Monoamine and amino acid content in brain regions of Brattleboro rats

Monoamine and amino acid content were measured in brain regions from 12 week old male, homozygous Brattleboro (DI,n=12) and Long-Evans control (LE,n=12) rats. Norepinephrine (NE) content was significantly elevated (16–25%) in the spinal cord, pons-medulla and anterior hypothalamus of DI rats when compared to LE controls. NE content of the neurointermediate lobe of pituitary in DI rats was almost twice that of LE controls. Serotonin content was also significantly elevated in the spinal cord, pons-medulla, anterior hypothalamus and forebrain of DI rats relative to the LE controls. Taurine content in DI rats was increased (31–42%) above that of LE rats in the anterior hypothalamus, striatum and forebrain. Glutamine content was also greater in DI rats than LE in the spinal cord, pons-medulla, anterior hypothalamus, striatum, hippocampus and forebrain. The changes in monoamine and amino acid content were discussed in relation to the cardiovascular and osmoregulatory deficits that are present in DI rats due to arginine vasopressin (AVP) deficiency. The possible role of AVP in modulating NE turnover was also discussed. The increase in brain TAU content in DI rats may be a physiological response to hypernatremia.     

Angiotensin II increases ACTH release in the absence of endogenous arginine-vasopressin

Recent studies from our laboratory indicate a primary central site of action of Angiotensin II (AII) to release ACTH. The present studies were designed to test whether AII is able to release ACTH in vivo in a similar fashion in intact, cannulated, freely moving Long-Evans (LE) or in vasopressin (AVP)-deficient, Brattleboro (DI) female rats. The in vivo response to AII was compared with that elicited by synthetic CRF. AII injected i.v. (0.4 or 2 micrograms/100 g BW) induced a significant, dose-related increase in plasma ACTH values 5 and 15 min after injection, in both LE and DI rats. CRF given to LE and DI rats at 0.4 micrograms/100 g BW elicited a larger increase in ACTH plasma values than a similar dose of AII, 5 or 15 min after the injection. Moreover, ACTH levels after CRF in DI rats were significantly greater than those obtained in LE controls. In vitro studies using dispersed anterior pituitary cells indicate that the response of cells from either LE or DI rats to AII or AVP (both at 10(-9) and 10(-8)M) was similar. Cells from DI donors were hyperresponsive to CRF (2 X 10(-11) and 10(-10)M) in terms of ACTH release when compared with the response of cells from LE rats. The present results suggest that the presence of AVP is not essential to mediate the central response to AII and that AII may act centrally to stimulate CRF release from the hypothalamus in vivo, which would then enhance ACTH output. The results in the DI rat indicate that the increased response to CRF may be an important compensatory mechanism involved in the regulation of adrenocortical function in the DI rat.     

Plasma ADH in normal Long-Evans rats and in Long-Evans rats heterozygous and homozygous for hypothalamic diabetes insipidus.

A radioimmunoassay for the measurement of arginine-vasopressin (AVP) in rat plasma is described. By precipitating plasma proteins with acetone prior to the assay, a blanc value of zero was obtained for plasma of rats with hypothalamic diabetes insipidus (DI). Under $\text{ad lib}$. water intake as well as after 48 hrs of dehydration, AVP concentrations in plasma of rats heterozygous for DI were by roughly 50 % lower than in plasma of normal control rats. In plasma of DI rats no AVP was measurable 24 hrs after withdrawal of water. These data support the notion that DI rats do not produce any AVP and that the heterozygous animals have partial ADH deficiency. - In normal and heterozygous rats, AVP concentrations in plasma obtained at 5 p.m. were lower than in plasma collected at 10 a.m. This might indicate a diurnal fluctuation of plasma AVP concentrations in the rat.     

Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol

We have tested the hypothesis that animals with reduced levels of arginine vasopressin (AVP) would show reduced tolerance to ethanol. Brattleboro rats either heterozygous or homozygous for the diabetes insipidus (DI) trait and normal Sprague-Dawley rats were exposed to ethanol vapor for 21 days. Two days later, tolerance was evaluated by monitoring body temperature reductions after intraperitoneal injection of 2 g/kg (20% w/v) ethanol. Under the same conditions of chronic ethanol exposure, Sprague-Dawley rats, but not Brattleboro rats, displayed tolerance to the hypothermic effects of intraperitoneal ethanol. This phenomenon did not appear to be related to differences in ethanol metabolism or blood alcohol levels in Brattleboro rats. These data support a possible role for AVP in the development or maintenance of tolerance.     

