DRD2 co-expression network and a related polygenic index predict imaging, behavioral and clinical phenotypes linked to schizophrenia.

Genetic risk for schizophrenia (SCZ) is determined by many genetic loci whose compound biological effects are difficult to determine. We hypothesized that co-expression pathways of SCZ risk genes are associated with system-level brain function and clinical phenotypes of SCZ. We examined genetic variants related to the dopamine D2 receptor gene DRD2 co-expression pathway and associated them with working memory (WM) behavior, the related brain activity and treatment response. Using two independent post-mortem prefrontal messenger RNA (mRNA) data sets (total N=249), we identified a DRD2 co-expression pathway enriched for SCZ risk genes. Next, we identified non-coding single-nucleotide polymorphisms (SNPs) associated with co-expression of this pathway. These SNPs were associated with regulatory genetic loci in the dorsolateral prefrontal cortex (P<0.05). We summarized their compound effect on co-expression into a Polygenic Co-expression Index (PCI), which predicted DRD2 pathway co-expression in both mRNA data sets (all P<0.05). We associated the PCI with brain activity during WM performance in two independent samples of healthy individuals (total N=368) and 29 patients with SCZ who performed the n-back task. Greater predicted DRD2 pathway prefrontal co-expression was associated with greater prefrontal activity and longer WM reaction times (all corrected P<0.05), thus indicating inefficient WM processing. Blind prediction of treatment response to antipsychotics in two independent samples of patients with SCZ suggested better clinical course of patientswith greater PCI (total N=87; P<0.05). The findings on this DRD2 co-expression pathway are a proof of concept that gene co-expression can parse SCZ risk genes into biological pathways associated with intermediate phenotypes as well as with clinically meaningful information.

Brain-derived neurotrophic factor (BDNF) Val(66)Met polymorphism differentially predicts hippocampal function in medication-free patients with schizophrenia.

A Val(66)Met single-nucleotide polymorphism (SNP) in the brain-derived neurotrophic factor (BDNF) gene impairs activity-dependent BDNF release in cultured hippocampal neurons and predicts impaired memory and exaggerated basal hippocampal activity in healthy humans. Several clinical genetic association studies along with multi-modal evidence for hippocampal dysfunction in schizophrenia indirectly suggest a relationship between schizophrenia and genetically determined BDNF function in the hippocampus. To directly test this hypothesized relationship, we studied 47 medication-free patients with schizophrenia or schizoaffective disorder and 74 healthy comparison individuals with genotyping for the Val(66)Met SNP and [(15)O]H(2)O positron emission tomography (PET) to measure resting and working memory-related hippocampal regional cerebral blood flow (rCBF). In patients, harboring a Met allele was associated with significantly less hippocampal rCBF. This finding was opposite to the genotype effect seen in healthy participants, resulting in a significant diagnosis-by-genotype interaction. Exploratory analyses of interregional resting rCBF covariation revealed a specific and significant diagnosis-by-genotype interaction effect on hippocampal-prefrontal coupling. A diagnosis-by-genotype interaction was also found for working memory-related hippocampal rCBF change, which was uniquely attenuated in Met allele-carrying patients. Thus, both task-independent and task-dependent hippocampal neurophysiology accommodates a Met allelic background differently in patients with schizophrenia than in control subjects. Potentially consistent with the hypothesis that cellular sequelae of the BDNF Val(66)Met SNP interface with aspects of schizophrenic hippocampal and frontotemporal dysfunction, these results warrant future investigation to understand the contributions of unique patient trait or state variables to these robust interactions.

Epistatic interactions of AKT1 on human medial temporal lobe biology and pharmacogenetic implications.

AKT1 controls important processes in medial temporal lobe (MTL) development and plasticity, but the impact of human genetic variation in AKT1 on these processes is not known in healthy or disease states. Here, we report that an AKT1 variant (rs1130233) previously associated with AKT1 protein expression, prefrontal function and schizophrenia, affects human MTL structure and memory function. Further, supporting AKT1's role in transducing hippocampal neuroplasticity and dopaminergic processes, we found epistasis with functional polymorphisms in BDNF and COMT--genes also implicated in MTL biology related to AKT1. Consistent with prior predictions that these biologic processes relate to schizophrenia, we found epistasis between the same AKT1, BDNF and COMT functional variants on schizophrenia risk, and pharmacogenetic interactions of AKT1 with the effects on cognition and brain volume measures by AKT1 activators in common clinical use--lithium and sodium valproate. Our findings suggest that AKT1 affects risk for schizophrenia and accompanying cognitive deficits, at least in part through specific genetic interactions related to brain neuroplasticity and development, and that these AKT1 effects may be pharmacologically modulated in patients.

