after SPS exposure. Immunofluorescence for AVP was also performed during the 7-day period following SPS exposure and after an additional forced swimming stress paradigm. We observed that the plasma concentrations of AVP, ACTH, and CORT were not altered by SPS; ACTH content in the pituitary and AVP mRNA expression in the supraoptic nucleus (SON) were significantly reduced by SPS. During the 7-day period following SPS, the intensity of immunoreactivity, the size of the soma, and the immunoreactive optical density of the dendrites of AVP neurons in the SON all increased. An apparent reduction in the intensity of AVP immunoreactivity was observed in the SON at 4 h after additional stress. Additional forced swimming led to a rapid increase in the dendritic AVP content only in the controls and not in the SPS-treated rats. These findings suggest that AVP is a potential biomarker for past exposure to severe stress and that alterations in AVP may affect the development of pathogenesis in stress-related disorders.     

The hippocampal corticosterone receptor system of the homozygous diabetes insipidus (Brattleboro) rat

The binding of [³H] corticosterone to hippocampal cytosol receptors of Brattleboro rats homozygous for diabetes insipidus (Ho-Di) and of normal Brattleboro rats (Ho-No) was investigated at 24 h after removal of the adrenals. The apparent maximal binding capacity of the Ho-Di hippocampal corticosterone receptor system was about 30% less than that of the Ho-No rats. Substitution of the vasopressin deficient rats with 1E pitressin tannate in oil partially restores the hippocampal corticosterone receptor level towards that of the control animals.     

Presence of estrogen receptor immunoreactivity in the oxytocin-containing magnocellular neurons projecting to the neurohypophysis in the guinea-pig.

The immunoperoxidase technique was used on adjacent sections of guinea-pig brain to compare precisely the distribution of estrogen receptor-immunoreactive cells and progesterone receptor-immunoreactive cells in the supraoptic nucleus and the paraventricular nucleus. Only estrogen receptor-immunoreactive neurons were found in the supraoptic nucleus. A large number of estrogen receptor-positive cells were observed in the periventricular magnocellular groups throughout the rostrocaudal extent of the paraventricular nucleus, whereas only a few progesterone receptor-immunoreactive cells were scattered in the anterior portion of this region. We used a combination of axonal tracing with double immunocytochemical detection to determine whether estradiol acts directly on the oxytocin-immunoreactive neurons which project to the neurohypophysis. Oxytocin-immunoreactive cells were found in the supraoptic nucleus, ventrally to the optic pathways, in subchiasmatic and retrochiasmatic areas, and in the anterior hypothalamic area. These cells were also retrogradely labeled by Granular Blue when this tracer was injected intravenously. In the paraventricular nucleus, the Granular Blue/oxytocin-positive cells were observed in the periventricular magnocellular groups whereas Granular Blue labeled neurons were found in both parvocellular and magnocellular components. We found that almost all the oxytocin-immunoreactive cells revealed estrogen receptor immunoreactivity. In conclusion, the comparative study of distribution of estrogen receptors and progesterone receptors in the guinea-pig supraoptic and paraventricular nuclei indicates that, in the supraoptic nucleus, only estrogen receptors are present and that, in the paraventricular nucleus, they are far more numerous than progesterone receptors. The present findings demonstrate that the magnocellular cells which contain estrogen receptors are oxytocinergic. In addition, these cells are retrogradely labeled pointing to a neurohypophysial projection. It is likely that estradiol controls the hypothalamo-neurohypophysial oxytocin system by direct action on the magnocellular neurons.     