Effects of water-deprivation on aldosterone production in Brattleboro male rats congenitally lacking vasopressin

Effects of water-deprivation on several metabolic parameters and on plasma aldosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE) as controls. Two separate experiments were performed over a period of 72 hours: 1) to determine the global effect of water-deprivation, water deprived rats were compared with hydrated animals, 2) to elucidate the specific effect of dehydration alone, water-deprived rats were compared with similar food-restricted, but water-supplied DI and LE rats. In hydrated animals, plasma aldosterone concentration was close to 50% less in DI rats than in LE rats. After 72 hours, plasma aldosterone values increased mainly because of dehydration and this increase was greater in DI rats than in LE rats. At the same time, plasma aldosterone concentration remained lower in DI rats compared to LE rats. The changes in plasma aldosterone concentration after dehydration and possible reasons for the impairment of aldosterone production in DI rats are discussed.     

Effects of water deprivation on corticosterone production in the male Brattleboro rat genetically deprived of vasopressin

Effects of 72 h water-deprivation on plasma corticosterone concentration have been investigated in male Brattleboro rats homozygous for hypothalamic diabetes insipidus (DI) and in male Long-Evans rats (LE), as controls. To determine the global effect of water deprivation, drinking water deprived rats were compared with hydrated animals. Because water deprived rats showed a depressed food intake, to elucidate the specific effect of dehydration alone, drinking water deprived rats were compared with similar food-restricted but water supplied animals. Increases in adrenal weights and in plasma corticosterone content, following 72 h water-deprivation, were greater in DI than in LE rats. In LE rats, they seemed to be the result of both dehydration and denutrition. Conversely in DI rats lacking vasopressin, dehydration alone increased neither adrenal weights nor plasma concentration of corticosterone; the whole plasma corticosterone content was reduced. So, in DI rats, the global response to drinking water deprivation was essentially due to food restriction, whose effect was partly suppressed by dehydration. Whatever the circumstances, plasma concentrations of corticosterone were higher in DI than in LE rats. Interrelationships between water deprivation, stress, vasopressin and glucocorticoids are discussed.     

Thermoregulatory, behavioral and seizure modulatory effects of AVP in the gerbil

Recent reports suggest that arginine vasopressin (AVP) may be an endogenous antipyretic peptide and a mediator of febrile convulsions [10,12]. The spontaneously seizing Mongolian gerbil was used to investigate the thermoregulatory, behavioral and seizure modulatory effects of AVP. Injection of AVP (1.0 and 5.0 μg IV and 0.01–1.0 mg/kg SC) caused dose-related falls in body temperature. Stereotypic scratching, terminated by a body shake, was observed after AVP (1.0–5.0 μg IV). However, such behavior was not observed after subcutaneous injection of AVP. AVP did not potentiate seizure induction in the gerbils but rather reduced the seizure incidence. The data demonstrate that AVP can reduce body temperature and cause specific behaviors, but it does not appear to play a role in the pathogenesis of seizures in the seizure sensitive strain of Mongolian gerbil.     

Production of corticosterone in male homozygous Brattleboro rats

Plasma concentration, metabolic clearance rate and in vitro adrenal production of corticosterone were measured in Brattleboro male rats homozygous for diabetes insipidus (DI) and in Long-Evans male rats (LE) as controls in resting conditions, under stress caused by pentobarbitone anesthesia and surgery and after three days water deprivation. In resting animals, plasma concentrations and in vitro adrenal production of corticosterone were higher in DI rats than in LE rats. Under pentobarbitone anesthesia and surgery, plasma concentrations and metabolic clearance rate of corticosterone were slightly but not statistically lower in DI rats; however, the in vivo production rate of corticosterone was significantly lower. After three days water deprivation, increasing plasma corticosterone level was consistently higher in DI than in Le rats. These results are not in favour of a reduced glucocorticoid activity of the adrenal of DI rats and of an important role played by vasopressin on the stimulation of the hypothalamopituitary adrenal activity at least in resting conditions; its role may depend upon stressful circumstances.     

