Attenuated stress response to acute lipopolysaccharide challenge and ethanol administration in vasopressin V1b receptor knockout mice

The arginine vasopressin (Avp) 1b receptor (Avpr1b) present on anterior pituitary corticotrophs is involved in the stimulation of adrenocorticotrophic hormone (ACTH) secretion, especially during times of stress. Corticotrophin-releasing hormone (CRH) is considered the major ACTH secretagogue during acute stress whereas Avp appears to be the more dominant mediator of the hypothalamic-pituitary-adrenal (HPA) axis response during chronic stress situations. To investigate the role of the Avpr1b in the HPA axis response to acute stress, we measured ACTH and corticosterone (CORT) plasma levels in Avpr1b knockout (KO) mice and wild-type controls in response to bacterial lipopolysaccharide (LPS) challenge and ethanol (EtOH) administration. Mice deficient in Avpr1b had markedly compromised plasma ACTH and CORT responses to acute (30 min) LPS, but normal ACTH and CORT response to more extended exposure (4 h) to the immune system activator. The plasma ACTH and CORT levels stimulated by intoxicating, sedative doses of EtOH (3.2 and 4 g/kg) were significantly decreased in the Avpr1b KO mice compared to wild-type littermates. Significantly higher EtOH-induced plasma ACTH and CORT secretion was measured in female than in male Avpr1b wild-type mice. There were no differences in the blood alcohol levels following acute EtOH administration in Avpr1b KO or wild-type mice of either gender. Our results clearly suggest that Avpr1b plays a significant role in the HPA axis response to acute immune stress and EtOH intoxication.     

The role of the arginine vasopressin Avp1b receptor in the acute neuroendocrine action of antidepressants

In times of stress the hypothalamic-pituitary-adrenal (HPA) axis is activated and releases two neurohormones, corticotropin-releasing hormone (Crh) and arginine vasopressin (Avp), to synergistically stimulate the secretion of adrenocorticotropin hormone (ACTH) from the anterior pituitary, culminating in a rise in circulating glucocorticoids. Avp mediates its actions at the Avp V1b receptor (Avpr1b) present on pituitary corticotropes. Dysregulation of the stress response is associated with the pathophysiology of depression and a major treatment involves increasing the availability of monamines at the synaptic cleft. Acute administration of selective serotonin reuptake inhibitors (SSRI) and tricyclic antidepressants (TCA) has previously been shown to activate the HPA axis. The present study was undertaken to evaluate the involvement of the Avpr1b in the HPA axis response to acute SC administration of an SSRI (fluoxetine 10mg/kg) and a TCA (desipramine 10mg/kg). We measured plasma ACTH and corticosterone (CORT) levels and neuropeptide mRNA expression in the hypothalamic paraventricular nucleus (PVN) of Avpr1b knockout (KO) mice and wild-type controls. Fluoxetine and desipramine administration significantly attenuated plasma ACTH and CORT levels in male and female Avpr1b KO mice when compared to their wild-type counterparts. Avp, oxytocin (Oxt) and Crh mRNA expression in the PVN did not change in fluoxetine-treated male Avpr1b KO or wild-type mice. In contrast, fluoxetine treatment increased PVN Avp mRNA levels in female Avpr1b wild type but not KO animals. PVN Oxt mRNA levels increased in fluoxetine-treated female mice of both genotypes. The data suggests that the Avpr1b is required to drive the HPA axis response to acute antidepressant treatment and provides further evidence of a sexual dichotomy in the regulation of PVN Avp/Oxt gene expression following antidepressant administration.     

