The vasopressin-deficient Brattleboro rat: lessons for the hypothalamo-pituitary-adrenal axis regulation

Adaptation to stress is indispensable to life and the hypothalamo-pituitary-adrenocortical axis is one of the major components of the adaptation. The hypothalamic component consists of corticotropin-releasing hormone and arginine vasopressin, with a questionable contribution of the latter. Vasopressin was more important in the regulation of the adrenocorticotropin secretion in the perinatal vasopressin-deficient Brattleboro rats than in adulthood, where its role depended on the nature of the stressor encountered. In adults, the vasopressin deficiency did not influence the development of chronic stress response. In the neonatal rats, the role of vasopressin was supported by the inhibitory action of a V1b antagonist and vasopressin antiserum. As the corticosterone response to stress did not follow the adrenocorticotropin levels, we assume the presence of an adrenocorticotropin independent adrenal gland regulation in the neonates. We have shown that the apparent dissociation of the corticosterone and adrenocorticotropin responses is not due to the different time course of the two hormone responses, to different level of the corticosterone binding globulin or to changes in the adrenal gland sensitivity. In vitro experiments point to the contribution of beta-adrenoceptors in the process. It was also confirmed by in vivo tests using the vasopressin-deficient Brattleboro pup as a model organism, where corticosterone levels may rise without adrenocorticotropin level changes. Another important question is the role of adrenocorticotropin beyond the corticosterone secretion regulation, which could be supposed, e.g., in cardiovascular events, immunological processes, and metabolism. We can conclude that Brattleboro rats gave us much information about the stress-axis regulation far beyond the role of vasopressin itself.     

A comparison of the febrile responses of the Brattleboro and Sprague-Dawley strains of rats to endotoxin and endogenous pyrogens

The febrile responses of homozygous (di/di) Brattleboro rats, to both intravenous endogenous pyrogen and to a lipopolysaccharide endotoxin, were compared with those of normal Sprague-Dawley rats. There were no detectable differences between the fever curves of the two strains in response to endogenous pyrogen. Brattleboro rats, which are deficient in the neuropeptide arginine vasopressin (AVP), displayed fevers that were both qualitatively and quantitatively indistinguishable from those of normal Sprague-Dawley rats that do not suffer from congenital diabetes insipidus. It is concluded that the absence of AVP-containing cells in Brattleboro rats is not an important factor in determining the nature of their febrile responses to endogenous pyrogen. More remarkable, however, were the divergent febrile responses of the two strains to intravenously injected endotoxin. Normal rats displayed hypothermic responses, whereas the Brattleboro rats became febrile. By 2 h after the injection of endotoxin, body temperatures in both strains had returned to normal. Three hours after the rats had been exposed to endotoxin, both strains were found to be totally refractory to endogenous pyrogen. However, when both strains of rats were tested to endogenous pyrogen 3 days later, their febrile responses were more than double the magnitude of their initial control responses. These alterations in the febrile responsiveness of rats occurring at different times after the injection of endotoxin appear to be related to the effects that endotoxin has on the cells of the reticuloendothelial system, over the same time course.     

Hyperreflexive behavior in Brattleboro rats

In two experiments we compared the size of the startle response elicited by shock and acoustic stimuli in animals having a congenital absence of vasopressin (Brattleboro rats) with closely related controls, which do synthesize vasopressin. The rats lacking vasopressin were hyperresponsive to both shock and acoustic stimuli. Inhibition of the acoustic startle by shock prestimuli also was greater in these animals. These results are consistent with reports which indicate that vasopressin attenuates responses to noxious stimuli.     

