Maternal neglect with reduced depressive-like behavior and blunted c-fos activation in Brattleboro mothers, the role of central vasopressin.

Early mother-infant relationships exert important long-term effects in offspring and are disturbed by factors such as postpartum depression. We aimed to clarify if lack of vasopressin influences maternal behavior paralleled by the development of a depressive-like phenotype. We compared vasopressin-deficient Brattleboro mothers with heterozygous and homozygous normal ones. The following parameters were measured: maternal behavior (undisturbed and separation-induced); anxiety by the elevated plus maze; sucrose and saccharin preference and forced swim behavior. Underlying brain areas were examined by c-fos immunocytochemistry among rest and after swim-stress. In another group of rats, vasopressin 2 receptor agonist was used peripherally to exclude secondary changes due to diabetes insipidus. Results showed that vasopressin-deficient rats spend less time licking-grooming their pups through a centrally driven mechanism. There was no difference between genotypes during the pup retrieval test. Vasopressin-deficient mothers tended to explore more the open arms of the plus maze, showed more preference for sucrose and saccharin and struggled more in the forced swim test, suggesting that they act as less depressive. Under basal conditions, vasopressin-deficient mothers had more c-fos expression in the medial preoptic area, shell of nucleus accumbens, paraventricular nucleus of the hypothalamus and amygdala, but not in other structures. In these areas the swim-stress-induced activation was smaller. In conclusion, vasopressin-deficiency resulted in maternal neglect due to a central effect and was protective against depressive-like behavior probably as a consequence of reduced activation of some stress-related brain structures. The conflicting behavioral data underscores the need for more sex specific studies.

Further neurochemical and behavioural investigation of Brattleboro rats as a putative model of schizophrenia.

Brattleboro (BRAT) rats are a mutant variant of the Long-Evans (LE) strain deficient in the neurohormone vasopressin. BRAT rats show behavioural alterations relevant to schizophrenia. In particular, BRAT rats show deficits in prepulse inhibition (PPI) and alterations in various measures of cognition. The aim of this study was to replicate the reported PPI deficits in BRAT rats and its reversal by antipsychotic drugs and to investigate other behavioural and neurochemical characteristics. Acoustic startle reactivity, PPI, spontaneous and amphetamine-induced locomotor activity (LMA) and ex-vivo steady state neurochemistry were measured in male homozygous BRAT rats and LE rats. The effects of antipsychotics on PPI deficits were also determined. Relative to LE, BRAT rats showed enhanced startle reactivity, hyperactivity to a novel environment, PPI deficits and decreased levels of dopamine and DOPAC (dihydroxyphenylacetic acid) in the frontal cortex. BRAT and LE rats showed similar levels of hyperactivity following amphetamine (0.26 mg/kg s.c.). PPI deficits were attenuated by acute clozapine (5-10 mg/kg s.c.), risperidone (0.1-1 mg/kg i.p.), haloperidol (0.1-0.5 mg/kg p.o.) and less robustly by olanzapine (0.3-3 mg/kg s.c.). Chronic administration of clozapine (5 mg/kg s.c., once daily) attenuated baseline hyperactivity and elevated PPI of both strains. Clozapine concentrations were higher in BRAT brains compared with LE rats. These data confirm the reported PPI deficit in BRAT rats and its reversal by antipsychotic drugs, suggesting BRAT rats may represent a potential model for identifying novel antipsychotic drugs.

Activity of oxytocinergic neurons in the supraoptic nucleus under stimulation of alpha2-adrenoceptors in Brattleboro rats.

Vasopressin (AVP) deficient homozygous Brattleboro rats exhibit severe osmotic challenges due to waterless chronic hypernatremia and hyperosmolality. We investigated the effect of xylazine, an alpha2-adrenoceptor agonist, on the activity of oxytocinergic (OXY) neurons in the supraoptic nucleus (SON) of homozygous (di/di), heterozygous (di/+), and control (+/+) rats. Ninety minutes after saline (0.1 mL/100 g b.w., i.p.) or xylazine injection (10 mg/kg, i.p.) rats were anaesthetized with pentobarbital (50 mg/kg i.p.) and sacrificed by transcardial perfusion with fixative. Activity of OXY neurons was evidenced by nuclear Fos protein immunoreactivity. Fos/OXY colabelings were analyzed on 40-mum thick coronal sections using computerized light microscope. As expected, plasma osmolality and water intake revealed high heterogenity within the di/di group of rats. Fos expression in SON of di/di rats was correlated with osmolality of each rat. In saline-treated rats, maximum activation of Fos reached around 4% in +/+, 20% in di/+ rats, and as much as 60% in di/di rats. Xylazine activated in SON about 70% of OXY-ergic neurons in +/+, 60% in di/+ rats, and more than 80% in di/di rats. The present findings indicate that in spite of the high spontaneous activity of SON OXY-ergic neurons due to the AVP deficiency in di/di rats, many of the silent OXY-ergic neurons in the SON remained acceptable for alpha2-adrenoceptor stimulation.

