Stress-Induced Oxytocin Release in the Rat After Lesion of the Paraventricular Nuclei; Possible Deficiency of Corticotrophin-Releasing Factor

Oxytocin is released under stressful conditions and corticotrophin-releasing factor (CRF) is known to be involved in mediating general ‘stress responses’. We therefore examined whether CRF neurons in the paraventricular nucleus participate in the stress-induced oxytocin release in the rat. CRF (0.02 to 2 nmol) injected into the third ventricle produced a dose-dependent rise in the plasma oxytocin concentration. The oxytocin release induced by CRF occurred without a change in blood pressure, and was not affected by dexamethasone pretreatment, which prevents adrenocorticotrophin release following CRF injection. Lesioning of the paraventricular nucleus reduced oxytocin release by immobilization stress, but did not alter the release of oxytocin in response to osmotic stimulation induced by intraperitoneal injection of hypertonic saline. Anti-CRF serum injection into the third ventricle reduced delayed oxytocin response to immobilization stress. These results are consistent with the hypothesis that CRF neurons in the paraventricular nucleus are involved in the oxytocin release during immobilization stress in the rat.     

Stress-Specific Regulation of Corticotropin Releasing Hormone Receptor Expression in the Paraventricular and Supraoptic Nuclei of the Hypothalamus in the Rat

Corticotropin releasing hormone (CRH), a major regulator of pituitary ACTH secretion, also acts as a neurotransmitter in the brain. To determine whether CRH is involved in the regulation of hypothalamic function during stress, CRH receptor binding and CRH receptor mRNA levels were studied in the hypothalamus of rats subjected to different stress paradigms: immobilization, a physical-psychological model; water deprivation and 2% saline intake, osmotic models; and i.p. hypertonic saline injection, a combined physical-psychological and osmotic model. In agreement with the distribution of CRH receptor binding in the brain, in situ hybridization studies using ³⁵S-labeled cRNA probes revealed low levels of CRH receptor mRNA in the anterior hypothalamic area, which were unaffected after acute or chronic exposure to any of the stress paradigms used. Under basal conditions, there was no CRH binding or CRH receptor mRNA in the supraoptic (SON) or paraventricular (PVN) nuclei. However, 2 h after the initiation of acute immobilization, CRH receptor mRNA hybridization became evident in the parvicellular division of the PVN, with levels substantially increasing from 2 to 4 h, decreasing at 8 h and disappearing by 24 h. Identical hybridization patterns of CRH receptor mRNA were found in the parvicellular PVN after repeated immobilization; levels were similar to those after 2 h single stress following immobilization at 8-hourly intervals for 24 h (3 times), and very low, but clearly detectable 24 h after 8 or 14 days daily immobilization for 2 h. On the other hand, water deprivation for 24 or 60 h and intake of 2% NaCI for 12 days induced expression of CRH receptor mRNA in the SON and magnocellular PVN, but not in the parvicellular pars of the PVN. Both parvicellular and magnocellular hypothalamic areas showed CRH receptor mRNA following i.p. hypertonic saline injection, single (4 h after) or repeated at 8-hourly intervals for 24 h (3 injections), or one injection daily for 8 or 14 days. Consistent with the expression of CRH receptor mRNA, autoradiographic studies showed binding of ¹²⁵I-Tyr-oCRH in the parvicellular division of the PVN after immobilization; in the magnocellular division of the PVN after osmotic stimulation, and in the PVN and SON after i.p. hypertonic saline injection. The data show that stress-specific activation of the parvicellular and magnocellular systems is associated with CRH receptor expression, and suggest a role for CRH in the autoregulation of hypothalamic function.     

