GPR88 in A2A receptor-expressing neurons modulates locomotor response to dopamine agonists but not sensorimotor gating.

The orphan receptor, GPR88, is emerging as a key player in the pathophysiology of several neuropsychiatric diseases, including psychotic disorders. Knockout (KO) mice lacking GPR88 throughout the brain exhibit many abnormalities relevant to schizophrenia including locomotor hyperactivity, behavioural hypersensitivity to dopaminergic psychostimulants and deficient sensorimotor gating. Here, we used conditional knockout (cKO) mice lacking GPR88 selectively in striatal medium spiny neurons expressing A2A receptor to determine neuronal circuits underlying these phenotypes. We first studied locomotor responses of A2A R-Gpr88 KO mice and their control littermates to psychotomimetic, amphetamine, and to selective D1 and D2 receptor agonists, SKF-81297 and quinpirole, respectively. To assess sensorimotor gating performance, mice were submitted to acoustic and visual prepulse inhibition (PPI) paradigms. Total knockout GPR88 mice were also studied for comparison. Like total GPR88 KO mice, A2A R-Gpr88 KO mice displayed a heightened sensitivity to locomotor stimulant effects of amphetamine and SKF-81297. They also exhibited enhanced locomotor activity to quinpirole, which tended to suppress locomotion in control mice. By contrast, they had normal acoustic and visual PPI, unlike total GPR88 KO mice that show impairments across different sensory modalities. Finally, none of the genetic manipulations altered central auditory temporal processing assessed by gap-PPI. Together, these findings support the role of GPR88 in the pathophysiology of schizophrenia and show that GPR88 in A2A receptor-expressing neurons modulates psychomotor behaviour but not sensorimotor gating.

Estrous cycle effects on behavior of C57BL/6J and BALB/cByJ female mice: implications for phenotyping strategies.

Systematic behavioral phenotyping of genetically modified mice is a powerful method with which to identify the molecular factors implicated in control of animal behavior, with potential relevance for research into neuropsychiatric disorders. A number of such disorders display sex differences, yet the use of female mice in phenotyping strategies has been a rare practice because of the potential variability related to the estrous cycle. We have now investigated the behavioral effects of the estrous cycle in a battery of behavioral tests in C57BL/6J and BALB/cByJ inbred strains of mice. Whereas the performance of BALB/cByJ female mice varied significantly depending on the phase of the estrous cycle in the open field, tail flick and tail suspension tests, the behavior of C57BL/6J females, with the exception of the tail suspension performance, remained stable across all four phases of the estrous cycle in all of the tests including open field, rotarod, startle reflex and pre-pulse inhibition, tail flick and hot plate. We also found that irrespective of the estrous cycle, the behavior of C57BL/6J females was different from that of BALB/cByJ groups in all of the behavioral paradigms. Such strain differences were previously reported in male comparisons, suggesting that the same inter-group differences can be revealed by studying female or male mice. In addition, strain differences were evident even for behaviors that were susceptible to estrous cycle modulations, although their detection might necessitate the constitution of large experimental groups.

Effects of social crowding on emotionality and expression of hippocampal nociceptin/orphanin FQ system transcripts in mice.

The novel nociceptin/orphanin FQ (N/OFQ) system was proposed to be an important component of neural circuits involved in stress-coping behaviour and fear. This study investigated whether variations between the mouse strains in vulnerability to social crowding stress might be linked to different regulation of N/OFQ system transcripts in mice. Three weeks old C57BL/6J (B6), BALB/cByJ (CBy) and 129S2/SvPas (129S2) male mice were housed individually or in crowded (7/cage) conditions and then tested as adults in a battery of anxiety tests (open field, elevated plus-maze and acoustic startle reflex tests). Both 129S2 and B6 mice displayed increased signs of anxiety under crowded housing, while CBy mice tended to show the opposite profile. Analysis of gene expression revealed a 10-fold increase of nociceptin precursor and 4-fold increase of the NOP receptor mRNAs contents in the hippocampus of CBy mice kept in crowded conditions compared to those housed individually. In B6 mice, mRNA level of the peptide precursor remained unchanged, while that of the receptor was increased by 2-fold under crowding compared to individual housing. No significant changes were detected in 129S2 mice. These findings show that social housing may be important environmental stress factor in mice depending on the strain. The possible involvement of central nociceptin mechanisms in behavioural resilience to social crowding stress is discussed.