Gender-specific regulation of the hypothalamo-pituitary-adrenal axis and the role of vasopressin during the neonatal period.

Studies in arginine vasopressin (AVP)-deficient Brattleboro rats suggest that AVP is the predominant secretagogue during the perinatal period. Here we tested the hypothesis that congenital lack of vasopressin differentially modifies the stress reactivity of male and female rat pups. Vasopressin-producing (heterozygous, AVP+) and AVP-deficient (AVP-) Brattleboro rat pups of both genders were used. In 10-day-old pups, 24-h maternal separation and single, as well as repeated, ether inhalation induced remarkable adrenocorticotropin (ACTH) elevation only in AVP+ pups, supporting the role of vasopressin in hypothalamo-pituitary-adrenal (HPA) axis regulation. Surprisingly, the corticosterone elevations were even more pronounced in AVP- pups, suggesting the possibility of an ACTH-independent corticosterone-secretion regulation. In the case of maternal separation, both the plasma ACTH and corticosterone levels were higher in females than in males, while in case of ether inhalation only the ACTH levels were higher in females. Gender did not influence the stress reactivity or the effect of the genotype. We conclude that the gender of the pups did not profoundly influence HPA axis activity (the mechanism seems to be the same), but in contrast to the general view, we suggest that the females have a more active HPA axis than the males already during the neonatal period. However, the resting corticosterone elevation-well known in adult females- is missing.

Gastric mucosal endogenous prostacyclin levels are different in Brattleboro rats compared with Wistar strain.

Homozygous Brattleboro rats were investigated and compared to normal (physiological) Wistar strain rats regarding their gastric mucosal endogenous prostacyclin (PG-I(2)) level. It seems that the Brattleboro animals have a significantly lower level of this important protective material. Wistar rats having an artificial pituitary stalk lesion (which is the artificial equivalent of homozygous Brattleboro animals) showed no differences in endogenous mucosal prostacyclin level compared to normal Wistar rats. Therefore, we concluded that this hitherto unknown property of the homozygous Brattleboro rats is genetically determined.

Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens.

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [(3)H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

Thiazide induces water absorption in the inner medullary collecting duct of normal and Brattleboro rats.

The reduction of urinary volume after the use of thiazide in the treatment of diabetes insipidus (DI) is known as the "paradoxical effect." Since enhanced proximal solute and water reabsorption only partially account for the reduction in urinary volume, an additional diuretic effect on nephron terminal segments was postulated. Thus the aim of our work was to investigate the effect of hydrochlorothiazide (HCTZ) on water transport in the inner medullary collecting duct (IMCD) of normal and Brattleboro rats. Osmotic water permeability (P(f)) and diffusional water permeability (P(dw)) were studied at 37 degrees C and pH 7.4 by the in vitro microperfusion technique. In the absence of antidiuretic hormone (ADH), HCTZ (10(-6) M) added to the perfused fluid enhanced P(f) from 6.36 +/- 0. 56 to 19.08 +/- 1.70 micro(m)/s (P < 0.01) and P(dw) from 38.01 +/- 4.52 to 52.26 +/- 4.38 x10(-5) cm/s (P < 0.01) in normal rats and also stimulated P(f) in Brattleboro rats from 3.53 +/- 1.41 to 11.16 +/- 1.13 micro(m)/s (P < 0.01). Prostaglandin E(2) (PGE(2)) (10(-5) M) added to the bath fluid inhibited HCTZ-stimulated P(f) (in micro(m)/s) as follows: control, 16.93 +/- 2.64; HCTZ, 29.65 +/- 5.67; HCTZ+PGE(2), 10.46 +/- 1.84 (P < 0.01); recovery, 16.77 +/- 4.07. These data indicate that thiazides enhance water absorption in IMCD from normal rats (in the absence of ADH) and from Brattleboro rats and that the HCTZ-stimulated P(f) was partially blocked by PGE(2). Thus we may conclude that the effect of thiazide in the treatment of DI occurs not only in the Na(+)-Cl(-) cotransport in the distal tubule but also in the IMCD.