Peptide YY-like immunoreactivity in sympathetic neurons of the rat

The occurrence of peptide YY-like peptides in parts of the sympathetic nervous system of the rat was studied by immunocytochemistry and immunochemistry plus analysis by high performance liquid chromatography. Peptide YY-immunoreactive neurons and nerve fibers were detected in the superior cervical ganglion. Co-localization studies indicated that peptide YY and neuropeptide Y immunoreactivities co-exist in a subpopulation of neurons of the superior cervical ganglion. Immunochemical analysis revealed peptide YY-immunoreactive material, distinct from neuropeptide Y, in extracts of the superior cervical ganglion. On reverse-phase high performance liquid chromatography, extracts of superior cervical ganglion revealed several peaks of peptide YY-like immunoreactive material, one of which eluted close to the position of authentic porcine peptide YY. Peptide YY-immunoreactive nerve fibers were also present in sympathetic target tissues including the auricula and atria of the heart, carotid body, submandibular salivary gland and the adrenal cortex. It is suggested that peptide YY and/or peptide YY-like peptides are present not only in endocrine cells, but also in a subpopulation of cell bodies and fibers of the peripheral sympathetic nervous system.     

Somatostatin immunoreactivity in esophageal premotor neurons of the rat

Esophageal peristalsis is coordinated by premotor neurons localized to the central subnucleus of the nucleus of the solitary tract (NTScen). These premotor neurons project directly to motoneurons within the compact formation of the nucleus ambiguus (NAc). Somatostatin immunoreactive terminals have been previously demonstrated encircling motoneurons in the (NAc) (Cunningham, E.T., Jr. and Sawchenko, P.E., J. Neurosci., 9 (1989) 1668–1682). We combined transsynaptic tracing with pseudorabies virus and immunohistochemistry to localize somatostatin to premotor neurons within the NTScen.     

Neural inputs from the uterus to the paraventricular magnocellular neurons in the rat

Extracellular action potentials were recorded from antidromically identified, tonically firing cells in the hypothalamic paraventricular nucleus (PVN) of ovariectomized, estrogen-treated female rats under urethane anesthesia. Genital or somatic sensory stimuli, or electrical stimulation of the nerves innervating the pelvis were applied. Uterine horn or vaginal distension each excited 33% of the neurons tested. Probing of the cervix had no effect. Hindpaw pinch produced excitation in 39% and inhibition in 11% of the neurons tested. Non-noxious somatic stimuli had no effect. Stimulation of the uterine afferent nerves, the hypogastric and pelvic nerves, excited 55% and 30% of the neurons tested, respectively. Stimulation of a somatic nerve of the hindleg, the sciatic nerve, activated 80% of the neurons tested. These results indicate that specific sensory afferents arrive at the PVN from the uterus; in addition, somatic afferents converge in this hypothalamic nucleus.     

Ectopic expression of non-catecholaminergic tyrosine hydroxylase in rat hypothalamic magnocellular neurons

Hypothalamic magnocellular neurons constitute a good model of neurochemical plasticity, because a single neuron can express various combinations of neuropeptides and enzymes under different physiological conditions. Tyrosine hydroxylase has been shown to occur ectopically in various non-catecholaminergic neurons. We investigated the expression of tyrosine hydroxylase and its possible role in the magnocellular neurons of the supraoptic and paraventricular nuclei in salt-loaded and lactating rats, using in situ hybridization and immunohistochemistry, alone or combined, in light and electron microscopy. Our results demonstrated that almost 25% of the magnocellular neurons in the supraoptic nucleus and 15% in the paraventricular nucleus expressed tyrosine hydroxylase in salt-loaded rats, and 10% in the supraoptic nucleus of two-day lactating rats. Double labelling showed that this tyrosine hydroxylase was essentially synthesized in magnocellular neurons expressing vasopressin. The ultrastructural localization of tyrosine hydroxylase was less homogeneous in the cytoplasm of magnocellular neurons than in periventricular neurons. In lactating and salt-loaded rats, magnocellular neurons were devoid of the catecholamine biosynthesis markers aromatic L-amino acid decarboxylase, L-3,4 dihydroxyphenylalanine, dopamine and GTP-cyclohydrolase I. Tyrosine hydroxylase expression did not increase after rats were injected with reserpine. Our results indicate that the phenotype of the magnocellular neurons expressing tyrosine hydroxylase in lactating and salt-loaded rats is non-catecholaminergic, and suggest that this tyrosine hydroxylase might be involved in osmoregulation.     