Auditory and visual prepulse inhibition in mice: parametric analysis and strain comparisons.

Prepulse inhibition (PPI) is a multimodal phenomenon where the prepulse and the startling stimulus can be presented in either the same or the different sensory modalities. The aim of the present study was to characterize intramodal and cross-modal PPI in mice. We first examined the effects of varying prepulse intensity and prepulse duration on auditory and visual PPI in three inbred mouse strains C57BL/6J, 129S2 and BALB/cByJ mice. Increasing the intensity (5-15 dB above the background) and the duration (1-25 milliseconds) of the acoustic prepulse increased auditory PPI, and maximum level of inhibition was reached with each prepulse intensity at specific prepulse duration (between 5 and 15 milliseconds). Varying the intensity (30-300 lux) and the duration (1-25 milliseconds) of the light flashes had similar impact on visual PPI level (optimal durations between 1 and 10 milliseconds). There were also marked strain differences in PPI performances, with 129S2 and BALB/cByJ mice displaying the highest and the lowest scores of auditory PPI, respectively. In contrast, opposite strain ranking was obtained for visual PPI. The temporal expression of PPI was then studied in the same mouse strains using a wide range of interstimulus intervals (2-2000 milliseconds between the prepulse offset and the pulse onset). The time-course of the auditory and the visual PPI were relatively comparable (bell-shaped curve) with optimal lead-times between 10 and 100 milliseconds, but the shape of the temporal function varied between the mouse strains depending on the prepulse modality. These findings demonstrate that PPI has many physiological and genetic determinants that vary greatly across temporal and intensity domain, as well as stimulus modality.

Functional consequences of reduction in NMDA receptor glycine affinity in mice carrying targeted point mutations in the glycine binding site.

We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration-response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carrying Grin1(D481N) and Grin1(K483Q) mutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutant Grin1(D481N) animals are viable and fertile and appear to develop normally. However, homozygous mutant Grin1(K483Q) animals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in either Grin1(D481N) or Grin1(K483Q) mice. Interestingly, in situ hybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits in Grin1(D481N) mice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity. Grin1(D481N) mice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype of Grin1(K483Q) animals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.

Influence of the 5-HT(2C) receptor antagonist SB242,084 on behaviour produced by the 5-HT(2) agonist Ro60-0175 and the indirect 5-HT agonist dexfenfluramine.

Ro60-0175 has been described as a selective agonist at the 5-HT(2C) receptor, yet it has only 10- fold higher affinity at the 5-HT(2C) compared to the 5-HT(2A) subtype, and equivalent affinity for the 5-HT(2B) receptor. The selective 5-HT(2C) receptor antagonist SB242,084 (0.5 mg kg(-1) i.p.), blocked the hypoactivity and penile grooming induced by Ro60-0175 (1 mg kg(-1) s.c.). The combination of SB242,084 (0.5 mg kg(-1) i.p.) and Ro60-0175 (3 - 10 mg kg(-1)) produced a completely different pattern of behaviours including wet-dog shakes, hyperactivity and back muscle contractions. These latter effects were blocked by the selective 5-HT(2A) receptor antagonist MDL100,907 (0.5 mg kg(-1) i.p.), but not the 5-HT(2B) receptor antagonist SB215,505 (3 mg kg(-1) p.o.). The indirect 5-HT releaser/reuptake inhibitor dexfenfluramine (1 - 10 mg kg(-1) i.p.) produced a mild increase in locomotor activity, penile grooming, and occasional back muscle contractions and wet-dog shakes. Pre-treatment with SB242,084 (0.5 mg kg(-1)), blocked the incidence of penile grooming, and markedly potentiated both the dexfenfluramine-induced hyperactivity, the incidence of back muscle contractions, and to a lesser extent wet-dog shakes. Some toxicity was also evident in animals treated with dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)), but not in any other treatment groups. The hyperactivity and toxicity produced by the dexfenfluramine (10 mg kg(-1))/SB242,084 (0.5 mg kg(-1)) combination was replicated in a further study, and hyperthermia was also recorded. Both hyperthermia and toxicity were blocked by MDL100,907 (0.5 mg kg(-1)) but not SB215,505 (3 mg kg(-1)). An attenuation of the hyperlocomotor response was also observed following MDL100,907. These findings suggest that 5-HT(2C) receptor activation can inhibit the expression of behaviours mediated through other 5-HT receptor subtypes.

