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Kisspeptin is the natural ligand of the G protein-coupled receptor -54 and plays a major role in gonadotropin-releasing hormone secretion in the hypothalamus. Kisspeptin-10 is an endogenous derivative of kisspeptin and has 10 -amino acids. Previous studies have demonstrated that central administration of kisspeptin-10 stimulates the secretion of arginine vasopressin (AVP) in male rats. We examined the effects of kisspeptin-10 on- excitatory synaptic inputs to magnocellular neurosecretory cells (MNCs) including AVP neurons in the supraoptic nucleus (SON) by obtaining in vitro whole-cell patch-clamp recordings from slice preparations of the rat brain. The application of kisspeptin-10 (100 nM-1 μM) significantly increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in a dose-related manner without affecting the amplitude. The kisspeptin-10-induced potentiation of the mEPSCs was significantly attenuated by previous exposure to the kisspeptin receptor antagonist kisspeptin-234 (100 nM) and to the protein kinase C inhibitor bisindolylmaleimide I (20 nM). These results suggest that kisspeptin-10 participates in the regulation of synaptic inputs to the MNCs in the SON by interacting with the kisspeptin receptor., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activity of these neurons. ATP plays a crucial role in the regulation of SON MNCs by activating the purinergic P2X and P2Y receptors. Recent reports of interaction between P2X receptors and pannexin channels have provided new insights into the physiology of the central nervous system; however, the function of pannexin channels has not been assessed in AVP neurons. In the present study, we examined the possible contribution of the pannexin channel in ATP-induced responses in SON AVP neurons. We used the whole-cell patch-clamp technique in isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein transgene. The ATP-induced current was inhibited in a concentration-dependent manner by pannexin channel blockers carbenoxolone and mefloquine, whereas the connexin channel blockers flufenamic acid and lanthanum had no effect. Multi-cell reverse transcriptase-polymerase chain reaction experiments confirmed the existence of pannexin-1 mRNA in AVP neurons. The involvement of the ATP-activated transient receptor potential vanilloid and acid-sensing ion channels was excluded. These results suggest that pannexin channels in SON AVP neurons are involved in the regulatory mechanisms of neuronal activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Accumulation of daily life stress (chronic stress) often causes functional gastrointestinal diseases. Central oxytocin plays an important role in attenuating stress responses and regulating positive social interactions. Adult male rats were either paired or singly housed 1 week prior to the stress loading. Solid gastric emptying was measured after 7 consecutive days of chronic heterotypic stress. To study whether endogenous oxytocin is involved in restoring the delayed gastric emptying after paired housing, an oxytocin antagonist was injected intracerebroventricularly (icv) before the gastric emptying study. CRF and oxytocin mRNA expression in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) was evaluated by real time RT-PCR. In singly housed rats, chronic heterotypic stress significantly delayed gastric emptying (35.1+/-2.8%, n=6, P

Body fluid balance requires the release of arginine vasopressin (AVP) from the neurohypophysis. The hypothalamic supraoptic nucleus (SON) is one of the major sites for the synthesis of AVP, and secretion of AVP is controlled by the electrical activities of magnocellular neurosecretory cells (MNCs), which in turn are regulated by neuronal excitatory glutamatergic and inhibitory GABAergic inputs and humoral factors such as plasma osmolality. Previous studies have shown that brain-derived neurotrophic factor (BDNF) mRNA was increased by osmotic stress in the rat SON. In the present study, the effects of BDNF on excitatory and inhibitory synaptic inputs were examined in the MNCs of rat SON, using the whole-cell patch-clamp technique in in vitro brain slice preparations. BDNF application caused a significant reduction in the frequency and amplitude of the spontaneous inhibitory postsynaptic currents of the MNCs without affecting the spontaneous excitatory postsynaptic currents. Next, whole-cell patch-clamp recordings from dissociated SON MNCs expressing AVP-enhanced green fluorescent protein (eGFP) transgene revealed that the amplitude of GABA-induced currents were significantly smaller after BDNF treatment. Moreover, multi-cell reverse transcriptase-polymerase chain reaction (RT-PCR) experiments revealed the expression of TrkB mRNA in AVP-eGFP neurons. These results suggest that BDNF in the rat SON may decrease the postsynaptic GABAergic activity and may be involved in the regulatory mechanisms of body fluid homeostasis.

In a previous study performed in mouse models of energetic challenge, there was evidence to suggest that the orphan G protein-coupled receptor GPCR101 may have a role in the regulation of energy balance. To further investigate this possibility, we utilised in situ hybridisation to determine the effect of energetic challenges experienced by pregnant and lactating rats on GPCR101 mRNA expression. In the rat hypothalamus, GPCR101 mRNA expression was detected in a number of hypothalamic nuclei. During pregnancy and lactation, GPCR101 mRNA level remained unchanged in most nuclei, but had increased in the supraoptic nucleus by the end of pregnancy and remained elevated during lactation. GPCR101 mRNA expression showed a similar pattern of expression in the rostral ventromedial parvocellular subdivision of the paraventricular nucleus. A common feature of these two nuclei is the production of the peptide oxytocin. Dual in situ hybridisation revealed GPCR101 and oxytocin mRNA co-expression in neurons of these two nuclei. In the supraoptic nucleus, in situ hybridisation revealed that the temporal regulation of oxytocin and GPCR101 mRNA expression were similar. In the paraventricular nucleus, although temporal changes in oxytocin mRNA expression were similar to GPCR101, the spatial expression of the two mRNA species was different; in contrast to GPCR101, oxytocin mRNA expression changed in both parvo- and magnocellular neurons during lactation. In conclusion, increased GPCR101 mRNA expression in supraoptic and paraventricular nuclei from late pregnancy to late lactation may reflect the functional importance of this receptor in the regulation of neurons of these nuclei during this period.

Inter-neuronal coupling is a relatively recently documented property of a wide variety of cell groups in the mammalian central nervous system. For many of these groups there is evidence that the coupling can be modulated by synaptic inputs. Incidence of dye coupling among vasopressin (VP) neurons of the rat supraoptic nucleus (SON) has been shown to increase in response to either activation of histamine H(1)-receptors or to increased NO production. Both of these effects involve activation of cGMP-dependent pathways. We tested the hypothesis that activation of H(1)-receptors resulted in downstream activation of NO synthase, which then mediated the H(1)-receptor effects. Putative VP neurons were intracellularly recorded and dye-injected in horizontal slices of hypothalamus, in which monosynaptic connections from the tuberomammillary nucleus (TM) were intact and electrically stimulated. Single-pulse TM stimulation evoked EPSPs and repetitive stimulation resulted in a nearly 3-fold increase in coupling incidence over unstimulated slices. TM-stimulated increased coupling was completely blocked by inhibitors of NO synthase (L-NAME) or of soluble guanylyl cyclase (ODQ or methylene blue), or pyrilamine, suggesting that the H(1)-receptor is not directly linked to guanylyl cyclase. Addition of the NO precursor, L-arginine or the NO donor, SNP, in combination with TM stimulation produced increases in coupling that were not significantly larger than those seen with stimulation alone, supporting the idea that a common pathway was used. We conclude that H(1)-receptors activate NO synthase via G-protein-coupled pathways and that NO working though its receptor, induces the downstream cGMP-dependent processes that result in increased inter-neuronal coupling.

