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Evaluating and coping with stressful social events as they unfold is a critical strategy in overcoming them without long-lasting detrimental effects. Individuals display a wide range of responses to stress, which can manifest in a variety of outcomes for the brain as well as subsequent behavior. Chronic Social Defeat Stress (CSDS) in mice has been widely used to model individual variation following a social stressor. Following a course of repeated intermittent psychological and physical stress, mice diverge into separate populations of social reactivity: resilient (socially interactive) and susceptible (socially avoidant) animals. A rich body of work reveals distinct neurobiological and behavioral consequences of this experience that map onto the resilient and susceptible groups. However, the range of factors that emerge over the course of defeat have not been fully described. Therefore, in the current study, we focused on characterizing behavioral, physiological, and neuroendocrine profiles of mice in three separate phases: before, during, and following CSDS. We found that following CSDS, traditional read-outs of anxiety-like and depression-like behaviors do not map on to the resilient and susceptible groups. By contrast, behavioral coping strategies used during the initial social stress encounter better predict which mice will eventually become resilient or susceptible. In particular, mice that will emerge as susceptible display greater escape behavior on Day 1 of social defeat than those that will emerge as resilient, indicating early differences in coping mechanisms used between the two groups. We further show that the social avoidance phenotype in susceptible mice is specific to the aggressor strain and does not generalize to conspecifics or other strains, indicating that there may be features of threat discrimination that are specific to the susceptible mice. Our findings suggest that there are costs and benefits to both the resilient and susceptible outcomes, reflected in their ability to cope and adapt to the social stressor.

Obesity is a persistent and pervasive problem, particularly in industrialized nations. It has come to be appreciated that the metabolic health of an individual can influence brain function and subsequent behavioral patterns. To examine the relationship between metabolic phenotype and central systems that regulate behavior, we tested rats with divergent metabolic phenotypes (Low Capacity Runner: LCR vs. High Capacity Runner: HCR) for behavioral responses to the conflict between hunger and environmental novelty using the novelty suppressed feeding (NSF) paradigm. Additionally, we measured expression of mRNA, for peptides involved in energy management, in response to fasting. Following a 24-h fast, LCR rats showed lower latencies to begin eating in a novel environment compared to HCR rats. A 48-h fast equilibrated the latency to begin eating in the novel environment. A 24-h fast differentially affected expression of cocaine-amphetamine regulated transcript (CART) mRNA in the nucleus accumbens (NAc), where 24-h of fasting reduced CART mRNA in LCR rats. Bilateral microinjections of CART 55–102 peptide into the NAc increased the latency to begin eating in the NSF paradigm following a 24-h fast in LCR rats. These results indicate that metabolic phenotype influences how animals cope with the conflict between hunger and novelty, and that these differences are at least partially mediated by CART signaling in the NAc. For individuals with poor metabolic health who have to navigate food-rich and stressful environments, changes in central systems that mediate conflicting drives may feed into the rates of obesity and exacerbate the difficulty individuals have in maintaining weight loss.

The role of the fibroblast growth factor (FGF) system in brain-related disorders has received considerable attention in recent years. To understand the role of this system in neurological and psychiatric disorders, it is important to identify the specific members of the FGF family that are implicated, their location and the various mechanisms they can be modulated. Each disorder appears to impact specific molecular players in unique anatomical locations, and all of these could conceivably become targets for treatment. In the last several years, the issue of how to target this system directly has become an area of increasing interest. To date, the most promising therapeutics are small molecule inhibitors and antibodies that modulate FGF receptor (FGFR) function. Beyond attempting to modify the primary players affected by a given brain disorder, it may prove useful to target molecules, such as membrane-bound or extracellular proteins that interact with FGF ligands or FGFRs to modulate signaling.

BACKGROUND: Despite strong evidence linking fibroblast growth factor 2 (FGF2) with anxiety and depression in both rodents and humans, the molecular mechanisms linking FGF2 with anxiety are not understood. METHODS: We compare 1) mice that lack a functional Fgf2 gene (Fgf2 knockout [KO]), 2) wild-type mice, and 3) Fgf2 KO with adult rescue by FGF2 administration on measures of anxiety, depression, and motor behavior, and further investigate the mechanisms of this behavior by cellular, molecular, and neuroendocrine studies. RESULTS: We demonstrate that Fgf2 KO mice have increased anxiety, decreased hippocampal glucocorticoid receptor (GR) expression, and increased hypothalamic-pituitary-adrenal axis activity. FGF2 administration in adulthood was sufficient to rescue the entire phenotype. Blockade of GR in adult mice treated with FGF2 precluded the therapeutic effects of FGF2 on anxiety behavior, suggesting that GR is necessary for FGF2 to regulate anxiety behavior. The level of Egr-1/NGFI-A was decreased in Fgf2 KO mice and was reestablished with FGF2 treatment. By chromatin immunoprecipitation studies, we found decreased binding of EGR-1 to the GR promoter region in Fgf2 KO mice. Finally, we examined anxiety behavior in FGF receptor (FGFR) KO mice; however, FGFR1, FGFR2, and FGFR3 KO mice did not mimic the phenotype of Fgf2 KO mice, suggesting a role for other receptor subtypes (i.e., FGFR5). CONCLUSIONS: These data suggest that FGF2 levels are critically related to anxiety behavior and hypothalamic-pituitary-adrenal axis activity, likely through modulation of hippocampal glucocorticoid receptor expression, an effect that is likely receptor mediated, albeit not by FGFR1, FGFR2, and FGFR3.

Individual differences in the locomotor response to novelty have been linked to basal differences in dopaminergic neurotransmission. Mesolimbic dopaminergic outputs are regulated by cholecystokinin (CCK), a neuropeptide implicated in anxiety. In turn, CCK expression is regulated by fibroblast growth factor-2 (FGF2), which has recently been identified as an endogenous regulator of anxiety. FGF2 binds to the high-affinity fibroblast growth factor receptor-1 (FGF-R1) to regulate the development and maintenance of dopamine neurons in the ventral tegmental area (VTA). However, the relationship between the FGF and CCK systems in the VTA is not well understood. Therefore, we utilized the selectively-bred low-responder (bLR; high-anxiety) and high-responder (bHR; low-anxiety) rats to examine the effects of repeated (21-day) FGF2 treatment on CCK and FGF-R1 mRNA in the rostral VTA (VTAr). In vehicle-treated controls, both CCK and FGF-R1 mRNA levels were increased in the VTAr of bLR rats relative to bHR rats. Following FGF2 treatment, however, bHR-bLR differences in CCK and FGF-R1 mRNA expression were eliminated, due to decreased CCK mRNA levels in the VTAr of bLR rats and increased FGF-R1 expression in bHR rats. Differences after FGF2 treatment may denote distinct interactions between the CCK and FGF systems in the VTAr of bHR vs. bLR rats. Indeed, significant correlations between CCK and FGF-R1 mRNA expression were found in bHR, but not bLR rats. Colocalization studies suggest that CCK and FGF-R1 are coexpressed in some VTAr neurons. Taken together, our findings suggest that the FGF system is poised to modulate both CCK and FGF-R1 expression in the VTAr, which may be associated with individual differences in mesolimbic pathways associated with anxiety-like behavior.