FMRF-NH2-like peptide is deficient in the pituitary gland of the Brattleboro rat

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. In the rat, F-8-F-NH2 immunoreactivity (IR) is highly localized in the neurohypophysis. In this study, F-8-F-NH2-IR was studied in the hypothalamo-neurohypophyseal system of an Arg8-vasopressin (AVP)-deficient animal, the Brattleboro (DI) rat, and the normal control Long-Evans (LE) strain. F-8-F-NH2-IR in the DI pituitary is below the level of detection in contrast to that in the LE (0.50 +/- 0.04 pmol/gland). Neuropeptide Y (NPY) levels are increased two-fold in the DI pituitary while AVP levels are below detection. The content of F-8-F-NH2-IR in the hypothalami and spinal cords of DI and LE rats is not statistically different, suggesting that the absence of F-8-F-NH2-IR in the Brattleboro pituitary is not due to a genetic defect in F-8-F-NH2 biosynthesis. The results of this study raise the question whether AVP could be involved in the regulation of F-8-F-NH2 immunoreactivity in the neurohypophysis.     

Vasopressin, corticosterone levels, and gastric ulcers during food-restriction stress.

Corticosterone levels and ulcers were compared in vasopressin-containing (LE) and vasopressin-deficient (DI) rats under ad lib and food-restricted conditions. In the ad lib situation, DI and LE rats had similar corticosterone levels and no ulcers. After 1 day of food restriction, the corticosterone levels were elevated in DI and LE rats, with a significantly higher level in LE rats. No ulcers were present in either strain. After 2 days of food restriction, the corticosterone levels were similar in DI and LE rats. The level in DI rats was comparable to that of the preceding day, but the level in LE animals dropped significantly from the previous day. Significant ulceration was evident in DI rats, but absent in LE rats. Following 3 days of food restriction, the corticosterone level in LE rats had returned to the ad lib level, whereas, for DI rats, an elevated level was maintained. There were no ulcers in LE rats, but they were present in DI rats. Thus LE and DI rats responded differently to the stress of food restriction. The mechanism underlying the response is most likely related to changes in the hypothalamic-pituitary-adrenocortical axis and its reaction to stress.     

Genetic selection for ethanol withdrawal severity: Differences in replicate mouse lines

We report an ongoing within-family selective breeding project for the severity of handling-induced withdrawal seizures in mice made physically dependent on ethanol by inhalation. Two Withdrawal Seizure Prone (WSP) and two Withdrawal Seizure Resistant (WSR) lines have been subjected to five generations of selection, and two control (WSC) lines are maintained. Each WSP line had more severe and each WSR line had less severe withdrawal convulsions than its respective WSC line. Differences relative to control lines were more pronounced in the WSP lines and were not due to differences in effective dose of ethanol. Heritabilities were higher in the WSP lines than in the WSR lines. These lines will be useful for studying physiological determinants of ethanol dependence and withdrawal.     

Increased sensitivity to NMDA is involved in alcohol-withdrawal induced cytotoxicity observed in primary cultures of cortical neurones chronically pre-treated with ethanol

Severe cellular damage and neuronal cell loss were previously observed in cultures of primary cortical neurones after chronic ethanol pre-treatment followed by ethanol-withdrawal. In this study, we investigated the circumstances and the possible cellular changes leading to alcohol-withdrawal induced neuronal cell death. When cultures were pre-treated with ethanol (25-200mM) once for 24 or 72h, the amount of the subsequent 24h alcohol-withdrawal induced cell death-estimated by measuring the release of lactate dehydrogenase (LDH)-was elevated only in cultures pre-treated with 200mM ethanol for 72h. On the contrary, as little as 50mM ethanol produced significant (P<0.01) increase in the withdrawal induced LDH-release in cultures pre-treated repeatedly with ethanol once daily for three consecutive days. When ethanol was re-added to the cultures during the withdrawal period, the LDH-release was dose-dependently reduced to the level of control. In ethanol pre-treated cultures N-methyl-D-aspartate (NMDA) (0.01-1mM) induced excitotoxicity as well as NMDA evoked elevation of cytosolic calcium ion concentration was increased. In contrast, the depolarising agent veratridine (0.01-1mM) produced similar extent of neuronal injury and elevation in cytosolic calcium ion concentration in control as in ethanol pre-treated cultures. According to these observations, repeated ethanol treatment appears to cause more robust adaptive changes in cultured neurones leading to more pronounced withdrawal induced cellular damage than chronic but single treatment does. In addition, the glutamatergic neurotransmission, especially the NMDA receptor system seems to be highly involved in the adaptive changes and in the cytotoxic effect of alcohol-withdrawal.     