Galanin-R1 receptor mRNA expression in the hypothalamus of the Brattelboro rat.

Using in situ hybridization the regulation of mRNA encoding the galanin receptor R1 was investigated in the mutant Brattelboro (diabetes insipidus) rat. We here report an increase of the galanin receptor R1 mRNA levels in the hypothalamic supraoptic and paraventricular nuclei of the mutant strains. The increase seemed to be confined to magnocellular neurons, since no changes were detected in galanin receptor R1 mRNA levels in the extra-hypothalamic nucleus of the olfactory tract. The results confirm that osmotic stimulation induces up-regulation of galanin receptor R1 mRNA levels. This may increase the sensitivity to galanin peptide, the endogenous ligand for this receptor.

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.

Identification and sequence analysis of arginine vasopressin mRNA in normal and Brattleboro rat aortic tissue.

Arginine vasopressin (AVP), a hormone of the hypothalamic-pituitary axis, was also localized in peripheral tissues. To explore AVP precursor gene expression at the vascular level, we have investigated gene transcripts by reverse transcription-polymerase chain reaction (RT-PCR) and sequencing in aortic tissue of normal rat and in the particular genetic condition of the homozygous (di/di) Brattleboro rat strain suffering from diabetes insipidus. In these rats, a gene deletion induces an unprocessed AVP precursor in the hypothalamus with undetectable immunoreactive AVP, in contrast to the detection of immunoreactive material at the vascular level. In normal rats, using primers complementary to exon 1 and 3 of the AVP neurophysin precursor gene, RT-PCR and sequencing revealed transcripts of the expected size from aorta, mesenteric artery and hypothalamus with normal, authentic sequences. Removal of aortic endothelium severely reduced the amounts of transcripts, suggesting their main endothelial origin. In Brattleboro rats, transcripts of similar size were obtained from aorta and hypothalamus and sequencing revealed the homozygous deletion (deltaG316) in both tissues, identical to that found in genomic DNA (deltaG1864). While sequence data from normal rats provide the first direct evidence for the presence of AVP precursor transcripts in rat aortic tissue, identification of the deleted sequence of transcripts in Brattleboro rat aorta suggests that tissue-specific mechanisms are operating for the expression of vasopressin neurophysin precursor in peripheral vascular tissue compared with the hypothalamus.

Renal excretory function in conscious Long Evans and vasopressin deficient (Brattleboro) rats after endothelin-A receptor inhibition.

All experiments were performed on conscious, freely moving male Long Evans as well as Diabetes incipidus (Brattleboro) rats (300-320 g). The endothelin-A (ETA) receptor antagonist BQ-123 (Neosystem) was administered through femoral vein cannula. Arterial blood pressure was measured trough femoral artery catheter. The bladder was cannulated for urine collection via a small suprapubic incision. After a 40 min control period BQ-123 infusion (16.4 nmol/kg/min, 25 microliters/min) was started and continued for 50 min. The effect of 32.8 nmol/kg/min BQ-123 infused in conscious Brattleboro rats was also investigated. Plasma and urine concentrations of sodium, potassium and chloride as well as osmolality were determined. Glomerular filtration rate (GFR) was estimated using the clearance of endogenous creatinine. Endothelin-A receptor inhibition by 16.4 nmol/kg/min BQ-123 infusion in conscious Long-Evans rats decreased urine flow rate by 38.4% (p < 0.02) and increased urine osmolality by 30.3% (p < 0.05). Sodium, potassium, chloride excretion did not alter. Endothelin-A receptor inhibition by 16.4 nmol/kg/min and by 32.8 nmol/kg/min BQ-123 infusion in conscious Brattleboro rats did not produce any change in urine flow rate, urine osmolality or excretion of the electrolytes studied. Endothelins acting via ETA receptors may function as an inhibitor of water reabsorption in the kidneys of conscious rats.