Approximately 50% of mood disorder patients exhibit hypercortisolism. Cortisol normally exerts its functions in the CNS via binding to mineralocorticoid receptors (MR) and glucocorticoid receptors (GR). Both MR and GR are highly expressed in human hippocampus and several studies have suggested that alterations in the levels of MR or GR within this region may contribute to the dysregulation in major depressive disorder (MDD). Studies have also shown functional heterogeneity across the hippocampus, with posterior hippocampus preferentially involved in cognitive processes and anterior hippocampus involved in stress, emotion and affect. We therefore hypothesize that GR and MR expression in hippocampus of control and MDD patients may vary not only with disease, but also with regional specificity along the anterior/posterior axis. Student's t-test analysis showed decreased expression of MR in the MDD group compared to controls in the anterior, but not the posterior hippocampus, with no significant changes in GR. Linear regression analysis showed a marked difference in MR:GR correlation between suicide and non-suicide patients in the posterior hippocampus. Our findings are consistent with previous reports of hippocampal corticosteroid receptor dysregulation in mood disorders, but extend those findings by analysis across the anterior/posterior axis of the hippocampus. A decrease in MR in the anterior but not posterior hippocampus of MDD patients emphasizes the important functional role of the anterior hippocampus in neuroendocrine regulation in humans.

Growth factors are critical in neurodevelopment and neuroplasticity, and recent studies point to their involvement in addiction. We previously reported increased levels of basic fibroblast growth factor (FGF2) in high novelty/drug-seeking rats (bred high responders, bHR) compared to low novelty/drug-seeking rats (bred low responders, bLRs). The present study asked whether an early life manipulation of the FGF system (a single FGF2 injection on postnatal day 2) can impact cocaine sensitization and associated neurobiological markers in adult bHR/bLR animals. Neonatal FGF2- and vehicle-treated bHR/bLR rats were sensitized to cocaine (7 daily injections, 15 mg/kg/day, i.p.) in adulthood. Neonatal FGF2 markedly increased bLRs' typically low psychomotor sensitization to cocaine (day 7 locomotor response to cocaine), but had little effect on bHRs' cocaine sensitization. Gene expression studies examined dopaminergic molecules as well as FGF2 and the FGFR1 receptor in cocaine naive animals, to investigate possible neurobiological alterations induced by neonatal FGF2 exposure that may influence behavioral response to cocaine. bLRs showed decreased tyrosine hydroxylase in the ventral tegmental area (VTA), decreased D1 and increased D2 receptor expression in the nucleus accumbens core, as well as decreased FGF2 in the VTA, substantia nigra, accumbens core, and caudate putamen compared to bHRs. Neonatal FGF2 selectively increased D1 receptor and FGF2 mRNA in the accumbens core of bLRs, which may contribute to their heightened cocaine sensitization. Our results suggest increased FGF2 in the mesodopaminergic circuit (as in baseline bHRs and neonatal FGF2-exposed bLRs vs. baseline bLRs) enhances an individual's susceptibility to cocaine sensitization and may increase vulnerability to drug seeking and addiction.

In this review, we propose a broader view of the role of the fibroblast growth factor (FGF) family in modulating brain function. We suggest that some of the FGF ligands together with the FGF receptors are altered in individuals with affective disorder and modulate emotionality in animal models. Thus, we propose that members of the FGF family may be genetic predisposing factors for anxiety, depression, or substance abuse; that they play a key organizing role during early development but continue to play a central role in neuroplasticity in adulthood; and that they work not only over extended time frames, but also via rapid signaling mechanisms, allowing them to exert an “on-line” influence on behavior. Therefore, the FGF family appears to be a prototype of “switch genes” that are endowed with organizational and modulatory properties across the lifespan, and that may represent molecular candidates as biomarkers and treatment targets for affective and addictive disorders.

Aggression frequently coincides with specific dimensions of emotionality, such as impulsivity, risk-taking, and drug abuse. Serotonergic (5-HTergic) neurotransmission contributes to the regulation of numerous neurobiological functions, and is thought to play a key role in modulating aggressive responses. The current study uses selectively-bred High (bHR) and Low (bLR) Responder rats that exhibit differences in emotionality and behavioral control, with bHRs exhibiting heightened novelty-induced exploration, impulsivity, and increased sensitivity to drugs of abuse, and with bLRs characterized by exaggerated depressive- and anxiety-like behaviors. Based on this behavioral profile we hypothesized that bHR rats exhibit increased aggression along with changes in testosterone and corticosterone secretion characteristic of aggression, and that these changes are accompanied by alterations in the expression of key genes that regulate 5-HTergic neurotransmission (Tph2 and Sert) as well as in the activation of 5-HTergic cell groups following aggressive encounter. Our data demonstrate that when compared to bLR rats, bHRs express increased baseline Tph2 and Sert in select brainstem nuclei, and when tested on the resident-intruder test they exhibited: 1) increased aggressive behavior; 2) potentiated corticosterone and testosterone secretion; and 3) diminished intrusion-induced c-fos expression in select 5-HTergic brainstem cell groups. The most prominent gene expression differences occurred in the B9 cell group, pontomesencephalic reticular formation, median raphe, and the gigantocellular nucleus pars α. These data are consistent with the notion that altered 5-HT neurotransmission contributes to bHRs' heightened aggression. Furthermore, they indicate that a specific subset of brainstem 5-HTergic cell groups contributes to the regulation of intrusion-elicited behavioral responses.

A missense polymorphism in the NRG1 gene, Val > Leu in exon 11, was reported to increase the risk of schizophrenia in selected families from the Central Valley region of Costa Rica (CVCR). The present study investigated the relationship between three NRG1 genetic variants, rs6994992, rs3924999, and Val > Leu missense polymorphism in exon 11, in cases and selected controls from an isolated population from the CVCR. Isolated populations can have less genetic heterogeneity and increase power to detect risk variants in candidate genes. Subjects with bipolar disorder (BD, n = 358), schizophrenia (SZ, n = 273), or unrelated controls (CO, n = 479) were genotyped for three NRG1 variants. The NRG1 promoter polymorphism (rs6994992) was related to altered expression of NRG1 Type IV in other studies. The expression of NRG1 type IV in the dorsolateral prefrontal cortex (DLPFC) and the effect of the rs6994992 genotype on expression were explored in a postmortem cohort of BD, SZ, major depressive disorder (MDD) cases, and controls. The missense polymorphism Val > Leu in exon 11 was not significantly associated with schizophrenia as previously reported in a family sample from this population, the minor allele frequency is 4%, thus our sample size is not large enough to detect an association. We observed however an association of rs6994992 with NRG1 type IV expression in DLPFC and a significantly decreased expression in MDD compared to controls. The present results while negative do not rule out a genetic association of these SNPs with BD and SZ in CVCR, perhaps due to small risk effects that we were unable to detect and potential intergenic epistasis. The previous genetic relationship between expression of a putative brain-specific isoform of NRG1 type IV and SNP variation was replicated in postmortem samples in our preliminary study.

We compared the response to repeated social defeat in rats selected as high (HR) and low (LR) responders to novelty. In experiment 1, we investigated the behavioral and neuroendocrine effects of repeated social defeat in HR-LR rats. By the last defeat session, HR rats exhibited less passive-submissive behaviors than LR rats, and exhibited higher corticosterone secretion when recovering from defeat. Furthermore, in the forced swim test, while HR defeated rats spent more time immobile than their undefeated controls, LR rats' immobility was unaffected by defeat. In experiment 2, we compared the effects of repeated social defeat on body, adrenal, thymus, and spleen weights in HR-LR rats; moreover, we compared the effects of repeated social defeat on stress related molecules gene expression in these two groups of rats. Our results show that HR rats exhibited a decrease in thymus weight after repeated social defeat that was not present in LRs. Analyses of in situ hybridization results found HR-LR differences in 5-HT(2a) mRNA levels in the parietal cortex and 5-HT(1a) mRNA levels in the dorsal raphe. Moreover, LR rats had higher glucocorticoid receptor (GR) mRNA expression than HR rats in the dentate gyrus, and repeated social defeat decreased this expression in LR rats to HR levels. Finally, hippocampal mineralcorticoid receptor (MR)/GR ratio was reduced in HR rats only. Taken together, our results show a differential response to social defeat in HR-LR rats, and support the HR-LR model as a useful tool to investigate inter-individual differences in response to social stressors.