High Voltage-Activated Ca2+ Currents in Rat Supraoptic Neurones: Biophysical Properties and Expression of the Various Channel α1 Subunits

The diversity of Ca2+ currents was studied in voltage-clamped acutely dissociated neurones from the rat supraoptic nucleus (SON), and the expression of the various corresponding pore-forming alpha1 subunits determined by immunohistochemistry. We observed the presence of all high voltage-activated L-, N-, P/Q- and R-type currents. We did not observe low-voltage-activated T-type current. The multimodal current/voltage relationships of L- and R-type currents indicated further heterogeneity within these current types, each exhibiting two components that differed by a high (-20 mV) and a lower (-40 mV) threshold potential of activation. L- and R-type currents were fast activating and showed time-dependent inactivation, conversely to N- and P/Q-type currents, which activated more slowly and did not inactivate. The immunocytochemical staining indicated that the soma and proximal dendrites of SON neurones were immunoreactive for Cav1.2, Cav1.3 (forming L-type channels), Cav2.1 (P/Q-type), Cav2.2 (N-type) and Cav2.3 subunits (R-type). Each subunit exhibited further specificity in its distribution throughout the nucleus, and we particularly observed strong immunostaining of Cav1.3 and Cav2.3 subunits within the dendritic zone of the SON. These data show a high heterogeneity of Ca2+ channels in SON. neurones, both in their functional properties and cellular distribution. The lower threshold and rapidly activating L- and R-type currents should underlie major Ca2+ entry during action potentials, while the slower and higher threshold N- and P/Q-type currents should be preferentially recruited during burst activity. It will be of key interest to determine their respective role in the numerous Ca2+-dependent events that control the activity and physiology of SON neurones     

κ-Opioid Receptor Activation Inhibits Post-Spike Depolarizing After-Potentials in Rat Supraoptic Nucleus Neurones In Vitro

Endogenous agonists acting at kappa-opioid receptors modulate the discharge activity of hypothalamic supraoptic nucleus vasopressin cells in vivo. Phasic activity in vasopressin cells is known to depend critically on intrinsic mechanisms involving post-spike depolarizing after-potentials and we hypothesized that inhibition of phasic bursting by an endogenous kappa-agonist may result from reducing the magnitude of depolarizing after-potentials. To investigate this possibility, intracellular sharp electrode recordings were obtained from supraoptic nucleus cells impaled in superfused explants of rat hypothalamus. Bath application of the selective kappa-agonist, U50,488H (0.1-1 microM), decreased the spontaneous firing rate of magnocellular neurosecretory cells (by 94. 0+/-4.5% at 1 microM, mean+/-SEM; P = 0.02, n = 4). U50,488H did not alter membrane potential (0.9+/-0.8 mV hyperpolarization at 1 microM, P = 0.17, n = 8) or input resistance (11.0+/-4.5% increase at 1 microM, P = 0.09, n = 5). U50,488H (0.1 and 1 microM, both n = 5) reduced depolarizing after-potential amplitude (by 29.9+/-9.3 and 78.0+/-10. 6%, respectively, P<0.001) in eight cells in which the baseline membrane potential was kept constant by dc-current injection and in which a depolarizing after-potential was evoked every 25-40 s by a brief (40-80 ms) train of 3-6 action potentials (the number of spikes in the trains was kept constant for each cell). Thus, kappa-opioid receptor activation reduces depolarizing after-potential amplitude in supraoptic nucleus cells and this may underlie the reduction in burst duration of vasopressin cells caused by an endogenous kappa-agonist in vivo.     

Response of Arginine Vasopressin-Enhanced Green Fluorescent Protein Fusion Gene in the Hypothalamus of Adjuvant-Induced Arthritic Rats

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.     

Estrogen-Sensitive Oxytocin Binding Sites are Differently Regulated by Progesterone in the Telencephalon and the Hypothalamus of the Rat

The localization at the cellular level and the regulation by progesterone of the estrogen-sensitive oxytocin binding sites was studied in the rat telencephalon and the hypothalamus by using quantitative film-autoradiography and histoautoradiography. Male rats (castrated or not) and ovariectomized females (estradiol supplemented or not) were used to characterize these sites and to precise their localization. They were detected in the striatal cell bridges, the olfactory tubercle, the principal nucleus of the bed nucleus of the stria terminalis and the medial nucleus of the amygdala of the telencephalon and in the medial preoptic, the ventromedial and the ventral premammillary nuclei of the hypothalamus. Estrogen administration in addition induced expression of oxytocin binding sites in the major island of Calleja, the anterior hypothalamic area and the terete nucleus. The density of the estrogen-sensitive oxytocin binding sites varied during the estrous cycle, but differently in the telencephalon and the hypothalamus. in the telencephalon it peaked at proestrus 9 h and was already decreased at proestrus 21 h, whereas in the hypothalamus it was similarly high at proestrus 9h and proestrus 21 h, suggesting the intervention of progesterone in the regulation of the hypothalamic estrogen-sensitive oxytocin binding sites.     

