Hypothalamo-neurohypophysial System Research Articles

Osmotic activation of the hypothalamo-neurohypophysial system reversibly downregulates the NMDA receptor subunit, NR2B, in the supraoptic nucleus of the hypothalamus

NMDA receptor activation produces a characteristic pattern of neuronal firing in magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) which has been associated with greater hormone release in vivo and in vitro. In addition, i.c.v. administered NMDA receptor blockers suppress the dehydration-induced rise in plasma vasopressin and drinking. To investigate the role of NMDA receptor subunits in the neuroendocrine functions of the magnocellular neuroendocrine cells of the hypothalamus, we examined the effects of osmotic stimulation on the protein expression of the NMDA receptor subunits, NR1 and NR2B, important in binding glycine and glutamate, respectively. Homogenates of SON, paraventricular nucleus of the hypothalamus (PVN), cortex and lateral hypothalamus from control rats and rats given 2% saline water to drink for 4–10 days were subjected to SDS–PAGE and Western blot analysis. This saline water drinking regimen produced a significant rise in plasma osmolality levels. NR1 and NR2B immunoreactivity was detected in SON, PVN, lateral hypothalamus and cortex but not in liver homogenates using subunit-specific polyclonal antibodies and quantified using computer-assisted densitometry. Mean NR2B immunoreactivity was significantly lower in SON (29%) and PVN homogenates (23%) from saline-treated rats than in those from control rats. In addition, the effect of dehydration on NR2B was regionally specific since no significant changes in NR2B expression were observed in homogenates of cortex and lateral hypothalamus. Rehydration allowed recovery of plasma osmolality as well as NR2B protein levels in the SON. These results suggest that changes in NMDA receptor subunit expression contribute to the plasticity manifested by in magnocellular neuroendocrine cells in response to osmotic activation of the hypothalamo-neurohypophysial system. In addition, our results indicate that NMDA receptors on SON and PVN MNCs may contribute to neuroendocrinological functions associated with body fluid homeostasis.

Evidence for a vasopressin system in the rat heart.

Traditionally, a hypothalamo-neurohypophysial system is thought to be the exclusive source of arginine vasopressin (AVP), a potent antidiuretic, vasoconstricting, and growth-stimulating neuropeptide. We have identified de novo synthesis of AVP in the heart as well as release of the hormone into the cardiac effluents. Specifically, molecular cloning of sequence tags amplified from isolated, buffer-perfused, and pressure-overloaded rat hearts allowed the detection of cardiac AVP mRNA. Subsequent experiments revealed a prominent induction of AVP mRNA (peak at 120 minutes, 59-fold, P<0. 01 versus baseline) and peptide (peak at 120 minutes, 11-fold, P<0. 01 versus baseline) in these isolated hearts. Newly induced vasopressin peptide was localized most prominently to endothelial cells and vascular smooth muscle cells of arterioles and perivascular tissue using immunohistochemistry. In addition to pressure overload, nitric oxide (NO) participated in these alterations, because inhibition of NO synthase by Nomega-nitro-L-arginine methyl ester markedly depressed cardiac AVP mRNA and peptide induction. Immediate cardiac effects related to cardiac AVP induction in isolated, perfused, pressure-overloaded hearts appeared to be coronary vasoconstriction and impaired relaxation. These functional changes were observed in parallel with AVP induction and largely prevented by addition of a V1 receptor blocker (10(-8) mol/L [deamino-Pen1, O-Me-Tyr2, Arg8]-vasopressin) to the perfusion buffer. Even more interesting, pressure-overloaded, isolated hearts released the peptide into the coronary effluents, offering the potential for systemic actions of AVP from cardiac origin. We conclude that the heart, stressed by acute pressure overload or NO, expresses vasopressin in concentrations sufficient to cause local and potentially systemic effects.