Nitric oxide modulates the firing rate of the rat supraoptic magnocellular neurons

In vitro, nitric oxide (NO) inhibits the firing rate of magnocellular neurosecretory cells (MNCs) of hypothalamic supraoptic and paraventricular nuclei and this effect has been attributed to GABAergic activation. However, little is known about the direct effects of NO in MNCs. We used the patch-clamp technique to verify the effect of l-arginine, a precursor for NO synthesis, and N(omega)-nitro-l-arginine methyl ester hydrochloride (l-NAME), an inhibitor of NOS, on spontaneous electrical activity of MNCs after glutamatergic and GABAergic blockade in Wistar rat brain slices. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10 muM) and dl-2-amino-5-phosphonovaleric acid (dl-AP5) (30 muM) were used to block postsynaptic glutamatergic currents, and picrotoxin (30 muM) and saclofen (30 muM) to block ionotropic and metabotropic postsynaptic GABAergic currents. Under these conditions, 500 muM l-arginine decreased the firing rate from 3.7+/-0.6 Hz to 1.3+/-0.3 Hz. Conversely, 100 muM l-NAME increased the firing rate from 3.0+/-0.3 Hz to 5.8+/-0.4 Hz. All points histogram analysis showed changes in resting potential from -58.1+/-0.8 mV to -62.2+/-1.1 mV in the presence of l-arginine and from -59.8+/-0.7 mV to -56.9+/-0.8 mV by l-NAME. Despite the nitrergic modulator effect on firing rate, some MNCs had no significant changes in their resting potential. In those neurons, hyperpolarizing after-potential (HAP) amplitude increased from 12.4+/-1.2 mV to 16.8+/-0.7 mV by l-arginine, but without significant changes by l-NAME treatment. To our knowledge, this is the first demonstration that NO can inhibit MNCs independent of GABAergic inputs. Further, our results point to HAP as a potential site for nitrergic modulation.     

Galanin coexists with vasopressin in the normal rat hypothalamus and galanin's synthesis is increased in the Brattleboro (diabetes insipidus) rat

Galanin is a peptide containing 29 amino acid residues, that is present in the median eminence, in the magnocellular neurons of the supraoptic (SON) and paraventricular nuclei (PVN) of the rat hypothalamus and in the posterior pituitary. We report here that: (1) immunoreactivity for galanin (GAL) and vasopressin coexist in the SON of normal rats, (2) levels of mRNA encoding preprogalanin are markedly elevated in the PVN and SON of Brattleboro (diabetes insipidus) rats, as determined by in situ hybridization histochemistry but (3) levels of GAL-like immunoreactivity (GAL-LI) are significantly reduced in the posterior pituitary of these rats, as determined by radioimmunoassay. We suggest that production and possibly secretion of the peptide GAL may be increased in the Brattleboro rat.     

Co-localization of interleukin-1 receptor type I and interleukin-1 receptor antagonist with vasopressin in magnocellular neurons of the paraventricular and supraoptic nuclei of the rat hypothalamus

Interleukin-1 receptor type I and interleukin-1 receptor antagonist were found in magnocellular neurons of the paraventricular and supraoptic nuclei of the rat hypothalamus by immunohistochemical detection. Double-labelling experiments revealed that both proteins occurred in vasopressin-containing neurons. A similar distribution pattern was observed in a group of vasopressin-positive accessory magnocellular neurons. Axons emanating from the interleukin-1 receptor type I- and interleukin-1 receptor antagonist-immunoreactive neuronal cell bodies could be seen within the hypothalamic nuclei, and varicosities expressing interleukin-1 receptor antagonist immunoreactivity were observed in the internal zone of the median eminence, as well as in the hypothalamo-pituitary projection. The co-localization of interleukin-1 receptor type I with vasopressin is in agreement with findings that interleukin-1 has a stimulatory effect on vasopressin synthesis and release. The hypothalamic neurons may serve as a source of interleukin-1 receptor antagonist to balance the effects of interleukin-1.