Diazepam Binding Inhibitor mRNA Research Articles

Circulating diazepam-binding inhibitor in infancy: Relation to markers of adiposity and metabolic health.

Diazepam-binding inhibitor (DBI) controls feeding behaviour and glucose homeostasis. Individuals born small-for-gestational-age (SGA) with excessive postnatal catch-up in weight are at risk for obesity and type 2 diabetes. To assess serum concentrations of DBI (0-2 years) in appropriate-for-gestational-age (AGA, n= 70) vs SGA infants (n= 33) with spontaneous catch-up and their relationship with endocrine-metabolic and adiposity markers. Longitudinal assessments included auxology, fasting glucose, insulin, insulin-like growth factor, high-molecular-weight adiponectin, DBI and body composition (absorptiometry). DBI was measured cross-sectionally in pregnant and non-pregnant women and in 2-day-old newborns. DBI mRNA expression levels were assessed in adult and neonatal tissues. Cord blood DBI concentrations were similar in AGA and SGA newborns and about fivefold higher than those in women. Serum DBI levels decreased by age 2 days, were higher in SGA vs AGA infants at age 2 years and associated negatively with markers of adiposity and insulin resistance and positively with high-molecular-weight adiponectin. DBI mRNA expression was lower in placenta than in other tissues. The increased DBI concentrations at birth are unrelated to prenatal growth. The higher DBI levels in SGA subjects at age 2 years may be related to catch-up growth or represent an adaptive mechanism to promote lipogenesis.

Endogenous Expression of ODN-Related Peptides in Astrocytes Contributes to Cell Protection Against Oxidative Stress: Astrocyte-Neuron Crosstalk Relevance for Neuronal Survival.

Astroglial cells are important actors in the defense of brain against oxidative stress injuries. Glial cells synthesize and release the octadecaneuropeptide ODN, a diazepam-binding inhibitor (DBI)-related peptide, which acts through its metabotropic receptor to protect neurons and astrocytes from oxidative stress-induced apoptosis. The purpose of the present study is to examine the contribution of the endogenous ODN in the protection of astrocytes and neurons from moderate oxidative stress. The administration of H2O2 (50μM, 6h) induced a moderate oxidative stress in cultured astrocytes, i.e., an increase in reactive oxygen species, malondialdehyde, and carbonyl group levels, but it had no effect on astrocyte death. Mass spectrometry and QPCR analysis revealed that 50μM H2O2 increased ODN release and DBI mRNA levels. The inhibition of ODN release or pharmacological blockage of the effects of ODN revealed that in these conditions, 50μM H2O2 induced the death of astrocytes. The transfection of astrocytes with DBI siRNA increased the vulnerability of cells to moderate stress. Finally, the addition of 1nM ODN to culture media reversed cell death observed in DBI-deficient astrocytes. The treatment of neurons with media from 50μM H2O2-stressed astrocytes significantly reduced the neuronal death induced by H2O2; this effect is greatly attenuated by the administration of an ODN metabotropic receptor antagonist. Overall, these results indicate that astrocytes produce authentic ODN, notably in a moderate oxidative stress situation, and this glio- and neuro-protective agent may form part of the brain defense mechanisms against oxidative stress injury.

Diazepam Enhances Production of Diazepam-Binding Inhibitor (DBI), a Negative Saliva Secretion Regulator, Localized in Rat Salivary Gland

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase–activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Acute food deprivation reduces expression of diazepam-binding inhibitor, the precursor of the anorexigenic octadecaneuropeptide ODN, in mouse glial cells

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.

