Antipsychotic drug-like facilitation of latent inhibition by a brain-penetrating neurotensin-1 receptor agonist.

Latent inhibition (LI) is a measure of cognitive gating and refers to reduced conditioned learning when there is pre-exposure to the conditioned stimulus (CS) before it is paired with the unconditioned stimulus (US). Dysregulation of LI is associated with some neuropsychiatric disorders, including schizophrenia, and the ability to facilitate LI in rodents is a reasonably good predictive test for antipsychotic drugs. Converging evidence supports neurotensin-1 receptor (NTS1) agonists as novel drugs for schizophrenia. Therefore, we investigated the ability of a brain-penetrating, selective NTS1 agonist, PD149163, to facilitate LI in heterozygous Brattleboro rats, a strain that exhibits naturally low LI. Conditioned taste aversion to flavored water (FW; 0.1% saccharin) was induced by pairing it with malaise-inducing injections of lithium chloride (LiCl). Prior to LiCl-FW pairing, rats received subcutaneous injections of saline, or PD149163 (100 µg/kg or 200 µg/kg). Half the rats in each drug group had been allowed to drink FW the day before the LiCl-FW pairing (pre-exposed rats). Two days after pairing, the amount of FW each rat consumed was recorded. LI, defined as significantly greater FW drinking in the pre-exposed group compared with the non pre-exposed group, was exhibited only among rats that received 200 µg/kg of PD149163. These results further support NTS1 agonists as potentially novel drugs for the treatment of schizophrenia.

Peripherally administered oxytocin modulates latent inhibition in a manner consistent with antipsychotic drugs.

BACKGROUND: Peripherally administered oxytocin (OT) has produced antipsychotic drug (APD)-like effects in animal tests that are predictive of APD efficacy. However, these effects have mainly been demonstrated using animal models of schizophrenia-like deficits in prepulse inhibition (PPI) of the startle reflex. Another schizophrenia-relevant abnormality that is the basis of a predictive animal test for APD efficacy is deficient latent inhibition (LI). LI is the normal suppression of a classically conditioned response when the subject is pre-exposed to the conditioned stimulus (CS) before it is paired with the unconditioned stimulus (UCS). Conditioned taste aversion (CTA), the normal avoidance of ingesting a food or liquid by animals when its taste is associated with an aversive experience, was used to test whether OT facilitates LI consistent with APDs. METHODS: Brown Norway rats, known to naturally display attenuated LI, were aversively conditioned on two consecutive exposures to flavored drinking water (0.1% saccharin) by pairing it with malaise-inducing lithium chloride injections. Concurrent with conditioning, rats received subcutaneous OT (0.02, 0.1, 0.5mg/kg) or saline. Some rats were pre-exposed to the flavored water prior to its aversive conditioning (pre-exposed) while others were not (non pre-exposed). Two days after aversive conditioning the amount of flavored water consumed during a 20-min session was recorded. RESULTS: As expected, LI, defined as greater consumption by pre-exposed vs. non pre-exposed rats was only weakly exhibited in Brown Norway rats and OT enhanced LI by reducing CTA in pre-exposed rats in a dose-dependent manner, with the 0.02 mg/kg dose producing the strongest effect. CONCLUSIONS: The facilitation of LI by OT is consistent with the effects produced by APDs and provides further support for the notion that OT has therapeutic potential for schizophrenia.

Enrichment of cis-regulatory gene expression SNPs and methylation quantitative trait loci among bipolar disorder susceptibility variants.