Immunocytochemical evidence for the presence of a mutant vasopressin precursor in the supraoptic nucleus of the homozygous Brattleboro rat

Summary CP-14, a tetradecapeptide from the predicted mutant vasopressin precursor in the homozygous Brattleboro rat was detected immunocytochemically in the supraoptic nucleus of homozygous Brattleboro but not normal rats. The staining was localized to the periphery of the perikarya. CP-14 immunoreactivity was not found in the neural lobes, paraventricular nuclei, accessory nuclei or suprachiasmatic nuclei of either homozygous Brattleboro or normal rats. Vasopressin immunoreactivity was found in the neural lobe and in the perinuclear region of neurons of the supraoptic, paraventricular, suprachiasmatic and accessory nuclei of normal rats. Vasopressin immunoreactivity was also found in homozygous Brattleboro rats, mainly in the ventral part of the supraoptic nucleus: densely stained solitary cells were found amongst other faintly stained perikarya. In both cell-types the staining was mainly in the periphery of the perikarya. No vasopressin immunoreactivity was detected in the paraventricular nuclei, suprachiasmatic nuclei, accessory nuclei or neural lobe of homozygous Brattleboro rats.CP-14 and vasopressin immunoreactivities were found to be co-localized; both were present in the periphery of the same perikarya of the supraoptic nuclei of homozygous Brattleboro rats. Differential staining was found with antioxytocin serum in both normal rats and homozygous Brattleboro rats: separate neurons were stained for either oxytocin or vasopressin and CP-14. Immunoreactive oxytocin was found mainly in the perinuclear region of the neurons from the supraoptic, paraventricular and accessory nuclei.     

Effect of vasopressin gene expression on the growth of Walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WA rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG x Brattleboro) F1 hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG x Brattleboro) F1 rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Signs of attenuated depression-like behavior in vasopressin deficient Brattleboro rats

Vasopressin, a peptide hormone functioning also as a neurotransmitter, neuromodulator and regulator of the stress response is considered to be one of the factors related to the development and course of depression. In the present study, we have tested the hypothesis that congenital deficit of vasopressin in Brattleboro rats leads to attenuated depression-like behavior in tests modeling different symptoms of depression. In addition, hypothalamic-pituitary-adrenocortical axis activity was investigated. Vasopressin deficient rats showed signs of attenuated depression-like behavior in forced swimming and sucrose preference tests, while their behavior on elevated plus maze was unchanged. Vasopressin deficiency had no influence on basal levels of ACTH and corticosterone and had only mild impact on hormonal activation in response to forced swimming and plus-maze exposure. However, vasopressin deficient animals showed higher level of dexamethasone induced suppression of corticosterone response to restraint stress and higher basal levels of corticotropin-releasing hormone mRNA in the hypothalamic paraventricular nucleus. In conclusion, present data obtained in vasopressin deficient rats show that vasopressin is involved in the development of depression-like behavior, in particular of the coping style and anhedonia. Moreover, behavioral and endocrine responses were found to be dissociated. We suggest that brain vasopressinergic circuits distinct from those regulating the HPA axis are involved in generating depression-like behavior.     

Renal effect of acute hypobaric pressure breathing in normal and diabetes insipidus rats

The role of blood volume regulatory mechanisms located in the low pressure system in the control of urinary excretion was studied using hypobaric pressure breathing in normal and diabetes insipidus (Brattleboro strain with a congenital lack of vasopressin) rats. Rats were placed in an altitude simulator chamber for 4 h. A pump maintained pressure reduced to 701, 577 and 472 mbar simulating respectively altitude of 3,000, 4,500 and 6,000 m. In normal rats, hypobaric breathing induced an increase in urine flow, urinary urea and K+ excretion and urinary pH but did not significantly modify creatinine and Na+ excretion. In diabetes insipidus rats, hypobaric breathing produced oliguria and an decrease in urea, creatinine, Na+, K+, Cl- urinary excretions. Since acute hypobaric pressure breathing induced opposed effects in normal and Brattleboro rats, it is suggested that this kind of experimental procedure which increases intrathoracic blood volume elicits a diuretic response through an inhibition of vasopressin release. These experiments confirm the main role of vasopressin in the control of central blood volume.     

