Investigation of social, affective, and locomotor behavior of adolescent Brattleboro rats reveals a link between vasopressin's actions on arousal and social behavior.

Arginine vasopressin (AVP) has recently been implicated in juvenile and adolescent social development. How AVP influences social development, however, is not understood. Adolescent homozygous Brattleboro rats (Hom), which lack AVP due to a mutation in the Avp gene, exhibit fewer active social behaviors (e.g., social play) but more passive social behaviors (e.g., huddling) than their wild type and heterozygous (Het) littermates, raising the possibility that AVP impacts social development through an arousal mechanism. Here, we test whether the atypical social phenotype of adolescent Hom rats is associated with altered behavioral arousal, social approach, or affective behaviors and whether Brattleboro mothers impact these behavioral phenotypes. Male and female Het and Hom adolescents born to Het or Hom mothers were tested in social interaction, open field, novelty-seeking, social approach, and marble burying tests. As reported previously, Hom rats played less and emitted fewer 50 kHz ultrasonic vocalizations while huddling more than their Het littermates. No genotype differences were detected in novelty seeking or social approach, nor were consistent differences found between offspring from Het and Hom mothers. However, Hom rats were less active in the open field and buried fewer marbles than Het rats indicating a hypoaroused, low anxiety phenotype. Open field activity correlated with levels of social play indicating that the effects of the Brattleboro mutation on arousal and social behavior are linked. These data demonstrate that chronic AVP deficiency impacts behavioral arousal during adolescence and support the hypothesis that AVP influences adolescent social development, in part, through its regulation of arousal.

Vasopressin deletion is associated with sex-specific shifts in the gut microbiome.

Brattleboro rats harbor a spontaneous deletion of the arginine-vasopressin (Avp) gene. In addition to diabetes insipidus, these rats exhibit low levels of anxiety and depressive behaviors. Recent work on the gut-brain axis has revealed that gut microbiota can influence anxiety behaviors. Therefore, we studied the effects of Avp gene deletion on gut microbiota. Since Avp gene expression is sexually different, we also examined how Avp deletion affects sex differences in gut microbiota. Males and females show modest but differentiated shifts in taxa abundance across 3 separate Avp deletion genotypes: wildtype (WT), heterozygous (Het) and AVP-deficient Brattleboro (KO) rats. For each sex, we found examples of taxa that have been shown to modulate anxiety behavior, in a manner that correlates with anxiety behavior observed in homozygous knockout Brattleboro rats. One prominent example is Lactobacillus, which has been reported to be anxiolytic: Lactobacillus was found to increase in abundance in inverse proportion to increasing gene dosage (most abundant in KO rats). This genotype effect of Lactobacillus abundance was not found when females were analyzed independently. Therefore, Avp deletion appears to affect microbiota composition in a sexually differentiated manner.

[Specificity of gene di (diabetes insipidus) expression in homologous inbred rat strains]. / Osobennosti ékspressii gena di (diabetes insipidus) v gomologichnykh inbrednykh liniiakh krys.

The diabetes insipidus mutation is displayed in homozygotes in the form of diabetes insipidus with water consumption from 30 to 100% of body weight per day. We developed two inbred sublines of the di/di Brattleboro rats as well as the recombinant inbred subline by integrating genes of August rats into the di/di mutant genome. Changes in the genetic background proved to have no effect on the quantitative parameters of the diabetes insipidus. The intensity of the secondary immune response and the content of tropomyosin in the medulla of the rat kidney can serve as additional marker traits of the di/di genotype.

Arginine vasopressin regulates CFTR and ClC-2 mRNA expression in rat kidney cortex and medulla.