Background The onset of major depressive disorder is likely precipitated by a combination of heredity and life stress. The present study tested the hypothesis that rats selectively bred on a trait related to emotional reactivity would show differential susceptibility or resilience to the development of depression-like signs in response to chronic mild variable intermittent stress (CMS). Methods Male Sprague–Dawley rats that were bred based on the trait of either high or low locomotor activity in response to a novel environment were exposed to 4 weeks of CMS or control conditions. Changes in hedonic behavior were assessed using weekly sucrose preference tests and anxiety-like behavior was evaluated using the novelty-suppressed feeding test. Results During 4 weeks of CMS, bred low responder (bLR) rats became anhedonic at a faster rate and to a larger degree than bred high responder (bHR) rats, based on weekly sucrose preference tests. Measures of anxiety-like behavior in the novelty-suppressed feeding test were also significantly increased in the CMS-exposed bLR rats, though no differences were observed between CMS-exposed bHR rats and their unstressed controls. Conclusions These findings present further evidence that increased emotional reactivity is an important factor in stress susceptibility and the etiology of mood disorders, and that bHR and bLR rats provide a model of resistance or vulnerability to stress-induced depression. Furthermore, exposing bHR and bLR rats to CMS provides an excellent way to study the interaction of genetic and environmental factors in the development of depression-like behavior.

Reactivity to environmental stressors influences vulnerability to neurological and psychiatric illnesses, but little is known about molecular mechanisms that control this reactivity. Since mice with forebrain-specific glucocorticoid receptor overexpression (GRov mice) display anxiety-like behaviors in novel environments and have difficulty adjusting to change in memory tasks, we hypothesized that these may be facets of a broader phenotype of altered reactivity to environmental demands. Male GRov and wild-type mice were tested in a multiple-trial object interaction test comprising environmental and object habituation and spatial and object novelty trials. Half the mice received restraint stress before testing. GRov mice exhibited more locomotor activity and, without stress, more object interaction than wild-type mice. Following acute stress, GRov mice no longer showed increased object exploration. While stress dampened responses to object novelty in both groups, GRov mice were particularly impaired in discrimination of spatial novelty post-stress. These data demonstrate that GRov leads to increased environmental reactivity, responsiveness to salience, and vulnerability to stress-induced cognitive deficits. They implicate forebrain glucocorticoid receptor (GR) in fine-tuning interactions with the environment and the interplay of emotional salience, coping abilities, and cognitive function.

We compared acute and chronic antinociceptive effects of morphine in animals with high reactivity (HR) vs. low reactivity (LR) to novelty. Antinociception was assessed by tail-flick test. Rats were i.p. injected with either saline or morphine (1.5 or 3mg/kg) every 12h for 7 days according to the treatment group. On day 1 of the experiment, LR animals in the 1.5mg/kg morphine group showed significantly higher tail-flick latency than HR. Moreover, significant tolerance to the antinociceptive effects of morphine at the used doses was observed in LR but not HR animals. However, effects of chronic morphine treatment on tail-flick latency in rat groups with similar morphine-induced acute antinociception were undistinguishable. The difference in tail-flick latency between HR and LR rats observed after acute 1.5mg/kg morphine injection was eliminated if beta-funaltrexamine (3mg/kg, i.p.) was administered 24h before the test, an indication that mu opioid receptors are responsible for the difference observed. Studies to anatomically characterize the difference in the acute analgesic effect of morphine in HR vs. LR animals did not however yield any significant difference in mu opioid receptor mRNA levels in locus coeruleus (LC), ventral periaqueductal gray (vPAG), nucleus raphe magnus (NRM) and nucleus reticularis paragigantocellularis (NRPG) between these two groups of animals. In conclusion, our results show that differences in novelty-seeking behavior can predict inter-individual variability in morphine-induced antinociception in rats. Such variability is dependent upon activation of mu opioid receptors, but does not correlate with mu opioid receptor expression in LC, vPAG or ventral medulla.

The fibroblast growth factor (FGF) system has previously been found to be altered in post-mortem brains of individuals with major depressive disorder (MDD). The present study tested whether the FGF system is altered following acute social defeat. Rats were exposed to four consecutive days of either a social defeat paradigm or novel cages. Animals were sacrificed after the last social defeat session and gene expression was assessed in the hippocampus by mRNA in situ hybridization. Molecular components of the FGF system were significantly downregulated following social defeat. Specifically, FGF2 and FGFR1 mRNA expression was decreased in various subfields of the hippocampus. Decreased tone of the FGF system following an acute social stressor is congruent with human post-mortem results of FGF system downregulation in depression. These findings suggest that modulating the FGF system may have therapeutic value in the treatment of MDD.

Age-related changes in the hippocampus increase vulnerability to impaired learning and memory. Our goal is to understand how a genetic vulnerability to cognitive impairment can be modified by aging and sex. Mice with a mutation in the cAMP response element binding (CREB) protein gene (CREB(alphadelta-) deficient mice) have a mild cognitive impairment and show test condition-dependent learning and memory deficits. We tested three ages of CREB(alphadelta-) deficient and wild-type (WT) mice in two Morris water maze (MWM) protocols: four trials per day with a 3-5 min inter-trial interval (ITI) (MWM4) and two trials per day with a 1 min ITI (MWM2). All CREB(alphadelta-) deficient mice performed well in the easier MWM4, except for the aged females that performed poorly. In the harder MWM2, young male and female and middle-aged male CREB(alphadelta-) deficient mice performed well, but aged male and all middle-aged and aged female CREB(alphadelta-) deficient mice were impaired. These results show that mice with a genetic vulnerability to impaired learning and memory exhibit increased vulnerability with age that is most apparent among females. Thus, a genetic predisposition to cognitive impairment may render females more vulnerable than males to such deficits with age.

kappa-opioid receptor antagonists such as nor-Binaltorphimine (nor-BNI) have been shown to produce antidepressant-like behavioral effects in animal models of depression. The aim of this study was to investigate further the duration of centrally administered nor-BNI-induced antidepressant-like actions measured by both behavior and brain-derived neurotrophic factor (BDNF) gene expression. In addition, antagonist studies were conducted to determine the role of opioid receptor subtypes and the time course of nor-BNI's pharmacological actions. Antidepressant-like behavioral effects were measured by decreased immobility in the rat forced swim test and BDNF mRNA expression was determined by in situ hybridization. Centrally administered nor-BNI (20 microg, i.c.v.) decreased immobility and increased BDNF mRNA expression in the hippocampus on day 1, not on days 3-14, post-administration. Systemic administration of selective mu-, delta- and kappa-opioid receptor antagonists did not block nor-BNI-induced antidepressant-like effects. In contrast, i.c.v. administration of nor-BNI 7 or 14 days earlier significantly blocked subsequent nor-BNI-induced decreased immobility and upregulation of BDNF mRNA expression. Although the duration of nor-BNI's antidepressant-like effects did not synchronize with that of its kappa-opioid receptor antagonist effects, this study is the first to show that centrally administered nor-BNI, like most clinically used antidepressants, can upregulate BDNF mRNA expression in the rat hippocampus. These findings further demonstrate that central kappa-opioid receptor mediates antidepressant-like effects of nor-BNI measured by both behavior and BDNF gene expression.