The subfornical organ (SFO) may act as a sodium- or osmoreceptor that drives hypothalamic and other nuclei to secrete vasopressin and to elicit drinking. However, in response to mild doses of hypertonic saline, Fos-like immunoreactivity (Fos-ir) is absent in the SFO whereas it is well expressed in the hypothalamic supraoptic (SON) and paraventricular (PVN) nuclei. This suggests that the hypothalamus may be activated in advance of the SFO. In this study, the fibers connecting the SFO and hypothalamus were disconnected by a wire knife cut so that Fos-ir could be examined in both the SFO and hypothalamus after an intragastric (ig) load of 0.5% of body weight of 0.6 M NaCl. Compared with Fos-ir in isotonic-loaded rats, Fos-ir after the hypertonic load was not significantly elevated in the SFO or median preoptic nucleus in sham-cut or knife-cut rats and was only slightly elevated in the OVLT in sham-cut rats. However, the hypertonic load in sham-cut rats greatly elevated Fos-ir in the SON and in the entire PVN, but this expression was reduced significantly by 30-50% in knife-cut rats. Thus, the connectivity between SFO and the hypothalamus is critical for the full expression of Fos-ir in the hypothalamus during moderate ig hypertonic saline loading even when the SFO itself does not yet express Fos-ir.

Agmatine, decarboxylated from arginine by arginine decarboxylase, is particularly prominent in the hypothalamus. The present study utilized the rat hypothalamo-neurohypophysial system to determine expression and "pre-synaptic" modulation of agmatine in the central nervous system (CNS). Under confocal-laser scanning, agmatine-like immunoreactivity (Agm-LI) was found enriched in arginine-vasopressin (AVP)-containing magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei (PVN). In addition, using electron microscopy, Agm-LI was found closely associated with large neurosecretory-like vesicles in neurohypophysial nerve terminals of posterior pituitary gland. Radioimmunoassay revealed that 10 and 30 microM agmatine concentration-dependently inhibited the depolarization-evoked AVP release from isolated neurohypophysial terminals. Using perforated patch-clamp, effects of agmatine on whole-terminal voltage-gated ion currents in the isolated neurohypophysial nerve terminals were examined. While it did not significantly affect either tetrodotoxin (TTX)-sensitive Na(+) or sustained Ca(2+)-activated K(+) channel currents, agmatine (1-40 microM) inhibited Ca(2+) channel currents in approximately 53% of the total nerve terminals investigated. The onset of inhibitory effect was immediate, and the inhibition was reversible and concentration-dependent with an IC(50)=4.6 microM. In the remaining (approximately 47%) neurohypophysial nerve terminals, only a higher (120 microM) concentration of agmatine could moderately inhibit Ca(2+) channel currents. The results suggest that: (1) endogenous agmatine is co-expressed in AVP-containing, hypothalamic magnocellular neurons of the SON/PVN and in neurohypophysial nerve terminals of posterior pituitary gland; (2) agmatine may serve as a physiological neuromodulator by regulating the voltage-gated Ca(2+) channel and, as a result, the release of AVP from neurohypophysial nerve terminals.

In the supraoptic nucleus (SON), the incidence of conducting gap junctions (gjs), as indicated by dye coupling, is low in cycling females, but dramatically elevated in nursing mothers. Functionally, this is consistent with the well-established presence of synchronous milk ejection bursts among oxytocin neurons only in the lactating rat. In situ hybridization data, however, revealed elevated gj mRNA expression on the last day of pregnancy, a time when burst firing by putative oxytocin neurons is absent. Using Lucifer Yellow dye coupling, we determined the incidence of high conductance gjs in SONs of proestrous, immediately prepartum, postpartum non-lactating, lactating day 1, and lactating day 9-10 rats. Results indicate that coupling incidence is high only at times when milk ejection bursts are known to occur, and that the elevated gj mRNA expression seen on the last day of pregnancy does not indicate conducting gjs. It is suggested that gj conductance states, but not gj expression, are modulated by plasma estradiol titers.

Using immunocytochemistry, in situ hybridization and image analysis, we attempted to compare the dynamical expression of tyrosine hydroxylase (TH) and vasopressin (VP) mRNAs and proteins in the magnocellular neurons of the supraoptic nucleus in rats drinking 2% NaCl for 1, 2 and 3 weeks. Three stages in the reaction of VPergic neurons have been distinguished. The initial stage (first week) showed a synchronous activation of TH and VP mRNAs and protein expression as well as an increased number of TH-immunoreactive neurons. The next stage (second week) was characterized by a further increase in the number of TH-immunoreactive neurons. The number of VPergic neurons also increased significantly. Although the TH and VP mRNAs levels fell during the second week of osmotic stimulation, the TH content increased significantly, and the VP content remained at the same level. During the last stage (third week), TH-immunoreactive neurons increased in number and were as numerous as VP-immunoreactive neurons in intact rats. These data suggest that, finally, all the VPergic neurons begin to synthesize TH. The concentrations of VP and TH mRNAs did not change during the third week of osmotic stimulation, while the VP and TH contents increased. Thus, our study shows that there is a correlation between TH expression and VP expression and suggests similar mechanisms for the regulation of VP and TH gene expression and synthesis during long-term osmotic stimulation.

The anterograde neuronal transport of Cholera toxin B subunit (CTB) was used in this study to label the termination of retinal afferents in the hypothalamus of the tree shrew Tupaia belangeri. Upon pressure-injection of the substance into the vitreous body of one eye, a major projection of the retinohypothalamic tract (RHT) was found to the hypothalamic suprachiasmatic nuclei (SCN). Although the innervation pattern was bilateral, the ipsilateral SCN received a somewhat stronger projection. Labeling was also found in the supraoptic nucleus and its perinuclear zone, respectively, mainly ipsilaterally as well as in the bilateral para- and periventricular hypothalamic regions without lateral predominance. In the raphe region, scattered fibers and terminals were seen in the dorsal and median raphe nuclei. CTB-immunoreactive structures were observed neither in the locus ceruleus nor in vagal nuclei. Our results, partly in contradiction to earlier studies using different tracing techniques in another tree shrew species (Tupaia glis), reveal that hypothalamic nuclei, in particular the SCN, are contacted by retino-afferent fibers which are thought to mediate the effects of light to the endogenous 'clock' and to parts of the neuroendocrine system.