The presence of both CFTR and ClC-2 proteins in the kidney suggest that they are involved in chloride transport along the nephron but their physiological roles in this organ are not known. To further understand the role of these chloride channels we studied Wistar rats subjected to dehydration for 2 days and also the homozygous Brattleboro rats, a strain of Long-Evans rats carrying an autosomal recessive mutation that leads to a deficiency of arginine-vasopressin (AVP) secretion in the plasma. The expression of CFTR was increased in the medulla of dehydrated Wistar rats and no variation was observed in the cortex. The expression of both ClC-2 and CFTR mRNAs was low in the renal cortex and medulla of the homozygous Brattleboro rats but returned to normal levels after AVP reposition. By the use of Madine-Darby canine kidney (MDCK) type I epithelial cells, it was observed that AVP (10(-8), 10(-7) and 10(-6) M) increased CFTR mRNA expression "in vitro" but no effect was observed when changes in the medium tonicity were caused by the addition of sucrose, NaCl, manitol or urea. The modulation of both CFTR and ClC-2 mRNA by AVP, the main hormone involved in the regulation of body fluid osmolality, suggests the participation of these two chloride channels in the renal tubule transcellular chloride transport modulated by AVP.

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.

Lack of vasopressin-independent upregulation of AQP-2 gene expression in homozygous Brattleboro rats.

Arginine vasopressin (AVP) plays an important role in the expression of aquaporin (AQP-2) in the collecting duct. The present study was undertaken to determine whether there is an AVP-independent regulation of AQP-2 gene expression in homozygous Brattleboro rats in which endogenous AVP is absent. Exogenous administration of 1-deamino-8-D-AVP produced an antidiuresis and expressed AQP-2 mRNA and AQP-2 protein in the renal medulla of the homozygous Brattleboro rats. Twelve hours of water deprivation produced severe dehydration in the homozygous Brattleboro rats, such that urinary osmolality increased from 200 to 649 mosmol/kgH(2)O. However, no increase in AQP-2 mRNA expression was observed after this dehydration, and the medullary tissue content and urinary excretion of AQP-2 also remained unchanged. Increases in AQP-2 mRNA expression and AQP-2 protein were evident in Long-Evans rats after 64 h of water deprivation, with a severity of dehydration almost equal to the 12-h dehydrated, homozygous Brattleboro rats. These results indicate the lack of an AVP-independent mechanism for upregulating AQP-2 mRNA expression in renal collecting duct cells.

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.

Regulation of pituitary V1b vasopressin receptor messenger ribonucleic acid by adrenalectomy and glucocorticoid administration.

Regulation of the number of pituitary vasopressin (VP) receptors plays an important role in controlling pituitary responsiveness during alterations of the hypothalamic pituitary adrenal axis. The mechanisms regulating these VP receptors were studied by analysis of the effects of adrenalectomy and glucocorticoid administration on V1b receptor (V1b-R) messenger RNA (mRNA) by Northern blot and by in situ hybridization in the rat. Adrenalectomy transiently decreased V1b-R mRNA levels by 18 h (77% and 62% for the 3.7-kb and 3.2-kb bands in the Northern blots, and 50% by in situ hybridization), returning to basal levels after 6 days. The decrease in V1b-R mRNA after 18 h adrenalectomy was fully prevented by dexamethasone (100 microg s.c.) but not by elimination of hypothalamic CRH and VP by paraventricular nucleus lesions or median eminence deafferentation. In sham-operated rats, dexamethasone increased receptor mRNA by 50% after 6 days. In contrast to Sprague-Dawley rats, in Brattleboro rats (di/di), which lack hypothalamic VP, adrenalectomy caused a sustained decrease in V1b-R mRNA levels (<50% of controls by 6 days). The data show that pituitary V1b-R mRNA is positively regulated by glucocorticoids and that the recovery of V1b-R mRNA levels after prolonged adrenalectomy is probably mediated by VP. In addition, the data suggest that the down-regulation of VP binding after long-term adrenalectomy is due to posttranscriptional events rather than to changes in V1b-R mRNA.

Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.

Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10[-2] U beta-galactosidase [beta-gal]), but not the mutated BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP secretion (3.0 +/- 1.4 pg/10[-2] U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13.5 +/- 4.0 pg/10[-2] U beta-gal). When a stop codon was inserted into the wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10[-2] U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05). In summary, (1) both VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region diminishes but does not abolish AVP processing and secretion; and (3) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild-type gene precursor. Thus, the CDI in BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.

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.

Homozygous Brattleboro rats display attenuated conditioned freezing responses.

In the present study we examined the influence of arginine vasopressin (AVP) on conditioned freezing behavior to aversive shock treatment by comparing the responses of Brattleboro homozygous (DI) rats, Brattleboro heterozygous (HZ) rats, and Long-Evans (LE) rats. Each animal was placed in a sound-attenuated shock chamber on the training day and given a series of 3 footshocks. On the following 4 consecutive days the rats were placed in the chambers where they had received their shock and levels of spontaneous freezing were evaluated. Levels of circulating vasopressin-associated neurophysin (NP) were subsequently determined in each rat strain. For each of the 4 test days, DI rats displayed significantly less freezing behavior when compared with LE rats and HZ rats. HZ rats displayed trends towards attenuated freezing responses when compared with LE rats. The data indicate that a relationship exists between the levels of central nervous system (CNS) and circulating AVP, and the amount of freezing displayed by each strain. These preliminary results suggest that vasopressin may be involved in appropriate autonomic and emotional responses to fearful stimuli in fear conditioning paradigms.

Ultrastructure of hypothalamic paraventricular neurons.

The hypothalamic paraventricular nucleus (PVN) plays a pivotal role in the regulation of endocrine processes and the modulation of autonomic functions. The multi-channel outputs of the nucleus are directed toward the anterior and posterior lobes of the pituitary, autonomic centers, and limbic structures. Counterbalancing the wide spectrum of efferent projections, the nucleus receives humoral signals from endocrine glands and neuronal afferents from several loci of the brain. The multiple functions of the nucleus are executed by neurons that are organized in distinct subnuclei and display complex chemotypes. The review demonstrates and discusses the organization, architecture, chemical composition, plasticity, and pathology of paraventricular neurons of the rat hypothalamus from the perspective of ultrastructural analysis. Electron microscopy--by its high resolution--offers a powerful tool that is suitable for revealing the structural background of physiological processes that modulate and govern the operation of paraventricular neurons.

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.

Vasopressin is involved in renal effects of high-protein diet: study in homozygous Brattleboro rats.

The present study was designed to test the possible role of vasopressin in the renal response to dietary protein. This possibility was suggested by the similarity of effects on renal function and morphology of chronic high-protein intake and chronic stimulation of urine concentration. Adult male Brattleboro rats, genetically unable to produce vasopressin, were fed high-protein (32% casein = HP, n = 8) or low-protein (10% casein = LP, n = 9) diet for 7 wk. Renal function was evaluated by clearance techniques based on 24-h urine collections in metabolic cages. The response to a single injection of the vasopressin analogue 1-desamino-8-D-arginine vasopressin (DDAVP) was also tested. Kidney weight and height of the different renal zones were assessed at the end of the study. HP diet increased urea excretion nearly sevenfold. Water intake increased by 57% (P less than 0.001) and urine flow rate by 71% (P less than 0.01). Urine osmolality rose from 104 to 181 mosmol/kgH2O (P less than 0.001). At variance with what occurs in rats with endogenous vasopressin (Sprague-Dawley; Bouby, N., et al. Kidney Int 34: 4-12, 1988), HP diet increased creatinine clearance per unit body weight by only 14% and did not change free water clearance, renal mass, and height of inner stripe of outer medulla. However, the rise in urine osmolality and drop in free water clearance after DDAVP were significantly greater in HP- than in LP-fed Brattleboro rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Arginine vasopressin gene regulation in the homozygous Brattleboro rat.