Corticosterone differentially modulates expression of corticotropin releasing factor and arginine vasopressin mRNA in the hypothalamic paraventricular nucleus following either acute or repeated restraint stress

Exposing rats to repeated restraint stress induces well-characterized adaptations in the expression of either corticotropin-releasing factor (CRF) or arginine-vasopressin (AVP) mRNA in the parvocellular neurons of the hypothalamic paraventricular nucleus (PVN). The effects of regulating corticosterone levels on this adaptation was studied in male rats. In intact rats, acute restraint stress increased the expression of CRF mRNA whilst AVP mRNA expression was no different to control. Repeated exposure resulted in habituation of CRF expression, whereas AVP mRNA increased above that seen in either non stressed or acutely stressed animals. In adrenalectomised rats with replacement pellets of corticosterone that replicated blood levels approximating to the daily trough (mean levels 37--65 ng/mL), basal CRF expression levels were raised, but the response to acute stress was still observed. However, the habituation seen in normal animals that had been repeatedly stressed was prevented, so that CRF mRNA levels continued to be raised after repeated stress. By contrast, the AVP response to both acute and repeated stress was unaltered in these low-dose corticosterone-treated rats compared with controls. Higher dose pellets, which resulted in blood levels around those of the daily maximum (mean 118--141 ng/mL) had the opposite effects. There was no change compared to intact rats in the expression of CRF mRNA following either acute or repeated stress, but the expected increase in AVP following repeated restraint was prevented. These experiments show that corticosterone has important modulating effects on the adaptive pattern of both CRF and AVP mRNA expression in the parvocellular PVN. The 'set-point' of corticosterone differs; for CRF, experiencing higher levels is necessary for subsequent adaptation to repeated restraint to occur, whereas for AVP a return to lower levels is necessary to allow this peptide to respond to repeated stress.     

The discovery of novel vasopressin V1b receptor ligands for pharmacological, functional and structural investigations

Until recently, pharmacological studies dealing with vasopressin receptor isoforms were severely hampered by the lack of selective agonists or antagonists that recognize the pituitary V(1b) vasopressin receptor. By contrast, many selective vasopressin-related compounds are available for characterization of the vasopressor (V(1a)) or antidiuretic (V(2)) vasopressin receptor subtypes. Recently, SSR149415, a selective nonpeptide molecule, was discovered with nanomolar affinity for mammalian V(1b) receptors and good selectivity for the other vasopressin and oxytocin receptor isoforms. This molecule exhibits potent antagonist properties both in vitro and in vivo. We also designed synthetic peptides derived from [deaminocysteine(1),arginine(8)]vasopressin (dAVP), modified in position 4 by various amino acid residues. Some of these, d[cyclohexylalanine(4)]AVP or d[lysine(4)]AVP, have a high affinity and an excellent selectivity for the human V(1b) receptor subtype. However, they exhibit a mixed V(1b)/V(2) pharmacological profile for the rat vasopressin receptor isoforms. Whatever the species considered, these peptides behave as agonists both in bioassays performed in vitro and in vivo. The d[cyclohexylalanine(4)]AVP was tritiated and represents the first selective radiolabelled ligand available for studying the human V(1b) receptors. The discovery of these new selective V(1b) agonists and V(1b) antagonist allows an accurate pharmacological characterization of all the vasopressin receptor isoforms. As emphasized in this review, attention to the vasopressin and oxytocin receptor species differences is of critical importance in studies with all vasopressin and oxytocin ligands.     

Overexpression of the V3 vasopressin receptor in transgenic mice corticotropes leads to increased basal corticosterone

The vasopressin V3 receptor (V3) is specifically expressed in pituitary corticotropes and mediates the stimulatory effect of vasopressin on adrenocorticotropic hormone (ACTH) release. The V3 gene is overexpressed in corticotrope pituitary tumours compared to normal pituitaries. We hypothesized that V3 overexpression might induce changes in corticotrope function and alter the regulation of the hypothalamic-pituitary-adrenal axis. Thus, we generated transgenic mice (POMV3) expressing the human V3 receptor in the pituitary under the control of rat pro-opiomelanocortin (POMC) promoter sequences. The transgene was efficiently transcribed and vasopressin binding was increased in both corticotropes and melanotropes. In-vitro ACTH release and inositol phosphate formation were unchanged in POMV3 pituitaries, but the responses to vasopressin were significatively increased. In vivo, basal circulating concentrations of ACTH in POMV3 mice were similar to those of controls but corticosterone concentrations were moderately increased. In addition, the levels of POMC mRNA in the transgenic pituitaries were comparable to those of control mice. Finally, POMV3 mice responded with a similar maximal increase of ACTH and corticosterone to a 20-min acute restraint stress. Together, these results show that hypophyseal V3 overexpression led to increased basal concentrations of corticosterone and suggest that the negative glucocorticoid feedback may be altered at the pituitary level.     