Atrial natriuretic peptide and its mRNA in heart and brain of vasopressin-deficient Brattleboro rats.

To understand the secretion and synthesis of atrial natriuretic peptide we measured immunoreactive atrial natriuretic peptide from plasma, heart tissues and brain areas, and ANP mRNA was determined from heart auricles and ventricles of vasopressin-deficient Brattleboro rats (DI) and from desmopressin treated Brattleboro rats (DI+DDAVP). Long-Evans rats (LE) served as controls. DI+DDAVP rats were given for 3 days sc. injections of 0.5 micrograms 1-desamino-8-D-arginine vasopressin in 1 mL saline twice a day. The rats were housed in single metabolic cages and urinary output and water intake were measured daily. All the body and organ weight parameters were similar in the three groups when the rats were killed. No change was seen in the plasma ANP level between the groups. The right ventricle of DI+DDAVP rats had significantly (P < 0.05) higher concentration of ANP than LE rats (15.8 +/- 4.4 vs. 3.4 +/- 0.6 ng mg-1 tissue). The left ventricle of DI and DI+DDAVP had significantly (P < 0.05) lower amounts of ANP mRNA than LE rats (0.5 +/- 0.2 vs. 1.3 +/- 0.2 and 0.5 +/- 0.1 vs. 1.3 +/- 0.2 arbitrary units). In the hypothalamus, the ANP concentration was significantly (P < 0.05) lower both in DI and in DI+DDAVP rats than in LE rats (9.3 +/- 1.3 vs. 14.5 +/- 1.6 and 6.1 +/- 0.6 vs. 14.5 +/- 1.6 pg mg-1 tissue). To conclude, although the water intake and urinary output of DI rats were changed towards normal with desmopressin treatment, the heart ventricular and hypothalamic ANP did not parallel the change.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of vasopressin mRNA in extrahypothalamic nuclei of the homozygous Brattleboro rat is not modulated by testosterone.

Previous studies from this and other laboratories have shown that levels of vasopressin (VP) mRNA are reduced in both hypothalamic magnocellular and extrahypothalamic nuclei of the homozygous Brattleboro rat (HOM) when compared to the normal Long-Evans (LE) and heterozygous Brattleboro rats (HET). Since extrahypothalamic VP gene expression is dependent on testosterone (T), we measured plasma T in HOM, HET and LE rats. The plasma T level of the intact HOM rat was not significantly different from intact LE or HET rats. Manipulation of circulating gonadal steroids by castration and T replacement was found to regulate the expression of VP mRNA in the bed nucleus of the stria terminalis and medial amygdala of LE and HET rats, but does not appear to modify the absence of VP mRNA in neurons in these nuclei in the HOM rat.

Dynamics of 7B2 and galanin expression in solitary magnocellular hypothalamic vasopressin neurons of the homozygous Brattleboro rat.

The homozygous Brattleboro rat (di/di), displaying a hypothalamic form of diabetes insipidus, synthesizes a vasopressin (VP) precursor with an abnormal C-terminus. The phenotypic expression of coexisting peptides in mutant magnocellular VP cells shows a differential pattern. 7B2 is one of the peptides which is not detectable, whereas there is a clear galanin expression. During postnatal life a small but increasing number of solitary post-mitotic VP neurons of the di/di rat undergoes a switch to a heterozygous phenotype. Here we report the presence of 7B2 and galanin in these heterozygous cells, which suggests that for the expression of 7B2, but not for that of galanin, the relative amount of mutant VP precursor must be diminished. Possible underlying mechanisms for this differential phenotypic expression of coexisting peptides are compartmentalization of precursor synthesis within the RER or a competition for sites involved in the translocation of the functionally reduced RER.

The amount of mutant vasopressin precursor in the supraoptic and paraventricular nucleus of Brattleboro rats increases with age.

In the homozygous diabetes insipidus (di/di) Brattleboro rat an aberrant vasopressin (VP) precursor is synthesized. An antiserum raised against a 14 amino acid sequence of this mutant precursor (CP-14) stains neurons in the supraoptic (SON) and paraventricular nucleus (PVN). In the present study it was shown that during life (i.e. from 16 days up to 83 weeks postnatally) the number of CP-14 SON cells and their content gradually increases. Only a few CP-14 SON cells are present in young pre-pubertal di/di rats. The staining becomes increasingly more intense after puberty. In contrast, the number of CP-14 PVN cells remains low in the post-pubertal period.