Effects of smoking during antipsychotic withdrawal in patients with chronic schizophrenia.

A number of studies have shown that patients with schizophrenia smoke more than other psychiatric patients and more than the general population. Also, medicated schizophrenics who smoke present more positive symptoms of schizophrenia than non-smokers. The objective of the present study was to assess the effect of smoking on ratings of psychopathology for 30 days following discontinuation of antipsychotic medication. The subjects were 101 treatment-resistant patients with schizophrenia who had been admitted to the inpatient service of Neuroscience Research Hospital (NRH), National Institute of Mental Health, between 1982 and 1994 to undergo studies involving discontinuation of antipsychotic medication. Patients were rated independently on a daily basis on the 22-item Psychiatric Symptom Assessment Scale (PSAS), an extended version of the Brief Psychiatric Rating Scale (BPRS). At baseline, ratings for Verbal Positive, Paranoia and Loss of Function were higher in smokers (n=65) than non-smokers (n=36), but a statistically significant difference was observed only for the Verbal Positive cluster. Analysis by gender revealed that male non-smokers had the lowest psychopathology ratings at baseline. There were no differences in Anxiety/depression, Behavior Positive, Deficit Symptoms or Mannerisms (a measure for abnormal involuntary movements). Following medication discontinuation, repeated-measure analysis demonstrated a 'time' effect for all the variables studied and a 'group' (smokers vs. non-smokers) effect for Verbal Positive, Paranoia, and Loss of Function. Post-hoc comparisons at individual time points showed significantly higher ratings for smokers at week 1 for Paranoia. No differences were observed at later time points. In conclusion, at baseline, smokers had more positive symptoms and were apparently more functionally impaired than non-smokers. This difference was no longer evident after a 30 day medication discontinuation period.

Treatment of tardive dyskinesia.

Although the new generation of atypical antipsychotic agents could some day eliminate concerns about tardive dyskinesia (TD), this disorder remains a significant clinical problem for both patients and physicians. Fortunately, many, if not most, cases of TD are mild. For patients with mild to moderate TD, therapeutic efforts are primarily directed at minimizing neuroleptic exposure or, when possible, changing to atypical agents. Most cases of TD do not seem to progress, suggesting that the risk of remaining on typical neuroleptics is probably small. Patients with moderate to severe forms of TD present greater challenges. These patients frequently require medication to suppress their dyskinesias. A variety of suppressive agents have been tried with limited success. No treatment strategy has emerged that is clearly superior or even successful in most patients. Increasing doses of typical neuroleptics may be useful for short-term suppression; however, the long-term efficacy and risk of this strategy have not been studied carefully. Data on atypical neuroleptics are scant. Clozapine's short-term suppressive effects seem, at best, weak, but patients may improve with long-term treatment. Medications with relatively few side effects that may have suppressive efficacy for some patients include calcium channel blockers, adrenergic antagonists, and vitamin E. Gamma-amino-butyric acid agonists agents and dopamine depleters are frequently useful, but have troubling side effects of their own. A variety of other medications have been employed, but are not well studied. For patients with tardive dystonia, anticholinergic agents or botulinum toxin has been particularly effective. Efforts to understand the neurobiology of TD may shed light on this persistent clinical conundrum.

New directions in the prevention and treatment of schizophrenia: a biological perspective.

Our current treatments for schizophrenia are, at best, palliative. With the exception of counseling those families with a known high risk for having schizophrenic offspring, no preventive measures are currently available. The not too distant future, however, promises to bring improvements in somatic treatments as well as the possible introduction of preventive measures. We are fully aware that current biological treatments work best when they are combined with psychosocial intervention, and expect that future biological treatments and preventions will also involve appropriate nonbiological considerations. Psychosocial treatments are covered elsewhere in this issue. Here we look at how modern genetics, pre- and perinatal factors, early and sustained intervention, and new medications are likely to decrease both the number of individuals with schizophrenia and the severity of the illness.

Pharmacological characterization of regulation of phosphoinositide metabolism by recombinant 5-HT2 receptors of the rat.