Expression of the arginine vasopressin gene in response to salt loading in oxytocin gene knockout mice

Accumulating evidence suggests that both oxytocin and arginine vasopressin (AVP) are vital components in the regulation of body fluid balance. However, the physiological role of oxytocin and possible cooperative interactions between oxytocin and AVP in sodium balance remain obscure, even though recent studies using oxytocin knockout (OTKO) mice suggested that oxytocin may contribute to the regulation of salt appetite. In the present study, we examined the effects of salt loading (drinking 2% NaCl for 5 days) on the expression of the AVP gene in the paraventricular (PVN) and supraoptic nuclei (SON) of wild-type, OTKO and heterozygous littermates using in situ hybridization histochemistry. In addition, the effects of salt loading on the expression of the oxytocin gene were also examined in wild-type and heterozygous mice. Under the non salt-loaded condition, the levels of AVP mRNA in the PVN and SON of OTKO mice were significantly decreased compared to those in wild-type mice. Nevertheless, the up-regulation of the expression of the AVP gene in response to salt loading was preserved in OTKO mice. The degree of the up-regulation in OTKO mice tended to be greater compared to those in wild-type mice, suggesting compensatory up-regulation of the expression of the AVP gene in OTKO mice after salt loading. The basal levels of oxytocin mRNA in the PVN and SON of heterozygous mice were significantly lower than those in wild-type mice. Salt loading caused an increase of oxytocin mRNA levels in the PVN and SON of both wild-type and heterozygous mice. The ratios of increase of oxytocin mRNA levels were very similar between wild-type and heterozygous mice, suggesting that the single remaining oxytocin gene in heterozygous mice responds normally to an osmotic cue. Finally, salt loading tended to increase the serum concentration of sodium regardless of genotype, and there were no genotype differences in both the control and salt-loaded groups. These results suggest ways in which oxytocin may play a cooperative role together with AVP in the regulation of sodium balance.     

Pituitary-adrenal responses to oxotremorine and acute stress in male and female M1 muscarinic receptor knockout mice: comparisons to M2 muscarinic receptor knockout mice

Both within the brain and in the periphery, M(1) muscarinic receptors function primarily as postsynaptic receptors and M(2) muscarinic receptors function primarily as presynaptic autoreceptors. In addition to classical parasympathetic effectors, cholinergic stimulation of central muscarinic receptors influences the release of adrenocorticotrophic hormone (ACTH) and corticosterone. We previously reported that oxotremorine administration to male and female M(2) receptor knockout and wild-type mice increased ACTH to a significantly greater degree in knockout males compared to all other groups, and that M(2) knockout mice of both sexes were significantly more responsive to the mild stress of saline injection than were wild-type mice. These results accord with the primary function of M(2) receptors as presynaptic autoreceptors. In the present study, we explored the role of the M(1) receptor in pituitary-adrenal responses to oxotremorine and saline in male and female M(1) knockout and wild-type mice. Because these mice responded differently to the mild stress of saline injection than did the M(2) knockout and wild-type mice, we also determined hormone responses to restraint stress in both M(1) and M(2) knockout and wild-type mice. Male and female M(1) knockout and wild-type mice were equally unresponsive to the stress of saline injection. Oxotremorine increased both ACTH and corticosterone in M(1) wild-type mice to a significantly greater degree than in knockout mice. In both M(1) knockout and wild-type animals, ACTH responses were greater in males compared to females, and corticosterone responses were greater in females compared to males. Hormone responses to restraint stress were increased in M(2) knockout mice and decreased in M(1) knockout mice compared to their wild-type counterparts. These findings suggest that M(1) and M(2) muscarinic receptor subtypes differentially influence male and female pituitary-adrenal responses to cholinergic stimulation and stress. The decreased pituitary-adrenal sensitivity to oxotremorine and restraint stress noted in M(1) knockout mice is consistent with M(1) being primarily a postsynaptic receptor. Conversely, the increased pituitary-adrenal sensitivity to these challenges noted in M(2) knockout mice is consistent with M(2) being primarily a presynaptic autoreceptor.     