The gut-brain peptide, cholecystokinin (CCK), inhibits food intake when injected either systemically or within the brain. To determine whether CCK's effect in the brain is anatomically specific, CCK-8 (0. 8, 4, 20, 100, 500 pmol) was microinjected into one of 14 different brain sites of rats, and its impact on subsequent food intake was measured. CCK-8 at 500 pmol significantly suppressed intake during the first hour post-injection following administration into six hypothalamic sites (anterior hypothalamus, dorsomedial hypothalamus, lateral hypothalamus, paraventricular nucleus, supraoptic nucleus, ventromedial hypothalamus) and two hindbrain sites (nucleus tractus solitarius, fourth ventricle). Although lower doses were sometimes effective (anterior hypothalamus, dorsomedial hypothalamus, nucleus tractus solitarius), there appeared to be no significant difference in potency among sites. Injections into the medial amygdala, nucleus accumbens, posterior hypothalamus, dorsal raphe, and ventral tegmental area were either ineffective or produced a delayed response. The higher doses required for most sites, as well as the widespread effectiveness of CCK-8 within the hypothalamus, suggest that spread of CCK-8 to adjacent brain sites, and (or) to the periphery, may have been required for anorexia to occur. Findings reported in an accompanying paper provide strong evidence that paraventricular nucleus injection of CCK-8 (500 pmol) did not increase plasma CCK-levels sufficiently to suppress feeding by a peripheral mechanism. Together, these results suggest that CCK may be acting as a neurotransmitter or neuromodulator within two different brain regions to produce satiety - one region which includes the nucleus tractus solitarius in the hindbrain, and another more distributed region within the medial-basal hypothalamus.

The subfornical organ (SFO) is a forebrain structure that converts peripheral blood-borne signals reflecting the hydrational state of the body to neural signals and then through efferent fibers conveys this information to several central nervous system structures. One of the forebrain areas receiving input from the SFO is the supraoptic nucleus (SON), a source of vasopressin synthesis and control of release from the posterior pituitary. Little is known of the transduction and transmission processes by which this conversion of systemic information to brain input occurs. As a step in elucidating these mechanisms, the present study characterized the ionic currents of dissociated cells of the SFO that were identified as neurons that send efferents to the SON. A retrograde tracer was injected into the SON area in eleven-day-old rats. After three days for retrograde transport of the label, the SFOs of these animals were dissociated and plated for tissue culture. The retrograde tracer was used to identify the soma of SFO cells projecting to the SON so that voltage-dependent ionic currents using whole-cell voltage clamp methods could be studied. The three types of currents in labeled SFO neurons were characterized as a 1) rapid, transient inward current that can be blocked by tetrodotoxin (TTX) characteristic of a sodium current; 2) slow-onset sustained outward current that can be blocked by tetraethylammonium (TEA) characteristic of a delayed rectifier potassium current; and 3) remaining outward current that has a rapid-onset and transient characteristic of a potassium A-type current., (Copyright 1999 Elsevier Science B.V.)

To elucidate the role of GABAB receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABAB agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the gamma-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABAB agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABAB antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca2+ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABAB receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABAB receptors inhibits electrical activity of SON neurons of both male and female rats and that GABAB receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA., (Copyright 1998 Elsevier Science B.V.)

Vasopressin was localized in the supraoptic nucleus (SON), paraventricular nucleus, amygdala, habenula, and the posterior pituitary. Microdensitometry analysis revealed an inversely proportionate relationship between the relative vasopressin levels in the SON and the posterior pituitary when exposed to varying periods of dehydration. Short term dehydrated bats displayed increased vasopressin in the SON and decreased levels in the posterior pituitary. Chronically dehydrated bats displayed decreased vasopressin in the SON with increased levels in the posterior pituitary., (Copyright 1998 Elsevier Science B.V.)

We measured expression of the oxytocin gene in the supraoptic nucleus (SON) during pregnancy, parturition and lactation to examine its relationship to states of accumulation or depletion of oxytocin stores and to conditions of strong excitation of oxytocin neurons. The primary transcript (heterogeneous nuclear RNA, hnRNA) of the oxytocin gene was measured using a 3H-cDNA probe against intron 1 for in situ hybridisation. Autoradiographs of the SON showed the hnRNA as discrete clumps of silver grains within the nucleus of each neuron. The number of cells expressing oxytocin hnRNA did not change during pregnancy but increased during parturition; 10-day lactating animals showed similar increases. Oxytocin mRNA was also measured by in situ hybridisation using a 3H- or 35S-labelled oligonucleotide probe against exon C: hybridisation was seen over the cytoplasm of supraoptic neurons, but no differences were measured between virgin, mid-pregnant, preparturient, parturient or 2-day lactating rats. The data suggest that enhanced oxytocin gene transcription is not necessary to increase oxytocin stores in pregnancy. However, acute stimulation of magnocellular oxytocin neurons at parturition, which strongly increases neuron activity and secretion, results in a rapid increase in the number of cells expressing oxytocin hnRNA, and increased expression is sustained in lactation.

Hypophysial stalk-transected (ST) and sham-operated animals were subjected to a chronic intermittent salt loading regimen (CISL) for 14 days beginning 1 day post surgery (dps). Animals were sacrificed at 15 and 36 dps. Three days after the termination of CISL, water consumption in ST + CISL animals decreased to the same level as that of sham-operated animals, while that of ST + water animals was maintained at a significantly higher level. The number of the surviving vasopressinergic neurons in the supraoptic nuclei of the ST + CISL group was significantly higher than that of ST + water group. CISL induced vasopressinergic axonal sprouting into the external zone of the median eminence, and formation of subependymal perivascular plexus. While CISL also enhanced regeneration of oxytocinergic axons into the external zone, it did not, however, have any effect on the number of oxytocinergic neurons surviving axotomy.

In the midbrain-hemitransected rats, 22 pairs of oxytocin neurons were recorded from 17 rats. Of the bursts on the lesioned side, the amplitude was smaller, the onset time was later and the burst span was shorter (P < 0.01). These results indicated that the amplitude and time course of the burst of oxytocin neurons mainly depend on suckling information from the nipples on their opposite side.

The aim of the present study was to compare the postlesional responses of vasopressin-producing (VP) and oxytocin-producing (OT) neurons of the supraoptic nucleus (SON) to transection of neurohypophysial axons. At different times after sectioning the median eminence of adult rats, immunocytochemical staining of both types of neuronal cell bodies and axons indicated that: (1) the number of OT neurons detected within the SON was only slightly decreased as compared with controls (-20%), whereas the number of VP neurons was severely decreased (-60%); and (2) the large majority of axonal sprouts that regenerated into the external layer of the median eminence were OT neurohypophysial axons. The injection of a retrograde tracer into various areas surrounding the SON further showed that numerous SON neurons could be retrogradely labeled when the injection was centered in the lateral hypothalamus dorsal to the SON. The immunocytochemical identification of these retrogradely labeled neurons demonstrated that most of them were OT neurons. When animals were subjected to median eminence transection and to a unilateral surgical cut placed in the lateral hypothalamus above the SON, the survival of both OT and VP neurons was dramatically reduced in the SON ipsilateral to the hypothalamic lesion, as compared to the contralateral SON. Taken together, these data indicate that OT (and to a lesser extent VP) neurons of the SON display collateral projections towards the lateral hypothalamus that protect them from retrograde degeneration following the lesion of their neurohypophysial projections.

