Homozygous Brattleboro Research Articles

Differential distribution of acetylcholinesterase activity among vasopressin- and oxytocin-containing supraoptic magnocellular neurons

Acetylcholinesterase activity was demonstrated histochemically at light- and electron-microscopic levels, in Vibratome sections of the supraoptic nucleus of fixed hypothalami derived from osmotically stimulated and unstimulated Long-Evans rats, from homozygous Brattleboro rats with hypothalamic diabetes insipidus, from lactating rats, from normal adult male house mice ( Mus musculus) and from mice with hereditary nephrogenic diabetes insipidus ( di/di). Reaction product was located in supraoptic magnocellular neurons; in dorsal and rostral aspects of the supraoptic nuclei lightly stained cells predominate, whereas in ventral and caudal regions densely staining perikarya predominate. Pre- and post-embedding immunocytochemical detection of oxytocin-neurophysin or vasopressin-neurophysin, combined with acetylcholinesterase histochemistry, showed that the lightly staining cells are oxytocinergic, and the densely staining cells vasopressinergic. Osmotic stimulation of the animals, either by substitution of drinking water for 3 days with 2.5% saline or by reason of genetic defects which result in diabetes insipidus, enhanced the acetylcholinesterase activity of the vasopressin neurons but had little effect on the weakly acetylcholinesterase-reactive oxytocin cells. Acetylcholinesterase activity was particularly marked in the hypertrophied abnormal magnocellular neurons of homozygous Brattleboro rats which do not release significant amounts of vasopressin. The increased acetylcholinesterase activity in osmotically stimulated animals cannot, therefore, be a function of vasopressin. Acetylcholinesterase activity was also detected in large multipolar neurons lying dorsolateral to the supraoptic nucleus, and in their fine axonal processes which project towards the supraoptic nucleus. A very few synaptic boutons surrounded by acetylcholinesterase reaction product were found in contact with magnocellular neuron basal dendrites. However, much of the punctate acetylcholinesterase reactivity observed at the light microscopic level and previously interpreted as representing the loci of cholinergic synaptic boutons was shown to be intracellular, and probably caused by acetylcholinesterase activity in some large, secondary lysosomes.

Anterior neocortical kindling in vasopressin-deficient rats

Kindling of seizures with stimulation of anterior neocortex was examined in control rats and in Brattleboro rats deficient in arginine-vasopressin (AVP). There were no significant differences between control rats, homozygous Brattleboro rats, and heterozygous Brattleboro rats in the rate and pattern of kindling of generalized seizures. Thus AVP is not critically involved in anterior neocortical kindling.

Mutant vasopressin precursor in the endoplasmic reticulum of the Brattleboro rat. Ultrastructural evidence from individual "vasopressin" cells localized with the light microscope by use of a new gold/silver method for immunostain enhancement.

This ultrastructural study demonstrates that the vasopressin immunoreactivity found in the occasional, densely stained cells in the hypothalamus of the homozygous Brattleboro rat is localized in the rough endoplasmic reticulum. 50-micron Vibratome sections were stained with anti-vasopressin serum by use of a peroxidase method with 3,3-diaminobenzidine as chromogen. The diaminobenzidine end-product has a specific capability to bind gold particles from a chloroauric acid solution and the bound gold was used to precipitate silver grains from a silver developer. The stained sections were flat embedded in resin and ultrathin sections were cut of areas containing the immuno-identified occasional cells. In these densely stained, vasopressin-immunoreactive cells of homozygous Brattleboro rats the rough endoplasmic reticulum was dilated. The lumen of the reticulum contained both end-products of diaminobenzidine and gold/silver grains, but some parts of the reticulum appeared unstained. No other cell organelles were immunostained and no secretory granules were found. In control rats, gold/silver deposits were found throughout the cytoplasm of vasopressin-immunoreactive cells. In these immunostained cells secretory granules were seen.

Plasma osmolality predicts extracellular fluid catechol concentrations in the lateral hypothalamus.