Microtubule-associated protein-2 in the hypothalamo-neurohypophysial system: low-molecular-weight microtubule-associated protein-2 in pituitary astrocytes

Microtubule-associated protein-2 is the most abundant microtubule-associated protein in the brain and is responsible for morphogenesis and maintenance of the nervous system. In the present experiments, we have examined the localization of microtubule-associated protein-2 in the hypothalamo-neurohypophysial system of the rat using western blots and immunohistochemistry. Two monoclonal antibodies against microtubule-associated protein-2, antibody C and AP20, were used: antibody C recognizes both the high- and low-molecular-weight isoforms of microtubule-associated protein-2; antibody AP20 specifically detects high-molecular-weight microtubule-associated protein-2 only. Western blots analysis revealed expression of high-molecular-weight microtubule-associated protein-2 in the whole brain, hippocampus and whole hypothalamus. While the supraoptic nucleus expressed only high-molecular-weight microtubule-associated protein-2, the adult posterior pituitary predominantly expressed low-molecular-weight microtubule-associated protein-2, which was also seen in the embryonic whole brain. Light microscopic immunohistochemistry revealed that both antibody C and AP20 intensely stained dendrites of the dendritic and somatic zones in the supraoptic nucleus. Double labeling with antibodies against microtubule-associated protein-2 and oxytocin (or vasopressin) demonstrated that microtubule-associated protein-2 was localized in dendrites of magnocellular neurons in the supraoptic nucleus. In the posterior pituitary, however, antibody C stained fine processes and cell bodies of astrocytes, which were identified by an antibody against glial fibrillary acidic protein. Antibody AP20 also stained fine processes of some astrocytes in the posterior pituitary, but the intensity of immunoreactivity with antibody AP20 was weaker than that with antibody C. This result suggests that microtubule-associated protein-2 in astrocytes of the posterior pituitary is predominantly of the low-molecular-weight type. Moreover, western blots revealed low-molecular-weight microtubule-associated protein-2 of the posterior pituitary at a molecular weight slightly higher than embryonically expressed low-molecular-weight microtubule-associated protein-2, indicating that low-molecular-weight microtubule-associated protein-2 in the posterior pituitary is possibly the isoform microtubule-associated protein-2d. The present results demonstrate that astrocytes in the posterior pituitary of adult rats still retain the ability to express the immature variant of microtubule-associated protein-2, low-molecular-weight microtubule-associated protein-2, and its expression is probably linked to structural plasticity.

Contribution of astrocytes to activity-dependent structural plasticity in the adult brain.

A striking example of the capacity of adult astrocytes to undergo reversible morphological changes in response to stimuli which enhance neuronal activity is offered by astrocytes of the adult hypothalamo-neurohypophysial system (HNS). The HNS is composed of magnocellular neurons secreting the neurohormones oxytocin and vasopressin from axon terminals in the neurohypophysis. Upon activation of HNS secretion, glial coverage of oxytocin neurons significantly diminishes and their surfaces become extensively juxtaposed. These glial changes are invariably accompanied by structural synaptic remodelling resulting in increased numbers of GABAergic, glutamatergic, and noradrenergic afferents. In the neurohypophysis, they result in an enhanced neurohemal contact area. HNS glia in the adult continue to display "embryonic" features that may allow such activity-dependent structural plasticity. For example, supraoptic astrocytes display a radial glia-like morphology and continue to express vimentin, together with GFAP. All HNS astrocytes secrete extracellular matrix glycoproteins, like tenascin-C; they also express high levels of polysialylated NCAM or PSA-NCAM and the glycoprotein F3, molecules considered essential for neuronal-glial interactions in the developing and lesioned CNS. HNS expression of most of these proteins does not visibly vary under different conditions of neurohormone secretion. We consider them as permissive factors, therefore, allowing HNS cells to undergo remodeling whenever the proper stimuli intervene. In the hypothalamic nuclei, one such stimulus is oxytocin itself which, in synergy with steroids, can induce neuronal-glial remodelling; adrenaline does so in the neurohypophysis.

Ultrastructural localisation of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamo-neurohypophysial system.

