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.     

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.     

Interaction of putative vasopressin receptors in rat brain and bovine pituitary gland with a vasopressin anti-idiotype antibody as revealed by immunoblotting

Vasopressin (AVP)-binding proteins were obtained from rat brain and the anterior and posterior lobes of bovine pituitary glands by (a) preparation of crude membranes, (b) solubilization of membrane proteins, (c) passage through an affinity column containing immobilized AVP, and (d) elution from the column with excess AVP. Gel electrophoresis revealed protein bands of 55 and 62 kilodaltons in rat brain, bovine posterior lobe and, to a far lesser extent, in the anterior lobe, which were similar to those previously identified in rat brain to be associated with AVP binding. Immunoblotting demonstrated that the 55 kilodalton bands of rat brain and bovine pituitary gland were selectively immunoreactive with an AVP anti-idiotype antibody. In addition, immunoreactivity occurred with a 62 kilodalton component of rat brain.     

Behavioral differences between vasopressin-deficient (Brattleboro) and normal Long-Evans rats

Behavioral comparisons were made between rats of the Brattleboro strain with hereditary hypothalamic diabetes insipidus (DI) and normal Long-Evans rats. Measurements were made of activity behavior in a lighted open field and in a darkened activity chamber. Subtle measurement specific differences in the activity behavior of DI rats were found which suggested altered emotion, motivation and/or attention in the DI rats. In terms of learned behavior, DI and normal rats displayed a similar degree of habituation to all within-session activity measures in both the open field and darkened activity chamber. In a passive avoidance test, DI rats exhibited a degree of avoidance behavior equivalent to that of normal animals. Thus, these studies provide evidence that the vasopressin-deficient rat is not defective in learning and memory processes. The data can be interpreted as suggesting that vasopressin may influence memory tasks by extrinsic modulation of related states of emotionality, motivation and/or attention rather than by direct involvement in the retrieval and consolidation of information.     

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.     

Effect of vasopressin on open field and activity behavior of the vasopressin-deficient (Brattleboro) rat

The effect of 1 and 5 micrograms AVP injections on open field and photoactivity chamber behavior of D.I. and normal Long-Evans animals was studied. Administration of 5 micrograms AVP (SC) resulted in a statistically significant depression of both open field and photochamber activity in the D.I. rat, but had a less pronounced effect on normal animals. However, 1 microgram AVP resulted in only minor alterations of activity in both D.I. and normal animals. In terms of learned behavior, D.I. and normal animals displayed similar within-session habituation when comparisons were made following the same treatment conditions. Thus, this study supports the hypothesis that vasopressin may influence memory tasks by modulation of related states of emotionality, motivation, and/or attention rather than by direct involvement in the retrieval and/or consolidation of information.     

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.     

Vasopressin neurons grafted into Brattleboro rats: Viability and activity

Blocks of the anterior hypothalamus containing vasopressin neurons were grafted from normal 17-day-old rat fetuses into the median eminence of adult female rats with a congenital deficiency of vasopressin neurons (Brattleboro strain rats). Immunocytochemical staining of the transplants 40 days after grafting demonstrated the presence of magnocellular neurons which stained positively for vasopressin and neurophysin. Axons from these neurons could be traced into the median eminence and the primary capillary plexus of the hypothalamo-hypophyseal portal system. Water consumption decreased by as much as 63% in animals carrying viable grafts. The observation that water consumption decreased and remained depressed in hosts carrying viable grafts along with the immunocytochemical data suggest that the transplanted neurons are synthesizing, storing, and releasing biologically active VP.     

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.     

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.     

Simultaneous monoamine histofluorescence and neuropeptide immunocytochemistry: III. Ontogeny of catecholamine varicosities and neurophysin neurons in the rat supraoptic and paraventricular nuclei

The ontogeny of the rat supraoptic (SON) and paraventricular (PVN) nuclei was studied using a combined fluorescence-immunocytochemical technique for the simultaneous localization of catecholamines (CA) and neurophysin (NP). NP neurons and CA varicosities were first detected in the SON and PVN at 17 days postcoitus. The development of NP neurons which included increases in immunoreactivity in both nuclei proceeded through fetal and neonatal stages, approaching maturity by 21–28 days postnatal; the maturation of the PVN lagged behind that of the SON. CA varicosities appeared to make contact with NP neurons beginning at 21–22 days postcoitus. An apparent increase in varicosity-perikaryal contacts with age was observed in both nuclei; by 14–21 days postnatal adult-like patterns were established. The prenatal dominance of NP stain relative to CA fluorescence may suggest a possible neurotrophic role for magnocellular neurons and/or their products upon ingrowing noradrenergic axons.     