Expression of somatostatin receptors is impaired in the cerebellum of developing Brattleboro rats.

Somatostatin (SRIF) receptors are expressed in the external granule cell layer of the rat cerebellum during early postnatal life. The aim of the present study was to investigate the distribution and biochemical characteristics of SRIF binding sites in the cerebellum of homozygous (vasopressin deficient) Brattleboro rats, which exhibit a selective impairment of their granule cell layer. This study has been conducted in 13-day-old rats by means of membrane-binding assay and autoradiography using [125I-Tyr0,DTrp8]S14 as a radioligand. In the cerebellum of homozygous Brattleboro rats, Scatchard plot analysis revealed the existence of a single class of SRIF receptors with similar Kd values as in Long-Evans or heterozygous Brattleboro rats (180-200 pM). Conversely, a marked reduction of the concentration of SRIF binding sites was observed in Brattleboro rats as compared to heterozygous or Long-Evans rats. In homozygous Brattleboro rats, autoradiographic studies revealed that the concentration of SRIF receptors was reduced in all lobules of the cerebellum as compared to Long-Evans. In addition, the magnitude of the decrease of receptor concentration was greater than the loss of granule cells observed in the homozygous Brattleboro rat. These results indicate that the expression of SRIF receptors by immature granule cells of the cerebellum is markedly reduced in Brattleboro rats. Whether the impairment of SRIF receptors in diabetes insipidus rats can directly be ascribed to vasopressin deficiency remains to be determined.

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

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

The role of the hypothalamic paraventricular nuclei for the regulation of pineal melatonin synthesis: new aspects derived from the vasopressin-deficient Brattleboro rat.

There is evidence for an involvement of the hypothalamic paraventricular nuclei (PVN) in the regulation of pineal melatonin synthesis in rats. Since electrical stimulation of the PVN or the systemic administration of arginine-vasopressin (AVP) result in a depression of the nocturnal melatonin surge, this neuropeptide appears to be pivotal for the transduction of PVN-efferent, pinealopetal signals. We therefore used an AVP-deficient animal model, the Brattleboro rat, to further investigate the mechanisms responsible for pineal regulation. Anesthetized adult male animals received 2 min of bilateral electrical stimulation of the PVN either during the day or at night. Thirty min later, pineal glands were removed and pineal N-acetyltransferase (NAT) activities and melatonin contents were determined. Stimulation resulted neither during the day nor at night in any significant alterations of pineal NAT activity or melatonin content when compared to control or sham-stimulated animals. These data further support the proposed modulatory role of AVP for the regulation of melatonin synthesis in the Epiphysis cerebri of genetically intact rats.

Lack of the suckling-induced prolactin release in homozygous Brattleboro rats: the vasopressin-neurophysin-glycopeptide precursor may play a role in prolactin release.

Suckling stimulus did not induce significant release of prolactin (PRL) in lactating homozygous Brattleboro rats, whereas it did it in heterozygous animals. Daily treatment of homozygous rats with vasopressin partly restored the PRL response to suckling. Findings suggest that vasopressin-neurophysin-glycopeptide precursor missing in homozygous Brattleboro rats may play a role in suckling-induced PRL release.

High metabolic activity in the dorsal vagal complex of Brattleboro rats.

Receptor densities for angiotensin II and atriopeptin are particularly high in the dorsal vagal complex (DVC) of the caudal medulla oblongata. Measurements of glucose metabolism in individual components of the DVC, compared with those in Long-Evans rats, revealed that the area postrema was activated selectively both in water-sated and water-deprived Brattleboro rats, which have high circulating levels of angiotensin II. Other parts of the DVC, including subnuclei of the nucleus of the solitary tract and the dorsal motor nucleus of the vagus nerve, as well as brainstem structures within efferent trajectories of the DVC, had elevated rates of glucose metabolism in Brattleboro rats deprived of water overnight and in Sprague-Dawley rats dehydrated for 120 h. The findings are consistent with neural activation by angiotensin II, as either a hormone or neurotransmitter, within subregions of the dorsal medulla oblongata having high densities of putative receptors and immunoreactive perikarya and fibers containing both angiotensin II and atriopeptin.