The distribution of the P2X(2) subtype of the purine receptor associated with the extracellular signalling activities of ATP was studied in the rat hypothalamo-neurohypophysial system at the electron microscope level. Receptors were labelled with ExtrAvidin-horseradish peroxidase preembedding immunocytochemistry using a polyclonal antibody against a fragment of an intracellular domain of the receptor. Immunoreactivity to P2X(2) receptors was localised in: (i) paraventricular and supraoptic nuclei-in subpopulations of endocrine neurones, neurosecretory and non-neurosecretory axons and dendrites; and (ii) the neurohypophysis-in pituicytes and subpopulation of neurosecretory axons. In both the hypothalamic nuclei examined, labelled asymmetric axo-dendritic synapses were commonly observed. These synapses involved either P2X(2)-labelled axon terminals (synaptic buttons) and unlabelled dendrites or labelled dendrites and unlabelled axon terminals. Axo-somatic synapses established by P2X(2)-positive axons on P2X(2)-positive endocrine cell bodies as well as on P2X(2)-negative somata were also observed. The functional significance of these findings is discussed.

The human hypothalamo-neurohypophysial system in health and disease

The present paper reviews the changes observed in the human supraoptic (SON) and paraventricular (PVN) nuclei, and their projections to the neurohypophysis, median eminence and to other brain areas in health and disease.

Chapter 2.1.2 Neurophysiology of magnocellular neuroendocrine cells: Recent advances

Magnocellular neuroendocrine cells of the hypothalamic paraventricular and supraoptic nuclei are responsible for most of the vasopressin and oxytocin in the peripheral blood as well as for central release of these peptides in selected brain areas. As the principal component of the hypothalamo-neurohypophysial system, these neurons have been a subject of continual study for half a century. The wealth of solid information from decades of in vivo studies has provided a firm basis for in vitro, brain slice and explant investigations of neural mechanisms involved in the control and regulation of vasopressin and oxytocin neurons. In vitro methods have revealed the presence and permitted the study of monosynaptic projections to supraoptic neurons from the olfactory bulbs, the tuberomammillary nuclei of the posterior hypothalamus and from the organum vasculosum of the lamina terminalis. Such methods have also facilitated the elucidation of the various ionic currents controlling neurosecretory cell activity as well as the roles of calcium binding proteins and release of calcium from internal stores. This review summarizes recent advances in our understanding of the afferent inputs that impinge upon these two cell types, and the cellular and molecular mechanisms intrinsic to these neurons that determine their activity patterns and, in part, their responses to incoming stimuli.

Chapter 2.2.1 The magnocellular neurons of the hypothalamo-neurohypophyseal system display remarkable neuropeptidergic phenotypes leading to novel insights in neuronal cell biology

For decades the magnocellular neurons of the hypothalamo-neurophypophyseal system (HNS), in which either vasopressin or oxytocin are produced and released into the bloodstream, have been playing a pivotal role in fundamental discoveries in the nervous system. The primary structure of vasopressin and oxytocin was the first of all neuropeptides to be published, i.e., in the 1950s by the Nobel prize laureate Du Vigneaud. Moreover, many trend-setting discoveries have their origin in the HNS, which abundantly expresses vasopressin and oxytocin, clearly displays its function and is relatively easily to manipulate. Examples are the phenomenon of coexpression of neuropeptides, patch-clamping of nerve endings, axonal transport of RNA, neuroglia interactions and the behavioral effects. An extraordinarily intriguing example is the homozygous Brattleboro rat, which lacks vasopressin by a germ-line mutation, and has disclosed many of the fundamental characteristics of peptidergic neurons, and neurons in general. In this chapter we will discuss a few of them, in particular the recent data on mutations in vasopressin RNA. It is to be expected that the HNS will retain its informative role in the next decades.