We conducted a systematic study of top susceptibility variants from a genome-wide association (GWA) study of bipolar disorder to gain insight into the functional consequences of genetic variation influencing disease risk. We report here the results of experiments to explore the effects of these susceptibility variants on DNA methylation and mRNA expression in human cerebellum samples. Among the top susceptibility variants, we identified an enrichment of cis regulatory loci on mRNA expression (eQTLs), and a significant excess of quantitative trait loci for DNA CpG methylation, hereafter referred to as methylation quantitative trait loci (mQTLs). Bipolar disorder susceptibility variants that cis regulate both cerebellar expression and methylation of the same gene are a very small proportion of bipolar disorder susceptibility variants. This finding suggests that mQTLs and eQTLs provide orthogonal ways of functionally annotating genetic variation within the context of studies of pathophysiology in brain. No lymphocyte mQTL enrichment was found, suggesting that mQTL enrichment was specific to the cerebellum, in contrast to eQTLs. Separately, we found that using mQTL information to restrict the number of single-nucleotide polymorphisms studied enhances our ability to detect a significant association. With this restriction a priori informed by the observed functional enrichment, we identified a significant association (rs12618769, P(bonferroni)<0.05) from two other GWA studies (TGen+GAIN; 2191 cases and 1434 controls) of bipolar disorder, which we replicated in an independent GWA study (WTCCC). Collectively, our findings highlight the importance of integrating functional annotation of genetic variants for gene expression and DNA methylation to advance the biological understanding of bipolar disorder.

A genome-wide association study of attempted suicide.

The heritable component to attempted and completed suicide is partly related to psychiatric disorders and also partly independent of them. Although attempted suicide linkage regions have been identified on 2p11-12 and 6q25-26, there are likely many more such loci, the discovery of which will require a much higher resolution approach, such as the genome-wide association study (GWAS). With this in mind, we conducted an attempted suicide GWAS that compared the single-nucleotide polymorphism (SNP) genotypes of 1201 bipolar (BP) subjects with a history of suicide attempts to the genotypes of 1497 BP subjects without a history of suicide attempts. In all, 2507 SNPs with evidence for association at P<0.001 were identified. These associated SNPs were subsequently tested for association in a large and independent BP sample set. None of these SNPs were significantly associated in the replication sample after correcting for multiple testing, but the combined analysis of the two sample sets produced an association signal on 2p25 (rs300774) at the threshold of genome-wide significance (P=5.07 × 10(-8)). The associated SNPs on 2p25 fall in a large linkage disequilibrium block containing the ACP1 (acid phosphatase 1) gene, a gene whose expression is significantly elevated in BP subjects who have completed suicide. Furthermore, the ACP1 protein is a tyrosine phosphatase that influences Wnt signaling, a pathway regulated by lithium, making ACP1 a functional candidate for involvement in the phenotype. Larger GWAS sample sets will be required to confirm the signal on 2p25 and to identify additional genetic risk factors increasing susceptibility for attempted suicide.

Further characterization of the predictive validity of the Brattleboro rat model for antipsychotic efficacy.

Our laboratory and others have reported that Brattleboro (BRAT) rats, a Long Evans (LE) strain with a single gene mutation, have inherent deficits in prepulse inhibition (PPI) homologous to those observed in schizophrenia patients and that these deficits are reversed by antipsychotic drugs (APDs). To further evaluate the potential predictive validity of BRAT rat PPI for APDs, we compared the effects of acute subcutaneous administration of the typical APD chlorpromazine to that of three psychotropic drugs without antipsychotic efficacy, the antidepressant imipramine, the anxiolytic diazepam and the anticonvulsant mood stabilizer valproic acid on male and female BRAT rat PPI. Male and female BRAT rats exhibited baseline (saline treatment) PPI that was not different from each other (21.1% and 21.3%, respectively) and low compared with those historically exhibited by LE rats (approximately 59%). Chlorpromazine facilitated PPI in male and female BRAT rats, whereas imipramine, diazepam, and valproic acid had no significant effect on PPI. These results suggest that PPI in the BRAT rat responds specifically to drugs with APD efficacy but not psychotropic drugs of different therapeutic families.

Sensorimotor gating in neurotensin-1 receptor null mice.