The medial septal area has been implicated in the control of the magnocellular neurosecretory cells of the hypothalamus, and in particular, in the regulation of neurons secreting oxytocin. The present study investigated the hypothesis that this medial septal pathway originates in the subfornical organ. Brief electrical stimulation of the subfornical organ or of the medial septum both evoked a transient rise in intramammary pressure equivalent to that caused by an i.v. injection of 1 mU oxytocin. The optimal frequency was 5-20 Hz for 5-10 s. Prolonged stimulation also elicited at its onset a single transient response, similar to that evoked by brief stimulation. Extracellular recordings were made from neurosecretory cells of the supraoptic nucleus identified by antidromic stimulation of the neural stalk and further classified as vasopressinergic and oxytocinergic by their reaction at the time of reflex milk ejection induced by suckling. Single-pulse stimulation of the subfornical organ rarely produced excitation, but short trains of stimuli evoked a large excitation in most oxytocinergic and vasopressinergic neurons. To delineate further the pathway from the subfornical organ to the magnocellular neurons, stimulations were combined with various lesions of the medial forebrain. The effects of stimulation of the subfornical organ were abolished after a section immediately rostral to the organ, and in most cases after lesion of the medial septum. Stimulation of the medial septum no longer had an effect after the subfornical organ had been lesioned a week prior to experiments, a period sufficient to allow degeneration of subfornical efferents. This study shows that the excitatory afferent input to the oxytocin and vasopressin-secreting neurons of the hypothalamus from the medial septal area originates in the subfornical organ. This input is not involved in the main afferent control of the milk ejection reflex since lesions of the subfornical organ and of the medial septum had no effect on the reflex. It is suggested, therefore, that the subfornical input to both oxytocin and vasopressin cells intervenes to facilitate synergistic action of both hormones in non-reproductive functions.

The present study compared Fos expression in identified hypothalamic magnocellular neurons in lactating and non-lactating female rats submitted to acute haemorrhage or 24 h of water deprivation, stimuli that induce the release of both oxytocin and vasopressin. Quantitative analysis of preparations doubly immunostained for Fos and either of the neuropeptides revealed that oxytocin and vasopressin neurons synthesise Fos in response to either stimulus but to a different degree, depending on the type of neuron, the type of stimulus, the location of the neurons and the reproductive state of the animal. Thus, in terms of number of cells, haemorrhage was significantly more potent than water deprivation in inducing Fos immunoreactivity in either type of neuron in the supraoptic, paraventricular and anterior commissural nuclei. However, the Fos reaction of vasopressin cells in response to either stimulus was greater than that of oxytocin cells in the supraoptic and paraventricular nuclei, and in the perifornical posterior nucleus and nucleus circularis in response to water deprivation. Moreover, when considering each neuronal population as a whole, it was obvious that Fos synthesis varied in relation to the location of the neurons in the different hypothalamic nuclei, suggesting the existence of functionally distinct neuronal subgroups. Finally, our analyses clearly indicated that Fos synthesis in either type of magnocellular neuron was closely linked to the reproductive state of the animal since after haemorrhage or water deprivation, the number of Fos-positive oxytocin cells in the supraoptic nucleus and Fos-positive vasopressin cells in the paraventricular nucleus was significantly less in lactating than in virgin rats.

N-Methyl-D-aspartate (NMDA) microinjection (1 mM, 0.2 microliter) into the hypothalamic supraoptic nucleus (SON) stimulated heart rate in urethane-anaesthetized rats. This effect was inhibited by coinjection of a competitive blocker of NMDA receptors, CPP (20 nmol) or by pretreatment with a sympathetic ganglionic blocker, chlorisondamine chloride (5 mg/kg i.p.), but not by prior hypophysectomy. Furthermore, the cardioexcitatory effect of intra-SON NMDA was inhibited by prior intra-SON injection of a competitive blocker of nitric oxide (NO) synthesis, NG-nitro-L-arginine methyl ester (40 nmol) or a blocker of the soluble guanylate cyclase, Methylene blue (20 nmol), and was mimicked by intra-SON injection of a calcium ionophore, A23187 (10 nmol), which stimulates NO production by raising intracellular free calcium levels. Finally, intra-SON microinjection of a membrane-permeating cGMP analog, 8-bromo-cGMP (20 nmol) stimulated heart rate in urethane-anaesthetized rats. The results point to a functional link between a sympathetically mediated cardiophysiological effect of NMDA receptor stimulation in the SON and activation of the NO/cGMP signal transduction pathway.

The supraoptic (SON) and paraventricular nucleus (PVN) of the human hypothalamus produce vasopressin (AVP) and oxytocin (OXT). Since in these nuclei no cells are lost during aging or Alzheimer's Disease (AD), factors are searched for which may be responsible for this remarkable stability. Earlier work in both rat and human indicated that the peptide synthesis of these neurons was activated in the oldest age groups as judged from increased neuronal and nuclear size and AVP plasma levels. The size of the Golgi Apparatus (GA) has proved to be a very sensitive parameter for the synthetic activity of these neurosecretory cells in animal experiments. In order to determine changes in the GA during aging and in Alzheimer's Disease, we applied a polyclonal antiserum against immunoaffinity purified MG-160, a sialoglycoprotein of the medial cisternae of the GA, on formalin-fixed and paraffin-embedded sections of the SON and PVN of patients ranging in age from 29 to 97 years. However, our standard fixation procedure masked antigenic sites resulting in a minimal immunocytochemical staining in most of the tissues examined. It appeared to be possible, however, to retrieve the antigen and to obtain an excellent staining of the GA by heating sections in a microwave oven before immunostaining. Following this procedure, an increase in size and intensity of the GA became apparent in individuals from about 70 years and older. In AD patients a similar increase in size and intensity of the immunostained GA was observed. Taken together, these results indicate that SON and PVN neurons are activated during the course of aging and also in AD and that this activation takes place at an earlier age than observed previously by other cellular parameters.

We made extracellular recordings from 113 spontaneously active neurons in the A1 area, after identifying the cells by antidromically activating them from the region of the supraoptic nucleus in urethane-anesthetized rabbits. We tested the response of these neurons to inputs from abdominal vagal, renal and somatic nerves. Electrical stimulation of the abdominal vagus nerve activated 64/85 neurons tested (75%), and had no effect on the remaining 25%. Latency was 195 +/- 25 ms, (conduction velocity 0.7 m/s). Stimulation of renal afferents had no effect on the discharge rate of 4 neurons tested. Stimulation of branches of the sciatic nerve inhibited 7/17 A1 area neurons tested, excited 4 and had no effect on 6 neurons. Stimulation of the central ear nerve inhibited 4/17 neurons tested, excited 6 and had no effect on 7 neurons. Gastric distension had no effect on 20/24 neurons tested. Lightly touching the animals back and legs had no effect on the discharge of 45/49 neurons tested. Similarly, painful stimuli failed to affect 44/49 neurons tested. Our results indicate that A1 area neurons, with projections to the region of the supraoptic nucleus, receive excitatory inputs from the abdominal vagus nerve. The visceral information transmitted to A1 cells by these abdominal vagal afferents is not yet determined, but acute gastric distention does not appear to be a physiological stimulus. A1 area neurons seem not to be involved in transmitting somatic information to the hypothalamus.