An arginine vasopressin V1b antagonist, SSR149415 elicits antidepressant-like effects in an olfactory bulbectomy model

Olfactory bulbectomized (OB) rats have been considered to serve as a useful animal model of depression in terms of behavioral, neurochemical, and neuroendocrine alterations, which reflect symptoms of patients with major depression. These behavioral and neurochemical changes in OB rats are normalized by the chronic administration of antidepressants. Recently, it has been reported that the compounds acting on stress-related peptide receptors such as an arginine vasopressin 1b (V(1b)) receptor antagonist and corticotropin-releasing factor (CRF) 1 receptor antagonists have antidepressant-like effects in several animal models. Here, the effects of acute and chronic (14 days) treatment with a V(1b) receptor antagonist (SSR149415) and a CRF1 receptor antagonist (CP-154,526) were examined in olfactory bulbectomy-induced hyperemotionality. Oral acute treatment with SSR149415 or CP-154,526 did not affect olfactory bulbectomy-induced hyperemotionality. In contrast, oral chronic treatment with SSR149415 (10 and 30 mg/kg) or CP-154,526 (10 mg/kg) significantly reduced hyperemotionality. The present results suggest that stress-related peptides such as arginine vasopressin and CRF might be implicated in olfactory bulbectomy-induced hyperemotionality. Furthermore, blockade of the V(1b) receptor or the CRF1 receptor may be useful in treating subjects suffering from chronic stressful conditions.     

Inhibition of vasopressin V1b receptor translation by upstream open reading frames in the 5'-untranslated region

The 5'-UTR of the vasopressin V1b receptor (V1bR) mRNA contains small open reading frames (ORF) located upstream (u) of the main ORF encoding the V1bR. The ability of the three proximal uORFs to be translated into peptides and their influence on V1bR translation was examined using fusion constructs of uORFs and V5 epitope, or ATG/ATA uORF mutations in the V1bR cDNA. In vitro translation and western blot analysis after transfection of uORF1-V5 or uORF2-V5 into cells revealed that uORF1 can be translated. As predicted by computer analysis, in vitro translation using a rabbit reticulocyte/canine microsome system, immunohistochemistry and western blot in membranes of transfected cells with uORF1-V5 revealed translocation of the uORF1 peptide into membrane fractions. In vitro translation of V1bR cDNA with mutations of the two uORFs proximal to the initiating methionine, uORFs 1 and 2 (Mut 1-2), or uORF2 (Mut 2) showed significantly increased translation of a 46 kDa band corresponding to the V1bR, compared with wild-type (WT) V1bR, an effect that was attenuated by cotranslation of uORF1-V5. Consistently, VP-induced inositol phosphate formation was higher in Chinese hamster ovay cells transfected with Mut 1-2 than with WT V1bR. Immunohistochemical and western blot analysis, using an antibody against uORF1, revealed peptide immunoreactivity in rat pituitary but not in liver. Pituitary uORF immunoreactivity increased following glucocorticoid administration. The present study shows that uORFs in the 5'-UTR of the V1bR mRNA inhibit V1bR translation, and suggests that translation of a 38-amino acid membrane peptide encoded by uORF1 exerts tonic inhibition of V1bR translation.     

Pituitary-adrenal responses to cholinergic stimulation and acute mild stress are differentially elevated in male and female M(2) muscarinic receptor knockout mice