BACKGROUND: Converging evidence has implicated endogenous neurotensin (NT) in the pathophysiology of brain processes relevant to schizophrenia. Prepulse inhibition of the startle reflex (PPI) is a measure of sensorimotor gating and considered to be of strong relevance to neuropsychiatric disorders associated with psychosis and cognitive dysfunction. Mice genetically engineered to not express NT display deficits in PPI that model the PPI deficits seen in schizophrenia patients. NT1 receptors have been most strongly implicated in mediating the psychosis relevant effects of NT such as attenuating PPI deficits. To investigate the role of NT1 receptors in the regulation of PPI, we measured baseline PPI in wildtype (WT) and NT1 knockout (KO) mice. We also tested the effects of amphetamine and dizocilpine, a dopamine agonist and NMDA antagonist, respectively, that reduce PPI as well as the NT1 selective receptor agonist PD149163, known to increase PPI in rats. METHODS: Baseline PPI and acoustic startle response were measured in WT and NT1 KO mice. After baseline testing, mice were tested again after receiving intraperatoneal (IP) saline or one of three doses of amphetamine (1.0, 3.0 and 10.0 mg/kg), dizocilpine (0.3, 1.0 and 3.0 mg/kg) and PD149163 (0.5, 2.0 and 6.0 mg/kg) on separate test days. RESULTS: Baseline PPI and acoustic startle response in NT1 KO mice were not significantly different from NT1 WT mice. WT and KO mice exhibited similar responses to the PPI-disrupting effects of dizocilpine and amphetamine. PD149163 significantly facilitated PPI (P < 0.004) and decreased the acoustic startle response (P < 0.001) in WT but not NT1 KO mice. CONCLUSIONS: The data does not support the regulation of baseline PPI or the PPI disruptive effects of amphetamine or dizocilpine by endogenous NT acting at the NT1 receptor, although they support the antipsychotic potential of pharmacological activation of NT1 receptors by NT1 agonists.

Neurotensin agonists block the prepulse inhibition deficits produced by a 5-HT2A and an alpha1 agonist.

RATIONALE: Neurotensin (NT) agonists have been proposed as potential antipsychotics based exclusively upon their ability to inhibit dopamine-2 (D2) receptor transmission. Several other pharmacological mechanisms have been implicated in enhancing the antipsychotic profile produced by D2 inhibition alone. These include inhibition of 5-HT2A and alpha1-adrenoceptors. Recently, we reported that systemic administration of the neurotensin agonist PD149163 blocks deficits in prepulse inhibition (PPI) of the startle reflex produced by the 5-HT2A receptor agonist DOI. This suggested that NT agonists could inhibit 5-HT2A modulation of neurotransmission. OBJECTIVE: To determine if other peripherally administered NT agonists shared this effect, we examined the effects of NT69L, another NT agonist, on DOI-induced PPI deficits. In addition, to determine if NT agonists also inhibit alpha1-adrenoceptor neurotransmission, we examined the effects of PD149163 and NT69L on PPI deficits induced by the alpha1-adrenoceptor agonist, cirazoline. METHODS: In the NT69L/DOI study, rats received subcutaneous (SC) injections of NT69L (0, 0.1, 1, or 2 mg/kg) followed 30 min later by SC saline or DOI (0.5 mg/kg). In the NT agonist/cirazoline studies, animals received SC injections of either PD149163 (0, 0.01, 0.1, or 1 mg/kg) or NT69L (0, 0.01, 0.1, or 1 mg/kg) followed 30 min later by SC saline or cirazoline (0.7 mg/kg). Animals were tested in startle chambers 20 min later. RESULTS: In all three experiments the PPI disruption produced by DOI and cirazoline was blocked by the NT agonists. CONCLUSIONS: These findings provide strong evidence that NT agonists inhibit 5-HT2A and alpha1-adrenoceptor modulation of neurotransmission, pharmacological effects that, in conjunction with their known inhibition of dopamine transmission, strengthen the antipsychotic potential of NT agonists.

The effects of systemic NT69L, a neurotensin agonist, on baseline and drug-disrupted prepulse inhibition.