The Brattleboro rat, which has an autosomally recessive form of diabetes insipidus, has been reported to have a marked defect in the regulation of arginine vasopressin (AVP) gene expression. However, it is not known whether this is a primary genetic defect or occurs secondary to the urinary water losses which occur in the absence of circulating AVP in the Brattleboro rat. This present study was therefore undertaken to study AVP gene regulation in the Brattleboro rat after chronic AVP treatment by osmotic minipump for 2 wk. In Brattleboro rats without AVP treatment, neither urinary osmolality (Uosm) nor hypothalamic AVP mRNA was significantly changed after 24 h of fluid deprivation (Uosm, 413 +/- 33 to 588 +/- 44, NS; AVP mRNA, 39.33 +/- 2.95 to 46.39 +/- 2.71 pg/micrograms total RNA, NS). In contrast, when Brattleboro rats were treated with AVP for 2 wk, the regulation of AVP gene occurred in response to 24 h of fluid deprivation. In these studies, hypothalamic AVP mRNA was significantly increased compared with the Brattleboro rats still receiving AVP with free access of water (28.9 +/- 3.5 vs. 65.0 +/- 3.3 pg/micrograms total RNA, P less than 0.001). Further studies in Long-Evans rats demonstrate a similar response to a comparable degree of fluid deprivation as Uosm and AVP mRNA were significantly increased after 72 h of fluid deprivation (Uosm, 1,505 +/- 186 to 5,460 +/- 560 mosmol/kg, P less than 0.001; AVP mRNA, 31.7 +/- 3.9 to 77.5 +/- 4.6 pg/micrograms total RNA, P less than 0.001). These results indicate that AVP-replaced homozygous Brattleboro rats can regulate AVP gene expression normally in response to fluid deprivation. This finding indicates that the defect in AVP gene regulation in the Brattleboro rat not receiving AVP replacement is a secondary phenomenon rather than a primary genetic defect.

Lack of effect of vasopressin replacement on renin hypersecretion in Brattleboro rats.

To determine how the vasopressin deficiency in homozygous Brattleboro rats with diabetes insipidus produces increased renin secretion, homozygous and heterozygous Brattleboro rats were infused through subcutaneously implanted Alzet minipumps for 1 wk with a dose of arginine vasopressin that restored plasma vasopressin to normal in the homozygous animals. In the homozygous animals, plasma renin activity (PRA) and the PRA response to immobilization remained elevated compared with Long-Evans controls. Propranolol reduced PRA to normal and markedly reduced the PRA response to immobilization. PRA was normal in heterozygous Brattleboro rats. The data indicate that the increased renin secretion in homozygous rats is a result of increased sympathetic activity, and because circulating vasopressin does not cross the blood-brain barrier, it seems likely that the increased sympathetic activity is central in origin.

Age-related development of a heterozygous phenotype in solitary neurons of the homozygous Brattleboro rat.

A single-base deletion in the single-copy vasopressin gene is the cause of diabetes insipidus in the homozygous Brattleboro rat (di/di). It results in the synthesis of an altered vasopressin precursor of which the axonal transport is blocked. Paradoxically, a small number of solitary hypothalamic neurons displays all the immunoreactivities of the wild-type vasopressin precursor (i.e., vasopressin, neurophysin, and a glycopeptide). In the present paper we provide evidence that these neurons have undergone a switch to a genuine heterozygous (di/+) phenotype; i.e., they contain the immunoreactivities of both the wild-type and the mutated vasopressin precursors. In the neural lobe, glycopeptide fibers are also present, showing that axonal transport of the wild-type precursor is restored. Moreover, the number of neurons displaying this di/+ phenotype increases markedly and in a linear way (from 0.1% up to 3% of the vasopressin cells) with age. These findings indicate that after mitotic division has ceased, genomic alterations occur in somatic neurons in vivo. The molecular event generating the di/+ phenotype in the di/di animal could involve a somatic intrachromosomal gene conversion between the homologous exons of the vasopressin and the related oxytocin genes.