Transfection of 5-HT2 receptor cDNA in 293 cells induced the expression of a protein binding domain, exhibiting the classical 5-HT2 receptor transduction mechanism. Both [3H]DOB and [3H]spiperone high affinity binding sites were present in membranes of sense but not of antisense, 5-HT2 receptor cDNA transfected cells. Addition of 1 microM 5-HT induced a time-dependent increase of phosphoinositide (PI) metabolism in sense but not in antisense, 5-HT2 receptor cDNA transfected cells. Graded concentrations of 5-HT and of different serotonergic agonists showed different potencies (DOI greater than 5-HT greater than quipazine greater than DOM greater than alpha-methyl-5-HT greater than 8-OH-DPAT greater than 2-methyl-5-HT greater than CGS-12066B) in stimulating turnover of PI in cells transfected with cDNA encoding for 5-HT2 receptors of the rat. The ability of different antagonists to inhibit 5-HT-stimulated turnover of PI bore a direct relationship with their potency to inhibit 5-HT2 receptor binding in cells transfected with 5-HT2 receptor cDNA (spiperone greater than ketanserin greater than ritanserin greater than mianserin greater than haloperidol). Preincubation of transfected 293 cells with pertussis toxin failed to modify either 5-HT- or DOI-induced activation of metabolism of PI. Pretreatment of transfected 293 cells with DOI (100 nM) for 2 hr or more, significantly reduced activation of turnover of PI elicited by graded doses of 5-HT. When the transfected 293 cells were exposed to DOI (100 nM) for 12 hr and the challenge was performed after a 2-hr wash-out period, the desensitization of the response to 5-HT was virtually abolished.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine modulates the effect of ovarian steroids on glutamic acid decarboxylase activity in the rat fallopian tube.

Glutamic acid decarboxylase (GAD) activity was measured in the oviduct of normal rats in diestrous and in rats ovariectomized (OVX) seven days before. OVX induced a significant decrease of GAD activity in the Fallopian tube. This effect was completely reversed by coadministration of estradiol benzoate + progesterone (E + P). Simultaneous injection of atropine, but not of alpha-methyl-para-tyrosine or labetalol, completely prevented the activation of GAD induced by ovarian sterois. Moreover, prostigmin significantly potentiated the action of E + P on GAD activity in the rat oviduct. These data clearly suggest the participation of acetylcholine in the mechanisms whereby ovarian steroids regulate GAD activity in the rat Fallopian tube.

The 5-HT2 receptor in brain: recent biochemical and molecular biological developments and new perspectives in its regulation.

Radioligand binding as well as molecular biological studies revealed an heterogeneity of serotonin (5-HT) receptors in the central nervous system. The early availability of specific antagonists for the serotonin-2 (5-HT2) receptor subtype (spiperone, ketanserin and ritanserin represented an important step towards the biochemical, physiological and, more recently, molecular characterization of 5-HT2 receptors in brain. Though they are unevenly distributed in different brain areas, they are highly expressed in the frontal cortex. Based on radioligand studies, either two different 5-HT2 receptors or one 5-HT2 receptor with two different affinity states might exist. Molecular biological studies revealed that the 5-HT2 receptor belongs to the G-protein receptor superfamily and the 5-HT2 receptor clone encodes a single-subunit protein containing approximately 450 amino acids arranged in seven interconnected transmembrane segments. Recent studies suggested that 5-HT itself might not represent the endogenous ligand for the 5-HT2 receptor. Isolation and purification of an endogenous peptide of mol. wt 6-8 kDa with affinity for [3H]ketanserin recognition sites further supports this possibility. The rapid advances in the molecular understanding of the 5-HT2 receptor and its putative endogenous ligand may have significant implications in the actual debate on the classification of the 5-HT2 receptor subtypes.

Semi-preparative purification of an endogenous ligand for brain serotonin-2 receptors by coil planet centrifuge counter-current chromatography.

A horizontal flow-through coil planet centrifuge equipped with a rotatory frame holding three sets of composite column assemblies was used for purification of an endogenous ligand (ketanserin binding inhibitor) for the [3H]-ketanserin (3H-KET) recognition site. The protein mixture containing the endogenous material was successfully resolved by using a two-phase solvent system consisting of 95% ethanol-31.5% ammonium sulphate (1:2). The active fractions on 3H-KET binding obtained after counter-current chromatography (CCC) were further purified through a C18 microBondapak reversed-phase high-pressure liquid chromatographic column. The introduction of this advanced CCC technique represents an important step in the application of CCC for the separation of polar proteins from protein mixtures.