Effect of vasopressin gene expression on the growth of walker 256 carcinosarcoma in rats

The growth pattern of the Walker 256 solid tumor has been studied in rats with different doses of the mutant vasopressin gene. In contrast to the vasopressin gene of normal WAG rats, that of mutant Brattleboro rats has a deletion in the coding region that blocks expression at the translation level. The mutation is inherited as a recessive character and is expressed in homozygous Brattleboro rats as diabetes insipidus with an increased water consumption because of the absence of vasopressin in the blood. (WAG × Brattleboro) F₁ hybrids have the same normal phenotype as WAG rats, including a low water consumption. Walker 256 carcinosarcoma, which is not strain-specific, intensely grows only in WAG and (WAG × Brattleboro) F₁ rats. In these groups, the growth of the tumor leads to the animal death within approximately 30 days after the inoculation of tumor cells. In Brattleboro rats, the carcinosarcoma grows less intensely: the tumor node somewhat increases only within the first two weeks, after which the tumor began to decrease and eventually disappears altogether. Both characters exhibit a 100% concordance at the individual level.     

Concordance between vasopressin gene expression and growth of walker 256 carcinosarcoma in rats

The growth features of Walker 256 carcinosarcoma in rats of different genotypes were investigated. The experiments has been carried out on rats of the inbred Brattleboro and WAG lines, as well as on their hybrids segregated during congenic translocation of the normal vasopressin gene to the genotype of the Brattleboro rats. Brattleboro rats do not express the vasopressin gene. It has been found that there are only two types of tumor growth dynamics. In rats of the inbred Brattleboro line and in homozygotes di/di, that were segregated by backcrossings of heterozygous offsprings from the original crossbreeding between (WAG × Brattleboro) F₁ × Brattleboro and the individuals with parental Brattleboro genotype, having grown to some extent the tumor regresses and disappears. In hybrid heterozygous siblings of di/+ genotype tumor grows linearly with time and always leads to fatal outcome. It has been found that, in the congenic procedure, the tumor regression trait is stably maintained and persistently inherited in lineage concordantly with the di/di genotype and, in rats with at least one allele of a normally expressed vasopressin gene, continuous and lethal tumor growth is always observed.     

Vasopressin stimulates endocytosis in kidney collecting duct principal cells

In target epithelia, a vasopressin-induced water permeability increase is accompanied by the appearance of intramembranous particle (IMP) clusters, probably representing water-permeable patches, in the apical plasma membrane of responding cells. In the collecting duct principal cell, we have previously shown that these clusters are located in clathrin-coated pits. To determine whether vasopressin induces the endocytic uptake of these membrane domains in principal cells, we have examined the uptake of horseradish peroxidase (HRP) by principal cells of normal rats, vasopressin-deficient Brattleboro rats, and vasopressin-treated Brattleboro rats, following intravenous injection of HRP. By quantitative electron microscopy, principal cells of Brattleboro homozygous rats were found to take up much less HRP into cytoplasmic vesicles than normal rats, and HRP uptake was increased to normal levels in vasopressin-treated Brattleboro rats. Many invaginating coated pits at the cell surface were loaded with HRP reaction product, indicating their participation in the observed endocytosis of HRP. We conclude that vasopressin stimulates endocytosis in collecting duct principal cells. Since we have already shown that IMP clusters are found in coated pits at the cell surface, the endocytic removal of these putative water-permeable patches from the apical membrane seems to occur via a clathrin-mediated mechanism in this tissue.     

Secretion of aldosterone and corticosterone by the adrenals of Brattleboro rats in response to ACTH administration

The secretions of aldosterone and corticosterone in response to administration of 0,5 mUI of (1,24) ACTH (synacthène-Ciba) were measured in the adrenal venous blood of 15 Brattleboro female rats genetically lacking vasopressin and in 15 Long-Evans female rats, pretreated with dexamethasone. The secretions of aldosterone and corticosterone increased according to a similar profile in the two groups of animals: maximum values were 20-30 min. after ACTH injection; however the steroidogenic secretion of the adrenal cortex was always about 50% less in the Brattleboro female rats than in Long-Evans female rats. This result suggests mainly that vasopressin may be involved in the mechanisms which control the in vivo production of aldosterone by the adrenal glomerulosa cells.     

