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.

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.

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.

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.

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.

Endocrine modulation of gamma-aminobutyric acidergic innervation in the rat fallopian tube.

The present study investigates the effect of different endocrine manipulations on the gamma-aminobutyric acid (GABA)-ergic system in the rat fallopian tube. Either hypophysectomy or ovariectomy induced a significant decrease of glutamic acid decarboxylase (GAD) activity and of GABA levels in in situ tubes. This effect was completely reversed by either gonadotropins or combined estrogen-progesterone administration, respectively. Estrogen or progesterone alone proved less effective than the administration of both steroids in counteracting the effect of ovariectomy on GAD activity. The in vitro incubation of ovariectomized rat fallopian tubes with estrogen-progesterone for 1 h failed to counteract the reduction of the GAd activity induced by surgical manipulation. The in vivo effect of estrogen-progesterone administration on the GABA-ergic system seems to be specific since steroid treatment induced the synthesis of an enzyme which was immunologically identical to the GAD present in the fallopian tube and brain of normal diestrous rat. Autotransplantation of the fallopian tube under the skin brought about a decrease of GAD activity similar to that obtained after ovariectomy. In this situation, however, estrogen-progesterone administration did not counteract the decrease of GAD activity induced by fallopian tube deafferentation. The present results demonstrate that an interaction between the GABA-ergic system and the hypothalamo-pituitary-gonadal axis seems to be operative at the level of the rat fallopian tube. However, the physiological meaning of this interrelationship between the endocrine and the peripheral nervous systems remains to be clarified.

Effect of single or repeated estrogen administration on tuberoinfundibular GABA neurons and anterior pituitary GABA receptors: biochemical and functional studies.

The effect of single or protracted administration of estradiol valerate on the hypothalamo-pituitary gamma-aminobutyric acid (GABA)ergic system and on plasma prolactin levels has been evaluated in female rats 2 months after the last (chronic treatment) or the single dose of the steroid. In the group of animals receiving one dose of estrogen, no modifications were detected in the activity of the tuberoinfundibular GABAergic neurons as implied by unchanged GABA accumulation either in the median eminence or the anterior pituitary after blockade of GABA catabolism with ethanolamine-O-sulphate. However, a complete disappearance of the low affinity population of GABA receptors in the anterior pituitary was observed. In this experimental condition, where baseline prolactin levels were 3-fold higher than in control rats, muscimol, a potent GABA agonist, was effective in significantly lowering plasma prolactin concentrations. Chronic estradiol valerate administration reduced GABA accumulation in the median eminence and the anterior pituitary at 4, but not at 2 h, after intracerebroventricular injection of ethanolamine-O-sulphate. Moreover, in this instance, a complete disappearance of the high affinity population of GABA receptors in the anterior pituitary was detected. Long-term estrogen administration induced also a 55-fold increase of plasma prolactin titers and muscimol was ineffective in reducing prolactin concentrations in plasma. The ability of muscimol to inhibit prolactin release only in single-estrogen-treated animals strongly suggests that the high affinity population of anterior pituitary GABA receptors is that involved in the mechanisms whereby GABA inhibits prolactin release from anterior pituitary.

Prolactin in cerebrospinal fluid increases the synthesis and release of hypothalamic gamma-aminobutyric acid.

The effect of intracerebroventricularly (i.v.t.)-injected rat prolactin (2 micrograms/rat) on the function of tuberoinfundibular gamma-aminobutyric acid (GABA)ergic neurones was assessed in adult male rats by measuring the activity of glutamic acid decarboxylase (GAD) in the mediobasal hypothalamus (MBH) and the concentrations of GABA in hypophysial portal plasma and in the anterior pituitary gland. Fourteen hours after i.v.t. injection of rat prolactin the activity of GAD in the MBH was significantly (P less than 0.05) increased and it remained elevated for at least 16 h after injection. The mean concentrations of GABA in hypophysial portal plasma and in the anterior pituitary were twice those found in vehicle-treated controls 16 h after administration of rat prolactin; no significant effects were observed at earlier time-periods. A significant (P less than 0.01) and long-lasting decrease in endogenous plasma prolactin concentrations was detected 2 h after the i.v.t. injection of rat prolactin and the concentrations remained suppressed for up to 16 h. The present results are consistent with the concept that the activity of tuberoinfundibular GABAergic neurones is regulated, at least in part, by circulating prolactin. The ability of prolactin to accelerate the synthesis and release of GABA in the MBH might constitute a short loop feedback system by which the hormone regulates its own secretion.

Neurochemical studies on GABAergic and aminergic systems in the rat brain following acute and chronic piracetam administration.

In view of the metabolic and behavioural effects of piracetam (P), a cyclic derivative of gamma-aminobutyric acid (GABA), in experimental animals and in man, it was of interest to investigate the effect of acute or chronic administration of the compound on the function of different brain neurotransmitter systems. P was ineffective in modifying either synthesis release, uptake or post- synaptic binding sites for GABA. Acute P injection decreased dose-dependently cGMP levels in the rat cerebellum. Moreover, this effect was not mediated through a GABAergic mechanism. An acute challenge with Piracetam 15 days after chronic treatment with the compound increased DOPAC levels in the striatum and counteracted haloperidol-induced PRL rise. Furthermore, chronic P administration increased normetanephrine levels in the cerebral cortex, an index of the release of norepinephrine at the synaptic level, and induced a desensitization of beta-adrenoceptors in this same brain area. In conclusion, besides the well documented effect of P on cholinergic neurons, P seems to exert its biological and behavioural effects through activation of catecholaminergic mechanisms.