The A1 noradrenaline (NA) cell group of the caudal medulla provides a direct excitatory input to supraoptic nucleus (SON) vasopressin (VP) cells. We have now investigated the possibility that NA released from A1 terminals acts presynaptically to modulate A1 input to VP cells. Initial experiments done in pentobarbitone anaesthetized rats established that SON application of NA excited VP cells but also depressed their response to activation of the A1 input. Moreover, the latter effect was mimicked by the alpha 2-adrenoceptor agonist clonidine and antagonised by intravenous administration of the alpha 2-adrenoceptor antagonist yohimbine. Further studies showed that yohimbine also prevented the relative decline in synaptic excitation of VP cells normally observed as A1 activation frequency increases. These data are consistent with the proposal that NA, released from the SON terminals of A1 NA cells, acts via alpha 2-adrenoceptors to depress A1 transmitter release and thus A1 influence on VP cells.

Anorexia nervosa is associated with vasopressin, oxytocin and serotonin abnormalities. Because of the relationship between exercise and anorexia nervosa, we explored the weight-loss syndrome produced by wheel running in food-deprived rats. Its effects on regional vasopressin and oxytocin concentrations were determined under basal conditions and following systemic fluoxetine. Weight-matched, exercised and unexercised rats served as controls. Fluoxetine caused abnormalities in suprachiasmatic vasopressin and dynorphin A content and in thymus oxytocin content that did not occur in weight-matched or exercised controls. No syndrome-specific anomalies occurred in the hypothalamo-neurohypophysial system or dorsal vagal complex (DVC). However, weight reduction and fluoxetine increased circulating vasopressin; moderate exercise caused fluoxetine-induced elevations in posterior pituitary vasopressin and oxytocin; and, unlike the other groups, fluoxetine increased DVC oxytocin in freely fed unexercised rats. It was concluded that syndrome-specific vasopressin and oxytocin abnormalities occur that are not secondary to weight loss or moderate exercise; that weight loss or fluoxetine increases circulating vasopressin; that moderate exercise alters neurohypophysial vasopressin and oxytocin content; and that weight loss or exercise inhibits a fluoxetine-stimulated increase in DVC oxytocin. Finally, it was argued that the fluoxetine abnormalities indicate possible serotonin dysfunction in the syndrome.

The protein, Fos, detected immunohistochemically, was used to identify neurons in the brain that were activated after hemorrhage in the conscious rat. Spinally projecting neurons in the paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM) were identified by the presence of rhodamine-labeled latex beads which had been previously injected into the upper thoracic spinal cord. On the experimental day, conscious rats underwent either (1) withdrawal of 4 ml of blood from a carotid cannula (n = 8) which reduced mean arterial pressure from 96.6 +/- 2.7 to 42.7 +/- 7.1 mmHg, (2) withdrawal of 2 ml of blood (n = 4) which did not affect mean arterial pressure. Animals that were not hemorrhaged were used as controls (n = 6). After the 4 ml hemorrhage, dense concentrations of Fos-positive cell nuclei were found in the lamina terminalis, supraoptic nuclei (SON), PVN and in the medulla. In contrast, the density of Fos-positive cells in 2 ml-hemorrhaged rats was not different from controls except in the SON and in the medial PVN in 2 of 4 rats. After the 4 ml hemorrhage 14.4 +/- 1.2% of the spinally projecting neurons in the PVN and 22.7 +/- 6.1% in the RVLM expressed Fos (P < 0.001 compared to control). After the 2 ml hemorrhage the proportion was 12.2 +/- 3.1% in the PVN (P < 0.001 compared control) but only 5.4 +/- 2.2% in the RVLM (P > 0.05 compared to control). The results suggest that spinally projecting neurons in the PVN and RVLM participate in the reflex responses to hemorrhage.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel pathway between the subfornical organ and the supraoptic nucleus involving the collateral branches of cell bodies situated in the lamina terminalis has been studied. Fluorogold was injected into the supraoptic nucleus and rhodamine-labelled microspheres into the subfornical organ of rats. Nineteen % of neurons in the median preoptic nucleus and 30% of neurons in the OVLT projecting to the subfornical organ also had axons extending to the supraoptic nucleus. These pathways may represent a novel trajectory for the rely of information from the lamina terminalis to the supraoptic nucleus.

We previously reported that food deprivation significantly decreased arginine-vasopressin (AVP) mRNA levels in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also greatly stimulated the pituitary-adrenocortical system in rats. In this study, we deprived adrenalectomized rats with subcutaneously implanted low-dose corticosterone pellets (ADX + B) of food for 3 days to investigate the involvement of corticosteroid feedback regulation in the food deprivation-induced decrease in AVP mRNA in both the SON and the PVN. The plasma corticosterone levels in these animals were maintained at low levels constantly over 24 h. The ACTH concentration in the morning plasma was markedly increased in the food-deprived ADX + B rats as compared to the fed ADX + B rats. Food deprivation significantly decreased the corticotropin-releasing hormone (CRH) content in the median eminence and increased the CRH and AVP content in the neurointermediate lobe of the pituitary. Semiquantitative in situ hybridization histochemistry revealed that AVP mRNA levels were decreased in the SON but, inversely, increased in magnocellular as well as parvocellular subdivisions of the PVN following food deprivation. These results suggest that: (1) AVP mRNA responds differently to food deprivation between the SON and the PVN; (2) the glucocorticoid feedback can exert on AVP mRNA in the PVN but not in the SON in the food-deprived rats; and (3) food deprivation affects the neurohypophysial levels of CRH and AVP.

Glutamate microinjection (1 M, 250 nl) into the hypothalamic supraoptic nucleus (SON) stimulated heat production in brown adipose tissue (BAT) and caused a rapid and sustained increase in interscapular BAT and core temperatures in urethane-anaesthetized rats. This effect was blocked by intraperitoneal pretreatment with a sympathetic ganglionic blocker, chlorisondamine chloride (2.5 mg/kg), or a beta-adrenergic receptor blocker, propranolol (2.5 mg/kg), but not by prior hypophysectomy or intracerebroventricular pretreatment with specific receptor blockers to vasopressin (d(CH2)5[Tyr(Me)2]AVP, 5 micrograms) or oxytocin (d(CH2(5)[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT, 5 micrograms). The results demonstrate that stimulation of SON cells with glutamate elicits a non-vasopressinergic/non-oxytocinergic neural signal that can bring about a sympathetically-mediated increase in BAT thermogenesis. Heat production in BAT is an important mechanism of thermal protection during cold stimulation, and there is evidence that osmotic stimulation can influence thermoregulation. SON neurons play a major role in osmoregulation via release of the peptide hormones vasopressin and oxytocin. The present results suggest the possibility that apart from releasing peptide hormones for osmoregulation, SON neurons might be involved in mediating the effect of osmotic stimulation on thermoregulatory responses involved in thermal adaptation.