Centrally administered neurotensin (NT) produces behavioral and biochemical effects that are very similar to the effects of antipsychotic drugs. Therefore, there is much interest in the potential use of NT agonists as antipsychotic drugs. We have previously reported that PD149163, a NT(8-13) analogue, produced effects on prepulse inhibition (PPI) of startle after systemic administration that were suggestive of an atypical antipsychotic-like drug profile. To determine if these effects are shared by other peripherally administered NT agonists, we tested the effects of NT69L, a recently developed NT agonist that penetrates the CNS, on drug-induced PPI deficits. In the first experiment, rats received subcutaneous (s.c.) injections of NT69L (vehicle, 0.08, 0.25, and 1.0mg/kg) followed 30min later by subcutaneous saline or D-amphetamine (2.0mg/kg). In the second experiment, NT69L injections were followed by saline or the non-competitive NMDA antagonist dizocilpine (0.1mg/kg). Both D-amphetamine and dizocilpine significantly decreased PPI as expected. In the first experiment, NT69L significantly increased PPI levels at baseline and after D-amphetamine. In the second experiment, NT69L attenuated PPI deficits produced by dizocilpine, without increasing baseline PPI. In addition, NT69L had no effect on startle magnitude. The effects of NT69L in these studies were similar in some ways to the effects of PD149163 and were also consistent with the preclinical effects of atypical antipsychotic drugs. These data provide further support for the notion that NT agonists may have use as novel antipsychotic drugs. Furthermore, the ability of NT69L and PD149163 to attenuate dizocilpine-disrupted PPI, an antipsychotic drug effect not mediated by dopamine, suggests that NT agonists may produce some of their antipsychotic-like effects by modulating neurotransmitter systems other than dopamine, such as serotonin, noradrenaline or glutamate.

SR146131, a cholecystokinin-A receptor agonist, antagonizes prepulse inhibition deficits produced by dizocilpine and DOI.

RATIONALE: Converging evidence has demonstrated that cholecystokinin (CCK) inhibits mesolimbic brain dopamine (DA) function via activation of CCK-A (CCK-1) receptors. These effects of CCK have stimulated interest in the potential use of CCK agonists as antipsychotic drugs. Most research on the antipsychotic-like drug effects of CCK has used CCK or CCK analogues that nonselectively activate both CCK-A and CCK-B (CCK-2) receptors, which may produce opposite effects. SR146131, a CCK-A selective nonpeptide agonist, has recently been developed (Sanofi-Synthelabo). OBJECTIVE: To determine whether SR146131 exhibits antipsychotic-like qualities in the prepulse inhibition (PPI) paradigm. METHODS: We performed experiments to determine whether SR146131 (vehicle, 0.01, 0.1, 1.0 mg/kg) would attenuate PPI deficits induced by amphetamine (2.0 mg/kg), an indirect dopamine agonist, and dizocilpine (0.1 mg/kg), a noncompetitive N-methyl-D-aspartate (NMDA) antagonist. Since SR146131 demonstrated significant effects on PPI disrupted by the noncompetitive NMDA antagonist, an effect associated with drugs that inhibit serotonin (5HT)2A transmission, we also tested the effects of SR146131 on PPI disruption produced by 2,5-dimethoxy-4-iodoamphetamine (DOI, 0.5 mg/kg), a direct 5HT2A agonist. RESULTS: SR146131 did not significantly affect startle magnitude, baseline PPI, or amphetamine-induced PPI deficits. However, it dose-dependently antagonized dizocilpine and DOI-induced PPI deficits. CONCLUSIONS: The lack of an effect of SR146131 on amphetamine-induced disruption of PPI suggests that a selective nonpeptide CCK-A agonist may not produce antipsychotic-like effects on dopamine transmission. However, the unexpected effects of SR146131 on dizocilpine and DOI-induced PPI deficits are consistent with the effects of drugs that inhibit transmission in the 5HT2A receptor system, including atypical antipsychotic drugs. Possible mechanisms underlying these findings are discussed.

Decreased haloperidol-induced potentiation of zif268 mRNA expression in the nucleus accumbens shell and the dorsal lateral striatum of rats lacking cholecystokinin-A receptors.