Desmopressin, but not vasopressin, decreases activity of the hypothalamo-neurohypophysial system in Brattleboro rats

The pituitary neural lobe of homozygous Brattleboro rats has high rates of glucose utilization not affected by chronic treatment with exogenous vasopressin, despite attenuation of polydipsia and polyuria. We evaluated whether this effect may result from the inability of vasopressin to affect the hypothalamo-neurohypophysial metabolism or from the development of resistance to chronic vasopressin treatment. We used the [14C]deoxyglucose method to compare 28-h effects of vasopressin treatment (5 U/kg, i.m., twice a day) with that of desmopressin (100 micrograms/kg, i.p., once a day), a long-lasting antidiuretic hormone, on glucose utilization of the hypothalamo-neurohypophysial system and related structures in conscious homozygous Brattleboro rats. Vasopressin and desmopressin reduced water intake, plasma osmolality and plasma Na+ concentration similarly. Vasopressin decreased glucose utilization in the supraoptic nucleus, subfornical organ and median preoptic nucleus, but did not alter activity in the paraventricular nucleus and neural lobe. Desmopressin decreased glucose utilization in all these structures. The results indicate that desmopressin has a more potent inhibitory action on the hypothalamo-neurohypophysial system than vasopressin over this short duration of treatment. The lack of response in the neural lobe from chronic treatment with vasopressin seems to be due to its inability to affect the paraventricular nucleus metabolism. The maintenance of metabolic activity in the paraventricular nucleus of vasopressin-treated Brattleboro rats suggests that this structure contributes importantly to the metabolism of neural lobe.     

Immunocytochemical staining of supraoptic neurons from homozygous Brattleboro rats by use of antibodies against two domains of the mutated vasopressin precursor

Summary By use of an antibody against the 14 amino acids in the mutated vasopressin precursor (CP-14) characteristic of the homozygous Brattleboro rat, an immunohisto- and-cytochemical study was performed on the supraoptic nuclei of homozygous Brattleboro rats. At the light-microscopic level, varying numbers of perikarya per section exhibited a positive reaction. The most intense staining was observed in a patchy manner on the peripheral portions of the cytoplasm, its central portion being stained less intensely. The antiserum did not react with the supraoptic perikarya of the Wistar rat. In the homozygous Brattleboro rat, antibodies against normal vasopressin only rarely resulted in a positive immunoreaction. However, when it was observed, incubation of the subsequent section with CP-14-antiserum suggested a co-localization of both peptides in the same perikaryon. At the ultrastructural level, CP-14 immunoreactivity was demonstrated on the secretory cisternae of the Golgi apparatus, on lysosome-like bodies and on parts of the rough endoplasmic reticulum. With the use of an antibody against normal vasopressin, immunoreactivity was confined to very limited areas of the rough endoplasmic reticulum. The oxytocin immunoreactivity in supraoptic perikarya of Brattleboro rats did not differ from that in the Wistar rat, either at the light- or at the electron-microscopic levels.     

The vasopressin precursor in the Brattleboro (di/di) rat

The vasopressin precursor in the rat hypothalamus has been studied, using trypsin to release desglycinamide vasopressin and coupling it to glycinamide (T & G treatment). The resulting amidated nonapeptide was detected and measured with a radioimmunoassay for vasopressin. The "vasopressin" produced in this way had the full immunoreactivity of the authentic peptide but eluted from an hplc column 1 min earlier and appeared to have a larger molecular weight. It was found that T&G treatment generated vasopressin immunoreactivity in extracts of the supraoptic nucleus (SON) of the Brattleboro rat in just the same way as it did in normal animals. Furthermore, this procedure produced vasopressin immunoreactivity in those hplc fractions from Brattleboro SON extracts that corresponded with the elution time of vasopressin precursor. Similar amounts of "vasopressin" could be generated from Brattleboro and normal SONs. These results support the suggestion that the Brattleboro SON synthesizes an aberrant vasopressin precursor which is not processed by the cell.     