Adrenocorticotrophic hormone (ACTH) and corticosterone responses to cholinergic stimulation are greater in male rats and mice than in females. To explore the role of M(2) muscarinic receptors in this sex difference, we administered the nonselective muscarinic agonist, oxotremorine, the acetylcholinesterase inhibitor, physostigmine, and saline (a mild stressor) to male and female M(2) receptor knockout (KO) and wild-type (WT) mice of the same genetic background. Because M(2) receptors function primarily as presynaptic autoreceptors, we hypothesized that their absence in M(2) KO mice would increase the sensitivity of hormone responses to cholinergic stimulation in these groups. Both male and female M(2) KO mice were significantly more responsive to the stress of saline injection than were their WT counterparts. Oxotremorine and physostigmine increased ACTH and corticosterone in all four groups, but to a significantly greater degree in KO males compared to WT males, KO females, and WT females. The increase in ACTH also was significantly greater in WT males compared to WT females. By contrast, the increase in corticosterone was significantly more in females compared to males, independent of genotype. Following pretreatment with the nonselective muscarinic antagonist, scopolamine, ACTH and corticosterone responses to oxotremorine and to saline in the M(2) KO mice were comparable with those of their WT counterparts. These findings suggest that the M(2) muscarinic receptor subtype influences male and female pituitary-adrenal responses following stimulation by both mild stress and cholinergic drugs. The M(2) receptor appears to regulate ACTH responses to cholinergic stimulation in males but not in females; however, other muscarinic receptors may be involved because corticosterone responses were higher in females compared to males. Because ACTH and corticosterone responses were greater in male and female M(2) KO mice, the M(2) receptor appears to dampen the stress response.     

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. These studies suggest that increased V1b receptor mRNA levels contribute to the VP receptor upregulation observed during repeated immobilization and i.p. hypertonic saline injection, whereas the lack of parallelism between V1b receptor mRNA and VP binding indicates that regulation of steady-state levels of V1b receptor mRNA is not a primary determinant in the control of pituitary VP receptor concentration during stress.     

Attenuated Stress Response to Acute Restraint and Forced Swimming Stress in Arginine Vasopressin 1b Receptor Subtype (Avpr1b) Receptor Knockout Mice and Wild‐Type Mice Treated with a Novel Avpr1b Receptor Antagonist

Arginine vasopressin (AVP) synthesised in the parvocellular region of the hypothalamic paraventricular nucleus and released into the pituitary portal vessels acts on the 1b receptor subtype (Avpr1b) present in anterior pituitary corticotrophs to modulate the release of adrenocorticotrophic hormone (ACTH). Corticotrophin‐releasing hormone is considered the major drive behind ACTH release; however, its action is augmented synergistically by AVP. To determine the extent of vasopressinergic influence in the hypothalamic‐pituitary‐adrenal axis response to restraint and forced swimming stress, we compared the stress hormone levels [plasma ACTH in both stressors and corticosterone (CORT) in restraint stress only] following acute stress in mutant Avpr1b knockout (KO) mice compared to their wild‐type controls following the administration of a novel Avpr1b antagonist. Restraint and forced swimming stress‐induced increases in plasma ACTH were significantly diminished in mice lacking a functional Avpr1b and in wild‐type mice that had been pre‐treated with Avpr1b antagonist. A corresponding decrease in plasma CORT levels was also observed in acute restraint‐stressed knockout male mice, and in Avpr1b‐antagonist‐treated male wild‐type mice. By contrast, plasma CORT levels were not reduced in acutely restraint‐stressed female knockout animals, or in female wild‐type animals pre‐treated with Avpr1b antagonist. These results demonstrate that pharmacological antagonism or inactivation of Avpr1b causes a reduction in the hypothalamic‐pituitary‐adrenal (HPA) axis response, particularly ACTH, to acute restraint and forced swimming stress, and show that Avpr1b knockout mice constitute a model by which to study the contribution of Avpr1b to the HPA axis response to acute stressors.     

Changes in hypothalamic-pituitary-adrenal function, body temperature, body weight and food intake with repeated social stress exposure in rats

These present studies aimed to compare changes in hypothalamic-pituitary-adrenal (HPA) activity and body temperature in response to acute social defeat, to repeated social stress and to novel restraint after repeated stress, as well as to assess effects on metabolic parameters by measuring body weight gain and food and water intake. We found that social defeat produced a marked increase in both adrenocorticotrophic hormone and corticosterone compared to placement in a novel cage. Similarly, body temperature was also increased during social defeat and during 30 min of recovery from defeat. We then examined the effects of 6 days of repeated social stress and observed minimal HPA responses to repeated social stress compared to control rats. These neuroendocrine responses were contrasted by robust increases in body temperature during stress and during recovery from stress during 6 days of repeated stress. However, in response to novel restraint, repeatedly stressed rats displayed facilitated body temperature responses compared to controls, similar to our previous findings with HPA activity. Food intake was increased during the light period during which defeat took place, but later intake during the dark period was not affected. Repeated stress decreased body weight gain in the dark period but food intake was increased overall during the 6 days of repeated stress in the light period. As a result, repeated stress increased cumulative food intake during the light period in the stressed rats but these relatively small increases in food intake were unable to prevent the diminished total weight gain in repeatedly stressed rats. Overall, the results demonstrate that, although acute social defeat has similar effects on temperature and HPA activity, repeated exposure to social stress has divergent effects on HPA activity compared to body temperature and that dampened weight gain produced by repeated social stress cannot be fully explained by changes in food intake.     