Ethanol and nitrous oxide produce withdrawal-induced convulsions by similar mechanisms in mice

Twenty generations of selective breeding were used to produce lines (strains) of mice which differ markedly from one another in ethanol physical dependence development as indexed by handling-induced convulsions (HIC) induced by withdrawal from ethanol. These withdrawal seizure prone (WSP) and withdrawal seizure resistant (WSR) selection lines now differ by over 10-fold in HIC scores after equivalent exposure to intoxicating levels of ethanol via inhalation. Since handling-induced convulsions can be readily elicited following withdrawal from nitrous oxide, we sought to determine if the very large differences in ethanol withdrawal-induced HIC bred into these selection lines would generalize to nitrous oxide. Following a 60 min exposure to 75% nitrous oxide (in O2), a greater than 10-fold difference in HIC scores, and a 2-fold difference in tremor incidence was seen upon withdrawal in WSP vs. WSR mice. These findings closely parallel those seen with ethanol, and demonstrate that a large degree of commonality exists in the genes and the mechanisms determining these withdrawal signs. HIC elicited by nitrous oxide withdrawal were readily suppressed by ethanol, and HIC elicited by ethanol withdrawal were promptly suppressed by 75% nitrous oxide in WSP mice. Nitrous oxide also suppressed HIC and tremor associated with nitrous oxide withdrawal.     

Effect of vasopressin administration and deficiency upon 3H-AVP binding sites in the CNS and periphery during development.

Arginine8-vasopressin (AVP, 40 micrograms/100 g b.wt., SC) was administered to male Long-Evans (LE) pups from day 1 to 7 of life and the pups were sacrificed on day 8 or 60. 3H-AVP binding was performed on membranes prepared from the liver, kidney, and septum. No significant changes were observed in the kidney or septum of animals 8 or 60 days old. However, the chronic AVP treatment did result in a significant increase in the density of 3H-AVP binding sites in the liver when compared to control day 8 pups (control 44 +/- 2 vs. AVP 56 +/- 3 fmol/mg protein), with no change in affinity. This effect was maintained into adulthood, as the day 60 AVP-treated LE rats also showed a significant increase in liver 3H-AVP binding sites compared to control (control 186 +/- 9 vs. AVP 239 +/- 14 fmol/mg protein), with no change in affinity. A comparison of 3H-AVP binding sites in 8-day-old LE, heterozygous Brattleboro (HET-BB), and homozygous Brattleboro rats (HOM-BB) was performed to assess the effect of complete (HOM-BB) and partial (HET-BB) VP deficiency on binding sites in the CNS and periphery. The liver again was the only tissue in which a change in 3H-AVP binding characteristics was noted. The HOM-BB rat (Bmax 144 +/- 6 fmol/mg protein) displayed a significant increase in AVP binding sites from the LE rat (Bmax 100 +/- 7 fmol/mg protein), while the 3H-AVP binding sites in the HET-BB rat liver (Bmax 69.8 +/- 9 fmol/mg protein) were significantly lower than LE rats. Thus hepatic AVP receptors appear most sensitive to the presence or absence of vasopressin during the early postnatal period.     

Effects of vasopressin replacement during food-restriction stress

The effects of subcutaneous injections of vasopressin in vasopressin-deficient (Brattleboro or DI) rats were observed during nonstress (habituation) and stress (food-restriction) conditions as compared to other rats. Four groups of animals were employed: 1) Long-Evans (LE) rats that were food restricted with no injections (normal control animals), 2) DI rats that were food restricted with no injections, 3) DI rats injected with vasopressin, and 4) DI rats injected with peanut oil (vehicle). The parameters studied were: body weight, food intake, water intake, and gastric ulcer formation. With respect to body weight, water intake, and ulcer formation, two sets of animals emerged. The vasopressin-injected DI rats resembled the LE control rats, whereas the peanut oil-injected DI rats were similar to the DI rats with no injections. The former set of animals showed a higher body weight, reduced water intake, and fewer gastric ulcers than the latter set of animals. Thus the vasopressin-injected DI rats and the LE control rats could cope with the stress of food restriction, but the peanut oil-injected DI rats and the DI rats with no injections could not.     