Bimodal modulation by nicotine of anxiety in the social interaction test: role of the dorsal hippocampus.

In conditions generating moderate levels of anxiety in the social interaction test (low light, unfamiliar arena or high light, familiar arena), parenteral administration of nicotine had bimodal actions, low doses (0.01 and 0.1 mg/kg i.p.) had anxiolytic effects and high doses (0.5 and 1.0 mg/kg i.p.) had anxiogenic effects. In test conditions where anxiety was lowest (low light, familiar arena) and highest (high light, unfamiliar arena), nicotine was without effect after intraperitoneal or hippocampal administration. Thus, nicotine plays a modulatory role in which the activity of other neurotransmitters is crucial to its expression. After bilateral administration to the dorsal hippocampus, nicotine (0.1-8.0 microg) had anxiogenic effects in conditions of moderate anxiety; mecamylamine (30 ng) was silent in these conditions, indicating no intrinsic tone. Our results show that the dorsal hippocampus is one area that can mediate anxiogenic effects in the social interaction test, but the brain region mediating anxiolytic effects remains to be identified.

Drug-induced potentiation of prepulse inhibition of acoustic startle reflex in mice: a model for detecting antipsychotic activity?

RATIONALE: Schizophrenic patients typically have impaired startle habituation (SH) and prepulse inhibition of the startle reflex (PPI). PPI can be disrupted in rats by psychomimetics, and drug-induced reversal of this deficit is considered to predict potential antipsychotic properties. Certain strains of mice, such as C57BL/6J, naturally display poor PPI. OBJECTIVE: To test whether mice spontaneously showing low levels of PPI might prove a useful tool for detecting novel antipsychotics. METHODS: PPI and SH were evaluated in four strains of mice: BALB/cByJ, MORO, 129/SvEv and C57BL/6J. The effects of antipsychotic [haloperidol (1, 3 and 6 mg/kg), clozapine (0.3, 1, 3 and 30 mg/kg) and risperidone (0.1, 0.3 and 1 mg/kg)] and non-antipsychotic [diazepam (3, 10 and 30 mg/kg), buspirone (1, 3 and 10 mg/kg), desipramine (3, 10 and 30 mg/kg), morphine (3, 10 and 30 mg/kg) and scopolamine (0.3, 1 and 3 mg/kg)] drug treatments were studied on PPI. RESULTS: Haloperidol (6 mg/kg), clozapine (3 and 30 mg/kg), and risperidone (1 mg/kg) all significantly enhanced PPI in C57BL/6J. All non-antipsychotics failed to improve PPI in this strain, except diazepam. Facilitation of PPI was also obtained in the other strains; however, clear interstrain differences were observed depending on the class of antipsychotic used and on the level of prepulse intensity. CONCLUSION: Antipsychotic-induced facilitation of PPI is clearly detected in mice naturally exhibiting poor levels of sensorimotor gating (e.g., C57BL/6J), but is also observed in other strains of mice. The use of this procedure as a potential screening test for detecting novel antipsychotic medications is discussed.

Modulation of behaviour on trials 1 and 2 in the elevated plus-maze test of anxiety after systemic and hippocampal administration of nicotine.