Beta‐amyloid peptide stimulates endozepine release in cultured rat astrocytes through activation of N‐formyl peptide receptors

Astroglial cells synthesize and release endozepines, a family of neuropeptides derived from diazepam-binding inhibitor (DBI). The authors have recently shown that beta-amyloid peptide (Abeta) stimulates DBI gene expression and endozepine release. The purpose of this study was to determine the mechanism of action of Abeta in cultured rat astrocytes. Abeta(25-35) and the N-formyl peptide receptor (FPR) agonist N-formyl-Met-Leu-Phe (fMLF) increased the secretion of endozepines in a dose-dependent manner with EC(50) value of approximately 2 microM. The stimulatory effects of Abeta(25-35) and the FPR agonists fMLF and N-formyl-Met-Met-Met (fMMM) on endozepine release were abrogated by the FPR antagonist N-t-Boc-Phe-Leu-Phe-Leu-Phe. In contrast, Abeta(25-35) increased DBI mRNA expression through a FPR-independent mechanism. Abeta(25-35) induced a transient stimulation of cAMP formation and a sustained activation of polyphosphoinositide turnover. The stimulatory effect of Abeta(25-35) on endozepine release was blocked by the adenylyl cyclase inhibitor somatostatin, the protein kinase A (PKA) inhibitor H89, the phospholipase C inhibitor U73122, the protein kinase C (PKC) inhibitor chelerythrine and the ATP binding cassette transporter blocker glyburide. Taken together, these data demonstrate for the first time that Abeta(25-35) stimulates endozepine release from rat astrocytes through a FPR receptor positively coupled to PKA and PKC.

Increase in diazepam binding inhibitor expression by sustained morphine exposure is mediated via μ‐opioid receptors in primary cultures of mouse cerebral cortical neurons

Our previous in vivo experiment demonstrates that chronic morphine treatment up-regulates diazepam binding inhibitor (DBI) transcripts in mouse cerebral cortex, although detailed mechanisms were unclear (Katsura et al. [1998] J. Neurochem. 71:2638-2641). This study sought to elucidate the precise mechanisms of DBI mRNA up-regulation by long-term treatment with morphine using primary cultures of mouse cerebral cortical neurons. A significant increase in DBI mRNA was observed after sustained exposure to 0.3 microM morphine for 2 days, and the maximal expression occurred after 2 days of exposure, whereas transient exposure to 0.3 microM morphine for 15 min, 1 hr, and 3 hr produced no changes in the expression. Continuous exposure to DAMGO also significantly increased DBI mRNA expression, which was completely abolished by a selective antagonist of mu-opioid receptors, beta-funaltrexamine (beta-FNA). The morphine-induced increase in DBI mRNA expression and its content were completely inhibited by naloxone and beta-FNA, and the inhibitory potential of naloxonazine was about half that of beta-FNA. On the other hand, kappa- and delta-opioid receptor antagonists showed no effects on the morphine-induced increase in DBI mRNA. In addition, both a calmodulin antagonist and a CaM II kinase inhibitor significantly suppressed the morphine-induced increase in DBI mRNA. These results indicate that the increase in DBI expression is induced by continuous activation of mu-opioid receptors but not of kappa- and delta-opioid receptors and is regulated by the calcium/calmodulin-related phosphorylation system.

Up-regulated l-type high voltage-gated calcium channels cause increase in diazepam binding inhibitor induced by sustained morphine exposure in mouse cerebrocortical neurons

Mechanisms of increase in diazepam binding inhibitor (DBI) mRNA expression in mouse cerebrocortical neurons after sustained morphine exposure were investigated. Increases in DBI and its mRNA expressions induced by sustained morphine (0.3 μM) exposure for 3 days were completely abolished by naloxone and nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. Increase in [ 3H]diltiazem binding to the particulate fractions from the morphine-treated neurons was due to increased B max value with no changes in K d value. Western blot analysis on l-type high voltage-gated calcium channel (HVCC) subunits revealed the increased expressions of α1C, α1D, and α2/δ1 subunits and decreased of β4 subunit expression, whereas expression of N- and P/Q-type HVCC subunits was not changed. These results indicate that morphine-induced increase in DBI mRNA expression is mediated via increased Ca 2+ entry through up-regulated l-type HVCCs.