Evidence suggests that endogenous cholecystokinin (CCK), a neuropeptide that modulates brain dopamine function, may contribute to the therapeutic and motor effects of antipsychotic drugs via activation of CCK-A receptors in the mesolimbic and nigrostriatal pathways, respectively. To determine if CCK modulates the effects of antipsychotic drugs through CCK-A receptors, we measured the haloperidol-induced zif268 mRNA response in the nucleus accumbens (NA) shell, NA core, and dorsal lateral striatum (DLS) in Otsuka Long Evans Tokushima Fatty (OLETF) rats that lack CCK-A receptors due to a spontaneous mutation. OLETF rats and normal Long Evans rats were treated with subcutaneous (s.c.) injections of saline or haloperidol (2 mg/kg). In situ hybridization was performed and zif268 mRNA expression was quantified. The haloperidol-induced expression of zif268 mRNA was significantly decreased in the DLS (P < 0.01) and the NA shell (P < 0.05), but not in the NA core, in OLETF rats compared to LETO rats. These data suggest that CCK-A receptor mechanisms may contribute to the therapeutic and the extrapyramidal motor effects associated with antipsychotic drug treatment.

Startle and sensorimotor gating in rats lacking CCK-A receptors.

Otsuka Long Evans Tokushima Fatty (OLETF) rats lack CCK-A receptors because of a genetic mutation. Previous studies have shown that CCK-A receptors seem to play a role in the regulation of prepulse inhibition (PPI) of the startle reflex, an operational measure of sensorimotor gating. This study investigated baseline and drug-disrupted PPI in OLETF rats and their non-mutant counterparts, Long Evans Tokushima Otsuka (LETO) rats. Baseline PPI did not differ significantly between the two rat genotypes but OLETF rats exhibited a higher acoustic startle response compared to LETO rats. Amphetamine (2 mg/kg), and the non-competitive NMDA antagonist, dizocilpine (0.1 mg/kg), disrupted PPI in LETO rats but not in the OLETF rats. Apomorphine (0.5 mg/kg) failed to disrupt PPI in both LETO and OLETF rats, and haloperidol (0.5 mg/kg) produced a comparable facilitation of PPI in both groups. In a separate study, OLETF rats were found to be less sensitive to the locomotor stimulating effects of amphetamine. These results suggest that CCK-A receptors play a significant role in the behavioral effects of amphetamine and dizocilpine. The PPI response of OLETF rats to amphetamine and dizocilpine is similar to normal rats pretreated with atypical antipsychotics, suggesting that CCK-A receptors may play an important role in the restoration of drug-disrupted PPI by antipsychotics.

Amphetamine-induced zif268 mRNA expression in the medial posterior nucleus accumbens in cholecystokinin-A receptor mutant rats.

Converging evidence supports a role for cholecystokinin (CCK) in modulating dopamine (DA)-mediated activity in the rat mesolimbic system. In particular, CCK co-localized with mesolimbic DA cells originating in the ventral tegmental area potentiates DA function in the medial posterior nucleus accumbens (mpNA) through CCK-A receptors. Recently, a strain of rats lacking the CCK-A receptor, Otsuka Long Evans Tokushima Fatty (OLETF), has been discovered making it possible to study the mesolimbic DA regulatory role of CCK-A receptors. Previous studies have shown that OLETF rats are less sensitive to amphetamine (AMPH)-induced behavioral effects compared to controls. To determine if this altered sensitivity is associated with decreased AMPH-induced postsynaptic activation in the mpNA in OLETF rats, we performed the following experiment. OLETF (CCK-A mutants) and Long Evans Tokushima Otsuka (LETO) rats (controls) were given subcutaneous injections of either saline or AMPH (5.0 mg/kg). One hour after injection all animals were sacrificed and activation of the mpNA was assessed using in situ hybridization with antisense probes for zif268 mRNA. AMPH treatment produced a significant up-regulation of zif268 mRNA expression in both OLETF and LETO rats (P

Differential regional zif268 messenger RNA expression in an escalating dose/binge model of amphetamine-induced psychosis.