RATIONALE: The elevated plus-maze provides a test situation in which distinctive states of anxiety are elicited on trials 1 and 2 and the dorsal hippocampus has previously been shown to mediate the anxiogenic effects of (-)-nicotine in the social interaction test. OBJECTIVE: To determine the effects of a wide dose range of (-)-nicotine on trial 1 and 2 in the plus-maze after systemic administration and whether the dorsal hippocampus is a site mediating the anxiogenic effect of nicotine. METHODS: (-)-Nicotine (0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 mg/kg) was injected IP 30 min before testing for 5 min in the plus-maze. Rats receiving dorsal hippocampal infusions received bilateral infusions of 0.5 microl of artificial CSF or (-)-nicotine (0.1, 1, 4 or 8 microg). The needle was left in place for 50 s after injection and testing took place 3 min later. Rats tested on trial 1 were naive to the plus-maze, those tested on trial 2 had received a previous 5-min undrugged exposure to the maze 48 h earlier. RESULTS: Low doses of (-)-nicotine (0.001, 0.005, 0.01, 0.05 and 0.1 mg/kg, IP) were without effect on either trial, but higher doses (0.5 and 1 mg/kg, IP) had anxiogenic effects on both trials, as shown by decreases in percentage time spent and percentage entries onto the open arms. Infusion of (-)-nicotine (0.1, 1, 4 and 8 microg) bilaterally into the dorsal hippocampus was without effect on trial 1, but 1 microg had an anxiolytic effect on trial 2, shown by an increased percentage time spent on the open arms. CONCLUSIONS: The results on both trials in the plus-maze after systemic administration of nicotine add to previous reports from the social interaction test that high doses of nicotine have anxiogenic effects. However, the effects of nicotine in the dorsal hippocampus are different in all three anxiety tests (anxiogenic in social interaction, ineffective on trial 1, anxiolytic on trial 2) showing that nicotinic cholinergic control in this brain region may vary depending on the state and/or type of anxiety generated by the test. The brain region(s) underlying the anxiogenic effects of IP nicotine on both trials in the plus-maze remain to be identified.

Impact of environmental housing conditions on the emotional responses of mice deficient for nociceptin/orphanin FQ peptide precursor gene.

Nociceptin/orphanin FQ (N/OFQ) is a newly discovered neuropeptide that has been implicated in the neurobiological regulation of the behavioral responses to stress and fear. To investigate the role of this peptide in the expression of stress/anxiety-related behaviors in mice, a gene targeting approach to disrupt N/OFQ in the pre-proN/OFQ gene was used. The impact of environmental housing conditions (single and social housing) was assessed on N/OFQ-knockout male and female mice in different experimental paradigms known to trigger distinctive types of stress and anxiety states. Neurological examination of homozygous mutant adult animals indicated that basic neurological functions (vision, audition, olfaction, tactile and pain sensitivity, motor performances) were normal. When housed individually, N/OFQ-knockout animals displayed responses similar to control animals in behavioral tests of emotional reactivity (behavioral despair, locomotor activity, light-dark preference, and acoustic startle tests). In contrast, increased emotional responses were detected when individually housed mice were crowded together (five per cage) under conditions of competitive access to food, water, space, and social contacts. Under those conditions, male mice deficient for N/OFQ developed greater home-cage aggression and increased fear/anxiety-like behaviors in the light-dark and acoustic startle tests, when compared to their wild-type littermates. Group-housed female mutants also showed higher level of anxiety in the acoustic startle test, but needed additional restrain stress to express detectable levels of anxiety in the light-dark test. These data indicate a clear environment-induced rise in fear reactions of N/OFQ-knockout mice. They further suggest that N/OFQ system is essential for development of adequate coping strategies to acute and chronic stress.

Intra-accumbens infusion of D(3) receptor agonists reduces spontaneous and dopamine-induced locomotion.