Sensation-seeking is a human personality trait associated with a greater propensity to use psychoactive substances. A rat model showing face validity of this human trait has been developed. The model is based on the variety of behavioral responses that rats exhibit in a novel and inescapable environment, with some animals (high-responders, HR) being highly active, and others (low-responders, LR) showing less exploration. More active rats (HR) also show increased drug-taking and decreased anxiety-like behavior. There is evidence that response to novelty may rely on differential 5-HT-mediated neurotransmission. This research focuses on the recently discovered 5-HT6 and 5-HT7 receptors which share affinity for neuroleptic drugs and hallucinogens. To date, emerging evidence suggests that 5-HT6 and 5-HT7 may be involved in cognition and mood regulation, respectively. To further our knowledge of their behavioral attributes, we compared patterns of gene expression for these receptors in the brains of HR and LR rats. As a control, gene expression for the 5-HT3 receptor was investigated because its contribution to anxiety and addiction is only weakly demonstrated. Transcript levels for 5-HT6 in the olfactory tubercle inversely correlated with the level of locomotion in a novel environment. Phenotype differences in mRNA signal for 5-HT6 showed a complex pattern in the dentate gyrus. LR rats were statistically higher in the most anterior region of the dentate gyrus, while HR rats were higher in median areas of the dentate gyrus. Levels of 5-HT7 transcript in HR rats were significantly lower than LR rats in pivotal areas for information trafficking, such as thalamo-cortical projection areas and dorsal hippocampus. By contrast, phenotype differences in 5-HT3 expression were not found in areas of the limbic cortex and mesolimbic system. Taken together, these results provide new insight into the potential contribution of 5-HT to novelty-seeking behavior and associated behaviors such as substance abuse.

Metabolic syndrome is characterized by obesity, elevated blood pressure (BP), insulin resistance, and hypercholesterolemia. Recently an animal model of this disorder has been proposed in rats selectively bred based on their performance on a treadmill-running task. Accordingly, low capacity runner (LCR) rats exhibited all of the diagnostic criteria for metabolic syndrome, including elevated BP, as compared to their high capacity runner (HCR) counterparts [U. Wisløff, S.M. Najjar, O. Ellingsen, P.M. Haram, S. Swoap, Q. Al-Share, M. Fernstrom, K. Rezaei, S.J. Lee, L.G. Koch, S.L. Britton, Cardiovascular risk factors emerge after artificial selection for low aerobic capacity, Science 307 (2005) 418-420]. Previous studies have highlighted the importance of GABAergic neurotransmission in the medullary cardiovascular-regulatory areas in the central control of BP. Thus, we hypothesized a dysregulation in GABAergic transmission in the medullary cardiovascular-regulatory nuclei of LCR rats. To begin testing this hypothesis we carried out experiments examining expression of the GABA synthetic enzymes, GAD65 and GAD67, mRNAs in the two rat strains via radioactive in situ hybridization. Our results showed GAD65 and GAD67 mRNAs were widely expressed throughout the brainstem; quantification revealed increased GAD65 mRNA expression in LCR animals in the caudal nucleus tractus solitarius (NTS) and rostral ventrolateral medulla (VLM) as compared to HCR rats. Conversely, no differences in the expression of GAD67 were detected in these regions. These data are consistent with the notion of altered GABAergic neurotransmission in the NTS and VLM in metabolic syndrome, and point to the importance of these regions in cardiovascular regulation.

Numerous studies have demonstrated that Sprague-Dawley rats exhibit a wide range of locomotor reactivity when placed in a novel environment. High Responder (HR) rats show exaggerated locomotor response to novelty, enhanced neuroendocrine stress reactivity, decreased anxiety-like behavior, and propensity to self-administer psychostimulants, compared to the less active Low Responder (LR) animals. Few studies have explored the early environmental factors which may underlie the HR-LR differences in emotional reactivity. Considering the enormous impact of maternal care on rodent neurodevelopment, we sought to examine maternal behavior in HR-LR dams to determine whether they exhibit differences which could contribute to their offspring's differential temperaments. Females, like males, can be classified as HR versus LR, showing marked differences in novelty-induced locomotor activity and anxiety-like behavior. HR-LR mothers behaved differently with their pups during the first two postpartum weeks. LR dams spent greater time licking and nursing their pups compared to HR dams, with the most prominent differences occurring during the second postpartum week. By contrast, when non-lactating HR-LR females were presented with orphaned pups, the pattern of maternal response was reversed. HR females were more responsive and showed greater maternal care of the novel pups compared to LR females, which were probably inhibited due to fear of the unfamiliar pups. This underscores the critical interplay between the female's emotional phenotype, her hormonal status and her familiarity with the pup as key factors in determining maternal behavior. Future work should explore neural and hormonal mechanisms which drive these HR-LR differences in maternal behavior and their impact on the development of the offspring.

Gene expression microarray analysis in postmortem brains is one of the fastest growing fields of psychiatric research. Here we show that common polymorphisms (SNPs) present on probe sets can masquerade as significant "gene expression" differences. After first observing this artifact in the Catechol-O-methyl transferase (COMT) gene, we replicate the finding in two additional genes predicted to show this artifact. Many Affymetrix chips contain thousands of SNPs that are both common and in the central probe region affecting hybridization, and thus have the potential to confound expression analysis.

Recent evidence now suggests the involvement of the fibroblast growth factor (FGF) system in mood disorders. Specifically, several members of the FGF family have been shown to be dysregulated in individuals with major depression. In this review, we will introduce the FGF system in terms of structure and function during development, in adulthood, and in various regions and cell types. We will also review the FGF system as a mediator of neural plasticity. Furthermore, this review will summarize animal as well as human studies. The majority of animal studies have focused on stress, environmental enrichment, pharmacological manipulations, and the hippocampus. By contrast, human studies have focused on volumetric measurements, antidepressant literature, and, most recently, post-mortem microarray experiments. In summary, a reduced tone in the FGF system might alter brain development or remodeling and result in a predisposition or vulnerability to mood disorders, including major depression.

Gene expression profiling of brain tissue samples applied to DNA microarrays promises to provide novel insights into the neurobiological bases of primate behavior. The strength of the microarray technology lies in the ability to simultaneously measure the expression levels of all genes in defined brain regions that are known to mediate behavior. The application of microarrays presents, however, various limitations and challenges for primate neuroscience research. Low RNA abundance, modest changes in gene expression, heterogeneous distribution of mRNA among cell subpopulations, and individual differences in behavior all mandate great care in the collection, processing, and analysis of brain tissue. A unique problem for nonhuman primate research is the limited availability of species-specific arrays. Arrays designed for humans are often used, but expression level differences are inevitably confounded by gene sequence differences in all cross-species array applications. Tools to deal with this problem are currently being developed. Here we review these methodological issues, and provide examples from our experiences using human arrays to examine brain tissue samples from squirrel monkeys. Until species-specific microarrays become more widely available, great caution must be taken in the assessment and interpretation of microarray data from nonhuman primates. Nevertheless, the application of human microarrays in nonhuman primate neuroscience research recovers useful information from thousands of genes, and represents an important new strategy for understanding the molecular complexity of behavior and mental health.

Nonpeptidic delta-opioid receptor agonists produce antidepressant-like effects in rodents, and compounds that inhibit the breakdown of endogenous opioid peptides have antidepressant-like effects in animal models. In this study, the behavioral effects of the enkephalinase inhibitor, RB101 (N-[(R, S)-2-benzyl-3-[(S)(2-amino-4-methyl-thio)-butyldithio]-1-oxopropyl]-l-phenylalanine benzyl ester), were examined. Specifically, the effects of RB101 on convulsive activity, locomotor activity, and antidepressant-like effects in the forced swim test were studied in Sprague–Dawley rats, and the opioid receptor types mediating these effects were examined by antagonist studies. In addition, the effects of RB101 on brain-derived neurotrophic factor (BDNF) mRNA expression were evaluated in relation to its antidepressant effects. RB101 produced delta-opioid receptor-mediated antidepressant effects (32 mg/kg i.v. and 100 mg/kg i.p.) and increased locomotor activity (32 mg/kg i.v.) in rats. RB101 did not produce convulsions or seizures and did not alter BDNF mRNA expression. In conclusion, RB101 has the potential to produce antidepressant effects without convulsions.

