Understanding how non-coding genomic polymorphisms affect gene expression.

The NIH Common Fund GTEx project is designed to serve as a data and post-mortem tissue resource to the research community. The project is testing the role of genomic variation in altering gene expression across a wide array of tissues in a large number of human post-mortem donors. Both data and tissue samples are available to the research community for additional studies.

Brain somatic mutations: the dark matter of psychiatric genetics?

Although inherited DNA sequences have a well-demonstrated role in psychiatric disease risk, for even the most heritable mental disorders, monozygotic twins are discordant at a significant rate. The genetic variation associated with mental disorders has heretofore been based on the search for rare or common variation in blood cells. This search is based on the premise that every somatic cell shares an identical DNA sequence, so that variation found in lymphocytes should reflect variation present in brain cells. Evidence from the study of cancer cells, stem cells and now neurons demonstrate that this premise is false. Somatic mutation is common in human cells and has been implicated in a range of diseases beyond cancer. The exuberant proliferation of cortical precursors during fetal development provides a likely environment for somatic mutation in neuronal and glial lineages. Studies of rare neurodevelopmental disorders, such as hemimegencephaly, demonstrate somatic mutations in affected cortical cells that cannot be detected in unaffected parts of the brain or in peripheral cells. This perspective argues for the need to investigate somatic variation in the brain as an explanation of the discordance in monozygotic twins, a proximate cause of mental disorders in individuals with inherited risk, and a potential guide to novel treatment targets.

Why has it taken so long for biological psychiatry to develop clinical tests and what to do about it?

Patients with mental disorders show many biological abnormalities which distinguish them from normal volunteers; however, few of these have led to tests with clinical utility. Several reasons contribute to this delay: lack of a biological 'gold standard' definition of psychiatric illnesses; a profusion of statistically significant, but minimally differentiating, biological findings; 'approximate replications' of these findings in a way that neither confirms nor refutes them; and a focus on comparing prototypical patients to healthy controls which generates differentiations with limited clinical applicability. Overcoming these hurdles will require a new approach. Rather than seek biomedical tests that can 'diagnose' DSM-defined disorders, the field should focus on identifying biologically homogenous subtypes that cut across phenotypic diagnosis--thereby sidestepping the issue of a gold standard. To ensure clinical relevance and applicability, the field needs to focus on clinically meaningful differences between relevant clinical populations, rather than hypothesis-rejection versus normal controls. Validating these new biomarker-defined subtypes will require longitudinal studies with standardized measures which can be shared and compared across studies--thereby overcoming the problem of significance chasing and approximate replications. Such biological tests, and the subtypes they define, will provide a natural basis for a 'stratified psychiatry' that will improve clinical outcomes across conventional diagnostic boundaries.

Social amnesia in mice lacking the oxytocin gene.

The development of social familiarity in rodents depends predominantly on olfactory cues and can critically influence reproductive success. Researchers have operationally defined this memory by a reliable decrease in olfactory investigation in repeated or prolonged encounters with a conspecific. Brain oxytocin (OT) and vasopressin (AVP) seem to modulate a range of social behaviour from parental care to mate guarding. Pharmacological studies indicate that AVP administration may enhance social memory, whereas OT administration may either inhibit or facilitate social memory depending on dose, route or paradigm. We found that male mice mutant for the oxytocin gene (Oxt-/-) failed to develop social memory, whereas wild-type (Oxt+/+) mice showed intact social memory. Measurement of both olfactory foraging and olfactory habituation tasks indicated that olfactory detection of non-social stimuli is intact in Oxt-/- mice. Spatial memory and behavioural inhibition measured in a Morris water-maze, Y-maze, or habituation of an acoustic startle also seemed intact. Treatment with OT but not AVP rescued social memory in Oxt-/- mice, and treatment with an OT antagonist produced a social amnesia-like effect in Oxt+/+ mice. Our data indicate that OT is necessary for the normal development of social memory in mice and support the hypothesis that social memory has a neural basis distinct from other forms of memory.