The present study investigated whether PD 128907 and 7-OH-DPAT, described as preferential dopamine (DA) D(3) receptor agonists, produce hypolocomotion by acting at postsynaptic dopaminergic receptors within the nucleus accumbens. Bilateral infusion of PD 128907 (1.5 and 3 microg/0.5 microl) induced a dose-dependent hypolocomotion, whereas its enantiomer, PD 128908, was inactive. Local infusion of 7-OH-DPAT and the preferential DA autoreceptor agonist, B-HT 920, at the same dose range also decreased spontaneous locomotion. In addition, both drugs induced yawning with B-HT 920 producing the greatest effect. In the second experiment, the ability of these agonists to reduce the locomotor activity induced by intra-accumbens injection of DA (10 microg/0.5 microl) was studied. Pretreatment with either PD 128907 or 7-OH-DPAT (3 microg) reduced DA-induced hyperactivity. Local infusion of B-HT 920 (3 microg) failed to antagonise the locomotor effects of DA. Altogether these findings suggest that PD 128907 and 7-OH-DPAT induce hypolocomotion by acting in part at postsynaptic DA receptors. The possible role of D(2) and/or D(3) receptors in the mediation of these effects is discussed.

Chronic fluoxetine in tests of anxiety in rat lines selectively bred for differential 5-HT1A receptor function.

Selective breeding for high and low sensitivity to the hypothermic response induced by the 5-HT1A receptor agonist 8- OH-DPAT has established two lines of rat (HDS and LDS, respectively) whose behavior differs in a model of depression and in the social interaction test of anxiety. The HDS line has a higher level of anxiety and, furthermore, does not display the usual anxiogenic response to intrahippocampal administration of 8-OH-DPAT. It was therefore hypothesized that this line of rat might be a useful model of high trait anxiety with a susceptibility to depression. We thus investigated whether chronic treatment with fluoxetine would result in an anxiolytic effect in the social interaction test in the LDS and HDS lines of rat. In both lines, acute fluoxetine (10 mg/kg) produced an anxiogenic effect in the social interaction test; when rats were tested 24 h after 14 days of fluoxetine treatment there were no anxiolytic effects in either line. In the social interaction test, chronic fluoxetine treatment did not change either the anxiogenic effect of 8-OH-DPAT (100 ng) injected bilaterally into the dorsal hippocampus in the LDS line or the lack of response in the HDS line. In the elevated plus-maze, chronic fluoxetine treatment resulted in a significant anxiogenic effect in the HDS line, but was without effect in the LDS line. Intrahippocampal 8-OH-DPAT was without effect in the plus-maze in either line. These results suggest that chronic treatment with fluoxetine did not modify the hippocampal 5-HT1A receptor in either line. The anxiogenic effects observed in the plus-maze in the HDS line after chronic fluoxetine might relate to line differences in 5-HT1A receptors in other brain regions.

A history of chronic morphine exposure during adolescence increases despair-like behaviour and strain-dependently promotes sociability in abstinent adult mice.

A crucial issue in treating opiate addiction, a chronic relapsing disorder, is to maintain a drug-free abstinent state. Prolonged abstinence associates with mood disorders, strongly contributing to relapse. In particular, substance use disorders occurring during adolescence predispose to depression later in adulthood. Using our established mouse model of opiate abstinence, we characterized emotional consequences into adulthood of morphine exposure during adolescence. Our results indicate that morphine treatment in adolescent mice has no effect on anxiety-like behaviours in adult mice, after abstinence. In contrast, morphine treatment during adolescence increases behavioural despair in adult mice. We also show that morphine exposure strain-dependently enhances sociability in adult mice. Additional research will be required to understand where and how morphine acts during brain maturation to affect emotional and social behaviours into adulthood.

Stimulation of nicotinic receptors in the lateral septal nucleus increases anxiety.

