Stephen J. Lolait

Vasopressin V1b receptor knockout reduces aggressive behavior in male mice

Increased aggression is commonly associated with many neurological and psychiatric disorders. Current treatments are largely empirical and are often accompanied by severe side effects, underscoring the need for a better understanding of the neural bases of aggression. Vasopressin, acting through its 1a receptor subtype, is known to affect aggressive behaviors. The vasopressin 1b receptor (V1bR) is also expressed in the brain, but has received much less attention due to a lack of specific drugs. Here we report that mice without the V1bR exhibit markedly reduced aggression and modestly impaired social recognition. By contrast, they perform normally in all the other behaviors that we have examined, such as sexual behavior, suggesting that reduced aggression and social memory are not simply the result of a global deficit in sensorimotor function or motivation. Fos-mapping within chemosensory responsive regions suggests that the behavioral deficits in V1bR knockout mice are not due to defects in detection and transmission of chemosensory signals to the brain. We suggest that V1bR antagonists could prove useful for treating aggressive behavior seen, for example, in dementias and traumatic brain injuries.

Distribution of mRNA encoding B78/apj, the rat homologue of the human APJ receptor, and its endogenous ligand apelin in brain and peripheral tissues.

The human APJ receptor is a G protein-coupled receptor which functions as an efficient alternative co-receptor for a number of human immunodeficiency virus type 1 and simian immunodeficiency virus strains. We have cloned the rat APJ receptor, which we term B78/apj, and have mapped the mRNA distribution of both the receptor and its natural ligand apelin in rat tissues. Northern blot analysis showed a similar pattern of expression for B78/apj and apelin mRNAs with hybridising transcripts seen in the lung, heart, skeletal muscle, kidney, brain and liver. In situ hybridisation histochemistry studies revealed intense B78/apj gene expression in the parenchyma of the lung, a sub-population of glomeruli in the kidney, the corpora lutea of the ovary and isolated cells of the anterior lobe of the pituitary. B78/apj mRNA had a striking and unique distribution within the central nervous system (CNS) where receptor expression was found in cells within the meninges around the brain, in the posterior magnocellular and medial parvocellular areas of the hypothalamic paraventricular nucleus and in the supraoptic nucleus. This hypothalamic distribution offers a possible specific role of this receptor in mediating neuroendocrine responses in the CNS.

Localisation of GPR30, a novel G protein-coupled oestrogen receptor, suggests multiple functions in rodent brain and peripheral tissues

Recently, the G protein-coupled receptor GPR30 has been identified as a novel oestrogen receptor (ER). The distribution of the receptor has been thus far mapped only in the rat central nervous system. This study was undertaken to map the distribution of GPR30 in the mouse brain and rodent peripheral tissues. Immunohistochemistry using an antibody against GPR30 revealed high levels of GPR30 immunoreactivity (ir) in the forebrain (e.g. cortex, hypothalamus and hippocampus), specific nuclei of the midbrain (e.g. the pontine nuclei and locus coeruleus) and the trigeminal nuclei and cerebellum Purkinje layer of the hindbrain in the adult mouse brain. In the rat and mouse periphery, GPR30-ir was detected in the anterior, intermediate and neural lobe of the pituitary, adrenal medulla, renal pelvis and ovary. In situ hybridisation histochemistry using GPR30 riboprobes, revealed intense hybridisation signal for GPR30 in the paraventricular nucleus and supraoptic nucleus (SON) of the hypothalamus, anterior and intermediate lobe of the pituitary, adrenal medulla, renal pelvis and ovary of both rat and mouse. Double immunofluorescence revealed GPR30 was present in both oxytocin and vasopressin neurones of the paraventricular nucleus and SON of the rat and mouse brain. The distribution of GPR30 is distinct from the other traditional ERs and offers an additional way in which oestrogen may mediate its effects in numerous brain regions and endocrine systems in the rodent.

Stress‐Specific Regulation of Corticotropin Releasing Hormone Receptor Expression in the Paraventricular and Supraoptic Nuclei of the Hypothalamus in the Rat