An analytical technique is described which permits the quantitation of picogram concentrations of 3-methoxy-4-hydroxyphenylethylene-glycol (MHPG) in acid hydrolyzed extracts of microdissected regions of the rat brain, and this procedure is used to determine if alterations in the activity of noradrenergic neurons are reflected by changes in the concentrations of MHPG in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the rat hypothalamus. MHPG was not detected in non-hydrolyzed samples of either the PVN or SON, but following acid hydrolysis (heating of samples at 94 degrees C for 5 min in 0.16 M perchloric acid) MHPG was detected in both of these regions. These results indicate that MHPG exists primarily as a conjugate in the PVN and SON. Neurotoxin-induced lesions of the ventral noradrenergic bundle decreased norepinephrine (NE) and MHPG concentrations in the PVN and SON, demonstrating that tissue levels of MHPG in these brain regions are dependent upon the presence of noradrenergic neurons. Electrical stimulation of the locus coeruleus increased MHPG concentrations in the PVN, but not in the SON, whereas electrical stimulation of the medial forebrain bundle increased MHPG concentrations in both of these regions. The alpha 2-adrenergic receptor antagonist idazoxan increased, while the alpha 2-adrenergic receptor agonist clonidine decreased MHPG concentrations in both the PVN and SON, but neither idazoxan nor clonidine altered NE concentrations in these regions. Immobilization of rats in the supine position increased MHPG concentrations in the PVN and SON, and this was accompanied by a decrease in NE concentrations in the SON.(ABSTRACT TRUNCATED AT 250 WORDS)

The subfornical organ, along with other regions of the lamina terminalis, may contain osmoreceptors and is likely to be a site of action of blood-borne angiotensin II. The neural pathways by which these stimuli lead to vasopressin secretion, have been suggested to extend from the subfornical organ to hypothalamic sites of vasopressin production either directly or via synapses in an intervening nucleus such as the median preoptic nucleus. In the present study, cholera toxin conjugated to horseradish peroxidase (CT/HRP) or colloidal gold (CT/Au) has been injected, respectively, into the subfornical organ and supraoptic nucleus of the same animal. The anterograde and retrograde transport of the toxin from these two sites has made possible the identification, at the ultrastructural level, of a synapse in the median preoptic nucleus interposed in the pathway between the subfornical organ and the supraoptic nucleus. Moreover, the presence of retrogradely transported CT/HRP and CT/Au in the same neurone in the median preoptic nucleus indicates that some neurones in this nucleus have axons with collateral branches to both the subfornical organ and supraoptic nucleus. Either or both of these pathways may transmit information related to the tonicity of the blood or circulating levels of angiotensin II to sites in the hypothalamus.

Membrane properties of rat magnocellular neuroendocrine cells (MNCs) were examined during intracellular recordings in vivo. Current-clamp experiments revealed sustained outward rectification positive to -55 mV and transient outward rectification of depolarizing responses elicited from negative holding potentials. Trains of impulses were associated with a progressive increase of spike duration. Such features, which were not observed in neighboring non-neuroendocrine cells, are similar to those of MNCs in slices or explants of rat hypothalamus. In contrast, however, cells recorded in vivo were characterized by intense synaptic inhibition and a lower specific impedance than commonly observed from MNCs impaled in vitro.

The time course and pattern of serotonin (5-HT) reinnervation in the rat organum vasculosum laminae terminalis (OVLT) following intracerebroventricular administration of 5,7-dihydroxytryptamine were examined by means of [3H]5-HT uptake radioautography. 5-HT axonal varicosities reappeared in the OVLT within 3 months post-lesion. Six months later, they were found to have preferentially reinvested the neurohemal contact area (juxtavascular zone) of the organ. Regenerated terminals further increased in number. At the end of a 16-month survival time, they displayed normal-looking distributional patterns, similar to those of age-matched control animals, and formed new synaptic junctions in the juxtaventricular zone. The cellular mechanisms possibly responsible for 5-HT recovery in the OVLT are discussed.

Catecholaminergic innervation of the hypothalamic vasopressin (VP)-secreting supraoptic nucleus (SON) was examined at selected intervals after deafferenting neurosurgical lesions, with respect to potential contribution of peripheral vascular sympathetic fibers. Young adult (3 months) and aged (20 months) male F344 rats were subjected to mechanical knife-cut lesion just caudal and medial to the SON, superior cervical ganglionectomy (SCGx), or both surgeries. SON and lesion sites were assessed at 4, 14, 30 or 45 days after surgery, by CA histofluorescence. Functional evaluation in rats subjected to chronic lesions consisted of monitoring water balance (water consumption and urine volumes, and urine osmolality and VP content) in individual rats for presurgical and postsurgical intervals. Histofluorescence evaluation showed that SCGx did not affect the overall SON fluorescence pattern, although a minor sympathetic contribution to that pattern was discerned by comparing SON in rats subjected to lesion alone vs SCGx + lesion. Morphological reinnervation of SON was accomplished at 30 days in young rats, and 45 days in aged rats, after both lesion and SCGx. In young rats, histofluorescence density 30 days after deafferentation was denser than the innervation pattern seen in intact (sham-lesioned) animals, while reinnervation at 45 days postsurgically in aged rats only approximated the presurgical pattern. Vasopressin excretion and corresponding water conservation measures were compromised by SON deafferentation at both ages; excreted VP levels and water balance did not rebound to presurgical values at chronic postsurgical intervals in either young or aged rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of adrenergic and excitatory amino acid antagonists on supraoptic nucleus (SON) neurosecretory cell responses to stimulation of the A1 noradrenaline (NA) cell group were examined in anaesthetized male rats. As in previous studies, delivery of cathodal pulses (100 microA, 1 ms pulses, 1 Hz) to the A1 region of the caudal ventrolateral medulla excited spontaneously active, antidromically identified neurosecretory cells, the majority of which were identified as arginine vasopressin (AVP) secreting on the basis of basal discharge patterns and responses to abrupt increases in arterial blood pressure. Administration of alpha- and beta-adrenoreceptor antagonists, by systemic or intracerebroventricular delivery of a bolus, or by direct pressure injection into the SON, did not alter neurosecretory cell responses to A1 stimulation, even when doses applied exceeded that required for blockade of excitations elicited by local application of NA. Application of the broad spectrum excitatory amino acid antagonist kynurenic acid (5-40 mM) blocked the excitatory effects of locally applied glutamate (100 microM) and transiently inhibited spontaneous activity, but failed to alter the excitatory effects of A1 region stimulation on SON cells. Identical effects were obtained with a selective kainate/quisqualate receptor antagonist. These data indicate that neurosecretory cell responses to activation of the A1 cell group are unaltered by antagonists of alpha- and beta-adrenoreceptors, or excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrical stimulation of the lateral olfactory tract (LOT) has been shown to excite, monosynaptically, supraoptic nucleus (SON) neurons in slices of hypothalamus taken from male, virgin female or lactating rats. Only in the last of these, however, did 10 min of 10 Hz stimulation produce an increase in the incidence of Lucifer yellow dye-coupling, an indicator of electrotonic interactions. This coupling is virtually exclusively dendrodendritic. Since virgin females that have been induced to show full maternal behavior have altered dendritic morphology reminiscent of lactating animals (but different from males and untreated virgins) we investigated the effects of LOT stimulation in slices from maternally behaving virgins. Similar to the data for lactating rats, electrical stimulation of this tract, the terminals of which release excitatory amino acid transmitter, increased the incidence of dye-coupling by 112% (P less than 0.01). Also similar to lactating rats, the coupling was dendrodendritic and the increase was due entirely to increasing the number of neurons coupled to the injected neuron. No increase in coupling was seen in stimulated slices from pup-exposed control rats. We conclude that the maternal behaviors engaged in by both real mothers and induced virgins 'primes' the supraoptic neurons to increase coupling in response to olfactory system stimulation. This priming may occur via olfactory and vomeronasal stimulation during such behaviors as sniffing and anogenital licking of the pups. That coupling increased in tissue from maternally behaving virgins comparably to that from nursing mothers further suggests that SON neurons may play a role in maternal behavior independent of its well-documented role in the milk ejection reflex.