Role of androgens and glucocorticoids in the regulation of diazepam-binding inhibitor mRNA levels in male mouse hypothalamus

In peripheral organs, gonadal and adrenal steroids regulate diazepam-binding inhibitor (DBI) mRNA expression. In order to further investigate the involvement of peripheral steroid hormones in the modulation of brain DBI mRNA expression, we studied by semiquantitative in situ hybridization the effect of adrenalectomy (ADX) and castration (CX) and short-term replacement therapy on DBI mRNA levels in the male mouse hypothalamus. Cells expressing DBI mRNA were mostly observed in the arcuate nucleus, the median eminence and the ependyma bordering the third ventricle. In the median eminence and the ependyma bordering the third ventricule, the DBI gene expression was decreased in ADX rats and a single injection of corticosterone to ADX rats induced a significant increase in DBI gene expression at 3 and 12 h time intervals without completely restoring the basal DBI mRNA expression observed in intact mice. In the arcuate nucleus, ADX and corticosterone administration did not modify DBI mRNA expression. CX down-regulated DBI gene expression in the ependyma bordering the third ventricle. The administration of dihydrotestosterone (3–24 h) completely reversed the inhibitory effect of CX. In the median eminence and arcuate nucleus, neither CX or dihydrotestosterone administration modified DBI mRNA levels. These results suggest that the effects of glucocorticoids on the hypothalamo–pituitary–adrenocortical axis and androgens on the hypothalamo–pituitary–gonadal axis are mediated by DBI.

Somatostatin down‐regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes

Endozepines, a family of regulatory peptides related to diazepam-binding inhibitor (DBI), are synthesized and released by astroglial cells. Because rat astrocytes express various subtypes of somatostatin receptors (sst), we have investigated the effect of somatostatin on DBI mRNA level and endozepine secretion in rat astrocytes in secondary culture. Somatostatin reduced in a concentration-dependent manner the level of DBI mRNA in cultured astrocytes. This inhibitory effect was mimicked by the selective sst4 receptor agonist L803-087 but not by the selective sst1, sst2 and sst3 receptor agonists L779-591, L779-976 and L797-778, respectively. Somatostatin was unable to further reduce DBI mRNA level in the presence of the MEK inhibitor U0126. Somatostatin and the sst1, sst2 and sst4 receptor agonists induced a concentration-dependent inhibition of endozepine release. Somatostatin and the sst1, sst2 and sst4 receptor agonists also inhibited cAMP formation dose-dependently. In addition, somatostatin reduced forskolin-induced endozepine release. H89 mimicked the inhibitory effect of somatostatin on endozepine secretion. In contrast the PLC inhibitor U73122, the PKC activator PMA and the PKC inhibitor calphostin C had no effect on somatostatin-induced inhibition of endozepine release. The present data demonstrate that somatostatin reduces DBI mRNA level mainly through activation of sst4 receptors negatively coupled to the MAPK pathway, and inhibits endozepine release through activation of sst1, sst2 and sst4 receptors negatively coupled to the adenylyl cyclase/PKA pathway.

Beta‐amyloid peptides stimulate endozepine biosynthesis in cultured rat astrocytes

Accumulation of beta-amyloid peptide (Abeta), which is a landmark of Alzheimer's disease, may alter astrocyte functions before any visible symptoms of the disease occur. Here, we examined the effects of Abeta on biosynthesis and release of diazepam-binding inhibitor (DBI), a polypeptide primarily expressed by astroglial cells in the CNS. Quantitative RT-PCR and specific radioimmunoassay demonstrated that aggregated Abeta(25-35), at concentrations up to 10(-4) m, induced a dose-dependent increase in DBI mRNA expression and DBI-related peptide release from cultured rat astrocytes. These effects were totally suppressed when aggregation of Abeta(25-35) was prevented by Congo red. Measurement of the number of living cells revealed that Abeta(25-35) induced a trophic rather than a toxic effect on astrocytes. Administration of cycloheximide blocked Abeta(25-35)-induced increase of DBI gene expression and endozepine accumulation in astrocytes, indicating that protein synthesis is required for DBI gene expression. Altogether, the present data suggest that Abeta-induced activation of endozepine biosynthesis and release may contribute to astrocyte proliferation associated with Alzheimer's disease.