A Role for Vasopressin in the Stress-Induced Inhibition of Gonadotrophin Secretion: Studies in the Brattleboro Rat

The effects of stress on the secretion of adrenocorticotrophin, corticosterone and luteinizing hormone (LH) in rats congenially lacking hypothalamic vasopressin (Brattleboro rats) and in normal controls of the parent strain (Long Evans) have been compared in an attempt to examine the role of vasopressin in the stress-induced depression of gonadotrophin secretion. In the Long Evans rats, stress (0.6 mg/100g histamine, ip) initiated, within 5 and 20 min respectively, significant (P <0.01, Student's t-test) increases in the plasma adrenocorticotrophin and corticosterone concentrations. It also caused a reduction in the serum LH concentration which was maximal at 5 min. By contrast, in the vasopressin deficient Brattleboro rats, stress had no effect on the serum LH concentration and produced only modest increases in pituitary adrenocortical activity compared with those in Long Evans controls. Pretreatment of both Long Evans and Brattleboro rats with dexamethasone (20μg/100 g ip, daily for 3 days) effectively abolished the pituitary-adrenal response to stress. The steroid treatment also prevented the stress-induced suppression of LH in the Long Evans rats; indeed, these animals, unlike the vehicle-treated controls, exhibited a rise in serum LH concentration within 5 min of exposure to stress. Stress did not affect the serum LH concentrations in steroid-treated Brattleboro rats. The results confirm previous reports that vasopressin is required for the full expression of the pituitary-adrenocortical response stress. They also provide novel evidence which suggests that vasopressin released in stress contributes to the impairment of gonadotrophin secretion.     

Osmotic Modulation in Glutamatergic Excitatory Synaptic Inputs to Neurons in the Supraoptic Nucleus of Rat Hypothalamus in vitro

To clarify influence of osmotic stimulation on the excitatory synaptic inputs to the neurosecretory cells of the supraoptic nucleus (SON), the blind patch technique was used in rat hypothalamic slice preparations. Stable whole-cell recordings were made from 22 neurons in the SON. To observe spontaneous excitatory postsynaptic currents (sEPSCs) in the SON neurons, membrane potentials were clamped between −50 and −90  mV. The effects of hypertonic stimulation on the frequency of the sEPSCs were tested in 18 SON neurons. Bath application of mannitol 30 or 60  mM increased the frequency of the sEPSCs. During the application of mannitol (60  mM), the frequency of the sEPSCs increased in 12 of 15 neurons without a change in amplitude. Hypertonic stimulation with NaCl (30  mM) had similar effects to that of mannitol. The increased frequency of miniature EPSCs (mEPSCs) during mannitol application persisted in the presence of TTX in all 8 SON neurons tested with no change in amplitude. Both the non-NMDA antagonist CNQX at 10–30  μM (n=6) and the non-selective glutamate antagonist kynurenic acid at 1  mM (n=3) almost completely blocked the EPSCs while the NMDA antagonist AP-5 at 10  μM had no effect on the frequency of the EPSCs in the 4 neurons tested. During application of CNQX, mannitol (60  mM) was added to the perfusion medium in 3 SON neurons. Under these conditions, mannitol had no effect on the frequency of EPSCs. We conclude that hypertonic stimulation directly influences glutamatergic inputs to the neurosecretory cells of the SON by an action on the presynaptic terminals and enhances the excitatory synaptic events.     