The lateral hypothalamus has an important role in regulating food and water intake. We have investigated the endogenous release of monoamines from the lateral hypothalamus during manipulations of plasma osmolality and circulating volume. Adult male Sprague-Dawley rats implanted with carbon paste in vivo electrochemical (EC) electrodes in the lateral hypothalamus were placed on a 72-h water deprivation schedule. Although the carbon paste EC electrode has an intrinsically ambiguous signal in which changes in ascorbic acid may appear as changes in catechol concentrations, pharmacologic studies in lateral hypothalamus indicated that the electrode most likely measured norepinephrine and possibly epinephrine. On the test day, the EC electrodes were scanned with linear sweep voltammetry from -0.2 to +0.4 V at a rate of 5 mV/s. Semiderivative signal processing showed catechol and hydroxyindole peaks at +0.11 and +0.23 V, respectively. Baseline recordings were made prior to rats drinking distilled water, 10% sucrose, 5% dextrose, 0.30% NaCl, 0.90% NaCl, or 10% d-mannitol. To control for the act of drinking, other implanted dehydrated rats were intraperitoneally injected with 5% dextrose, 0.30% NaCl, or 0.90% NaCl. To dissociate the effects of osmolality and circulating volume on the EC response, hydrated rats implanted with EC electrodes were subcutaneously injected with 12% NaCl or intraperitoneally injected with 35% polyethylene glycol. Other rats subjected to water deprivation and osmotic challenges were decapitated and trunk blood was collected for measurements of plasma osmolality and hematocrit. Similar experiments were conducted using homozygous Brattleboro rats which lack arginine vasopressin (AVP) but which preserve normal plasma osmolality with prodigious drinking.(ABSTRACT TRUNCATED AT 250 WORDS)

Vasopressin-stimulated turnover of phosphatidylinositol in the decentralised superior cervical ganglion of the rat

Vasopressin (AVP) receptors have been demonstrated in the superior cervical ganglion (SCG) linked to the turnover of phosphatidylinositol (PI). We have investigated the effect of changes in autonomic function on the turnover of PI in the rat SCG in response to AVP and carbachol. Decentralisation of the SCG for 3 days causes a significant decrease in the response to AVP, while the response to carbachol is unaffected. The decrease in response to AVP is still present 21 days after decentralisation. A similar pattern of changes is present in ganglia from the homozygous Brattleboro rat. Pretreatment with reserpine has no effect on the response to AVP. The results suggest that AVP may be involved in ganglionic transmission in the autonomic nervous system, but that full expression of AVP-induced turnover of PI is dependent on an intact preganglionic input.

Elevated serum oxytocin of the vasopressin-deficient Brattleboro rat is present throughout life and is not sensitive to treatment with vasopressin.

The postnatal developmental course of the enhanced OT serum level of the vasopressin-deficient (homozygous) Brattleboro rat was investigated radioimmunochemically together with the response to treatment with Pitressin tannate. Compared with heterozygous Brattleboro (control) pups, in which serum OT appeared to have an adult value from birth onwards (about 10 pmol/l), homozygous rats had approximately 2-fold enhanced OT serum level throughout early development. Between day 55 and adulthood the levels of OT rose further to 40-50 pmol/l. A 3-day treatment with Pitressin tannate both in the period before or after the age (day 16) at which the polyuria of the homozygous Brattleboro mutant can be revealed, failed to reduce the serum OT. It was therefore concluded that the high OT serum levels in the vasopressin-deficient Brattleboro rat are not induced by osmotic imbalance, but probably originates from functional teratological aspects of the mutation.

Immunoreactive vasopressin and oxytocin in hypothalamo-hypophysial portal blood of the Brattleboro and Long-Evans rat: effect of adrenalectomy and dexamethasone.

Immunoreactive vasopressin and oxytocin were measured in the hypothalamo-hypophysial portal blood of both Long-Evans and homozygous Brattleboro rats. Adrenalectomy caused an increase in vasopressin immunoreactivity in portal blood in the Long-Evans strain, whilst administration of dexamethasone to these adrenalectomized animals resulted in a reduction in portal vasopressin immunoreactivity to levels below those seen in sham-operated animals. This vasopressin immunoreactivity co-eluted with synthetic vasopressin on high-pressure liquid chromatography (HPLC), and diluted in parallel in radio-immunoassay. In Brattleboro rats, however, although vasopressin-like immunoreactivity was detected, the portal concentration did not vary with the adrenal status of the animal, nor did it show the characteristics of standard vasopressin on HPLC or in immunoassay. Oxytocin was present in the portal blood of both Long-Evans and Brattleboro rats at similar very high concentrations, but did not vary in response to adrenalectomy. These results are consistent with a role for vasopressin, but not oxytocin, in the hypothalamic response to adrenalectomy and glucocorticoid feedback. Neither vasopressin immunoreactivity nor oxytocin appear to subserve this role in the homozygous Brattleboro rat.