Expressions of diazepam binding inhibitor in some selected brain regions of chronic morphine dependent rats at different time-points

目的 研究地西泮结合抑制因子(DBI)基因在慢性吗啡依赖大鼠部分脑区不同时点表达水平的变化.方法30只雄性SD大鼠随机分为研究组20只和生理盐水对照组10只.研究组:腹腔注射盐酸吗啡,2次/d,起始剂量为5 mg·kg-1·次-1,逐日递增5 mg·kg-1·次-1,至第10天为50 mg·kg-1·次-1,对照组同期注射相同体积的生理盐水.研究组于末次注射后3 h(依赖组)及72h(戒断3 d组)、第6天(戒断6 d组)、第10天(戒断10 d组)分别处死5只,对照组于末次注射后3h及72h分别各处死5只,灌注、取脑、冰冻切片并选取含有海马CA1区(HIPCA1)、中脑腹侧被盖区(VTA)、伏隔核(Nac)、前额叶皮质(PFC)、杏仁核(AMG)、中脑导水管(PAG)等结构的脑片,利用组织原位杂交技术检测DBI mRNA在各脑区的表达,进行组间比较.结果 吗啡成瘾组在HIPCA1、VTA、Nac、PFC、AMG、PAG和LC的DBI mRNA表达依次为:102.7±6.7、139.6±12.8、137.1±9.3、144.3±10.8、153.6±10.6、122.1±8.5和100.0±8.8,显著高于对照组,并均在戒断的第3天达到峰值,依次为139.7±12.4、181.1±10.2、159.3±13.3、186.6±7.6、195.1±7.0、176.9±14.7,随后出现下调,在戒断第6天各脑区仍高于对照组,在戒断第10天,除了 PFC仍为145.3±9.3,明显高于对照组(P <0.05)外,其他脑区与对照组无显著差异.结论DBIm RNA在慢性吗啡处理大鼠部分脑区的表达上调,可能参与了慢性吗啡依赖过程。

Conditioned place preference associates with the mRNA expression of diazepam binding inhibitor in brain regions of the addicted rat during withdrawal

The diazepam binding inhibitor (DBI) modulating the functions of the GABAA receptors is involved in the maladaptation of neural system during using opiate, but its role in opiate dependence is not fully understood. Using conditioned place preference (CPP) rat model and in situ hybridization technique, we examined the correlation between opiate dependence and the mRNA expression of DBI. We found that chronic morphine treatment enabled CPP and increased the DBI mRNA expression in crucial brain regions of addiction. Withdrawal for 3 days caused significant physical signs and further increased the DBI mRNA expression. Both the DBI mRNA and CPP expression remained significantly high but physical signs were at control level in the animals withdrawal for 6 days. Remarkably, the DBI mRNA expressions in the CA1 region of the hippocampus (CA1), ventral tagmental area (VTA), nucleus accumbens (NAc) and amygdala (AMG) were positively correlated to CPP during the periods from withdrawal for 3 days to withdrawal for 6 days. These findings suggest that DBI may play a role in both physical and psychological dependence of opiates.

Continuous exposure to nitric oxide enhances diazepam binding inhibitor mRNA expression in mouse cerebral cortical neurons

Effects of sustained exposure to nitric oxide (NO) formed by long-term activation of N-methyl- d-aspartate (NMDA) receptors and liberated from a long-lasting NO generator, DETA NONOate, on diazepam binding inhibitor (DBI) and its mRNA expressions were examined using mouse cerebral cortical neurons. Long-term exposure to NMDA increased DBI mRNA expression, and NO synthase inhibitors dose-dependently inhibited this increase. DETA NONOate dose-dependently increased DBI mRNA expression when exposing the neurons to this agent for 3 days and a maximal enhancement of the expression was found at 100 μM of the NO generator. In addition, a significant increase in DBI mRNA expression was observed 1 day after the exposure to 100 μM DETA NONOate, and the maximal expression was observed 2 days after the exposure, whereas transient exposure for less than 3 h to 100 μM DETA NONOate produced no changes in the expression. DETA NONOate (100 μM)-induced increase in DBI mRNA expression was completely abolished by concomitant exposure to hemoglobin. DBI content was also dose-dependently increased by DETA NONOate after the exposure for 3 days. The inhibition of cGMP formation by 1 H-[1,2,4] oxadiazolo [4,3-α]quinoxalin-1-one (ODQ) showed no affects on the DETA NONOate-induced expression, suggesting that the increased expression of DBI mRNA is mediated via processes independent of cGMP. These results indicate that continuous exposure of the neurons to NO is an essential factor for increasing DBI mRNA expression in the neurons.