Biochemical and functional aspects on the control of prolactin release by the hypothalamo-pituitary GABAergic system.

A growing body of biochemical, immunohistochemical, and autoradiographic evidence indicates the presence of two different GABAergic systems in the mediobasal hypothalamus: one intrinsic, the tuberoinfundibular GABAergic system, and the other extrinsic, whose cell bodies are located outside the mediobasal hypothalamus and which projects to this area and establishes synaptic contacts with aminergic and peptidergic neurons involved in endocrine function. This particular anatomical configuration provides a rational basis to explain the dual action of GABA (inhibitory and stimulatory) on prolactin release. Different studies aimed at identifying the precise role of GABA on prolactin function have demonstrated that this system can be modulated, at the pre- and/or post-synaptic level, by different experimental maneuvers in which prolactin secretion is physiologically and pharmacologically altered. GABA mainly appears to be involved in feedback mechanisms preventing an exaggerated prolactin output during specific physiological situations. The ability of clinically tested, direct GABAmimetic compounds to lower prolactin secretion in the rat points towards a clinical usefulness of these drugs in particular spontaneous or induced neuroendocrine disorders. However, the possibility of a widespread use of this type of compounds is hampered by the lack of potent, specific and non-toxic GABA agonists suitable for clinical purposes.

GABAergic mechanism involved in sinoaortic denervation in rats.

GABAergic neurotransmission in sinoaortic denervated (SAD) rats with that in sham-operated animals 15 days after operation. In sham-operated rats, glutamic acid decarboxylase (GAD) and 4-amino-butyrate-2-oxoglutarate aminotransferase (GABA-T) activities were higher in dorsal than in ventral regions of pons and medulla oblongata and a higher GAD activity was observed in anterior than in posterior hypothalamus. Fifteen days after SAD, GAD and GABA-T activities were significantly reduced in dorsal pons and in anterior hypothalamus whereas GABA-T activity was increased in ventral medulla oblongata. The results indicate the involvement of GABAergic neurotransmission in the deafferentation of the nucleus tractus solitarii by sinoaortic denervation. GABA hypothalamic inputs could be involved in the baroreflexes.

Studies on the GABAergic system in cardiovascular control in normotensive and in sinoaortic denervated rats.

The cardiovascular effects of i.v. gamma-amino-beta-hydroxybutyric acid (GABOB) were investigated in rats anaesthetized with urethane. GABOB produced a dose-dependent hypotensive response. Treatment with GABA-A receptor antagonists prevented the GABOB response while the GABA stimulation by diazepam enhanced this response. The beta 1-adrenoceptor antagonist reduced the GABOB-induced hypotension but beta 2-adrenoceptor antagonists did not affect it. Picrotoxin, bicuculline or diazepam produced an increase in basal blood pressure. Fifteen days after sinoaortic denervation in rats the glutamic acid decarboxylase and the aminobutyric acid transaminase (GABA-T) activities were significantly reduced in dorsal pons and in anterior hypothalamus whereas GABA-T activity was increased in ventral medulla oblongata. Our results demonstrate that GABOB stimulates GABA-A receptors in anaesthetized rats and thus exerts a neuromodulatory effect on cardiovascular function. GABAergic neurotransmission participates in the sinoaortic deafferentation in rats.

Effect of THIP and SL 76002, two clinically experimented GABA-mimetic compounds, on anterior pituitary GABA receptors and prolactin secretion in the rat.

In the present study, the ability of three direct GABA agonists, muscimol, THIP and SL 76002 to displace 3H-GABA binding from anterior pituitary and medio-basal hypothalamus membranes was evaluated. Further, the effect of both THIP and SL 76002 on baseline prolactin levels or after stimulation of hormone release with haloperidol has been also studied. Either muscimol, THIP or SL 76002 have shown to posses 7-, 7- and 3-fold higher affinity, respectively, for the central nervous system than for the anterior pituitary 3H-GABA binding sites. Moreover, THIP and SL 76002 have demonstrated to be respectively, 25- and 1000- fold less potent than muscimol in inhibiting 3H- GABA binding at the level of the anterior pituitary and about 25- and 2700- fold less potent at the level of the medio-basal hypothalamus. Under basal conditions, either THIP or SL 76002 were ineffective to reduce prolactin release. However, after stimulation of prolactin secretion through blockade of the dopaminergic neurotransmission with haloperidol (0.1 mg/kg), both THIP (10 mg/kg) and SL 76002 (200 mg/kg) significantly counteracted the neuroleptic-induced prolactin rise with a potency which is in line with their ability to inhibit 3H-GABA binding in the anterior pituitary. The present results indicate that both compounds inhibit prolactin release under specific experimental situations probably through a GABAergic mechanism. In view of the endocrine effects of these GABA-mimetic compounds, the possibility arises for an application of these type of drugs in clinical neuroendocrinology.