Effects of oestrogen upon nitric oxide synthase NADPH-diaphorase activity in the hypothalamo–neurohypophysial system of the rat

An understanding of the interaction between oestrogen and the nitric oxide synthase/nitric oxide system is important for determining the roles of nitric oxide in central nervous control of osmotic homeostasis and certain aspects of reproduction. The effects of oestrogen on nitric oxide synthase and nitric oxide synthase activity were investigated in the magnocellular neurosecretory system. Ovariectomized female rats were injected subcutaneously with 17β-estradiol benzoate either 10 μg daily for 4 days (short-term low-dose) or 200 μg daily for 21 days (long-term high-dose). In the neurohypophysis the density of NADPH-diaphorase staining—a marker for nitric oxide synthase activity—was increased after both short-term low-dose and long-term high-dose estradiol treatment, but no difference in nitric oxide synthase immunoreactivity was observed after either treatment. In the magnocellular supraoptic and paraventricular nuclei, short-term low-dose oestrogen treatment did not induce any detectable changes in nitric oxide synthase gene expression, the proportion of nitric oxide synthase-immunoreactive neurons, or the proportion of NADPH-diaphorase-positive neurons. Long-term high-dose oestrogen treatment also had no effect on nitric oxide synthase gene expression or immunoreactivity, but caused a reduction of the proportion of NADPH-diaphorase-positive neurons in the supraoptic nucleus and a reduction in the intensity of this histochemical staining. Qualitatively similar changes were observed in the magnocellular part of the paraventricular nucleus. The results provide, for the first time, evidence of a complex interaction between oestrogen and nitric oxide synthase in the neuroendocrine system in which nitric oxide synthase activity is regulated differently in the magnocellular cell bodies and axonal terminals and in which the activity of the enzyme rather than its expression is controlled.

Something fishy in the rat brain: molecular genetics of the hypothalamo-neurohypophysial system.

The brain peptides vasopressin and oxytocin play crucial roles in the regulation of salt and water balance. The genes encoding these neurohormones are regulated by cell-specific and physiological cues, but the molecular mechanisms remain obscure. New strategies, involving the introduction of rat transgenes into rats, are being used to address these issues, but the complexity of the rat genome has hampered progress. By contrast, the pufferfish, Fugu rubripes, has a "junk-free" genome. The oxytocin homologue from Fugu, isotocin, has been introduced into rats and is expressed in oxytocin neurons, where it is upregulated by physiological perturbations that upregulate the oxytocin gene. The Fugu and rat lineages separated 400 million years ago, yet the mechanisms that regulate the isotocin and oxytocin genes have been conserved. Fugu genome analysis and transgenesis in the physiologically tractable rat host are a powerful combination that will enable the identification of fundamental components of the neural systems that control homeostasis.

Role of non-NMDA receptors in osmotic and glutamate stimulation of vasopressin release: effect of rapid receptor desensitization.

Previous studies demonstrated that the increase in vasopressin (VP) release and induction of VPmRNA content by osmotic stimulation was blocked by kynurenic acid, a non-specific antagonist of excitatory amino acid (EAA) receptors. In order to identify the type of EAA receptor involved, perifused explants of the hypothalamo-neurohypophyseal system (HNS) were exposed to a ramp increase in osmolality (40 mOsm over 6 h achieved by increasing NaCl) in the presence and absence of 10 microM 6,7-dinitroquinoxaline-2,3-dione (DNQX), an antagonist of non-n-methyl-d-aspartate (NMDA) excitatory amino acid receptors. Vasopressin release and VP mRNA content were significantly increased by exposure to the osmotic stimulus. 6,7-dinitroquinoxaline-2,3-dione inhibited osmotically stimulated VP release (F=16.65, P=0.0008) without significantly reducing basal release. It also prevented the osmotically stimulated increase in VP mRNA content (P <0.05). Although these results implicated glutamate, the primary endogenous ligand for EAA receptors, in the regulation of VP, exogenous glutamate was ineffective in stimulating VP release from HNS explants in either low-Mg2+ or Mg2+-replete medium. However, blockade of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor desensitization with cyclothiazide (100 microM) caused a marked increase in VP release in response to 100 microM glutamate, and blockade of kainate receptor desensitization with concanavalin A resulted in a small, but significant increase in VP release in response to 1 mM glutamate. These results support a role for non-NMDA receptor activation in osmotic regulation of VP release.