Individual differences in novelty-induced exploratory activity identify rats which can serve as a model of human sensation-seeking, risk-taking behaviour. Experimentally naïve rats, when exposed to mild stress of a novel environment, exhibit variability in their exploratory activity. Some rats display high rates of locomotor reactivity to novelty (high responders (HR)), and others display low rates (low responders (LR)). The LRHR phenotype is a reliable predictor of drug-taking behaviour and is linked to differences in hippocampal glucocorticoid receptor mRNA expression. In this study, we investigated whether the LRHR phenotype is associated with differences in the quantitative morphology of the hippocampal field CA3, dentate gyrus molecular layer, granule cell layer and mossy fibres. LRs and HRs showed no significant differences in the volumes of CA3 and dentate molecular layer volume or the number of dentate granule cells. However, LRs had a significantly larger suprapyramidal mossy fibre terminal field volume when compared to HRs. The infrapyramidal mossy fibres did not differ between phenotypes. Also, we found a LRHR phenotype-independent significant negative correlation between molecular layer volume per granule cell and the total number of granule cells. These findings implicate the SP-MF in vulnerability for risk-taking behaviour, and we propose that LR and HR hippocampi may differ in the way novelty information is processed.

Dexamethasone is commonly used to limit the severity of chronic lung disease in premature infants with severe respiratory distress syndrome. Recent literature has demonstrated an association between dexamethasone exposure in critically ill premature neonates and later development of cerebral palsy. However, the majority of children exposed to dexamethasone in the neonatal period do not develop cerebral palsy or global developmental delay, and other more subtle effects of early life glucocorticoid exposure may go unnoticed. Presently, little is known regarding possible effects of early dexamethasone exposure on development of neuropeptide systems that are sensitive to glucocorticoid modulation. One such system is the pain-related opioid system that interacts with the stress-related limbic-hypothalamic pituitary adrenal (LHPA) axis. In the present study, a neonatal rat model was used to expose newborn rats to dexamethasone. Using a within-litter design, on postnatal days P3 through P6, pups were either handled, or they received a daily intramuscular injection of saline or dexamethasone. Adult animals were sacrificed on day of life P120, their brains were removed and quick-frozen. Using in situ hybridization histochemistry, mRNA expression of the opioid receptor-like (ORL1) receptor was measured in the paraventricular nucleus of the hypothalamus (PVN) and the hippocampal formation. In dexamethasone-treated adult male rats, ORL1 mRNA expression was increased in the PVN and dentate gyrus, but decreased in area CA1, when compared to handled and vehicle controls. These results suggest that prolonged glucocorticoid receptor (GR) occupation in the neonatal period leads to permanent alterations in ORL1 expression in the LHPA stress axis of the adult rat.

Beta-endorphin (beta-End) and its precursor, proopiomelanocortin (POMC), are expressed in testis and beta-End stimulates testosterone secretion locally. We measured POMC expression in Syrian hamster testis following transfer from long days (LDs) to short days (SDs). We used RT-PCR to amplify partial-length hamster POMC cDNA to generate a probe for Northern analysis. We also used rat beta-End antiserum for radioimmunoassay analysis. SD exposure for 2 weeks decreased POMC mRNA and beta-End, but not testis weight or testosterone. We conclude that POMC signaling may play a role in seasonal regulation of testicular function.

Estrogen receptor beta (ERbeta) has been previously mapped in the rat central nervous system. This study aims to explore the regulation of ERbeta mRNA as it is expressed in the intact and cycling female rat brain. Young adult female rats (90+ day, N=20) were screened for estrous phases via vaginal cytology and sacrificed. Brains and blood were collected and processed for in situ hybridization and estradiol (E2) and progesterone (P4) hormone assays, respectively. ERbeta mRNA levels exhibited significant correlations with ovarian steroid ratios (E2/P4) in various brain regions, including the bed nucleus of stria terminalis, the medial nucleus of amygdala, and the anteroventral periventricular nuclei but not the paraventricular and the supraoptic nuclei or the preoptic area of the hypothalamus. No regulatory changes were detected in the cortex. Specifically, in the affected regions, higher P4 levels were significantly correlated with higher ERbeta mRNA expression. In contrast, there was a tendency for higher E2 levels to be correlated with lower ERbeta mRNA expression, but this tendency reached significance only in the bed nucleus of stria terminalis. These results suggest that ERbeta mRNA is regulated in the intact and cycling female rat hypothalamic as well as extrahypothalamic brain regions, and the circulating ovarian hormones play a critical role.

DNA microarrays are powerful tools for the analysis of the organization and regulation of the brain, in both illness and health. Such messenger RNA expression methods are outgrowths of a marriage between the several genome sequencing projects and a wide variety of physical, chemical, optical, and electronic systems. The advantages of microarray analyses include the ability to study the regulation of several genes or even the entire genome in a single experiment. However, there are substantive issues associated with the use of these tools that need to be considered before drawing conclusions about the genomic regulation of the brain. These issues include the loss of most anatomic (i.e., cellular and circuit) specificity, only fair sensitivity, lack of absolute quantitative data, poor comparability between studies, and high variability in sample values, to mention the most obvious. In this review we point to some of the solutions proposed for these problems and novel techniques and approaches for newer methods. Among these are methods for making arrays more sensitive, including nonarray messenger RNA expression systems. The future of this field and its links to deeper protein and cell biology are both emphasized.

Nitric oxide (NO) has been implicated in the modulation of mitochondrial respiration, membrane potential, and subsequently in apoptosis. Although the presence of a mitochondrial NO synthase (mtNOS) has been described, there is no direct evidence in vivo of the presence of NO within mitochondria. It was the aim of this study to demonstrate the in vivo production of NO within mitochondria. Using the novel fluorometric NO detection system, 4,5-diaminofluorescein diacetate (DAF-2/DA), we observed the presence of NO production in PC12 and COS-1 cells by conventional and confocal fluorescence microscopy. Part of the overall NO signal was colocalized within a subpopulation of mitochondria, labeled with the potential-dependent probe MitoTracker red. These findings demonstrate for the first time that the subcellular distribution of NO production is consistent with the presence of a mitochondrial NOS. Our results provide a new tool to directly study the modulatory role of NO in mitochondrial respiration and membrane potential, in vivo.

The parabrachial nucleus (PB) is a major relay of noxious and non-noxious visceral sensory information from the nucleus of the solitary tract, spinal cord, and spinal trigeminal nucleus to the forebrain. The nucleus of the solitary tract, spinal cord, and trigeminal dorsal horns contain many enkephalin- and dynorphin-immunoreactive neurons that project to the PB. To study the role of mu-opioid receptors in relaying these inputs, we examined the distribution of mu-opioid receptor immunoreactivity in the PB. The most intense staining was in the external lateral parabrachial subnucleus (PBel), including dendrites extending from the PBel into the lateral crescent subnucleus. Because the Pbel is a major source of projections to the amygdala, we combined retrograde tracing from the central nucleus of the amygdala with immunohistochemistry for mu-opioid receptors. These experiments showed that mu-opioid receptors are expressed by Pbel neurons that project to the amygdala, including those Pbel neurons whose dendrites extend into the lateral crescent subnucleus. These results indicate that mu-opioid receptors in the PB may mediate or modulate nociceptive information relayed to the amygdala from medullary or spinal cord neurons that terminate not only in the Pbel, but also in the adjacent lateral crescent parabrachial subnucleus.