Physiological regulation of neurohypophyseal κ-opiate receptors

Quantitative autoradiography was used to examine the characteristics of κ-opiate receptor binding in neural lobe sections from dehyrated rats and water-sated homozygous Brattleboro rats. The density of κ-sites was decreased by 40% and 44%, respectively, after 5 days of water deprivation or hypertonic saline. Both the density and affinity of κ-receptor sites were lower in homozygous Brattleboro rats. We suggest that κ-receptor down-regulation occurs as a result of elevated release of opioid peptides induced by chronic osmotic stimulation.

Alterations in atrial natriuretic peptide receptors in rat brain nuclei during hypertension and dehydration.

We have studied the localization, kinetics, and regulation of receptors for the circulating form of the atrial natriuretic peptide (99-126) in the rat brain. Atrial natriuretic peptide receptors were discretely localized in the rat brain, with the highest concentrations in circumventricular organs, the choroid plexus, and selected hypothalamic nuclei involved in the production of the antidiuretic hormone vasopressin and in blood pressure control. Spontaneously (genetic) hypertensive rats showed much lower numbers of atrial natriuretic peptide receptors than normotensive controls in the subfornical organ, the area postrema, the nucleus of the solitary tract, and in the choroid plexus. These changes are in contrast with those observed for receptors of angiotensin II, another circulating peptide with actions opposite to those of the atrial natriuretic peptide. In acute dehydration after water deprivation, as well as in chronic dehydration such as that present in homozygous Brattleboro rats, there was an up-regulation of atrial natriuretic peptide receptors in the subfornical organ. Thus, circumventricular organs contain atrial natriuretic peptide receptors that could respond to variations in the concentration of circulating peptide. The localization of atrial natriuretic peptide receptors and the alterations in their regulation present in hypertensive and dehydrated rats indicate that these brain receptors are related to fluid regulation, including the secretion of vasopressin, and to cardiovascular function. Atrial natriuretic peptide receptors in the choroid plexus may be related to the formation of cerebrospinal fluid.

Prenatal development of the Brattleboro rat is influenced by genotype and lysine vasopressin treatment of the mother.

Homozygous (Hom) Brattleboro rats suffer from severe diabetes insipidus (DI) as a consequence of the lack of arginine-vasopressin (AVP) in the brain. Compared with heterozygous (Het) AVP-synthesizing Brattleboro rats, Hom rats show disturbed body and brain development. In this study breeding experiments with Het and Hom rats were performed to determine whether prenatal conditions might contribute to the developmental disturbances in Hom pups. For this purpose Het and Hom females were mated with Hom and Het males, respectively. In addition lysine-vasopressin (LVP) was administered to half of the pregnant females, since this has previously been shown to stimulate birth weight of Hom pups. On day 1 postnatally the body and brain weight of Hom pups of nontreated Hom mothers was significantly smaller than that of the Het litter mates, whereas no difference was found between the weight of Het or Hom pups of nontreated or LVP-treated Het mothers. These results indicate an important role of the genotype of the mother in prenatal development of Hom pups. LVP administration failed to diminish the growth deficits, but increased protein and DNA content of the cerebellum of both Het and Hom pups. Notwithstanding the improved prenatal growth of Hom pups from Het mothers, postnatally retarded development was still observed: at 1 month of age there was a significant difference between the body, brain and cerebellar weight of Het and Hom pups from Het mothers. It was therefore concluded that the prenatal situation of the Hom mother, i.e. AVP-deficiency, significantly contributes to the developmental disturbance of the Hom pup, but also that growth impairment is linked to the presence of the mutation in the Hom pups themselves.

Specificity of vasopressin binding to the posterior pituitary gland in the rat. An autoradiographic study.