Dystrophins in neurohypophysial lobe of normal and dehydrated rats: immunolocalization and biochemical characterization.

Dystrophin, utrophin and dystroglycan are present not only in muscle but also in brain. In muscle, they link the extracellular matrix to the cytoskeleton. Their function in brain is not understood. Here we show their presence in the hypothalamo-neurohypophysial system which secretes the neurohormones oxytocin and vasopressin. Using immunocytochemistry, we showed that dystrophins are present in the neurohypophysis of control rats. After water deprivation, immunoreactivity dramatically decreased and appeared in axonal swellings in the hypothalamic tract. Dystrophin immunostaining can be ascribed to dystrophin and/or utrophin as well as the DMD (Duchenne Muscular Dystrophy) gene short products Dp140 and Dp71 as revealed by Western immunoblots of synaptosomes isolated from neurohypophyses of control rats. In synaptosomes isolated from rats under water deprivation, the immunoreactivity entirely disappeared. Further biochemichal characterization of isolated neurosecretory granules (NSG) showed that Dp140 and Dp71 are enriched in the NSG stored in the swellings of the neurohypophysis whereas the NSG of the nerve endings are devoid of these proteins. In addition we observed that the presence of beta-dystroglycan and actin correlates with the presence of dystrophins. Our data favor a direct implication of the dystrophins and/or utrophin, dystroglycan and actin in the neurosecretory processes of the hypothalamo-neurohypophysial system.

Dopaminergic control of angiotensin II-induced vasopressin secretion in vitro.

Because dopamine influences arginine vasopressin (AVP) release, the present studies were designed to ascertain the dopamine receptor subtype that potentiates angiotensin II-induced AVP secretion in cultured hypothalamo-neurohypophysial explants. Dopamine (a nonselective D1/D2 agonist), apomorphine (a D2 >> D1 agonist), and SKF-38393 (a selective D1 agonist) dose dependently increased AVP secretion. Maximal AVP release was observed with 5 microM dopamine, 307 +/- 66% . explant-1 . h-1, 1 microM SKF-38393, 369 +/- 41% . explant-1 . h-1, and 0.1 microM apomorphine, 374 +/- 67% . explant-1 . h-1. Selective D1 antagonism with 1 microM SCH-23390 blocked AVP secretion to values no different from basal. Domperidone (D2 antagonist), phenoxybenzamine (nonselective adrenergic antagonist), and prazosin (alpha1-antagonist) failed to prevent release. D1 antagonism also prevented AVP secretion to 1 microM angiotensin II [angiotensin II, 422 +/- 87% . explant-1 . h-1 vs. angiotensin II plus SCH-23390, 169 +/- 28% . explant-1 . h-1 (P < 0.05)], but D2 and alpha1-adrenergic blockade did not. In contrast, AT1 receptor inhibition with 0.5 microM losartan blocked angiotensin II- but not dopamine-induced AVP release. AT2 antagonism had no effect. Although subthreshold doses of the agonists did not increase AVP secretion (0. 05 microM dopamine, 133 +/- 44% . explant-1 . h-1; 0.01 microM SKF-38393, 116 +/- 26% . explant-1 . h-1;and 0.001 microM angiotensin II, 104 +/- 29% . explant-1 . h-1 ), the combination of dopamine and angiotensin II provoked a significant rise in AVP [420 +/- 83% . explant-1 . h-1 (P < 0.01)]. Similar results were observed with SKF-38393 and angiotensin II, and the AVP response was blocked to basal levels by either D1 or AT1 antagonism. These findings support a role for D1 receptor activation to increase AVP release and mediate angiotensin II-induced AVP release within the hypothalamo-neurohypophysial system. The data also suggest that the combined subthreshold stimulation of receptors that use distinct intracellular pathways can prompt substantial AVP release.