This article provides an overview and general explanation of the rapidly developing area of gene chips and expression array technology. These are methods targeted at allowing the simultaneous study of thousands of genes or messenger RNAs under various physiological and pathological states. Their technical basis grows from the Human Genome Project. Both methods place DNA strands on glass computer chips (or microscope slides). Expression arrays start with complementary DNA (cDNA) clones derived from the EST data base, whereas Gene Chips synthesize oligonucleotides directly on the chip itself. Both are analyzed using image analysis systems, are capable of reading values from two different individuals at any one site, and can yield quantitative data for thousands of genes or mRNAs per slide. These methods promise to revolutionize molecular biology, cell biology, neuroscience and psychiatry. It is likely that this technology will radically open up our ability to study the actions and structure of the multiple genes involved in the complex genetics of brain disorders.

Psychoneuroendocrinology represents an interdisciplinary approach to integrating basic findings from several research disciplines encompassing psychiatry, psychology, neurology, neurobiology, neurosciences, and endocrinology. This field is aimed at elucidating endocrine functions and their dysfunctions. Insights into the control and regulation of endocrine functions by the central nervous system were well under way in the early part of this century. 76 At the same time, inroads in psychiatry provided compelling evidence that several diseases of the mind had their roots in organic disorders of the brain. Together with accumulating evidence indicating direct hormonal effects upon the brain, 20 and the realization that psychologic status (the mind or psyche) can dramatically influence neuroendocrine functions, 40 the emerging discipline of psychoneuroendocrinology emphasized the importance of exploring the inter-relationships between mind, brain, and hormones. A remarkably clear example of the power that psychologic status can exert over somatic neuroendocrine functions is the phenomenon of psychologic dwarfism. 27 As the name implies, one of the main features of dwarfism is abnormal regulation of the growth hormone system leading to unusually short stature in children. The discovery that this type of dwarfism could be traced mainly to severe adverse emotional conditions characterized by either the lack of, or abusive, primary caretaker/child relationship was of particular interest. 64 , 65 Most afflicted children removed from such adverse situations are observed to resume relatively normal growth within days, including restoration of growth hormone levels. This, and other psychosocial phenomena, exemplifies the importance of understanding the interplay between psychologic and organic factors to ultimately explain endocrine functioning. Psychosocial and emotional experiences are believed to play an essential role not only in the origin of psychologic dwarfism, but also in several other types of endocrine disorders. 40 Stress, which is also recognized to be triggered by negative emotional experiences, has been strongly implicated in the endocrine dysregulation of glucocorticoid hormonal levels, a comorbid symptom often observed in various affective disorders. 43 , 89 Although stress is a concept recognized by most civilizations around the world and is blamed as a culprit in diverse illnesses, it has remained an elusive concept difficult to define scientifically and to agree upon. 47 , 86 In a broad sense, stress has been used as a multifaceted term, which has included the stimulus or event reaching the organism (stressor), the perception and evaluation of an experience or situation as stressful, and the physiologic/endocrine responses elicited by this experience. 2 , 84 , 86 Although much is known about the physiologic and endocrine responses elicited by stressors and the various situations that can evoke such responses, our understanding of the cognitive processes involved in the evaluation of situations as stressful, and their neurophysiologic underpinnings, is not as clearly established. Moreover, this evaluation process, heavily based on cognitive styles and prior experiences, has been assumed to be an important factor underlying some of the large individual differences observed in humans and animals in response to similar stressful experiences. 2 A complete understanding of the effects of stress on endocrine functioning not only requires knowledge of the basic neuroendocrine mechanisms regulating the secretion of glucocorticoids via the hypothalamo-pituitary-adrenocortical (HPA) axis, but importantly requires knowledge of the interaction between this neuroendocrine system and the neurophysiologic evaluative functions that are pivotal in experiencing stress. The focus of this article is aimed at describing the animal models used in our laboratory to study some of the cognitive processes presumed to be implicated in the human experience of stress. The authors first highlight the basic constitutive elements of the HPA axis that control glucocorticoid secretion. Second, the authors describe the central nervous system circuits that are assumed to regulate the activity of the HPA axis, both anatomically and functionally. Finally, the authors discuss some of the findings pertinent to the involvement of psychologic variables in the regulation of the HPA axis in different animal models of stress.

It is now well established that opiate drugs, such as heroin and morphine exert their primary effects by mimicking naturally occurring substances, termed the opioid peptides or endorphins. Over the last 25 years, we have learned a great deal about the basic biology of the endogenous opioid system, both in terms of receptors and endogenous ligands. We have come to appreciate the molecular and biochemical complexity of this system, its widespread anatomy, and its diverse functions. These functions subsume a ‘housekeeping role’ that include a modulatory role of gastrointestinal, endocrine and autonomic functions; a sensory role, particularly prominent in inhibiting responses to noxious stimuli; an emotional role, evident in the powerful rewarding and addicting properties of endogenous and exogenous opioids; and a cognitive role, manifest in opioid modulation of learning and memory. Although we have learned a great deal about the pre-and post-synaptic elements, tissue-specific expression and molecular functions of this system, it is unclear if we have discovered all the relevant players or if more endogenous ligands and receptors will be identified. The issue of the number of preand postsynaptic elements and their relationship relates to a feature of this system that seems paradoxical and must be resolved if we are to understand fully its functioning and its relevance to drug abuse. Let us call this the ‘apparent paradox’ and return to it later in this chapter. We need a solid understanding of the endogenous

Background: Disturbances of the limbic–hypothalamic–pituitary–adrenal axis and the serotonin system are commonly found in depressive illness. Studying the effect of stress on these two neurobiological systems may give us important clues into the pathophysiology of affective illness and help us understand how stress and mood disorders are related. Methods: We studied the effect of chronic unpredictable stress and antidepressant treatment on serotonin 1A (5-HT 1A ), glucocorticoid (GR), and mineralocorticoid (MR) receptor levels in rat hippocampus, using in situ hybridization and receptor autoradiography. We also used in situ hybridization to quantify hippocampal 5-HT 1A , GR, and MR messenger (mRNA) levels in a small group of suicide victims with a history of depression, compared to matched controls (n = 6). Results: We found that rats subjected to chronic unpredictable stress showed a significant elevation of basal plasma corticosterone compared to nonstressed rats. Chronic stress also caused a decrease in 5-HT 1A mRNA and binding in the hippocampus. In addition, chronic stress produced alterations on the MR/GR mRNA ratio in this same region. The decreases in 5-HT 1A mRNA and binding, as well as the MR/GR alterations, were prevented in animals that received imipramine or desipramine antidepressant treatment. Zimelidine was unable to reverse stress-induced increases in corticosterone, and was only partially successful in preventing the stress-induced receptor changes in the hippocampus. Suicide victims with a history of depression showed changes that were very similar to the changes found in chronic stress. Conclusions: Alterations in hippocampal 5-HT 1A levels and in the MR/GR balance may be one of the mechanisms by which stress may trigger and/or maintain depressive episodes.