The present study investigated the role of nicotinic receptors in the lateral septum in the modulation of anxiety. The effects of direct injections of nicotine into the lateral septum were first investigated in two tests of anxiety, social interaction and elevated plus-maze tests. Intra-septal injection of nicotine (1 and 4 microgram) induced consistent anxiogenic effects in both tests. The reversal of nicotinic effects with mecamylamine was then studied in the social interaction test. Intra-septal injection of mecamylamine at a low dose (15 ng) induced an anxiolytic effect, suggesting the presence of intrinsic cholinergic tone increasing anxiety. At higher doses (30-50 ng), mecamylamine was without effect in the social interaction test, but blocked the anxiogenic effects of nicotine (4 microgram). These findings provide further evidence for the role of the lateral septum in the modulation of anxiety and suggest that cholinergic projections to this brain area facilitate anxiety through nicotinic receptors.

Stimulation of benzodiazepine receptors in the dorsal hippocampus and median raphé reveals differential GABAergic control in two animal tests of anxiety.

The effects of pharmacological challenges to the benzodiazepine receptors in the dorsal hippocampus and median raphé nucleus were investigated in the social interaction and the elevated plus-maze tests of anxiety in rats. In the social interaction test, bilateral administration of midazolam (1 and 2 micrograms), into the dorsal hippocampus had anxiolytic effects; flumazenil (500 ng) was silent, but was able to antagonize the anxiolytic effects of midazolam (2 micrograms). In the social interaction test, midazolam was also anxiolytic when infused into the median raphé nucleus; flumazenil (100 and 500 ng) increased locomotor activity, but did not change anxiety measures. As an anatomical control, midazolam (1 and 2 micrograms) was infused into the adjacent pontine reticular nucleus, and was without effect. In contrast to the social interaction test, local infusion of midazolam (1 and 2 micrograms) and flumazenil (100 and 500 ng) into either the dorsal hippocampus or the median raphé nucleus failed to change anxiety measures in the elevated plus-maze (trials 1 and 2). These results show that stimulation of the benzodiazepine receptors in the hippocampus or the median raphé nucleus leads to anxiolytic effects in the social interaction test, but not in the elevated plus-maze. It would therefore appear that the two tests detect different types of anxiety that are differentially modulated by GABAA-benzodiazepine receptors in the dorsal hippocampus and the median raphé nucleus.

Reducing gene expression in the brain via antisense methods.

This unit presents protocols that employ antisense oligodeoxynucleotides to reduce expression of target proteins in the brain. These oligonucleotides are generally designed to inhibit synthesis of a specific protein by hybridization to its mRNA. Because oligonucleotides show very poor penetration into the central nervous system (CNS) after systemic administration, they are either injected into the cerebrospinal fluid (CSF) or infused directly into the brain parenchyma. In this unit, the procedure most commonly used for delivering oligonucleotides continuously into CSF is outlined. In addition, a procedure is described for continuous infusion of oligonucleotides into a specific brain region, using the substantia nigra as an example.

A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat.

The biochemical and behavioral effects of a nonpeptidic, selective, and brain-penetrant agonist at the ORL1 receptor are reported herein. This low molecular weight compound [(1S,3aS)-8- (2,3,3a,4,5, 6-hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4. 5]decan-4-one] has high affinity for recombinant human ORL1 receptors and has 100-fold selectivity for ORL1 over other members of the opioid receptor family. It is a full agonist at these receptors and elicits dose-dependent anxiolytic-like effects in a set of validated models of distinct types of anxiety states in the rat (i.e., elevated plus-maze, fear-potentiated startle, and operant conflict). When given systemically, the compound has an efficacy and potency comparable to those of a benzodiazepine anxiolytic such as alprazolam or diazepam. However, this compound is differentiated from a classical benzodiazepine anxiolytic by a lack of efficient anti-panic-like activity, absence of anticonvulsant properties, and lack of effects on motor performance and cognitive function at anxiolytic doses (0.3 to 3 mg/kg i.p.). No significant change in intracranial self-stimulation performance and pain reactivity was observed in this dose range. Higher doses of this compound (>/=10 mg/kg) induced disruption in rat behavior. These data confirm the notable anxiolytic-like effects observed at low doses with the orphanin FQ/nociceptin neuropeptide given locally into the brain and support a role for orphanin FQ/nociceptin in adaptive behavioral fear responses to stress.