The Effect of Pinealectomy on Osmotically Stimulated Vasopressin and Oxytocin Release and Fos Protein Production within the Hypothalamus of the Rat

Osmotically stimulated vasopressin and oxytocin release were measured in pinealectomized and sham operated male rats infused with hypertonic sodium chloride. Neuronal activation in the hypothalamic regions associated with oxytocin and vasopressin release was investigated by quantitative assessment of Fos protein production. The osmotically stimulated release of both vasopressin and oxytocin was significantly lower in pinealectomized animals as compared to sham operated controls. The slope of regression lines between plasma osmolality and hormone concentrations in the sham animals showed a 1.0±0.1  pmol per mosm/kg rise in vasopressin and 2.0±0.4  pmol per mosm/kg rise in oxytocin whilst in the pinealectomized animals these values were significantly lower at 0.4±0.1  pmol vasopressin per mosm/kg and 0.8±0.2pmol oxytocin per mosm/kg. The osmotic thresholds for hormone release were unaffected by pinealectomy. Fos production was also significantly lower in the supraoptic nucleus and organ vasculosum of the lamina terminalis in the pinealectomized rat at 62±20 and 59±9 Fos immunoreactive cells/section as compared to corresponding values of 202±31 and 123±20 Fos immunoreactive cells/section in the shams. These observations suggest that reduced hormone release in the pinealectomized animal is due to lowered responsiveness of central osmoregulatory mechanisms and that melatonin may therefore influence the activation of the magnocellular system.     

Role of Anterior Peri-Third Ventricular Structures in the Regulation of Supraoptic Neuronal Activity and Neurohypophysical Hormone Secretion in the Rat

Neurohypophysical hormone release, and the electrical activity of single neurons of the supraoptic nucleus, were monitored in urethane-anaesthetized rats. Immediately after electrolytic lesions of the region anterior and ventral to the third ventricle (AV3V region), supraoptic neurons showed little spontaneous activity and their responses to ip injection of hypertonic saline were severely impaired; corresponding deficits were found in the secretion of both oxytocin and vasopressin. Similar deficits in oxytocin secretion were also found in rats following electrolytic lesions which destroyed all or part of the subfornical organ; however the effects of the lesions were not additive: rats with lesions of both the AV3V region and the subfornical organ region showed a similar degree of impairment of osmotically stimulated oxytocin secretion to rats with lesions of either site alone. Such deficits might occur either as a result of destruction of osmoresponsive projections to the magnocellular nuclei, or as a result of destruction of an afferent input which is essential for the full expression of the innate osmosensitivity of supraoptic neurons. To test the latter possibility, supraoptic neurons in AV3V-lesioned rats were activated by continuous application of glutamate, and then tested with ip injection of hypertonic saline. Five of seven cells tested responded significantly to the hyperosmotic stimulus, though the responses were significantly weaker than observed in sham-lesioned rats. We suggest that the innate osmosensitivity of supraoptic neurons does contribute to their responses to systemic osmotic stimulation, but that expression of this innate osmosensitivity requires inputs from the AV3V region and/or the subfornical organ, some of which may also be osmoresponsive. Electrical stimulus pulses applied to the AV3V region influenced the electrical activity of most supraoptic neurons strongly: the predominant response was a short-latency, short-duration inhibition followed by long-latency, long-duration excitation. Whereas intracerebroventricular administration of the angiotensin II antagonist saralasin reduced spontaneous or osmotically induced activity of supraoptic neurons, the neuronal responses to AV3V stimulation were impaired only with relatively high doses of saralasin. We conclude that angiotensin ll-sensitive neurons are an important component of the afferent pathways that sustain the excitability of supraoptic neurons, but that angiotensin is probably not the major transmitter of the projection from the AV3V region to the supraoptic nucleus.     

Effects of Novelty Stress on Vasopressin and Oxytocin Secretion by the Pituitary in the Rat