Corticotropin releasing hormone (CRH), a major regulator of pituitary ACTH secretion, also acts as a neurotransmitter in the brain. To determine whether CRH is involved in the regulation of hypothalamic function during stress, CRH receptor binding and CRH receptor mRNA levels were studied in the hypothalamus of rats subjected to different stress paradigms: immobilization, a physical‐psychological model; water deprivation and 2% saline intake, osmotic models; and i.p. hypertonic saline injection, a combined physical‐psychological and osmotic model. In agreement with the distribution of CRH receptor binding in the brain, in situ hybridization studies using 35S‐labeled cRNA probes revealed low levels of CRH receptor mRNA in the anterior hypothalamic area, which were unaffected after acute or chronic exposure to any of the stress paradigms used. Under basal conditions, there was no CRH binding or CRH receptor mRNA in the supraoptic (SON) or paraventricular (PVN) nuclei. However, 2 h after the initiation of acute immobilization, CRH receptor mRNA hybridization became evident in the parvicellular division of the PVN, with levels substantially increasing from 2 to 4 h, decreasing at 8 h and disappearing by 24 h. Identical hybridization patterns of CRH receptor mRNA were found in the parvicellular PVN after repeated immobilization; levels were similar to those after 2 h single stress following immobilization at 8‐hourly intervals for 24 h (3 times), and very low, but clearly detectable 24 h after 8 or 14 days daily immobilization for 2 h. On the other hand, water deprivation for 24 or 60 h and intake of 2% NaCI for 12 days induced expression of CRH receptor mRNA in the SON and magnocellular PVN, but not in the parvicellular pars of the PVN. Both parvicellular and magnocellular hypothalamic areas showed CRH receptor mRNA following i.p. hypertonic saline injection, single (4 h after) or repeated at 8‐hourly intervals for 24 h (3 injections), or one injection daily for 8 or 14 days. Consistent with the expression of CRH receptor mRNA, autoradiographic studies showed binding of 125I‐Tyr‐oCRH in the parvicellular division of the PVN after immobilization; in the magnocellular division of the PVN after osmotic stimulation, and in the PVN and SON after i.p. hypertonic saline injection. The data show that stress‐specific activation of the parvicellular and magnocellular systems is associated with CRH receptor expression, and suggest a role for CRH in the autoregulation of hypothalamic function.

Social motivation is reduced in vasopressin 1b receptor null mice despite normal performance in an olfactory discrimination task

In this study, we characterized more thoroughly the social behavior of vasopressin 1b receptor null (V1bR-/-) mice. We confirmed that V1bR-/- males exhibit less social aggression than their wild-type (V1bR+/+) littermates. We tested social preference by giving male subjects a choice between pairs of soiled or clean bedding. In general, V1bR+/+ mice spent significantly more time engaged in chemoinvestigation of these social stimuli than V1bR-/- mice. Male V1bR+/+ mice preferred female-soiled bedding over male-soiled bedding, male-soiled bedding over clean bedding, and female-soiled bedding over clean bedding. In contrast, V1bR-/- males failed to exhibit a preference for any bedding. This difference in behavior is not explained by an anosmic condition as there were no differences between V1bR-/- and V1bR+/+ mice in their abilities to detect a cookie buried in clean bedding, or in their ability to perform in an operant conditioning task using a fully automated liquid dilution olfactometer. In the latter task, male V1bR-/- mice were fully capable of discriminating between male and female mouse urine. The latencies to learn this task did not differ between the two genotypes. Thus, a V1bR-/- males ability to differentiate between male and female chemosensory cues appears no different than that of a V1bR+/+ males. We propose that the V1bR plays an important role in social motivation, perhaps by coupling the processing, integration, and/or interpretation of chemosensory cues with the appropriate behavioral response.

Molecular neurobiology and pharmacology of the Vasopressin/Oxytocin receptor family

Summary1. VP and OT mediate their wealth of effects via 4 receptor subtypes V1a, V1b, V2, and OT receptors.2. We here review recent insights in the pharmacological properties, structure activity relationships, species differences in ligand specificity, expression patterns, and signal transduction of VP/OT receptor.3. Furthermore, the existence of additional VP/OT receptor subtypes is discussed.

Regulation of Pituitary Vasopressin V1b Receptor mRNA during Stress in the Rat

Previous studies have shown a parallel relationship between pituitary vasopressin (VP) receptor content and responsiveness of the corticotroph during chronic stress. The regulation of pituitary VP receptors was further studied by analysis of V1b VP receptor mRNA levels in pituitaries of rats subjected to chronic immobilization, i.p. hypertonic saline injection (physical stress paradigms associated with increased pituitary responsiveness), and water deprivation, or to 2% saline in the drinking water (osmotic stress paradigms associated with decreased pituitary responsiveness). Northern blot hybridization with a 363 bp 32P‐labelled fragment of the rV1b receptor cDNA coding sequence revealed two bands of about 3.7 and 3.2 Kb, whereas a probe directed to the 5′ untranslated region recognized only the 3.7 Kb band. Repeated i.p. hypertonic saline injection, 3 times in 24 h at 8 h intervals, or daily for 8 days, increased the intensity of the 3.7 Kb band by 155 ± 17.5% (P<0.01) and 118 ± 14.6% (P<0.01), respectively, while the 3.2Kb band increased by 122 ± 39.3% (P<0.01) only after 3 times injection. Smaller increases of 39 ± 11 and 33 ± 9% (P<0.05) in the 3.7 Kb band were found after repeated immobilization 3 times in 24 h and 2 h for for 8 days respectively. In situ hybridization studies confirmed significant increases (P<0.05) in V1b receptor mRNA levels after 8 and 14 days repeated immobilization (63 ± 19% and 83 ± 10%) or i.p. hypertonic saline injection (110 ± 13% and 73 ± 20%). In response to acute stress, V1b receptor mRNA increased by 77 ± 5% (3.7 Kb band) after 4 h immobilization for 1 h, whereas both bands were reduced by 49 ± 5% and 45 ± 5%, 4 h after a single i.p. hypertonic saline injection. The decrease in V1b receptor mRNA following a single i.p. hypertonic saline injection was prevented by pretreatment with a V1 receptor antagonist, suggesting that increased VP secretion may account for this effect. In spite of the decrease in V1 b receptor mRNA following i.p. hypertonic saline injection, VP binding in pituitary membrane rich fractions, and VP‐stimulated inositol phosphate formation in quartered hemipituitaries were increased by 24 and 39%, respectively. V1b receptor mRNA levels were unchanged or decreased following prolonged osmotic stimulation.