Specific binding sites for vasopressin (AVP) were compared in the kidney and posterior pituitary gland from normal and homozygous Brattleboro rats using autoradiography with [3H]-AVP. The specificity of ligand binding was assessed by displacement with AVP analogues differing in specificity and potency. In the kidney, specific [3H]-AVP binding was found in the medulla, with much less in the cortex, in both normal and Brattleboro rats. Binding of [3H]-AVP was displaced most effectively by dDAVP and least by AVP-OH, in line with their relative antidiuretic potencies. In the pituitary gland, specific AVP binding was much higher in the posterior lobe than in the intermediate or anterior lobes. This posterior lobe AVP binding was greatly reduced in Brattleboro rats, even in animals given exogenous AVP to restore their water balance. It was unlikely that neurophysin binding was responsible since there was no correlation between [3H]-AVP displacement and neurophysin binding in a series of analogues slightly modified at the N or C terminus to affect selectively neurophysin or receptor binding. We suggest that specific AVP receptors may be present in the posterior pituitary gland, possibly on or in the AVP terminals themselves, associated with the normal synthesis, packaging and/or transport of AVP granules to the neural lobe.

Aggravation by morphine and D-aspartic acid of pyelonephritis induced by i.v. inoculation of Staphylococcus aureus in rats.

Experimental pyelonephritis was induced by intravenous inoculation of Staphylococcus aureus in homozygous Brattleboro diabetes insipidus (Hom Brattleboro DI), heterozygous Brattleboro (Het Brattleboro) and Wistar rats. One group of rats from each strain was implanted with morphine-containing pellet three days before inoculation. Another series of groups received D-aspartic acid (D-ASP) intraperitoneally, starting three days before inoculation throughout the experiments. Owing to the inhibition by morphine or D-ASP of food intake, another control group from each strain was subjected to food restriction. Pyelonephritis development on the tenth day of inoculation was evaluated by the determination of viable bacteria in urine and total kidney tissue, and pathomorphological lesions in kidney. Hom Brattleboro DI rats appeared more resistant. Morphine or D-ASP significantly increased the findings in three strains of rat.

Dehydration reduces κ-opiate receptor binding in the neurohypophysis of the rat

Quantitative autoradiography was used to examine the effects of dehydration on κ-opiate receptor binding in the neural lobe of the pituitary in rats. Dehydration was produced by 5 days of water deprivation or ingestion of 2% saline. Brattleboro rats homozygous for diabetes insipidus were used as a model of chronically disturbed water balance. Slide-mounted pituitary sections were incubated with [ 3H]bremazocine using κ-receptor-selective assay conditions. Binding in the neural lobe was quantified by densitometry of film autoradiographs. Specific [ 3H]bremazocine binding in neural lobe sections of control rats was 45.4 fmol/mg wet weight. In contrast, binding in neural lobe sections of water-deprived, saline-treated, and water-sated homozygous Brattleboro rats was lower by 50%, 35% and 37%, respectively. We suggest that chronic dehydration elevates levels of endogenous neurohypophyseal dynorphin and produces a down-regulation of κ-opiate receptors in the neurohypophysis, a change in affinity of the receptors for κ-agonists, or both.

Studies on desglycinamide arginine vasopressin and scopolamine in a modified/lever-touch autoshaping model of learning/memory in rats

Vasopressin administration has been reported to improve acquisition and retard extinction of both conditioned avoidance and food-reinforced behavioral tasks. In the present experiment the effects of a vasopressin analog (DGAVP) and scopolamine (SCOP) were tested in an autoshaped lever-touch model of learning and memory. Rats were food-deprived to 80% of original body weights and tested in modular cages which contained a retractable lever that was presented on a random interval 48 sec schedule. The lever retracted after 15 sec or when it was touched, at which time one 45 mg food pellet was delivered. Subcutaneous injection of 10 μg/kg DGAVP 1 hr prior to acquisition and extinction sessions did not alter responding compared to saline controls. DGAVP at doses of 10, 20, and 30 μg/kg also failed to affect responding in a more difficult task which included an 8 sec delay between lever retraction and reinforcement. Homozygous Brattleboro rats, which are deficient in vasopressin, did not differ from normal heterozygous littermates in the acquisition of the lever-touch response. Intraperitoneal injection of SCOP (0.1–0.8 mg/kg) 30 min prior to testing caused a dose-related impairment of acquisition compared to saline controls, but did not alter responding in animals which had previously acquired the lever-touch response. These data suggest that manipulations of vasopressin do not affect, while SCOP impairs, the acquisition of a positively reinforced lever-touch response in rats.