Effects of novel environmental stimuli on vasopressin and oxytocin secretion by the pituitary were studied in dehydrated male rats. As the novel environmental stimuli, rats were transferred to an experimental room, placed in a box painted black and given a pure tone auditory stimulus of 2 kHz. Exposure of rats to the novel environmental stimuli for a period of 2 min decreased plasma concentrations of vasopressin and increased plasma levels of adrenocorticotrophic hormone (ACTH) and prolactin, but did not significantly change the plasma level of oxytocin. The stimuli, however, became ineffective for producing the suppressive vasopressin response as the period of exposure was prolonged to more than 5 and up to 30 min, although the prolonged stimuli were still effective for inducing facilitatory ACTH and prolactin responses. After repeated exposures of rats to the environmental stimuli once a day for 5 or 10 days, the stimuli became disabled from producing the suppressive vasopressin response. However, the rats were still capable of responding to the novel stimuli of another kind. All these data suggest that novelty stress suppresses vasopressin secretion but does not change oxytocin secretion. In order to test the possibility that glucocorticoids expectedly secreted by the adrenals in response to the stress might have suppressed vasopressin secretion, a large amount of dexamethasone was administered to the rat before testing. Dexamethasone pretreatment depressed plasma levels of ACTH and vasopressin as reported previously and blocked the facilitatory ACTH response to the novelty stress. However, dexamethasone treatment did not affect the suppressive vasopressin response to the novelty stress. Thus, it is likely that the suppressive vasopressin response to novelty stress does not primarily depend upon endogenous glucocorticoids.     

Inward Rectification (Ih) in Immunocytochemically-ldentified Vasopressin and Oxytocin Neurons of Guinea-Pig Supraoptic Nucleus

Intracellular recordings of magnocellular neurons from the supraoptic nucleus of guinea-pigs were made with KCI/K citrate- and biocytin-filled electrodes. Fifty of 99 cells exhibited a time-dependent inward rectification (TDR). The TDR was activated during hyperpolarizing current pulses to membrane potentials more hyperpolarized than −75 mV. In voltage-clamp recordings, an inward current appeared at voltage steps more hyperpolarized than −75 mV, with properties similar to the slow inward rectifier (I_h) described in other tissues. The I_h was blocked by 2 mM CsCI. BaCI₂ (100 to 500 μM) did not block the I_h. Immunocytochemical identification of the recorded cells revealed that both vasopressin (AVP)- and oxytocin (OT)- containing neurons exhibited an I_h.     

Noradrenaline, Dopamine and Serotonin Release in the Paraventricular and Supraoptic Nuclei of the Rat in Response to Intravenous Cholecystokinin Injections

Microdialysis sampling was used to measure noradrenaline, dopamine and serotonin release in the supraoptic and paraventricular nuclei of urethane-anaesthetized rats following intravenous injection of 20μg/kg cholecystokinin. This dose of cholecystokinin stimulates oxytocin release from the posterior pituitary, while slightly inhibiting vasopressin release. Dialysis probes were placed in the paraventricular nucleus, and into dorsal or ventral regions of the supraoptic nucleus. Samples were collected at 10-min intervals in each animal before, during and after two injections of cholecystokinin, and following a control injection of 0.9% NaCI. The injections of cholecystokinin stimulated significant increases in the concentrations of noradrenaline, dopamine and serotonin in the paraventricular nucleus and of noradrenaline and serotonin in the dorsal supraoptic nucleus region. Conversely, in the ventral supraoptic nucleus region a significant reduction in noradrenaline release was observed, but dopamine and serotonin concentrations were not significantly affected. The control injections did not alter noradrenaline, dopamine or serotonin release.     

Stress-Induced Suppression of Pulsatile Luteinising Hormone Release in the Female Rat: Role of Vasopressin

Insulin-induced hypoglycaemic (IIH) stress evokes the release of arginine vasopressin (AVP) and suppresses luteinising hormone (LH) pulses in a number of species, a phenomenon augmented by the presence of oestradiol (E2). The aim of this study was to test the hypothesis that AVP not only disrupts pulsatile LH secretion in the female rat, but specifically mediates the effect of IIH stress on suppressing LH release. The role of E2 in augmenting the disruptive effect of AVP on LH secretion was also addressed. Rats were ovariectomized (OVX) and fitted with intracerebroventricular (i.c.v. ) and intravenous (i.v.) cannulae. For experiments requiring comparisons of neuroendocrine responses in the presence and absence of E2, animals were implanted subcutaneously with E2 or oil-filled capsules respectively. AVP (5 microg) administered via the i.c.v. cannula suppressed LH secretion by decreasing LH pulse amplitude without affecting LH pulse frequency, an effect that was blocked by central administration of an AVP antagonist (25 microg). This inhibitory response was evident only in E2-replaced OVX rats, thus suggesting a sensitizing influence of the gonadal steroid. In the AVP-deficient Brattleboro rats, IIH stress did not interrupt pulsatile LH secretion as demonstrated in Long Evans and Wistar controls. While these data might suggest a pivotal role for AVP in stress-induced suppression of LH release, central administration of an AVP antagonist did not prevent the interruption of LH pulses in response to IIH stress. Furthermore, it would appear that AVP is not primarily involved in hypoglycaemic stress-induced suppression of pulsatile LH secretion since central administration of very high doses of AVP resulted in a suppression of LH pulse amplitude and not frequency, while hypoglycaemic stress caused an interruption of LH pulses.     