Sex differences in neurotransmission parameters in response to repeated mild restraint stress exposures in intact male, female and ovariectomised female rats

The present study determined whether a repeated mild restraint stress exposure would differentially alter neuronal activity in male and female rats to gain insights into neurobiological substrates involved in sex differences in stress-induced behavioural responses. In our first set of experiments, we used Western blot analysis to determine whether alterations in several synaptic proteins were elicited by the repeated stress treatment. We found bidirectional changes in synaptophysin levels in female cerebral cortex and hippocampus that diverged between intact and ovariectomised females. There were persistent elevations in spinophilin levels in the male, but not female, hippocampus following the repeated mild restraint stress exposure. By contrast, levels of the NMDA receptor scaffolding protein, PSD-95, were altered only in intact female cerebral cortex and ovariectomised female hippocampus. We next used immunohistochemical evaluation of Fos expression as a marker for neuronal activation. We found significant increases in Fos immunoreactivity in all sex conditions across multiple brain regions in response to the repeated mild stress. Fos protein induction was greatest in the frontal cortex, piriform cortex and amygdala, with the degree of induction varying by sex condition. Fos induction was dramatically higher in amygdala and piriform cortex only in intact females following repeated stress compared to a single restraint stress exposure, suggestive of sensitisation rather than habituation. By contrast, the frontal cortex of intact and ovariectomised females showed habituation to the repeated stressor. Males displayed modest sensitisation in both the frontal cortex and dentate gyrus with no changes in other brain areas. Taken together, these findings show that exposure to a mild repeated stress results in sex differences in synaptic adaptations and patterns of brain activation that likely contribute to observed sex differences in stress-induced behaviours. This approach provides valuable insights into interactions between the hormonal milieu and responses to a repeated mild stress, and further supports the importance of considering hormonal status in treatment of stress-related disorders.     

Conditioned place preference to morphine in cannabinoid CB1 receptor knockout mice

Recent reports have suggested an involvement of the brain cannabinoid system in the morphine-reward pathway. To address this question we evaluated whether CB1 receptor knockout mice would show a conditioned place preference to morphine. CB1 receptor knockout mice developed a strong place preference to 4 and 8 mg/kg morphine, similar to that in wild-type Swiss-Webster mice. This data thus does not support a contribution of the brain cannabinoid system to morphine reward.     

Localization of vasopressin (V1a) receptor binding and mRNA in the rhesus monkey brain

Centrally released arginine vasopressin (AVP) has been associated with various behavioural and cognitive effects, such as scent marking, aggression, and memory, which are believed to be mediated by the V1a subtype of the vasopressin receptor. Although the distribution of V1a receptors is conserved in a few brain regions, the pattern of expression of this receptor is, in general, highly species-specific. We have used receptor autoradiography with the linear V1a receptor ligand (125I-Phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2) to characterize the pattern of receptor binding in the rhesus monkey brain. Brain sites of V1a receptor synthesis were defined using in-situ hybridization. The regions of highest V1a receptor density included the prefrontal, cingulate, pyriform, and entorhinal cortex, as well as the presubiculum and mamillary bodies. In addition, V1a receptor binding and mRNA were detected in several regions reported to have V1a receptor in most rodents, including the amygdala, bed nucleus of the stria terminalis, lateral septum, hypothalamus and the brainstem. The distribution is consistent with a role for vasopressin in higher cognitive functions, especially memory, in primates.     