Thyroliberin and the daily rhythm of vasopressin and oxytocin release from the hypothalamo-neurohypophysial system

The daily rhythm of neurohypophysial hormones release was investigated in rats given intracerebroventricularly (i.c.v.) thyroliberin (200 ng/10 μl—once daily over 4 days). In animals injected i.c.v. with vehicle solution (0.15 M sodium chloride) plasma vasopressin concentration was seen to rise over the hours of daylight, decreasing during the first part of the night; plasma oxytocin was the lowest in the morning and increased somewhat at midnight. The content of neurohypophysial vasopressin and oxytocin were highest in the morning; during the light hours they decreased progressively to minimum values at 18:00–19:00. Hypothalamic vasopressin content reached the maximum value at the midnight but hypothalamic oxytocin content was highest in the morning. Manifold injections of thyroliberin to rats resulted in a distinct decrease of neurohypophysial vasopressin and oxytocin content at the beginning of the day (06:00–07:00). Maximal values for neurohypophysial vasopressin at midday and also during the first phase of the night were observed; the same for oxytocin in the middle of the dark period was noted. The high increase of vasopressin and oxytocin plasma levels at the beginning of the day has been observed; the minimum for both vasopressin and oxytocin plasma concentrations during the dark period (24:00–01:00) has been observed. In the hypothalamus, thyroliberin impaired the basic rhythm of vasopressin as well as oxytocin release over the light hours and intensified distinctly this process during the night hours (i.e. a decrease of hypothalamic neurohormones content during the light period and its increase over the night hours). It is supposed that thyroliberin may considerably influence the rhythm of the vasopressin and oxytocin release from the hypothalamo-neurorohypophysial system.

N-methyl-D-aspartic acid stimulation of vasopressin release: role in osmotic regulation and modulation by gonadal steroids.

Previous experiments demonstrated that excitatory amino acids participate in the osmotic regulation of vasopressin secretion, but the specific involvement of N-methyl-D-aspartic acid (NMDA) receptors was not evaluated. This was demonstrated in the present studies. NMDA stimulated vasopressin release from perifused explants of the hypothalamo-neurohypophyseal system (HNS), and osmotic stimulation of vasopressin release was inhibited by MK-801 (10 microM) and AP5 (100 microM) NMDA receptor antagonists. The effective concentration of NMDA was dependent upon the Mg2+ concentration of the perifusate with stimulation observed at 1 microM NMDA in Mg2+-replete compared with 5 microM in low-Mg2+ medium. Previous experiments also demonstrated that estradiol and dihydrotestosterone (DHT) inhibited osmotically stimulated vasopressin secretion, and a nongenomic mechanism of action was suggested by the ability of steroids conjugated to bovine serum albumin to replicate the effect. Experiments were performed to explore the potential role of NMDA receptors in this mechanism. Estradiol (50 pg/ml) and DHT (3 ng/ml) inhibited NMDA stimulated vasopressin release in perifused HNS explants. These results suggest a role of NMDA receptors in the mediation of vasopressin secretion in osmotically stimulated release. Furthermore, estradiol and DHT may exert their inhibitory effect on osmotically stimulated vasopressin release via the NMDA receptor.

Regulated Expression of the Cell Adhesion Glycoprotein F3 in Adult Hypothalamic Magnocellular Neurons