Corticotropin-releasing factor receptors in rat forebrain: autoradiographic identification.

Corticotropin-releasing factor (CRF) receptors were identified in rat forebrain by autoradiography with an iodine-125-labeled analog of ovine CRF substituted with norleucine and tyrosine at amino acid residues 21 and 32, respectively. High-affinity receptors for CRF were found in discrete areas of rat forebrain, including laminae I and IV of the neocortex, the external layer of the medium eminence, the lateral nucleus of the amygdala, and the striatum. These results are consistent with earlier findings on the immunohistochemical distribution of CRF and suggest that endogenous CRF has a physiological role in regulating activity of the central nervous system.

Neuroendocrine bases of monogamy.

A number of studies have implicated the neurohypophyseal peptides oxytocin and vasopressin in the central mediation of complex social behaviors, including affiliation, parental care and territorial aggression. Research on a monogamous rodent, the prairie vole (Microtus ochrogaster), suggests that these neuropeptides are also involved in the control of several behaviors associated with monogamy, including pair bonding, paternal care and mate guarding. Comparative studies using several species of vole have identified species-specific patterns of oxytocin- and vasopressin-receptor expression in the brain that appear to be associated with a monogamous versus non-monogamous social structure. Molecular studies suggest that changes in the regulation of oxytocin- and vasopressin-receptor gene expression underlie these species differences in receptor distribution and might provide a mechanism for the evolution of monogamy in voles.

Neuropeptides and the evolution of social behavior.

Comparative studies over the past year have revealed two new insights into the role of neuropeptides in the evolution of social behaviors. First, across vertebrate taxa, certain neuropeptide effects appear to be gender-specific. Second, species variations in receptor gene structure can alter neuropeptide receptor distribution and thereby contribute to species differences in social behavior.

Oxytocin in the medial amygdala is essential for social recognition in the mouse.

Oxytocin (OT) knock-out mice fail to recognize familiar conspecifics after repeated social exposures, despite normal olfactory and spatial learning abilities. OT treatment fully restores social recognition. Here we demonstrate that OT acts in the medial amygdala during the initial exposure to facilitate social recognition. OT given before, but not after, the initial encounter restores social recognition in OT knock-out mice. Using c-Fos immunoreactivity (Fos-IR) as a marker of neuronal activation in this initial encounter, we found similar neuronal activation in the wild-type (WT) and OT knock-out mouse in olfactory bulbs, piriform cortex, cortical amygdala, and the lateral septum. Wild-type, but not OT knock-out mice exhibited an induction of Fos-IR in the medial amygdala. Projections sites of the medial amygdala also failed to show a Fos-IR induction in the OT knock-out mice. OT knock-out, but not WT, mice showed dramatic increases in Fos-IR in the somatosensory cortex and the hippocampus, suggesting alternative processing of social cues in these animals. With site-specific injections of OT and an OT antagonist, we demonstrate that OT receptor activation in the medial amygdala is both necessary and sufficient for social recognition in the mouse.

Facilitation of affiliation and pair-bond formation by vasopressin receptor gene transfer into the ventral forebrain of a monogamous vole.

Behaviors associated with monogamy, including pair-bond formation, are facilitated by the neuropeptide vasopressin and are prevented by a vasopressin receptor [V1a receptor (V1aR)] antagonist in the male prairie vole. The neuroanatomical distribution of V1aR dramatically differs between monogamous and nonmonogamous species. V1aR binding is denser in the ventral pallidal region of several unrelated monogamous species compared with nonmonogamous species. Because the ventral pallidum is involved in reinforcement and addiction, we hypothesize that V1aR activation in this region promotes pair-bond formation via a mechanism similar to conditioning. Using an adeno-associated viral vector to deliver the V1aR gene, we increased the density of V1aR binding in the ventral pallial region of male prairie voles. These males exhibited increased levels of both anxiety and affiliative behavior compared with control males. In addition, males overexpressing the V1aR in the ventral pallidal region, but not control males, formed strong partner preferences after an overnight cohabitation, without mating, with a female. These data demonstrate a role for ventral pallidal V1aR in affiliation and social attachment and provide a potential molecular mechanism for species differences in social organization.