Widespread alterations in central noradrenaline,dopamine, and serotonin systems in the Brattleboro rat not related to the local absence of vasopressin

A comprehensive study of monoamine transmitter and metabolite concentrations measured by HPLC was undertaken in female (vasopressin-deficient) Brattleboro rats as compared to Long Evans rats. Noradrenaline was significantly increased in 8 out of 13 dissected brain regions, whereas concentrations of the metabolite 3-methoxy-4-hydroxyphenylglycol were not altered. The increases were not restricted to areas which are normally innervated by vasopressin-containing neurons. Serotonin was increased in 6 and dopamine in 4 regions and this was accompanied in some areas by increases in the metabolites 5-hydroxyindolacetic acid and dihydroxyphenylacetic acid. Only in the striatum, cerebellum, and the medulla-pons no changes could be detected in any of the compounds of interest. These results show that the long term absence of vasopressin in Brattleboro rats appears to be associated with increases in monoamine transmitter contents and decreased metabolite/transmitter ratios. The regional distribution of these changes does not bear any relationship to the regional distribution of vasopressin cell bodies or nerve endings.     

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

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

Granule populations in oxytocin and abnormal perikarya of the supraoptic nucleus of homozygous Brattleboro rats: Effects of colchicine administration

Summary Magnocellular neurones in the supraoptic nucleus of the homozygous Brattleboro rat, which are unable to produce vasopressin, were investigated by immunocytochemistry to identify both the oxytocin cells and the abnormal neurones, which in normal animals would produce vasopressin. The abnormal cell profiles were significantly more rounded than those of the oxytocin cells. Both cell types showed evidence of hyperactivity, but the Golgi apparatus was more extensive in the oxytocin cells, probably as a result of the failure of the abnormal cells to produce vasopressin and its neurophysin and the resultant reduction in hormone packaging. Neurosecretory granules (NSG) 160 nm in diameter were found in the oxytocin perikarya but were absent from the abnormal cell bodies. In addition, a population of small dense granules (SDG) 100 nm in diameter was observed in both types of neurone, in numbers equal to the NSG in oxytocin cells.Injection of a low, non-lethal dose of the axonal transport inhibitor colchicine resulted in a rapid and equal accumulation of both NSG and SDG in oxytocin perikarya and of SDG in the abnormal perikarya after one day. The effects of colchicine were reversed 2–3 days after administration. The SDG, which may contain a co-transmitter or co-hormone substance, are thus produced at a similar rate to NSG, and appear to be transported from the perikarya for subsequent release at the nerve endings.     

Immuno-cytochemical demonstration of the inability of the homozygous Brattleboro rat to synthesize vasopressin and vasopressin-associated neurophysin

Summary Immuno-enzyme cytochemical investigations have shown that, (1) the hypothalamic supraoptic and paraventricular nuclei of the Brattleboro rat, as in the normal rat, contain separate neurons which produce oxytocin + neurophysin; (2) the hereditary inability of the Brattleboro rat to synthesize vasopressin and its associated neurophysin is due to a biochemical defect of separate neurophysin-vasopressin neurons in the supraoptic and the paraventricular nuclei. These observations strongly support the hypotheses that (1) vasopressin and its associated neurophysin are formed via a common precursor, and (2) the initial point of intracellular appearance of the hereditary defect in the Brattleboro rat lies in the synthesis of this precursor, which occurs on ribosomes.Moreover, observations have demonstrated that, in the Brattleboro rat, in addition to the hereditary inability of the hypothalamic magnocellular neurosecretory system to synthesize vasopressin, there also exists a similar hereditary defect in the hypothetical parvicellular suprachiasmatic-median eminence neurosecretory system.This paper is dedicated to Professor Dr. W. Bargmann, in honour of his 70th birthday.Presented in part at the meeting of the Belgian Society of Endocrinology May 17, 1975 (Vandesande et al., 1975d).     

The effects of lactation on impulsive behavior in vasopressin-deficient Brattleboro rats

Vasopressin (AVP)-deficient Brattleboro rats develop a specific behavioral profile, which—among other things—include altered cognitive performance. This profile is markedly affected by alterations in neuroendocrine state of the animal such as during lactation. Given the links between AVP and cognition we hypothesized that AVP deficiency may lead to changes in impulsivity that is under cognitive control and the changes might be altered by lactation. Comparing virgin and lactating AVP-deficient female Brattleboro rats to their respective controls, we assessed the putative lactation-dependent effects of AVP deficiency on impulsivity in the delay discounting paradigm. Furthermore, to investigate the basis of such effects, we assessed possible interactions of AVP deficiency with GABAergic and serotonergic signaling and stress axis activity, systems playing important roles in impulse control. Our results showed that impulsivity was unaltered by AVP deficiency in virgin rats. In contrast a lactation-induced increase in impulsivity was abolished by AVP deficiency in lactating females. We also found that chlordiazepoxide-induced facilitation of GABAergic and imipramine-induced enhancement of serotonergic activity in virgins led to increased and decreased impulsivity, respectively. In contrast, during lactation these effects were visible only in AVP-deficient rats. These rats also exhibited increased stress axis activity compared to virgin animals, an effect that was abolished by AVP deficiency. Taken together, AVP appears to play a role in the regulation of impulsivity exclusively during lactation: it has an impulsivity increasing effect which is potentially mediated via stress axis-dependent mechanisms and fine-tuning of GABAergic and serotonergic function.     