L-type high voltage-gated calcium channels cause an increase in diazepam binding inhibitor mRNA expression after sustained exposure to ethanol in mouse cerebral cortical neurons

Mechanisms for increase in diazepam binding inhibitor (DBI) mRNA expression after sustained exposure to ethanol (EtOH) were investigated. Increases in 30 mM KCl-induced [ 45Ca 2+] influx and DBI mRNA expression after EtOH (50 mM) exposure for 3 days were completely abolished by nifedipine, but not by ω-agatoxin VIA and ω-conotoxin GIVA. These results indicate that EtOH-induced increase in DBI mRNA expression is mediated via increased Ca 2+ entry through up-regulated L-type high voltage-gated calcium channels.

Psychological stress, but not physical stress, causes increase in diazepam binding inhibitor (DBI) mRNA expression in mouse brains

Effects of conditioned emotional stimuli (CES), which induce psychological stress, on the expression of cerebral diazepam binding inhibitor (DBI) mRNA in mouse were examined using a communication box. Cerebral DBI mRNA expression significantly increased in a time-dependent manner after the application of CES. The maximal enhancement of DBI mRNA expression was observed 2 days after the application of CES, and this increase faded out over 7 days after the treatment. Flunitrazepam (1 mg/kg), an agonist for central benzodiazepine (BZD) receptors, completely abolished the CES-induced elevation of cerebral DBI contents and its mRNA expressions. These results indicate that cerebral DBI is enhanced by psychological stress, which is regulated by central BZD receptors.

Withdrawal from nicotine facilitates diazepam binding inhibitor mRNA expression in mouse cerebral cortex

Changes in diazepam binding inhibitor (DBI) mRNA expression after withdrawal from nicotine were examined. Withdrawal from nicotine Increased DBI mRNA expression in cerebral cortices derived from nicotine-dependent mice and in the neurons continuously exposed to nicotine (0.1 μM). These results indicate that withdrawal from nicotine after its long-term exposure induces steep increase of DBI mRNA expression as reported previously in ethanol- and morphine-dependent animals.

NMDA receptor activation enhances diazepam binding inhibitor and its mRNA expressions in mouse cerebral cortical neurons

Effects of N-methyl- d-aspartate (NMDA) on diazepam binding inhibitor (DBI) and its mRNA expression in mouse cerebral cortical neurons were examined. A significant increase in DBI mRNA expression was observed 1 day after the exposure to 0.1 μM NMDA and the maximal expression occurred 2 days after the exposure, whereas transient exposure to 0.1 μM NMDA for 15 min, 1 and 3 h produced no changes in the expression. Similarly, no changes in the expression were found by the concomitant exposure to NMDA and MK-801, a NMDA receptor antagonist, for 72 h subsequent to the incubation with NMDA alone for 3 h. Such NMDA-induced increases in DBI mRNA expression were dose-dependently inhibited by MK-801. Moreover, neuronal DBI content significantly increased by treatment with NMDA, which was completely abolished by MK-801. These results indicate that continuous activation of NMDA receptors is an essential factor for increasing DBI expression in the neurons.

Cellular localization of the diazepam binding inhibitor (DBI) in the gastrointestinal tract of mice and its coexistence with the fatty acid binding protein (FABP).