Serotonergic responsivity in obsessive-compulsive disorder. Effects of chronic clomipramine treatment.

Clomipramine is a potent serotonin reuptake blocker that decreases the symptoms of obsessive-compulsive disorder (OCD). To investigate whether clomipramine treatment in OCD affects brain serotonergic responsiveness, metachlorophenylpiperazine (mCPP), a selective serotonin agonist, and placebo were given under double-blind conditions to nine patients with OCD before and after treatment with clomipramine. Unlike our previous observations of a marked transient increase in obsessional symptoms and anxiety following 0.5 mg/kg of mCPP, readministration of mCPP after four months of treatment with clomipramine did not significantly increase obsessional symptoms and anxiety. Similarly, the hyperthermic effect of mCPP observed before treatment was eliminated after treatment with clomipramine. These findings are consistent with the development of adaptive subsensitivity to the serotonergic agonist mCPP during clomipramine treatment. A similar alteration in the response to endogenous serotonin may mediate clomipramine's antiobsessional effects.

Serotonergic responsivity in obsessive-compulsive disorder. Comparison of patients and healthy controls.

To examine the "serotonin hypothesis" of obsessive-compulsive disorder (OCD), we studied the behavioral and neuroendocrine effects of metachlorophenylpiperazine (mCPP), a serotonergic agonist, in patients with OCD and healthy controls. Twelve patients and 20 controls were given a single dose of 0.5 mg/kg of mCPP, administered orally under double-blind, placebo-controlled, random-assignment conditions. Following mCPP, but not following placebo, patients with OCD experienced a transient but marked exacerbation of obsessive-compulsive symptoms. Moreover, compared with healthy controls, patients exhibited greater other behavioral (but not endocrinologic or thermal) changes after mCPP. These findings are consistent with a special role for the neurotransmitter serotonin in OCD psychopathology.

Anxiety and cerebral blood flow during behavioral challenge. Dissociation of central from peripheral and subjective measures.

To investigate the relationship between anxiety and regional cerebral blood flow, we administered behavioral challenges to 10 patients with obsessive-compulsive disorder while measuring regional cerebral blood flow with the xenon 133 inhalation technique. Each patient was studied under three conditions: relaxation, imaginal flooding, and in vivo (actual) exposure to the phobic stimulus. Subjective anxiety, obsessive-compulsive ratings, and autonomic measures (heart rate, blood pressure) increased significantly, but respiratory rate and PCO2 did not change across the three conditions. Regional cerebral blood flow increased slightly (in the temporal region) during imaginal flooding, but decreased markedly in several cortical regions during in vivo exposure, when anxiety was highest by subjective and peripheral autonomic measures. These results demonstrate that intense anxiety can be associated with decreased rather than increased cortical perfusion and that ostensibly related states of anxiety (eg, anticipatory and obsessional anxiety) may be associated with opposite effects on regional cerebral blood flow.

Clomipramine in obsessive-compulsive disorder. Further evidence for a serotonergic mechanism of action.