F3, a glycoprotein of the immunoglobulin superfamily implicated in axonal growth, occurs in oxytocin (OT)-secreting and vasopressin (AVP)-secreting neurons of the adult hypothalamo-neurohypophysial system (HNS) whose axons undergo morphological changes in response to stimulation. Immunocytochemistry and immunoblot analysis showed that during basal conditions of HNS secretion, there are higher levels of this glycosylphosphatidyl inositol-anchored protein in the neurohypophysis, where their axons terminate, than in the hypothalamic nuclei containing their somata. Physiological stimulation (lactation, osmotic challenge) reversed this pattern and resulted in upregulation of F3 expression, paralleling that of OT and AVP under these conditions. In situ hybridization revealed that F3 expression in the hypothalamus is restricted to its magnocellular neurons and demonstrated a more than threefold increase in F3 mRNA levels in response to stimulation. Confocal and electron microscopy localized F3 in secretory granules in all neuronal compartments, a localization confirmed by detection of F3 immunoreactivity in granule-enriched fractions obtained by sucrose density gradient fractionation of rat neurohypophyses. F3 was not visible on any cell surface in the magnocellular nuclei. In contrast, in the neurohypophysis, it was present not only in secretory granules but also on the surface of axon terminals and glia and in extracellular spaces. Taken together, our observations reveal that the cell adhesion glycoprotein F3 is colocalized with neurohypophysial peptides in secretory granules. It follows, therefore, the regulated pathway of secretion in HNS neurons to be released by exocytosis at their axon terminals in the neurohypophysis, where it may intervene in activity-dependent structural axonal plasticity.

Increased basal activity of the hypothalamo-pituitary-adrenal axis during pregnancy in rats bred for high anxiety-related behaviour.

In order to test the hypothesis that prenatal hormones influence the emotional maturation of the offspring, the hypothalamo-pituitary-adrenal (HPA) axis activity was studied at the end of pregnancy in two rat breeding lines differing consistently in their innate anxiety-related behaviour in the elevated plus-maze. Virgin and pregnant rats were fitted with a chronic jugular vein catheter and tested 5 days later. The high basal level of anxiety-related behaviour (HAB) described in males and females of the HAB breeding line persists in pregnancy as indicated by a significantly reduced number of entries into and time spent on the open arms of the elevated plus-maze between days 18 and 20 of pregnancy compared with pregnant rats of the breeding line with low anxiety-related behaviour (LAB). In general, an increase in anxiety was found in both breeding lines in pregnancy compared with the respective virgin controls. With respect to HPA axis activity, increased basal levels of adrenocorticotropin (ACTH) and corticosterone have been found in pregnant rats of the HAB line compared with pregnant LAB rats. ACTH and corticosterone secretion in response to emotional and complex physical stressors (exposure to the elevated plus-maze and forced swimming, respectively) did not differ between virgin and pregnant rats of either breeding line. However, independent of the inborn emotionality of the animals, a general attenuation in the HPA axis response to stressors and to exogenous CRH could be confirmed in pregnant rats. The basal and stress-induced activity of the hypothalamo-neurohypophysial system secreting oxytocin and vasopressin was also tested, and no differences were found relating to the emotionality or reproductive state of the animals except for a reduced vasopressin secretion in pregnant HAB rats after forced swimming. The elevated basal activity of the HPA axis, including enhanced circulating concentrations of corticosterone in pregnant HAB rats, may influence both the neuroendocrine and emotional development of their offspring. Thus, the passing-on of maternal behavioural characteristics via prenatal, hormonal 'imprinting' has to be considered as a possible contribution to emotional maturation during an individual's development.

Attenuated neuroendocrine responses to emotional and physical stressors in pregnant rats involve adenohypophysial changes.