Possible influence of tachykinins on body fluid homeostasis in the rat

The effect on water intake, urine flow and vasopressin release of intracranial injections of substance P, physalaemin and eledoisin was studied in Wistar and Brattleboro, homozygous and heterozygous, rats. The tachykinins strongly inhibited water intake both in Wistar and in Brattleboro, homozygous and heterozygous, rats. Physalaemin and eledoisin reduced urine flow in Wistar and heterozygous, but not in homozygous, Brattleboro rats. Substance P never affected urine elimination. Physalaemin and eledoisin produced a dose-dependent, long lasting release of vasopressin in Wistar rats. Substance P did not affect the release of vasopressin. The results suggest that both substance P and physalaemin could influence brain mechanisms which control water intake, acting as thirst inhibitors, and that physalaemin could also participate in body fluid control by conserving water through vasopressin release.     

Oxytocin as well as vasopressin potentiate ovine CRF in vitro

We have investigated the effects of synthetic oxytocin and vasopressin on corticotropin release induced by the 41-residue ovine corticotropin-releasing factor (oCRF) in vitro. Segments of the anterior pituitary glands obtained from male and female Wistar or from female hetero- and homozygous Brattleboro rats were used. Ovine CRF (0.1-2.5 nmol/l) stimulated corticotropin release by pituitaries of Wistar rats and this effect was augmented two- to threefold in the presence of arginine vasopressin (0.09-0.9 mIU/ml) or oxytocin (0.9-90 mIU/ml). A similar phenomenon was demonstrated in Brattleboro rats. These data favor the hypothesis that oxytocin might have a physiological role in the regulation of pituitary-adrenocortical function in homozygous Brattleboro rats which lack vasopressin.     

Immunocytochemical identification of dynorphin-containing vesicles in Brattleboro rats

Vasopressin and its carrier protein, vasopressin-associated neurophysin, are co-packaged together with an opioid peptide, dynorphin, into 160 nm diameter neurosecretory vesicles in the normal rat hypothalamo-neurohypophysial system. The homozygous Brattleboro rat lacks vasopressin and vasopressin-associated neurophysin, but contains substantial amounts of dynorphin in the vasopressin-deficient neurosecretory cells. We used post-embedding electron microscopic immunocytochemistry to determine the subcellular location of dynorphin in Brattleboro rats. The results show that dynorphin is present within 100 nm neurosecretory vesicles in homozygous Brattleboro cell bodies and axons, and within 160 nm vesicles in heterozygous (control) neurosecretory cell bodies and axons. Oxytocin-associated neurophysin is present in a separate population of magnocellular neurons in both homozygous and heterozygous rats, and is contained within 160 nm vesicles in both cases. Therefore, the absence of synthesis of the vasopressin prohormone results in a dramatic reduction of neurosecretory vesicle size, despite the continued synthesis and packaging of dynorphin peptides.     

Increased atrial natriuretic peptide (6-33) binding sites in the subfornical organ of water deprived and Brattleboro rats

Binding sites for rat atrial natriuretic peptide (6-33) (ANP) were quantitated in the subfornical organ of chronically dehydrated homozygous Brattleboro rats unable to synthesize vasopressin; heterozygous Brattleboro rats, their controls, Long Evans rats and Long Evans rats after 4 days of water deprivation. Brain sections were incubated in the presence of 125I-ANP and the results analyzed by autoradiography coupled to computerized microdensitometry and comparison to 125I-standards. Brattleboro rats and water deprived Long Evans rats presented a higher number of ANP binding sites than their normally hydrated controls. Our results suggest a role of ANP binding sites in the subfornical organ in the central regulation of fluid balance and vasopressin secretion.