The diazepam binding inhibitor (DBI), initially isolated as an endogenous 10-kDa polypeptide from the brain, has the ability to displace ligands from benzodiazepine binding sites on gamma-aminobutyric acid (GABA) receptors. However, DBI is widely distributed outside the brain, with the highest expression in the intestine. The present in situ hybridization study revealed the cellular expression of DBI mRNA throughout the gastrointestinal tract of mice, showing it to be intensely expressed in the spinous layer in the stratified squamous epithelium of the oral cavity, esophagus and forestomach, in surface mucous cells in the glandular stomach, and in columnar (absorptive) cells of the intestinal villi. A precise identification of DBI-expressing cell types was confirmed immunohistochemically, although the expressing cells detectable by the two histochemical methods differed slightly in their extension. Noteworthily, DBI always coexisted with the fatty acid binding protein (FABP), which participates in the uptake and metabolic processing of long chain fatty acids. In addition to the biochemical finding that DBI is identical with the acyl-CoA binding protein (ACBP), the distributional patterns of DBI and its colocalization with FABPs suggests its involvement in the absorption and metabolism of lipid in the epithelia of the digestive tract.

Mechanism for increase in expression of cerebral diazepam binding inhibitor mRNA by nicotine: involvement of L-type voltage-dependent calcium channels

We investigated the mechanisms underlying the increase in diazepam binding inhibitor (DBI) and its mRNA expression induced by nicotine (0.1 μM) exposure for 24 h using mouse cerebral cortical neurons in primary culture. Nicotine-induced (0.1 μM) increases in DBI mRNA expression were abolished by hexamethonium, a nicotinic acetylcholine (nACh) receptor antagonist. Agents that stabilize the neuronal membrane, including tetrodotoxin (TTX), procainamide (a Na + channel inhibitor), and local anesthetics (dibucaine and lidocaine), dose-dependently inhibited the increased expression of DBI mRNA by nicotine. The nicotine-induced increase in DBI mRNA expression was inhibited by L-type voltage-dependent Ca 2+ channel (VDCC) inhibitors such as verapamil, calmodulin antagonist (W-7), and Ca 2+/calmodulin-dependent protein kinase II (CAM II kinase) inhibitor (KN-62), whereas P/Q- and N-type VDCC inhibitors showed no effects. In addition, nicotine exposure for 24 h induced [ 3H]nicotine binding to the particulate fractions of the neurons with an increased B max value and no changes in K d. Under these conditions, the 30 mM KCl- and nicotine-induced 45Ca 2+ influx into the nicotine-treated neurons was significantly higher than those into non-treated neurons. These results suggest that the nicotine-stimulated increase in DBI mRNA expression is mediated by CAM II kinase activation resulting from the increase in intracellular Ca 2+ through L-type VDCCs subsequent to the neuronal membrane depolarization associated with nACh receptor activation.

Diazepam binding inhibitor (DBI) gene expression in the brains of socially isolated and group-housed mice

Diazepam binding inhibitor (DBI), a putative endogenous polypeptide ligand for benzodiazepine (BZD) receptors, has been shown to act as an inverse BZD receptor agonist in the brain. We previously suggested that the social isolation stress-induced decrease in pentobarbital sleeping time in mice was partly due to an increase in the activity of endogenous substances with an inverse BZD receptor agonist-like property such as DBI. In this study, we examined whether the DBI gene expression is affected by socially isolated stress. Consistent with the previous findings, the in situ hybridization result showed very strong signals of DBI mRNA around the regions of the third ventricle, especially the lining cells, the arcuate nucleus of the hypothalamus and the cerebellum, in both socially isolated and group-housed animals. Unexpectedly, however, semi-quantitative experiments with reverse transcription polymerase chain reaction technique revealed that socially isolated mice had significantly less expression of DBI mRNA in the hypothalamus than group-housed animals, and no difference in the expression in the other brain areas was observed between two animal groups. We discuss the relationship between the decrease of DBI mRNA expression in the hypothalamus and the decrease of GABA(A) receptor function following long-term social isolation in mice.