Effects of prenatal ethanol exposure and sex on the arginine vasopressin response to hemorrhage in the rat

AVP synthesis, storage, and osmotically stimulated release are reduced in young adult rats exposed prenatally to ethanol (PE). Whether the reduced release of AVP to the osmotic stimulus is due to impairment of the vasopressin system or specifically to an osmoreceptor-mediated release is not known. The present experiments were done, therefore, to determine whether a hemorrhage-induced AVP response would also be diminished in PE-exposed rats. Pregnant rats were fed either a control liquid diet [no prenatal ethanol (NPE)] or a liquid diet with 35% of the calories from ethanol from days 7-21 of pregnancy. Offspring were weaned at 3 wk of life. At 11 wk of age, femoral arterial catheters were surgically placed, and blood volumes were determined at 12 wk. Three days later, two hemorrhages of 10% of the blood volume were performed with samples taken before and 10 min after the hemorrhages. After a 20% blood loss, plasma AVP was 19% higher in NPE rats than in the PE rats despite no differences in mean arterial blood pressure (MABP). Also, hypothalamic AVP mRNA and pituitary AVP content were reduced in PE rats. Furthermore, confirming an earlier report of sex differences in AVP release, the hemorrhage-induced hormone response was twofold greater in female rats than male rats, regardless of previous ethanol exposure. These studies demonstrate that the AVP response to hemorrhage is reduced in PE rats independently of differences in MABP. The data are compatible with a theory of a reduced number of hemorrhage-responsive vasopressinergic neurons capable of stimulated AVP release in PE rats.     

Chronic ethanol exposure potentiates the locomotor-activating effects of corticotropin-releasing factor (CRF) in rats

The effects of chronic exposure (21 days) to ethanol vapors on locomotor response to intracerebroventricular (i.c.v.) administration of corticotropin releasing factor (CRF) was investigated in male Wistar rats. Responses to CRF were tested during chronic exposure, 1 1/2 hours following removal of ethanol vapors, and two weeks after withdrawal of ethanol. A greater sensitivity to the locomotor-activating effects of CRF was found in ethanol-treated rats as compared to their controls during ethanol exposure (P less than 0.001) and 90 min following removal of ethanol vapors (P less than 0.001) but not two weeks following withdrawal. These results support clinical findings of a reversible activation in the hypothalamic-pituitary-adrenal (HPA) axis in alcoholism. In addition, it appears that chronic exposure to ethanol can also modify central neuronal systems specifically responsive to the locomotor activating effects of CRF.     

Effect of chronic vasopressin treatment on alcohol drinking of Brattleboro HZ and DI rats

Preference for alcohol was determined for three groups of male and female rats, 100–150 days old, comprised of: (1) Long Evans (LE); (2) LE-derived Brattleboro heterozygous (HZ); and (3) Brattleboro homozygous (DI) animals afflicted with diabetes insipidus due to vasopressin deficiency. Each alcohol drinking test was run over 11 days during which food, water and an ethyl alcohol solution, increased in concentration from 3% to 25%, were freely available. Following an initial preference screen, 100 milli-units of vasopressin tannate in oil was administered subcutaneously, during a second preference test, once per day to each animal. This treatment ameliorated the polydipsia-polyuria syndrome characteristic of the DI sub-strain of Brattleboro rat. Administration of the peptide to both the LE or HZ animals exerted no effect on g/kg intake nor on the proportional measure of alcohol to water. However, in the DI rat of either gender, vasopressin reduced the mean absolute gram intake of alcohol over concentrations to resemble that of the other LE and/or HZ groups. These results demonstrate that vasopressin serves to normalize the intake of alcohol in the DI rat by virtue of the elimination of the diabetic condition. However, since vasopressin fails to alter alcohol consumption of the HZ and LE rats, it would appear that this neuroactive peptide may play only a minor role in the CNS mechanisms governing the voluntary selection of alcohol.