1. The responsiveness of the rat hypothalamo-pituitary-adrenal (HPA) axis and hypothalamo-neurohypophysial system (HNS) to emotional (elevated plus-maze) and physical (forced swimming) stressors and to administration of synthetic corticotrophin-releasing hormone (CRH) was investigated during pregnancy and lactation. In addition to pregnancy-related adaptations at the adenohypophysial level, behavioural responses accompanying the neuroendocrine changes were studied. 2. Whereas basal (a.m.) plasma corticosterone, but not corticotrophin (adrenocorticotrophic hormone; ACTH), levels were increased on the last day (i.e. on day 22) of pregnancy, the stress-induced rise in both plasma hormone concentrations was increasingly attenuated with the progression of pregnancy beginning on day 15 and reaching a minimum on day 21 compared with virgin control rats. A similar attenuation of responses to both emotional and physical stressors was found in lactating rats. 3. Although the basal plasma oxytocin concentration was elevated in late pregnancy, the stress-induced rise in oxytocin secretion was slightly lower in day 21 pregnant rats. In contrast to vasopressin, oxytocin secretion was increased by forced swimming in virgin and early pregnant rats indicating a differential stress response of these neurohypophysial hormones. 4. The blunted HPA response to stressful stimuli is partly due to alterations at the level of corticotrophs in the adenohypophysis, as ACTH secretion in response to CRH in vivo (40 ng kg-1, i.v.) was reduced with the progression of pregnancy and during lactation. In vitro measurement of cAMP levels in pituitary segments demonstrated reduced basal levels of cAMP and a lower increase after CRH stimulation (10 nM, 10 min) in day 21 pregnant compared with virgin rats, further indicating reduced corticotroph responsiveness to CRH in pregnancy. 5. The reduced pituitary response to CRH in late pregnancy is likely to be a consequence of a reduction in CRH receptor binding as revealed by receptor autoradiography. [125I] CRH binding in the anterior pituitary was significantly reduced in day 11, 17 and 22 pregnant rats compared with virgin controls. 6. Anxiety-related behaviour of the animals as revealed by the time on and entries into the open arms of the elevated plus-maze was different between virgin and pregnant rats with decreased number of entries indicating increased anxiety with the progression of pregnancy (except on pregnancy day 18). The emotional behaviour, however, was not correlated with the neuroendocrine responses. 7. The results indicate that the reduced response of the HPA axis to stressors described previously during lactation is already manifested around day 15 of pregnancy in the rat and involves physiological adaptations at the adenohypophysial level. However, alterations in stressor perception at higher brain levels with the progression of pregnancy may also be involved.

Effects of L-NAME on cerebral metabolic, vasopressin, oxytocin, and blood pressure responses in hemorrhaged rats.

NG-nitro-L-arginine methyl ester (L-NAME; 250 micrograms/5 microliters), an inhibitor of NO synthase, or the vehicle artificial cerebrospinal fluid (aCSF; 5 microliters) was administered intracerebroventricularly to conscious rats hemorrhaged (0.7 ml/min) to a 20% volume depletion. Hypotension was maximal 5 min after hemorrhage ended, with compensatory recovery to basal levels 20 min later, regardless of drug treatment. L-NAME, however, elevated (P < 0.05) blood pressure (vs. aCSF controls) 40-45 min after intracerebroventricular administration. In normovolemic rats, L-NAME produced a significant pressor response and increased plasma levels of vasopressin (VP) and oxytocin (OT). After hemorrhage, both hormone levels increased, but only OT was further enhanced by L-NAME. Thus centrally produced NO tonically inhibits OT and VP secretion under basal normovolemic conditions and selectively inhibits OT release during hypovolemia. Hemorrhage increased the rates of glucose utilization in the neural lobe, indicative of enhanced efferent neural functional activity. L-NAME further enhanced the metabolic activity in the entire hypothalamoneurohypophysial system of hemorrhaged animals. Several other brain structures involved in the regulation of blood pressure and the stress response were also metabolically affected by the hemorrhage and L-NAME.

Evidence for nitric oxide (NO) actions throughout the forebrain osmoresponsive circuit.

Alterations in neuronal nitric oxide synthase (nNOS) mRNA and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining within the hypothalamo-neurohypophysial system (HNS) during dehydration2,6,7,10 or lactation1,3,11 predict a regulatory role for NO. Available data suggests an inhibitory action of NO on neurohormone release2,4,5,8,12 though it may have both positive and negative actions at different levels of the HNS. We have looked for evidence for NO interactions between the HNS and its afferent inputs by measuring activity of nNOS containing neurones in the lamina terminalis during hormone demand.