Defective regulation of vasopressin gene expression in Brattleboro rats.

The Brattleboro rat has severe diabetes insipidus due to an autosomal recessive trait resulting in the inability to synthesize detectable amounts of hypothalamic vasopressin. To determine whether this abnormality is due to a regulatory defect in the Brattleboro rat's vasopressin gene, we studied changes in the hypothalamic content of vasopressin mRNA in normal Long-Evans and homozygous Brattleboro rats subjected to osmotic stress and correlated these changes with systemic responses to water deprivation. We report that the Brattleboro rat does have a marked defect in the regulation of vasopressin gene expression consisting of an inability to increase hypothalamic vasopressin mRNA content in response to severe osmotic stress.

Functional adaptation of thick ascending limb and internephron heterogeneity to urine concentration

The kidney is able to adapt to a variety of chronic changes in excretory needs, for example, to high or low sodium or potassium intake [1] and to acidosis and alkalosis [2]. One of the main functions the mammalian kidney assumes is the concentration of solutes in the urine. The diversity of mammals affords examples of renal adaptation to water conservation. Comparison among animals living in fresh water, in temperate regions, or inhabiting desert areas reveals several differences in kidney morphology and function [3]. Recent experiments have shown, not only among different species but within a given species as well, remarkable functional adaptation of the kidney to chronic variations in urine concentration. Urine concentration can be altered for sustained periods of time by modifying either antidiuretic hormone (ADH) or fluid intake. The Brattleboro rat offers a unique model in which the effects of the total absence of ADH can be studied [4]. By studying homozygous Brattleboro rats with diabetes insipidus (DI) and comparing their kidney function and morphology with that of heterozygous rats of the same strain, normal Long Evans rats (the strain from which the Brattleboro are derived), and DI rats chronically receiving antidiuretic hormone, we were able to demonstrate several features of the renal adaptation to water conservation. Several of these features were also observed in normal rats in response either to an increase or to a decrease in water intake for several weeks. These changes are described in the first part of this paper. The second part will address the mechanisms by which these changes are induced, and how they improve the urine concentrating process.

Reduced responsiveness to acute salt-loading of vasopressin-neurons and oxytocin-neurons in the heterozygous Brattleboro rat.

Heterozygous Brattleboro (HZ) rats exhibit a partial genetic deficiency in hypothalamic vasopressin (VP) production. The effects of this abnormality of HZ rats on the capacities of VP-neurons and oxytocin (OT)-neurons to respond to an acute salt-load were examined. Acute salt-loading was induced by intravenous infusion of 18% saline in conscious, chronically catheterized animals and the activities of VP-neurons and OT-neurons were interpreted from plasma concentrations of VP-associated neurophysin, [VP-RNP] and OT-associated neurophysin, [OT-RNP] at different time periods throughout the infusion. Plasma sodium concentration ([Na+]), plasma osmolality (Posm) and mean arterial pressure (MAP) were also monitored. Salt-loading produced significant rises in [VP-RNP] and [OT-RNP]. These rises were accompanied by increases in plasma [Na+], Posm and MAP. Releases of OT-RNP were approximately four times greater than those of VP-RNP. The responsiveness of VP-neurons to increases in Posm in the HZ rat was approximately one-half of that observed for the Long-Evans (LE) rat. Furthermore, the responsiveness of OT-neurons in these animals was approximately one-half of that for LE rats and one-third of that for homozygous Brattleboro (DI) rats. The changes in MAP during salt-loading do not appear to be different for HZ and LE rats. Hence, while VP may be involved in the rise in blood pressure during infusion of hypertonic saline, there is not a direct correlation between plasma levels of VP-RNP (and presumably VP) and rises in blood pressure.

Exogenous vasopressin modulates activity of oxytocin neurons in homozygous Brattleboro rats.