The technique of in situ hybridization with specific ribonucleotide probes was used to determine the distribution patterns of mRNA encoding the alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor (AR) subtypes in rat brain and spinal cord. The expression pattern of alpha 1a-AR mRNA has not been reported previously, and was found to be widespread throughout the rat central nervous system. High levels were found in regions of the olfactory system, several hypothalamic nuclei, and regions of the brainstem and spinal cord, particularly in areas related to motor function. Regions expressing moderate levels of mRNA for this receptor were the septum, bed nucleus of the stria terminalis, cerebral cortex, amygdala, cerebellum and pineal gland. Low expression levels were detected in the hippocampal formation. Most nuclei in the basal ganglia and thalamus expressed extremely low or undetectable levels of alpha 1a-AR mRNA. The expression patterns of the alpha 1b- and alpha 1d-AR mRNAs were similar to those described using oligonucleotide probes in earlier studies. High expression of alpha 1b-AR mRNA was noted in the pineal gland, most thalamic nuclei, lateral nucleus of the amygdala and dorsal and median raphe nuclei. Moderate expression levels were noted throughout the cerebral cortex, and in some olfactory, septal, and brainstem regions. The distribution of alpha 1d-AR mRNA was the most discrete of the three receptors examined. Expression was strong in the olfactory bulb, cerebral cortex, hippocampus, reticular thalamic nucleus, regions of the amygdala, motor nuclei of the brainstem, inferior olivary complex and spinal cord. Comparison of the distributions of the alpha 1a-, alpha 1b- and alpha 1d-AR mRNA suggests unique functional roles for each of these receptors.

The elicitation and reduction of fear were indexed with fear-potentiated startle and corticosterone release and induction of the immediate-early gene c-fos as a marker of neural activity in male Sprague-Dawley rats. Conditioning consisted of pairing one stimulus with footshock, which was withheld when the conditioned stimulus was preceded by a different modality stimulus, the conditioned inhibitor. On the test day, approximately 60% of the rats were used for c-fos in situ hybridization, and were presented with either the conditioned stimulus alone, the conditioned inhibitor alone, a compound of the two stimuli, or no stimuli, and killed 30 min following the presentation of 10 such stimuli. The remaining rats were tested with the fear-potentiated startle paradigm. Rats displayed reliable fear-potentiated startle and corticosterone release to the conditioned stimulus, and both measures were reduced when the conditioned stimulus was preceded by the conditioned inhibitor. The ventral bed nucleus of the stria terminalis, septohypothalamic nucleus, some tegmental nuclei, and the locus coeruleus had particularly high c-fos induction in rats that received the conditioned inhibitor, providing one of the first functional indication that these nuclei might be important in behavioural or endocrine inhibition. Conditioning specific c-fos induction in the three groups that received a stimulus on the test day was observed in many hypothalamic areas, the medial geniculate body and the central gray, structures previously involved in fear and anxiety. The cingulate, infralimbic and perirhinal cortex, nucleus accumbens, lateral septum, dorsal endopiriform nucleus, and ventral tegmental area had higher c-fos induction in rats presented with the fearful conditioned stimulus, confirming previous studies. The amygdala and hippocampus of conditioned rats did not show higher c-fos induction than in rats repeatedly exposed to the context. Many regions displayed c-fos messenger RNA induction in the control condition, suggesting that processes other than fear and anxiety participate in c-fos induction.

It is unclear how receptor/ligand families that are evolutionarily closely related achieve functional separation. To address this question, we focus here on the newly discovered Orphanin FQ, a peptide homologous to the opioid peptide Dynorphin, and its receptor, the Orphanin FQ receptor, which is highly homologous to the opioid receptors. In spite of this high degree of homology in terms of both ligands and receptors, there is little direct cross-talk between the Orphanin FQ system and the endogenous opioid system. Thus, the opioid peptides show either relatively low affinity or no affinity toward the Orphanin FQ receptor; conversely, Orphanin FQ has no affinity toward any of the opioid receptors. We sought to investigate the molecular basis of such discrimination by attempting to reverse it and endowing the Orphanin FQ receptor with the ability to bind opioids. We report that by mutating as few as four amino acids, we can produce a receptor that recognizes pro-Dynorphin products with very high affinity and yet still binds Orphanin FQ as well as the wild-type receptor. This suggests that the Orphanin FQ receptor has developed features that specifically exclude the opioids and that these features are distinct from those required for the high affinity binding of its own endogenous ligand.

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined mu, delta and kappa 1 receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned mu receptor (335-398) to examine the distribution of the mu receptor-like protein with immunohistochemical techniques. mu receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known mu receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.

Within the large family of G-protein-coupled receptors, a picture is emerging which contrasts the binding of small ligands and the binding of peptides to the seven-helix configuration of the proteins. Because of its unique richness in both peptide and non-peptide ligands, the opioid receptor family offers several advantages for achieving a better understanding of similarities and differences in ligand/receptor interactions across different classes of agonists and antagonists. Since multiple, naturally occurring, ligands interact with the multiple receptors with varying degrees of selectivity, this family is also an excellent model for examining the structural basis of selectivity. Thus, the molecular basis of binding affinity and selectivity of the κ and the δ opioid receptors was investigated by the construction of four κ/δ chimeric receptors. The pharmacological profiles of these chimeras as well as those of the wild type κ and δ receptors were determined by their binding with several different categories of opioid ligands. A linear model was used to deduce the relative contribution of each corresponding pairs of κ-δ receptor segments to the binding of a given ligand. The results show that the κ and δ receptors bind the same opioid core differently and achieve their selectivity through different mechanisms. In addition, the interaction of a peptide ligand with a receptor appears to be different from that of a small ligand. Furthermore, these results point to a particularly important role of the second extracellular loop and the top half of transmembrane domain 4 in the binding of prodynorphin products. Together, the results suggest that these peptide receptors can be bound and activated via multiple binding pockets as a function of their own topography and the nature of the interacting ligand.

Localization and regulation of mineralocorticoid receptor (MR) heteronuclear RNA (hnRNA) was assessed in rat hippocampus using an intron-directed in situ hybridization approach. The presence of hnRNA in the cell nucleus reflects recent gene transcriptional events and can be used as an index of neuronal transcriptional activation or inhibition. In the present study, sections incubated with an MR intron probe labeled cells in all regions known to contain MR mRNA. Signal generated by the intron probe was localized specifically to the nuclear compartment, consistent with recognition of hnRNA. Analysis of distribution across hippocampus indicated that MR hnRNA was particularly abundant in dentate gyrus (DG). In contrast, MR mRNA shows similar levels of expression across all hippocampal subfields. Exposure of animals to acute restraint markedly reduced MR hnRNA levels in CA1 and DG 60 min and 120 min following stress exposure, consistent with reduced gene transcription. These data support the hypothesis that stress-induced glucocorticoid secretion can rapidly affect transcription of steroid receptor genes. MR mRNA levels did not decrease over comparable time periods, suggesting either a significant lag-time between transcriptional changes and changes in cytoplasmic mRNA pools or stimulus-driven alterations in post-transcriptional RNA processing.

The effects of glucocorticoid hormones on oxytocin receptors in rat hippocampus were investigated. Oxytocin receptor autoradiography (using 0.1 and 1.2 nM concentrations of [125I]OVTA) revealed a significant (P < 0.02) decrease in oxytocin receptor binding in adrenalectomized animals 7 days after the surgery. Corticosterone replacement at the time of adrenalectomy prevented the decrease in oxytocin binding. The findings were significant in hippocampus and subiculum. These findings suggest regulation of oxytocin receptors, and possibly oxytocin-regulated behaviors by glucocorticoids.