Enhanced up-regulation of corticotropin-releasing hormone gene expression in response to restraint stress in the hypothalamic paraventricular nucleus of oxytocin gene-deficient male mice

The neuropeptide oxytocin is released not only into the blood, but also within the brain in response to various stressors. Accumulating evidence suggests that central oxytocin may play a major role in the regulation of neuroendocrine responses to stress. In the present study, using the oxytocin knockout mouse model, we tested whether oxytocin might act to attenuate stress-induced up-regulation of corticotropin-releasing hormone (CRH) mRNA expression in the brain. The expression of CRH mRNA in the paraventricular nucleus (PVN) after 4 h of restraint stress was examined in oxytocin gene-deficient (OTKO), wild-type and heterozygous male mice using in situ hybridization histochemistry. We found that basal levels of CRH mRNA were not different among the three genotypes. Although restraint stress resulted in a significant increase of CRH mRNA expression in the PVN regardless of genotype, the degree of stress induced-up-regulation was significantly higher in OTKO mice than in wild-type mice. The effects of restraint stress on the expression of the arginine vasopressin (AVP) and the oxytocin genes were also examined. Unlike CRH mRNA, basal expression (in nonstressed control groups) of AVP mRNA in OTKO mice, as well as oxytocin mRNA in heterozygous mice, was significantly lower in the PVN and the supraoptic nucleus than in wild-type mice. After restraint stress, the expression of AVP mRNA was significantly increased in the PVN of OTKO mice compared to the nonstressed control group, whereas the expression of both AVP and oxytocin mRNA were unchanged in the PVN and the supraoptic nucleus of wild-type and heterozygous mice. Finally, in a separate set of mice, restraint stress-induced Fos expression was also examined in several brain regions involved in stress response, including the lateral septum, the bed nucleus of the stria terminalis (BNST), the medial preoptic area, the PVN, the medial and central amygdala using immunohistochemistry. After 90 min of restraint stress, the number of Fos-expressing cells significantly increased in all brain regions examined regardless of genotype. However, the number of stress-induced Fos-expressing cells in the BNST and the medial amygdala of OTKO mice was significantly lower than in wild-type mice. Collectively, the findings in the present study suggest that oxytocin may regulate stress-induced CRH gene expression in the PVN. Furthermore, neuronal activity in the BNST and the medial amygdala may be involved in this neuroendocrine regulatory system.     

Behavioral and biochemical responses to d-amphetamine in MCH1 receptor knockout mice

The melanin-concentrating hormone (MCH) system is anatomically and functionally interlaced with the mesocorticolimbic dopamine system. Therefore, we investigated whether MCH(1) receptor knockout (KO) mice are more susceptible than wild-type (WT) mice to psychostimulant-induced locomotor stimulation and sensitization, dopamine receptor-mediated phosphorylation events and c-fos expression within the frontal cortex and ventral striatum. MCH(1) receptor KO mice have 20% higher basal locomotor activity, are hypersensitive to the locomotor activating effects of d-amphetamine (1 mg/kg), and develop behavioral sensitization to a regimen of repeated d-amphetamine administration that does not induce sensitization in WT mice. In addition, d-amphetamine-mediated regulation of p44-mitogen activated protein kinase (MAPK) phosphorylation within the frontal cortex was significantly enhanced in MCH(1) receptor KO mice, when compared with WT mice. No significant genotype difference in the effects of d-amphetamine on MAPK phosphorylation events within the ventral striatum, phosphorylation at Ser(897) of the NR1 subunit of the NMDA receptor or Ca(2+) and cyclic AMP response-element binding-protein (CREB) at Ser(133) in the frontal cortex was detected. d-Amphetamine (3 mg/kg) increased c-fos expression within the frontal cortex in MCH(1) receptor KO mice, but not WT mice. There were no d-amphetamine-induced changes in c-fos expression within the ventromedial striatum in KO or WT mice. Overall, MCH(1) receptor KO mice are hypersensitive to the behavioral and molecular effects of the dopaminergic psychostimulant d-amphetamine. Increased frontal cortical MAPK phosphorylation and c-fos expression in MCH(1) receptor KO mice indicates that the MCH(1) receptor may be an important target for treating neuropsychiatric disorders characterized by frontal cortex dysfunction, including depression, attention deficit hyperactivity disorder (ADHD) and schizophrenia.