APJ receptor mRNA expression in the rat hypothalamic paraventricular nucleus: Regulation by stress and glucocorticoids

The apelin receptor (APJ receptor, APJR) has recently come to prominence following the isolation and identification of its endogenous ligand, apelin, from bovine stomach tissue extracts. Investigation of APJR mRNA expression has revealed a hypothalamic distribution similar to that of vasopressin suggesting that the apelin–APJR system may be involved in the regulation of the hypothalamic‐adrenal‐pituitary (HPA) stress axis. To investigate whether APJR is involved in the regulation of hypothalamic function during stress, APJR mRNA expression levels were measured by in situ hybridization in the hypothalamus of rats subjected to acute and repeated restraint stress. Acute stress caused an increase in APJR mRNA expression in the hypothalamic parvocellular paraventricular nucleus (pPVN) while repeated restraint stress induced a sustained up‐regulation of pPVN APJR mRNA expression in intact rats. Removal of endogenous glucocorticoids by adrenalectomy also resulted in an increased expression of APJR mRNA in the PVN, suggesting a negative regulation of APJR mRNA expression by glucocorticoids. The role of glucocorticoids in mediating these stress‐induced changes was investigated by analysing the effects of acute and repeated restraint stress on APJR mRNA levels in adrenalectomized rats. In these rats, APJR mRNA expression levels did not change above the already elevated levels of adrenalectomized‐control rats. These data suggest that acute and repeated stress exert a stimulatory influence on APJR mRNA expression at the hypothalamic level that may be dependent on basal levels of circulating glucocorticoids, and further suggest a role for APJR in the regulation of hypothalamic function.

The vasopressin Avpr1b receptor: molecular and pharmacological studies

The distribution, pharmacology and function of the arginine vasopressin (Avp) 1b receptor subtype (Avpr1b) has proved more challenging to investigate compared to other members of the Avp receptor family. Avp is increasingly recognised as an important modulator of the hypothalamic–pituitary–adrenal (HPA) axis, an action mediated by the Avpr1b present on anterior pituitary corticotrophs. The Avpr1b is also expressed in some peripheral tissues including pancreas and adrenal, and in the hippocampus (HIP), paraventricular nucleus and olfactory bulb of the rodent brain where its function is unknown. The central distribution of Avpr1bs is far more restricted than that of the Avpr1a, the main Avp receptor subtype found in the brain. Whether Avpr1b expression in rodent tissues is dependent on differences in the length of microsatellite dinucleotide repeats present in the 5′ promoter region of the Avpr1b gene remains to be determined. One difficulty of functional studies on the Avpr1b, especially its involvement in the HPA axis response to stress, which prompted the generation of Avpr1b knockout (KO) mouse models, was the shortage of commercially available Avpr1b ligands, particularly antagonists. Research on mice lacking functional Avpr1bs has highlighted behavioural deficits in social memory and aggression. The Avpr1b KO also appears to be an excellent model to study the contribution of the Avpr1b in the HPA axis response to acute and perhaps some chronic (repeated) stressors where corticotrophin-releasing hormone and other genes involved in the HPA axis response to stress do not appear to compensate for the loss of the Avpr1b.

Abnormal fluid homeostasis in apelin receptor knockout mice.

The apelinergic system, comprised of apelin and its G protein-coupled receptor (APJ; APLNR as given in MGI Database), is expressed within key regions of the central nervous system associated with arginine vasopressin (AVP) synthesis and release as well as in structures involved in the control of drinking behaviour, including the magnocellular neurones of the hypothalamus, circumventricular organs, and the pituitary gland. This localisation is indicative of a possible functional role in fluid homeostasis. We investigated a role for APJ in the regulation of fluid balance using mice deficient for the receptor. Male APJ wild-type and knockout (APJ−/−) mice were housed in metabolic cages to allow determination of water intake and urine volume and osmolality. When provided with free access to water, APJ−/− mice drank significantly less than wild-types, while their urine volume and osmolality did not differ. Water deprivation for 24 h significantly reduced urine volume and increased osmolality in wild-type but not in APJ−/− mice. Baseline plasma AVP concentration increased comparably in both wild-type and APJ−/− mice following dehydration; however, APJ−/− mice were unable to concentrate their urine to the same extent as wild-type mice in response to the V2 agonist desmopressin. Analysis of c-fos (Fos as given in MGI Database) mRNA expression in response to dehydration showed attenuation of expression within the subfornical organ, accentuated expression in the paraventricular nucleus, but no differences in expression in the supraoptic nucleus nor median pre-optic nucleus in APJ−/− mice compared with wild-type. These findings demonstrate a physiological role for APJ in mechanisms of water intake and fluid retention and suggest an anti-diuretic effect of apelin in vivo.