The cysteine lysosomal proteases comprise a large family of highly conserved enzymes which are essential for intracellular protein turnover. These proteases are very efficient in their ability to degrade components of the extracellular matrix, and have been implicated in processes of cell growth, malignant transformation and inflammation. There is also a growing body of evidence for their involvement in the metabolism of the amyloid precursor protein. The production of insoluble /IA4 amyloid peptide is thought to be one of the key events that lead to the development of Alzheimer's pathology. To see the physiological role these enzymes play in the brain, we studied the relative abundance and distribution of the messenger RNAs for three lysosomal cysteine proteases, cathepsins B and L and cathepsin S, by in siru hybridization histochemistry in rat brain. All three enzymes are capable of degrading components of the extracellular matrix but they have different substrate preferences and resistances to neutral pH. We found that the mRNAs for cathepsins B, L, and S have different expression patterns in brain. Cathepsin B mRNA shows the highest level of expression. It has a wide distribution, and is preferentially expressed in neurons. The expression patterns of cathepsin B and cathepsin L mRNA overlap in many brain regions; in some areas they complement each other. Cathepsin B and L mRNAs are highly expressed in the choroid plexus, a structure that is instrumental in brain development. Both transcripts are also abundant in the neuropeptide synthesizing hypothalamic nuclei. Cathepsin S mRNA has wide expression pattern throughout brain, in grey and white matter. A great number of cells that express cathepsin S have microglial morphology. Regions that are known to contain the highest amounts of the amyloid precursor protein express highest levels of cathepsin B and cathepsin L mRNA. Also, all three transcripts are highly represented in regions that are most prone to degeneration in Alzheimer's disease. These results suggest a role for these lysosomal hydrolases released from degenerating cells in the development of Alzheimer's pathology. The mammalian lysosomal cysteine proteases, also known as cathepsins, are members of the papain family of cysteine proteases. It is a large group of enzymes with remarkable homology between species throughout the plant and animal kingdom." The members of this family are mainly implicated in intracellular protein turnover.8,26 Due to the ability of these enzymes to degrade constituents of the extra- cellular matrix, they have also been implicated in processes of cell growth, tumor invasiveness and inflammation.44 Although the biochemistry of differ- ent members of this family is fairly well studied, their physiological role and the regulatory mechanisms that govern their action in normal and pathological conditions are poorly understood. Cathepsin B and cathepsin L are two prominent members of the papain family with broad substrate specificity. Their expression is highly elevated in cells that have undergone malignant transformation.36 Rat

The distribution of cells expressing μ-receptor mRNA and μ-receptor binding sites were compared in brain and spinal cord tissue sections using a combination of in situ hybridization and receptor autoradiographic techniques. μ-Receptor mRNA was visualized with a 35S-labeled cRNA probe directed to transmembrane III–VI of the rat μ-receptor, while μ-receptor binding sites were labeled with the μ-selective ligand [3H]DAMGO. A high correspondence between the μ-receptor mRNA and receptor binding distributions was observed in the nucleus of the accessory olfactory bulb, anterior olfactory nuclei, striatal patches of the nucleus accumbens and caudate-putamen, endopiriform nucleus, claustrum, diagonal band of Broca, globus pallidus, ventral pallidum, bed nucleus of stria terminalis, most thalamic nuclei, medial and posteriocortical medial amygdala, lateral, dorsomedial, posterior and mammillary nuclei of the hypothalamus, presubiculum, subiculum, rostral interpeduncular nucleus, median raphe, inferior colliculus, parabrachial nucleus, locus coeruleus, central grey, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis, nucleus cuneatus, and the dorsal motor nucleus of vagus. Differences in μ-receptor mRNA and receptor binding distributions were observed in several regions, including the olfactory bulb, cortex, hippocampus, superior colliculus, spinal trigeminal nucleus, cochlear nucleus and spinal cord, and may be due to μ-receptor transport to presynaptic terminals.

Conventional in situ hybridization methods have been useful in characterizing the anatomical distribution of cells in the central nervous system that express dopamine D2 receptor mRNA. However, due to the large size of the D2 mRNA pool, this method may be insensitive to changes in D2 gene transcription. We have developed a method of hybridizing a 35S-labelled cRNA probe to an intron in the D2 receptor gene in order to measure the amount of primary transcript or heteronuclear RNA (hnRNA) in D2-expressing cells. Introns are found uniquely in hnRNA and are thought to be short-lived intermediates. Thus, monitoring introns could represent a more direct measure of D2 gene transcription. The anatomical distribution of the D2 hnRNA is similar to the distribution of D2 mRNA in the rat brain. D2 heteronuclear RNA was found in the nuclei of cells in the caudate putamen, nucleus accumbens, hippocampus, olfactory tubercle, substantia nigra, ventral tegmental area, and zona incerta. Other regions that contain D2 mRNA, but do not demonstrate intronic signal, include the globus pallidus, prefrontal, cingulate, entorhinal, and piriform cortex, septum, and amygdala. However, these areas have low amounts of D2 mRNA and may contain levels of D2 hnRNA that are below detection. Heteronuclear RNA quantitation by solution hybridization followed by RNase protection was performed on striatum, substantia nigra, cerebral cortex, hippocampus, hypothalamus, and pituitary using a D2 intron 7/exon 8 border probe. These results corroborate the distribution of hnRNA revealed with intronic in situ hybridization. In addition, protection assays were able to detect hnRNA in areas that express low levels of D2 like the cortex, hippocampus and hypothalamus. hnRNA/mRNA ratios calculated from intron/exon border probe protection assays were not equivalent for all the tissue areas studied, indicating that transcription and/or hnRNA half lives may differ between tissues that express D2 receptors. The combined use of intronic in situ hybridization and intron/exon border protection assay as an index of D2 gene transcription and RNA processing provides more information than measuring the mRNA pool alone. It may also prove to be a more useful measure of gene regulation, allowing for evaluation of gene responses to acute treatments.

In the central nervous system, adrenal steroids bind to two different types of corticosteroid receptors: glucocorticoid (GR) or mineralocorticoid (MR). In vitro biochemical and autoradiographic techniques have been used to infer GR and MR protein abundance in the hippocampus. Adrenalectomy (ADX) is routinely performed to measure the normal receptor number in absence of corticosterone (B), which would otherwise interfere with the binding reaction, The developing rodent has low basal B levels until the third week of life. We were interested in whether removal of circulating B may have a greater impact in the developing hippocampus than in the adult animal. In this study we examined the effect of a 14-h ADX on hippocampal GR and MR binding capacity (B(max)) by standard binding techniques and on gene expression by in situ hybridization. ADX was performed on Day 6, 10, 14, 18, 22, 28, 35, and 45 and on adult animals. GR B(max) increased from Day 6 to adult levels by Day 22 (d6 = 159.0 +/- 27; d22 = 369.1 +/- 43; a = 344.8 +/- 23, fmol/mg protein +/- SE). In contrast, MR B(max) had adult levels on Day 6 and increased above these until Day 45, when it decreased and approached adult concentrations (d6 = 83.2 +/- 22; d45 = 123 +/- 23; a = 76.9 +/- 13, fmol/mg protein +/- SE). The greatest absolute increase for both receptors occurred between Days 22 and 45 and correlated with increases in GR and MR gene expression. Moreover, age- and region-specific changes were evident in the developing hippocampus. In addition, the adult animal also exhibited an MR mRNA upregulation after 14 h of adrenalectomy. We propose extreme caution when interpreting GR and MR B(max) values obtained after short-term adrenalectomy in both adult and developing animals since upregulation of these genes is evident in this short time frame.

The effects of chronic cocaine exposure on dopamine DI and D2 receptor gene expression in the human brain were studied in postmortem samples from chronic cocaine abusing and matched control subjects. U~ing in situ hybridization and receptor autoradiography to examine messe ger ribonucleic acid (RNA ) and binding sites, respectively, neither D I nor Dz receptor expresshTn was found to be changed in the nucleus accumbens, caudate, putamen, or substantia nigra of the cocaine-exposed subjects. Although chronic cocaine exposure can produce alterations in dopaminergic neurotransmission, sustained compensatory changes in dopamine receptor expression do not appear to occur in the human.