Data from several previous studies link clomipramine's potent serotonergic effects to its clinical efficacy in reducing the symptoms of obsessive-compulsive disorder (OCD). To investigate this relationship further, we administered the serotonin (5-HT) receptor antagonist, metergoline, and placebo to ten patients with OCD in a crossover study carried out under double-blind, random-assignment conditions. In a previous study of untreated patients with OCD, we found no differences in the behavioral response to single-dose administration of metergoline or placebo. In the present study, patients with OCD receiving clomipramine hydrochloride on a long-term basis (with an average 40% lessening in OC symptoms) responded to a four-day period of administration of metergoline with significantly greater self- and observer-rated anxiety compared with the four-day placebo period. Obsessive-compulsive symptoms also tended to be greater during the metergoline phase, with significant drug-time interactions for both OC symptoms and anxiety peaking on day 4 of the metergoline phase. As anticipated, metergoline lowered plasma prolactin concentrations (providing evidence of physiologically significant 5-HT antagonism) but did not alter plasma clomipramine concentrations. These data further support the hypothesis that clomipramine's therapeutic behavioral effects in OCD are mediated via serotonergic mechanisms.

Obsessive-compulsive disorder. A double-blind trial of clomipramine and clorgyline.

Patients with obsessive-compulsive disorder who met DSM-III criteria and who had been ill for at least one year were studied in a double-blind, randomized, crossover comparison of the tricyclic antidepressant clomipramine hydrochloride and the monoamine oxidase inhibitor clorgyline hydrochloride. No significant improvement was evident after four weeks of treatment with placebo prior to the crossover study. Treatment with clomipramine was associated with significant improvement after both four and six weeks in measures of obsessions, anxiety, and depression. Antiobsessional responses to clomipramine did not depend on presence of depression. Improvement was correlated with plasma concentrations of clomipramine, but not with the plasma concentrations of any of its metabolites. No significant improvement was evident for the entire group with clorgyline treatment, although the conditions of individual patients did respond to the drug.

Increased plasma MHPG in dexamethasone-resistant depressed patients.

Severely depressed patients frequently show inadequate suppression of serum cortisol levels by dexamethasone. In a study of 15 depressed patients, we found a robust correlation between plasma levels of cortisol and 3-methoxy-4-hydroxy-phenylglycol after dexamethasone administration. These results suggest that dexamethasone resistance and adrenergic activation reflect parallel responses to illness-related stress in some depressed patients.

The sleep of patients with obsessive-compulsive disorder.

Fourteen patients with obsessive-compulsive disorder (OCD) were studied with all-night sleep EEG recordings. Nine of these patients reported abnormal sleep patterns before the polygraphic study. Analysis of the sleep records disclosed significantly decreased total sleep time with more awakenings, less stage 4 sleep, decreased rapid-eye-movement (REM) efficiency, and shortened REM latency compared with those of a group of age- and sex-matched normal subjects. These abnormalities generally resembled those of an age-matched group of depressed patients, although significant differences remained. These findings suggest that such sleep abnormalities as shortened REM latency may not be entirely specific for primary affective illness. They also point to a possible biological link between OCD and affective illness.

A benzodiazepine receptor-mediated model of anxiety. Studies in nonhuman primates and clinical implications.

beta-Carboline-3-carboxylic acid ethyl ester (beta-CCE) binds with high affinity to brain benzodiazepine receptors and has potent behavioral and physiologic effects in primates. Dose-related increases in behavioral agitation, plasma cortisol level, BP, and heart rate were observed after administration of doses between 50 and 500 micrograms/kg of beta-CCE to rhesus monkeys. All of these effects were blocked by pretreatment with diazepam. Pretreatment with clonidine hydrochloride and propranolol hydrochloride, both of which have been reported to have anxiolytic actions in man, attenuated only selective aspects of the response to beta-CCE. The behavioral, endocrine, and physiologic effects of low doses of beta-CCE in monkeys are similar to those observed in anxious patients or normal subjects under anxiety-provoking or stressful situations. Administration of benzodiazepine receptor active antagonists such as beta-CCE to primates may, therefore, provide a valid and reproducible model of human anxiety that could be used to investigate specific biologic aspects of anxiety disorders.

Regional induction of c-fos-like protein in rat brain after estradiol administration.