We studied the effects of chronic replacement with arginine vasopressin (AVP) or 1-desamino-D-arginine vasopressin (DDAVP), as well as acute replacement with AVP or DDAVP, on the responsiveness of oxytocin (OT) neurons as indexed by plasma oxytocin-associated neurophysin concentration [( OT-RNP]) during acute salt loading in conscious, chronically catheterized homozygous Brattleboro (DI) rats. Salt loading was carried out on days 5 and 12 of AVP (3,000 ng/day) or DDAVP (50 ng/day) treatment or 60 min after intraperitoneal injection of 1 microgram AVP or 25 ng DDAVP. All vasopressin treatments did not significantly alter the basal [OT-RNP]. In response to infusion of 18% saline, there were corresponding significant increases in plasma osmolality (Posmol) and [OT-RNP] in all animals. The increases in [OT-RNP] in vasopressin-treated DI rats were markedly reduced compared with those observed earlier for untreated DI animals despite similar rises in Posmol. The slopes of the relationship between delta [OT-RNP] and delta Posmol were 9.0 and 9.8 fmol X ml-1 X mosmol-1 X kg for chronically AVP-treated DI rats, 8.9, and 8.8 fmol X ml-1 X mosmol-1 X kg for chronically DDAVP-treated DI animals, 10.7 fmol X ml-1 X mosmol-1 X kg for acutely AVP-treated DI rats, and 8.3 fmol X ml-1 X mosmol-1 X kg for acutely DDAVP-treated animals compared with that of 34.9 fmol X ml-1 X mosmol-1 X kg for untreated DI rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute infusion of lithium chloride raises blood pressure in the conscious rat

The effects of acute infusion of lithium chloride (LiCl) were studied on mean arterial pressure (MAP) and magnocellular activity as shown by the concentrations of vasopressin-associated neurophysin ([VP-RNP]) and concentrations of oxytocin-associated neurophysin ([OT-RNP]) in plasma in conscious Long-Evans rats. Chronically-cannulated rats were infused intravenously at 10 μ1/100 g body wt/min with 13% LiCl for 20 min (total dose = 6.16 mequiv./kg body wt) or 0.65% LiCl for 60 min (total dose = 0.92 mequiv./kg body wt). Effects of 13% LiCl on mean arterial pressure were also examined in vasopressin-deficient homozygous Brattleboro rats. For Long-Evans rats, infusion of 13% Lid produced rapid and significant ( P < 0.001) increases in mean arterial pressure, the concentration of lithium in plasma ([Li +]), plasma osmolality ( P osmol). [VP-RNP], [OT-RNP] and significant decreases in heart rate and sodium concentration in plasma ([Na +]). For similar changes in plasma osmolality, lithium had a greater effect than sodium on mean arterial pressure, [VP-RNP], [OT-RNP]. For the 20 min infusion of 13% LiCl, there was a significant relationship ( P < 0.033) between ΔMAP and log Δ[VP-RNP] with a slope of 11.9 mmHg fmol −1 ml −1 ( r = 0.5678). Unlike that of Long-Evans rats, infusion of 13% LiCl only did not produce significant changes of mean arterial pressure in Brattleboro rats. For Long-Evans rats, infusion of 0.65% LiCl resulted in more gradual and smaller elevations of blood pressure, [Li +] and smaller decreases in heart rate with no significant changes in plasma osmolality, [Na +], [VP-RNP] and [OT-RNP]. The relationship between ΔMAP and log [Li +] over the 60 min of infusion of 0.65% LiCl was highly significant ( P < 0.001) with a slope of 15.0 mmHg mequiv. −1 1 −1 ( r = 0.6815). The data show that acute intravenous infusion of LiCl raised the blood pressure in the conscious Long-Evans rat and this effect of Li may involve mechanisms that are independent as well as dependent of the activity of vasopressin-containing neurons.

Immunoreactive arginine vasopressin in the rat thymus.

Immunoreactive arginine vasopressin (ir-AVP), coeluting with authentic nonapeptide on reverse phase HPLC, is present in the thymus of Sprague-Dawley, Long-Evans, and homozygous (di/di) Brattleboro rats, and BALB/c mice. By immunohistochemistry, ir-AVP positive cells are sparse, and do not appear to be lymphocytes. Adrenalectomy and dexamethasone administration to intact rats produces an identical response in terms of thymic ir-AVP, with a rise after 1-2 days followed by a fall to levels below baseline after 8 days. The rise 2 days after adrenalectomy, and the fall 8 days later, were both prevented by administration of aldosterone, but not by corticosterone or dexamethasone to adrenalectomized animals. The role(s) of thymic ir-AVP, and the physiological significance of its mineralocorticoid dependence, remain to be established.