Possible involvement of ovarian mechanisms other than estrogen-progesterone secretion in the regulation of glutamic acid decarboxylase activity of the rat fallopian tubes.

This study was performed to clarify the physiological role of the ovary in regulating the glutamic acid decarboxylase (GAD) activity in rat Fallopian tubes. To this purpose, GAD activity of the oviduct was evaluated in the following experimental conditions: immature or adult castrated (CX) rats; immature or adult CX rats treated with graded doses of estradiol benzoate (EB) or a fixed dose of EB and progesterone; adult CX rats bearing Silastic implants able to produce steady state estradiol plasma levels in the range of diestrous values; and prepubertal rats treated with ovulatory or anovulatory doses of exogenous gonadotropins (PMS and hCG). Moreover, the possible fluctuations of both gamma-aminobutyric acid (GABA) concentrations and GAD activity in the Fallopian tubes were studied during the estrous cycle. The results show that the prepubertal rat oviduct possesses a GABA content and a GAD activity analogous to those of normal diestrous rats. The GAD activity measured with the CO2 formation method was well correlated with the formation of labeled GABA, indicating that tubes of prepubertal rats are able to form the neurotransmitter by means of specific decarboxylation of glutamate. GAD activity, but not GABA levels, was increased over control values by the administration of exogenous gonadotropins. The role of the ovary in both adult and prepubertal rats to regulate this enzymatic activity is further stressed by the results of the experiments performed in CX animals which showed that ovariectomy produced a 4- to 5-fold decrease in GAD activity independent of the age of the animals. However, implantation of Silastic estradiol-containing capsules in adult CX animals or the administration of EB for 5 days in a dose range from 0.001-6.4 micrograms/day to adult ovariectomized animals and from 0.001-0.2 microgram/day to prepubertal animals did not modify GAD activity in spite of marked peripheral estrogenization of the animals evidenced by increases in uterine weight. Moreover, no variation of the enzymatic activity was observed at puberty (assessed by the age at vaginal opening). The administration of progesterone (0.2 mg) plus EB (0.01 microgram) did not produce any significant variation in GAD activity. GABA content and GAD activity of the tubes did not change during the estrous cycle. We, therefore, believe that other ovarian, still unidentified, secretions might be involved in the regulation of GAD activity in rat Fallopian tubes.

Effects of repeated tiapride administration on anterior pituitary dopamine receptors and prolactin release in the rat.

The substituted benzamides tiapride and sulpiride, and the classic neuroleptic haloperidol, were studied in the rat to assess their interaction with the anterior pituitary (AP) dopamine (DA) receptors both in vitro ([3H]spiperone binding) and in vivo prolactin-PRL-release). Tiapride weakly inhibited [3H]spiperone binding in both pituitary and striatal membranes with affinity 5-7 times lower than sulpiride and 400-300 times lower than haloperidol. All three drugs were more potent in displacing [3H]spiperone from striatum than from AP. In vivo, tiapride produced weak and transient stimulation of PRL release reaching a full effect at 2 mg/kg i.p. Similar doses of sulpiride produced longer-lasting effects. Haloperidol was more potent than both benzamides. In prolonged treatments (15 or 60 days), tiapride, given twice daily at 0.5 mg/kg i.p., did not modify [3H]spiperone binding in either AP or striatum, nor did it induce significant changes of basal PRL levels. The challenge with a low threshold dose of TIA (0.2 mg/kg ip) produced similar increases of PRL release in the group either treated with TIA or saline. The data indicate that the benzamides examined have low potency for interaction with DA receptors in pituitary and striatum. In particular, tiapride displayed weaker affinity for AP-DA receptors than the other drugs and induced only slight stimulation of PRL levels. Results from repeated tiapride administration indicate that the drug, at a clinically relevant dose, is unable to modify either kinetic characteristics of DA receptors in the pituitary or plasma PRL levels.