Dissociation of ACTH-Secretory Mechanisms in Rat Pituitary Cells: Evidence that Basal and Vasopressin-Stimulated Secretion Act via a Mechanism Distinct from that of Corticotrophin-Releasing Factor

To study the relationship between basal, corticotrophin-releasing factor- (CRF) and vasopressin-stimulated adrenocorticotrophic hormone (ACTH) secretion by rat anterior pituitary cells, dissociated anterior pituitary cells were seeded into tissue culture dishes and treated overnight with a cytotoxic conjugate specific for CRF-target cells. Immediately after extensive washing, or 1, 3, 6, 9 or 12 days later, cellular ACTH content, basal secretion and secretion in response to CRF or vasopressin were measured. ACTH content and basal secretion rate increased over time in both cytotoxic conjugate-pretreated and vehicle-pretreated cell populations. Compared with vehicle-pretreated cells, basal ACTH secretion was higher in cytotoxic conjugate-pretreated populations by Day 3 and reached an apparent maximum by Day 6. In such cells, net ACTH secretion post-vasopressin decreased as basal secretion increased; by Day 6 no vasopressin-stimulated secretion was seen. In cytotoxic conjugate-pretreated cells, the response to CRF was initially completely eliminated; however, as ACTH content and secretion increased with time, a small recovery of the response to CRF was observed on Days 3 and 6. In vehicle-pretreated cells, ACTH secretion in response to vasopressin increased in parallel with basal secretion. The response to CRF increased progressively over Days 1 to 6 as well; this response was more closely related to the increases observed in ACTH content. The shift in responsiveness of the cytotoxic conjugate-pretreated cells over time, from vasopressin-responsive to CRF-responsive, further demonstrates the dissociation of the mechanisms of the ACTH secretory responses to CRF and vasopressin. In addition, the increase in unstimulated secretion at the expense of the response to vasopressin in cytotoxic conjugate-treated cells is consistent with a common pathway for vasopressin-stimulated and basal release of ACTH.     

Sequential Exposure to Estrogen and Testosterone (T) and Subsequent Withdrawal of T Increases the Level of Arginine Vasopressin Messenger Ribonucleic Acid in the Hypothalamic Paraventricular Nucleus of the Female Rat

The hypothalamic peptides arginine vasopressin (AVP) and oxytocin (OT) have been implicated as mediators of socio-sexual behaviors in addition to their roles in osmolar homeostasis (AVP), milk ejection and uterine contractility (OT). Within 24  h of parturition, OT and AVP messenger ribonucleic acid (mRNA) levels increase in the hypothalamic paraventricular, and to a lesser degree, the supraoptic nucleus (PVN and SON) of the rat. We previously reported that the prepartum increase in OT mRNA is related to the spontaneous decline in progesterone levels prior to parturition. We also reported that increases in PVN and SON OT mRNA can be induced by exposing the ovariectomized rat to a steroid regimen that mimics the steroid milieu of pregnancy, namely sequential estrogen and progesterone and subsequent progesterone withdrawal. Levels of PVN and SON AVP mRNAs were not affected by progesterone withdrawal in late pregnant rats or the steroid regimen that increased OT mRNA in ovariectomized rats. These observations suggest that other factors, perhaps hormonal, may influence AVP mRNA levels. A decline in testosterone coincident with waning progesterone levels also occurs prepartum. Since peak levels of AVP mRNA prepartum coincide with the prepartum decline in testosterone, we questioned whether declining testosterone levels are important for the increase in AVP mRNA levels.