Amphetamine-induced psychosis is most often associated with a high-dose multiple binge pattern of stimulant abuse. To simulate these conditions in rats, we used an escalating dose/binge administration paradigm. Animals were pretreated with escalating doses of amphetamine (1.0-8.0mg/kg) over four days, then exposed to nine daily binges (8.0mg/kg every 2h; four injections/day). Other animals received either multiple injections of saline, saline followed by acute amphetamine (8.0mg/kg) or single daily injections of amphetamine (8.0mg/kg) in parallel with the escalating dose/binge treatment. One hour after the last injection, all animals were decapitated and regional brain activation patterns were assessed using in situ hybridization with antisense probes for zif268. Acute amphetamine resulted in a significant elevation of zif268 messenger RNA in both the nucleus accumbens and dorsal striatum. However, whereas after single daily amphetamine treatment this index was no longer elevated above control levels in the dorsal striatum, multiple binge exposures were required for the nucleus accumbens to return to baseline. Agranular insular cortex and medial olfactory tubercle zif268 messenger RNA expression was also markedly increased after acute amphetamine treatment but, unlike the nucleus accumbens and dorsal striatum, this increase was not significantly attenuated by either single daily injection or multiple binge treatment. Zif268 messenger RNA expression in the lateral nucleus of the amygdala also remained elevated above baseline after binge treatment. The possible relationships of these changes in zif268 messenger RNA regional expression patterns to the development of psychosis in high-dose stimulant abusers are discussed.

Dopamine transporter mRNA is up-regulated in the substantia nigra and the ventral tegmental area of amphetamine-sensitized rats.

Converging evidence supports a significant role for dopamine (DA) in the development of behavioral sensitization and it has been suggested that changes in either DA transporter (DAT) or D2 autoreceptors contribute to the effects of stimulant treatment. To determine if alterations in DAT or D2 autoreceptor mRNA are long-lasting and parallel the time course of amphetamine (AMPH)-induced behavioral sensitization we performed the following experiment. Two groups of rats were used for mRNA analysis by in situ hybridization. They were given either single daily injections of saline or AMPH (2.5 mg/kg) for 5 days and sacrificed 7 days later. Two groups pretreated in a similar manner were used to test for behavioral sensitization. Pretreatment with AMPH which resulted in a sensitization response profile after AMPH challenge also produced a significant up-regulation of DAT mRNA in both the ventral tegmental area (VTA) (P = 0.01) and substantia nigra (SN) (P < 0.05) compared to the saline controls, whereas there were no significant group differences in D2 mRNA in either the SN or the VTA. The possible role of these changes in behavioral sensitization is discussed.

Hippocampal glucocorticoid receptor mRNA is up-regulated by acute and down-regulated by chronic amphetamine treatment.

Repeated administration of amphetamine-like stimulants to rats results in enhanced behavioral responsiveness to subsequent administration of these drugs. Recent evidence suggests corticosterone may play a role in the development of sensitization perhaps through the down-regulation of glucocorticoid receptor (GR). To test this hypothesis further we examined the effects of five daily injections of amphetamine (AMPH) (2.5 mg/kg) on GR mRNA of adult Sprague-Dawley rats. Two other groups received saline for 4 days and then either saline or AMPH on the fifth day. All animals were killed 24 h after the last treatment and in situ hybridization was performed with an antisense mRNA GR probe. Quantification of hippocampal GR was accomplished by computer analysis of digitized images of CA1 and dentate gyrus. Acute AMPH produced a significant up-regulation of GR mRNA in CA1 and a nonsignificant trend towards up-regulation in the dentate gyrus. Repeated exposure to AMPH resulted in a significant down-regulation in CA1, and a nonsignificant trend towards down-regulation in dentate gyrus. These data support a role for hippocampal GR mRNA in the development of behavioral sensitization.