Mitral cells of the main and accessory olfactory bulbs have been shown to project monosynaptically to the supraoptic nucleus (SON) via the lateral olfactory tract (LOT) which uses excitatory amino acid transmitters. Data collected during characterization of these projections suggested that synaptic activation of SON neurons via LOT stimulation in slices influenced the incidence of dye-coupling. The present study pursued this suggestion using horizontally cut slices from male, virgin female and lactating rats. Neurons were confirmed to be excited by electrical stimulation of the tract, injected with Lucifer yellow, and synaptically activated for 10 min at 10 Hz (n = 92). Another 94 neurons were similarly confirmed and injected, but received no further stimulation. In an additional 8 slices, injected neurons were antidromically activated for 10 min at 10 Hz. Analyses done on 194 injected neurons from the 3 groups showed that synaptic activation resulted in a significant (P less than 0.01) increase in the incidence of coupling only in tissue from lactating rats. This increase was entirely due to larger numbers of cells being coupled dendrodendritically to the injected cells in the stimulated slices. Antidromic activation did not influence coupling. Increased coupling occurred among both oxytocin and vasopressin cell types. This is the first report of increased coupling resulting from synaptic activation in mammalian CNS. Changes seen only in lactating rats may be related to their altered SON ultrastructural morphology (i.e. dendritic bundling). Strong olfactory and vomeronasal input associated with some maternal behaviors may increase neuronal coupling and enhance hormone release in response to other incoming stimuli (e.g. suckling, dehydration).

A series of experiments were performed to study the effects of learning upon proteins in the split-brain pigeon. With this preparation, one side of the brain was trained in a color discrimination task while the other side served as a naive control. Proteins from both sides of the brain were separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed to measure protein levels or protein synthesis. No differences were found between trained and naive telencephalic hemispheres or between trained and naive optic lobes in the steady-state protein levels or in protein synthesis.

Lesions of the tissue surrounding the preoptic recess (AV3V region) have severe effects on body fluid homeostasis; these include acute adipsia and failure of the antidiuretic response. Because neurosecretory cells in supraoptic nuclei comprise the major source of antidiuretic hormone (ADH) in this species, we have previously observed the fine structure of supraoptic nuclei in rats with AV3V lesions. Paraventricular nuclei are the other major source of ADH in rats. Therefore, in this investigation we compared the fine structure of paraventricular nuclei in rats which had received AV3V lesions 3 days earlier with that of control rats which had received sham lesions and either had drinking water available or had water withheld for 3 days. Degenerating axons and axon terminals were present in paraventricular nuclei of lesioned rats. The degenerating terminals were in axodendritic and less often in axosomatic synapses. Morphometric evaluation revealed that neurosecretory cells did respond to the dehydrated state of the adipsic-lesioned animals, but the response was significantly attenuated compared to that which occurred in sham-lesioned rats deprived of water for 3 days. It appears that AV3V lesions damage afferent connections and impair the response of neurosecretory cells to dehydration in paraventricular as well as supraoptic nuclei. However, in paraventricular nuclei the response is not completely prevented by AV3V lesions during the adipsic period as was observed in supraoptic nuclei. The presence of a response in paraventricular nuclei may be at least partially stimulated by reduced body fluid volume. Information from volume receptors would be carried from the medulla to paraventricular nuclei by ascending pathways which are not affected by AV3V lesions.

Neural activity of the suprachiasmatic nucleus (SCN) in male albino rats was recorded under restricted daily feeding schedule. Circadian rhythm in multiple unit activity from the SCN showed essentially no interference with daily food cyclicity, while significant re-distribution of motor activities occurred during such a schedule. Since the central pacemaker is presumed to be unaffected by food availability, these results provide more support to the widespread notion that the SCN is an anatomical locus of the circadian pacemaker in rodents.

In anesthetized cats recordings were made from hypothalamo-neurohypophysial neurons in a supraoptic nucleus (SON) of the hypothalamus. The region of the nucleus tractus solitarius in the medulla, identified electrophysiologically as the site of termination of the first relay neurons of the sinus and aortic nerves, was stimulated with single or short trains of pulses (2-3 at 200 Hz). Out of 133 SON neurons 67 were affected by such stimuli. In 14 cells (21% of 'responsive' neurons) the stimulus produced profound inhibition of SON neuron activity after a latency of 10-30 msec. In another 8 neurons (12%) the inhibitory effect was observed after a longer latency of over 100 msec. An increase in intensity of stimulus merely prolonged or increased the inhibitory effect without changing the response qualitatively. The other 45 (67%) SON neurons were excited by stimulation of the nucleus tractus solitarius. In a small proportion of these neurons (5 cells, 7%) the stimulus evoked discharges, even in spontaneously silent neurosecretory cells, after a latency of 10-20 msec with little fluctuation. In the remaining 40 neurons, i.e. 60% of the 'responsive' neurons, the excitatory effect was observed after a latency of 40-120 msec. Again, changes in intensity of stimulation did not alter the nature of this response. The results indicate that both 'fast' as well as 'slow' pathways between the nucleus tractus solitarius and SON neurons exist and impulses travelling through the latter pathway from the carotid sinus or aortic nerve affect the larger proportion of SON neurons.

The number of specific binding sites for 125I-labeled human prolactin (hPrl) in crude membrane fractions from rat liver is sexually differentiated. Livers from female rats specifically bound much more of added [125I]hPrl than male liver membrane fractions which bound little, if any, Prl. Hypophysectomy of female animals eliminated hepatic binding of Prl; the effect was reversed by a pituitary implant under the kidney capsule. Treatment of male rats with estradiol valerate induced hepatic hPrl receptors, but only in the presence of an intact pituitary. Anterior hypothalamic deafferentation at the retrochiasmatic level in male rats increased the hepatic hPrl receptor concentration to the female level 3-4 days following the operation. This induction of hPrl receptors was also seen in the absence of the gonads. A transection rostral to the suprachiasmatic nucleus had no effect on the concentration of hPrl receptors in male animals. Our results demonstrate that [125I]hPrl binds specifically to rat liver membranes and that the number of binding sites is regulated by the hypothalamo-pituitary system. The regulatory brain centre appears to be located in the anterior hypothalamus or adjacent areas. The receptor-inducing pituitary factor might possibly be related to the so-called 'feminizing factor' involved in pituitary control of liver enzymes that metabolize drugs and steroids.