Ultrastructural characteristics of vasopressin-containing neurons in the paraventricular nucleus of the hypothalamus

Summary Vasopressin-containing neurons, identified by immunocytochemistry, are located predominantly in the posterior magnocellular division of the paraventricular nucleus of the rat hypothalamus. By electron microscopy, the immunoreaction product is seen within the cell bodies and neuronal processes. In the perikarya and dendritic processes, the immunoreactive material is associated primarily with neurosecretory granules. Axonal processes, identified by their content of microtubules and accumulation of neurosecretory granules, show the immunoreaction product in association with both of these organelles. Afferent axo-dendritic, axo-somatic and putative axo-axonic synapses with immunostained vasopressinergic neurons can be identified. The presynaptic profiles do not contain immunoreactive material. This study contributes to the ultrastructural characterization of vasopressinergic neurons in the paraventricular nucleus and of their afferent synaptic input.Supported by NIH Grants HD-12956 and 2SO7RR05403     

Catecholamine distribution and relationship to magnocellular neurons in the paraventricular nucleus of the rat

Summary The distribution of catecholamine synthesizing enzymes within the paraventricular nucleus of the rat hypothalamus is elucidated immunocytochemically by use of antibodies to tyrosine hydroxylase, dopamine -hydroxylase, and phenylethanolamine-N-methyltransferase. Tyrosine hydroxylase-immunostained cell bodies are localized in the periventricular stratum and adjacent parvocellular regions, but rarely in magnocellular subnuclei of the paraventricular nucleus. Tyrosine hydroxylase-immunostained fibers are present in greatest density in the periventricular zone, and moderate density in the parvocellular and magnocellular subnuclei. Dopamine -hydroxylase-immunostained fibers are remarkably dense in the posterior magnocellular division of the paraventricular nucleus, especially in the dorso-lateral portion where vasopressin-containing cells predominate. Noradrenergic fiber input to these magnocellular neurons is likely since phenylethanolamine-N-methyltransferase-immunostained fibers are sparse in magnocellular subnuclei of the paraventricular nucleus. Dual immunocytochemical staining of thick and thin tissue sections demonstrates with clarity an anatomical association of dopamine -hydroxylase-immunostained fibers and magnocellular neurons. Dopamine -hydroxylase-immunostained and phenylethanolamine-N-methyltransferase-immunostained fibers are dense in the medial parvocellular component of the paraventricular nucleus; distinct features of both antisera are presented.     

Coexistence of NADPH-diaphorase with vasopressin and oxytocin in the hypothalamic magnocellular neurosecretory nuclei of the rat

Coexistence of NADPH-diaphorase with vasopressin and oxytocin was studied in the magnocellular neurosecretory nuclei of the rat hypothalamus by use of sequential histochemical and immunocytochemical techniques in the same sections. Coexistence was found in all the nuclei examined (supraoptic, paraventricular, circular, fornical, and in some isolated neurons located in the hypothalamic area between the paraventricular and supraoptic nuclei). The ratios of neurons expressing both markers (NADPH-diaphorase and vasopressin, NADPH-diaphorase and oxytocin) in each of the nuclei were very similar. Although further studies must be carried out, the partial coexistence found in all nuclei suggests that NADPH-diaphorase is probably not related to general mechanisms involving vasopressin and oxytocin, but rather in specific functions shared by certain hypothalamic neuronal cell populations.     

Establishment of internal-image anti-idiotype monoclonal antibodies to a human antibody to lung cancer

 Internal-image anti-idiotype antibodies are expected to enhance anticancer effector mechanisms in vivo. The objective of this study was to establish hybridomas producing anti-idiotype monoclonal antibodies against a human monoclonal antibody (hmAb) 4G12 that reacts strongly with lung squamous cell carcinomas. BALB/c female mice 6 weeks old were immunized with 4G12. Splenocytes were hybridized with P3U1 cells and hybrid cells secreting anti-4G12 hmAb were cloned. Two clones reacted with 4G12 hmAb but not with 3H12 IgM hmAb, human IgM, human serum or fetal calf serum. These two Ab2 antibodies (IgG1κ) 2B12 and 2H1 demonstrated 91.5% and 90.3% inhibition in their reactivity with radiolabelled 4G12 on PC10 cells, indicating that 2B12 and 2H1 antibodies were of the Ab2β type. In criss-cross inhibition assays, the binding of 2B12 or 2H1 to 4G12 was not inhibited by 2H1 or 2B12. Thus 2B12 and 2H1 were thought to recognize the different epitopes on the antigen-binding sites. Antisera against 2B12 and 2H1 demonstrated specific reactivity to PC10 cells. The two Ab2β antibodies, 2B12 and 2H1, express internal images of lung squamous cell carcinoma recognized by the 4G12 antibody and may be useful for cancer immunotherapy. Received: 20 September 1996 / Accepted: 2 January 1997