The protooncogene c-fos is induced in rat brain by various forms of physiological stimulation. In this study immunocytochemical staining for a peptide fragment of the c-fos protein was used to assess estradiol's effects on c-fos in rat brain. After estradiol benzoate (100 micrograms/kg) administration to ovariectomized rats, the number of cells staining for c-fos-like protein increased in the anterior medial preoptic area, the medial preoptic area, the medial amygdala nucleus, and the ventromedial nucleus of hypothalamus, all regions rich in estradiol-concentrating cells. This increase peaked between 12-48 h (depending on the region) after estradiol administration and was not observed in several areas with a lower density of estradiol-concentrating cells. In the most affected region, the anterior medial preoptic area, estradiol effects were dose dependent and not altered by progesterone administration. It is not yet clear whether estradiol induces c-fos expression in brain directly or whether c-fos is part of a cascade of mechanisms by which estradiol regulates gene expression.

A selective oxytocin antagonist attenuates progesterone facilitation of female sexual behavior.

Although previous studies have demonstrated that exogenous administration of oxytocin (OT) enhances sexual receptivity in female rats, there is no compelling evidence that endogenous OT has a physiological role in the regulation of female sexual behavior. In the current studies we centrally administered d(CH2)5[Tyr(Me)2Thr4,Tyr-NH2(9)]ornithine vasotocin (or OTA), a selective OT receptor antagonist, to block endogenous OT in ovariectomized females primed with different levels of gonadal steroids. After OTA administration (100-1000 ng), females primed with estradiol benzoate (EB; 1 microgram) and progesterone (P; 250 micrograms) showed reductions in both receptive and proceptive behaviors. These effects of OTA were also evident, though less striking, in females primed with higher doses of EB (10 micrograms) and P (250 micrograms), but significant OTA effects were absent in females primed with EB (10 micrograms) alone. Thus, OTA appeared to attenuate P's facilitation of sexual behavior. Surprisingly, these behavioral effects of OTA administration were not apparent immediately, but emerged only when OTA was given with P 4-6 h before behavioral testing. To determine if these delayed, but lasting, behavioral effects were associated with OTA occupancy of the OT receptor, we measured OT receptor binding ex vivo using receptor autoradiography. Six hours after intracerebroventricular administration of OTA (1000 ng), OT receptor binding was reduced at least 75% in the ventromedial nucleus of the hypothalamus relative to control levels of binding. Thus, those OT receptors previously implicated in the regulation of sexual receptivity appear to be significantly blocked throughout the period of OTA's behavioral effects. Together, these studies lend support to the hypothesis that endogenous OT has a physiological role in the regulation of female sexual behavior.

The regulation of oxytocin receptor binding in the ventromedial hypothalamic nucleus by testosterone and its metabolites.

Oxytocin (OT) receptor binding in the ventromedial hypothalamic nucleus is regulated by testosterone (T) in male rats. However, T is metabolized in the brain, and many of the central effects of T are mediated by its metabolites. The experiments reported here were designed to determine whether T affects OT receptor binding directly or through the action of its metabolites 17 beta-estradiol and 5 alpha-dihydrotestosterone. Adult male rats were either sham operated or castrated and treated 1 week later with T propionate (TP), 17 beta-estradiol benzoate (EB), dihydrotestosterone benzoate (DHTB), DHTB plus EB, or oil. OT receptor binding was assessed autoradiographically using [125I]d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT. In addition, seminal vesicle weights were measured as an index of androgenic activity. These experiments showed that TP and DHTB plus EB increased OT receptor binding in the ventromedial hypothalamic nucleus to the levels in intact males. Treatment with EB alone partially reinstated binding to the levels in intact males, while DHTB treatment was without effect. Castrated males treated with either TP or DHTB had seminal vesicle weights comparable to those of gonadally intact males and greater than those of animals in all other steroid conditions, indicating that sufficient levels of circulating steroids were attained in these groups. These data suggest that the induction of hypothalamic OT receptor binding by T is the result of the combined actions of estradiol and dihydrotestosterone. However, the mechanism underlying this interaction is unknown.