A sensitive technique was developed for measuring continuously the summated neural activity of the rat hypothalamo-neurohypophysial tract. Large compound potentials of 4-9 mV peak amplitude were recorded from the fibre tract in response to electrical stimulation of the neurohypophysis. It was estimated that all fibres in the tract were electrically activated and contributed to the compound potential. It was shown that a decrease of peak amplitude could be used as a measure of changes in endogenous neural activity. The decrease in peak amplitude during an increase of endogenous neural activity was due to fatigue and temporal spread of the compound potential, and also to collision of antidromic and orthodromic action potentials. Decrease of the compound potentials was related to the intensity of vaginal distension, haemorrhage and plasma hyperosmolality, and it correlated with neurohypophysial hormone release. This method may usefully complement hormone assays and single cell analysis.

Small lesions of the tissue surrounding the anterior ventral third ventricle (AV3V) cause adipsia, but there is no compensatory antidiuretic response. Therefore, the fine structure of the supraoptic nucleus and neural lobe, the major sites of synthesis and release of antidiuretic hormone (ADH), were compared in rats rendered adipsic by AV3V lesions 3 days earlier, rats deprived of water for 3 days and rats drinking normally. In sham-lesioned rats which were deprived of water, neuronal somas in the supraoptic nucleus show signs of stimulated secretory activity. However, the neuronal somas of supraoptic nuclei of rats which did not drink because they were made adipsic by AV3V lesions resemble those of normally hydrated controls. Neural lobes of water deprived animals contain a sharply reduced number of neurosecretory granulated vesicles and reduced apposition of glial processes with the perivascular connective tissue compared to those of normally hydrated rats. In contrast, neural lobes of rats with AV3V lesions contain large accumulations of densely packed neurosecretory vesicles, as well as abundant dense bodies and multilamellar bodies which may be evidence of increased crinophagy, and they have increased interposition of glial processes between axon endings and the perivascular connective tissue. In rats with AV3V lesions the severe dehydration due to adipsia was unable to stimulate release of ADH. The accumulation of neurosecretory vesicles in the neural lobe indicates that transport of ADH to the neural lobe was not impaired in this time period, but that exocytosis of ADH-containing neurosecretory vesicles in the neural lobe was blocked.

Experiments were done in urethane anesthetized rats to identify single units in the region of the parabrachial nucleus (PBN) projecting directly to 'cardiovascular' responsive sites in either the paraventricular nucleus of the hypothalamus (PVH) or the supraoptic commissure and nucleus (SOC-SON) region. Fifty-five single units were antidromically activated in the ipsilateral PBN by electrical stimulation of either the PVH (n = 27) or SOC-SON region (n = 28) with latencies corresponding to conduction velocities of 0.3-5.1 m/s. The axons of PBN units projecting to the PVH conducted at significantly slower velocities (0.5 +/- 0.04 m/s) than those projecting to the SOC-SON region (1.6 +/- 0.25 m/s). These data suggest that ascending fibers from the PBN to the PVH are unmyelinated, whereas those to the SOC-SON region are primarily a little myelinated. In addition, since the PBN is known to receive cardiovascular and visceral afferent inputs, it is suggested that these neurons likely function in relaying this afferent information to hypothalamic areas involved in autonomic regulation.

The existence of monosynaptic connections between neurons in the paraventricular nucleus (PVN) of the hypothalamus and the intermediolateral cell column (ILC) of the spinal cord was studied by electrophysiological techniques in chloralose-anesthetized cats. Sympathetic preganglionic discharges (recorded from the 2nd or 3rd thoracic white ramus) were evoked by microstimulation of certain regions in or near the PVN with short train of pulses and below 50 microA current. By recording responses of 'identified' and 'non-identified' neurosecretory cells in the PVN and supraoptic nucleus (SON) to stimulation of the ILC of the thoracic cord, it was possible to identify antidromically evoked action potentials in 9 out of 297 neurons tested. Among them, 2 neurons were also antidromically excited by the pituitary stalk stimulation, 5 were orthodromically excited by the same stimulus and the remaining 2 were not excited by the stalk stimulation. Our results indicate that some PVN neurons, though small in number, send axons directly to the ILC of the cord, and that a very few neurons among these also send their axons to the pituitary gland.

The functional role of the ascending projection from A1 noradrenergic neurons of the caudal ventrolateral medulla to the supraoptic nucleus of the hypothalamus was investigated by examining the effects of electrical stimulation of the A1 region on the activity of supraoptic neurons deemed to be vasopressinergic or oxytocinergic on the basis of basal firing patterns and responsivity to baroreceptor activation. A1 stimulation enhanced the activity of all putative vasopressin-secreting supraoptic neurons tested. This effect appeared to be selective in that no putative oxytocin-secreting neurons were excited by A1 stimulation. Destruction of the supraoptic noradrenergic terminal plexus by local application of the neurotoxin 6-hydroxydopamine abolished the facilitatory effects of A1 stimulation but did not noticeably alter basal activity patterns, nor the influence of baroreceptor inhibitory pathways. These findings suggest a facilitatory role for noradrenergic afferents in regulating the activity of neurohypophysially-projecting vasopressin neurons of the supraoptic nucleus.

(1) The effects of arterial baroreceptor stimulation on the activity of 'identified' neurosecretory cells in supraoptic nuclei (SON) of the hypothalamus were investigated in anesthetized cats. (2) Stimulation of baroreceptors by distension of an 'isolated' carotid sinus greatly inhibited SON activity. A linear relationship was found to exist between stimulus intensity (an increase in the sinus pressure) and the degree of inhibition of SON neuron activity. This inhibitory effect was abolished by section of the sinus nerve. (3) The inhibitory effect of baroreceptors on SON neuron activity was most pronounced in the first 5 sec during stimulation, and the effect became less when the stimulus lasted for a long period. At the end of stimulation there was a transient reversal of the response. (4) The excitation of aortic baroreceptors by occluding descending aorta strongly inhibited SON neuron activity. (5) Occlusion of carotid arteries augmented the SON neuron activity. Electrical stimulation of the sinus nerve caused an excitation or an inhibition of SON neurons, depending on the stimulus intensities. Apparently these stimuli produced different degrees of excitation in baro- and chemoreceptors. (6) Combination of chemo- and baroreceptor stimulations revealed that the excitatory effect exerted by chemoreceptor stimulation on SON neurons was reversed or blocked by baroreceptor activation, indicating the powerful inhibitory influence of baroreceptors.