Caitlin E. McOmish

Phospholipase C-β1 knockout mice exhibit endophenotypes modeling schizophrenia which are rescued by environmental enrichment and clozapine administration

Phospholipase C-β1 (PLC-β1) is a rate-limiting enzyme implicated in postnatal-cortical development and neuronal plasticity. PLC-β1 transduces intracellular signals from specific muscarinic, glutamate and serotonin receptors, all of which have been implicated in the pathogenesis of schizophrenia. Here, we present data to show that PLC-β1 knockout mice display locomotor hyperactivity, sensorimotor gating deficits as well as cognitive impairment. These changes in behavior are regarded as endophenotypes homologous to schizophrenia-like symptoms in rodents. Importantly, the locomotor hyperactivity and sensorimotor gating deficits in PLC-β1 knockout mice are subject to beneficial modulation by environmental enrichment. Furthermore, clozapine but not haloperidol (atypical and typical antipsychotics, respectively) rescues the sensorimotor gating deficit in these animals, suggesting selective predictive validity. We also demonstrate a relationship between the beneficial effects of environmental enrichment and levels of M1/M4 muscarinic acetylcholine receptor binding in the neocortex and hippocampus. Thus we have demonstrated a novel mouse model, displaying disruption of multiple postsynaptic signals implicated in the pathogenesis of schizophrenia, a relevant behavioral phenotype and associated gene–environment interactions.

PLC-β1 knockout mice as a model of disrupted cortical development and plasticity: Behavioral endophenotypes and dysregulation of RGS4 gene expression

The complexity of the genetics underlying schizophrenia is highlighted by the multitude of molecular pathways that have been reported to be disrupted in the disorder including muscarinic, serotonergic, and glutamatergic signaling systems. It is of interest, therefore, that phospholipase C‐β1 (PLC‐β1) acts as a point of convergence for these pathways during cortical development and plasticity. These signaling pathways, furthermore, are susceptible to modulation by RGS4, one of the more promising candidate genes for schizophrenia. PLC‐β1 knockout mice were behaviorally assessed on tests including fear conditioning, elevated plus maze, and the Y maze. In situ hybridization was used to assess RGS4 expression. We found that PLC‐β1 knockout mice display abnormal anxiety profiles on some, but not all measures assessed, including decreased anxiety on the elevated plus maze. We also show memory impairment and a complete absence of acquisition of hippocampal‐dependent fear conditioning. Furthermore, at a molecular level, we demonstrate dramatic changes in expression of RGS4 mRNA in selective regions of the PLC‐β1 knockout mouse brain, particularly the CA1 region of the hippocampus. These results validate the utility of the PLC‐β1 knockout mouse as a model of schizophrenia, including molecular and cellular evidence for disrupted cortical maturation and associated behavioral endophenotypes.

Enviromimetics: exploring gene environment interactions to identify therapeutic targets for brain disorders

There is a growing awareness of the central role played by environmental factors in many of the most debilitating neural disorders. Epidemiological studies have suggested a complex balance between genetic and environmental factors in the pathogenesis of neurological and psychiatric conditions. The use of accurate animal models, combined with experimental manipulations such as environmental enrichment, has shown that increased sensory, cognitive and motor stimulation has beneficial effects in a range of CNS disorders, including Huntingtons, Alzheimers, Parkinsons and other neurodegenerative diseases. Various studies have identified molecular, structural and functional correlates of this experience-dependent plasticity. The authors propose that the molecular systems which mediate the therapeutic effects of environmental enrichment may provide novel targets for pharmacotherapies. More specifically, they elaborate a theoretical framework for the development of ‘enviromimetics’, therapeutics that mimic or enhance the beneficial effects of environmental stimulation, targeted towards a wide range of nervous system disorders.

Clozapine-induced locomotor suppression is mediated by 5-HT2A receptors in the forebrain.

The need for safer, more effective therapeutics for the treatment of schizophrenia is widely acknowledged. To optimally target novel pharmacotherapies, in addition to establishing the mechanisms responsible for the beneficial effects of antipsychotics, the pathways underlying the most severe side effects must also be elucidated. Here we investigate the role of serotonin 2A (5-HT2A), serotonin 2C (5-HT2C), and dopamine 2 receptors (D2) in mediating adverse effects associated with canonical first- and second-generation antipsychotic drugs in mice. Wild-type (WT) and 5-HT2A knockout (KO) mice treated with haloperidol, clozapine, and risperidone were assessed for locomotor activity and catalepsy. WT mice showed a marked reduction in locomotor activity following acute administration of haloperidol and high-dose risperidone, which was most likely secondary to the severe catalepsy caused by these compounds. Clozapine also dramatically reduced locomotor activity, but in the absence of catalepsy. Interestingly, 5-HT2A KO mice were cataleptic following haloperidol and risperidone, but did not respond to clozapines locomotor-suppressing effects. Restoration of 5-HT2A expression to cortical glutamatergic neurons re-instated the locomotor-suppressing effects of clozapine in the open field. In sum, we confirm that haloperidol and risperidone caused catalepsy in rodents, driven by strong antagonism of D2. We also demonstrate that clozapine decreases locomotor activity in a 5-HT2A-dependent manner, in the absence of catalepsy. Moreover, we show that it is the cortical population of 5-HT2A that mediate the locomotor-suppressing effects of clozapine.

Environmental Enrichment Ameliorates Behavioral Impairments Modeling Schizophrenia in Mice Lacking Metabotropic Glutamate Receptor 5

Schizophrenia arises from a complex interplay between genetic and environmental factors. Abnormalities in glutamatergic signaling have been proposed to underlie the emergence of symptoms, in light of various lines of evidence, including the psychotomimetic effects of NMDA receptor antagonists. Metabotropic glutamate receptor 5 (mGlu5) has also been implicated in the disorder, and has been shown to physically interact with NMDA receptors. To clarify the role of mGlu5-dependent behavioral expression by environmental factors, we assessed mGlu5 knockout (KO) mice after exposure to environmental enrichment (EE) or reared under standard conditions. The mGlu5 KO mice showed reduced prepulse inhibition (PPI), long-term memory deficits, and spontaneous locomotor hyperactivity, which were all attenuated by EE. Examining the cellular impact of genetic and environmental manipulation, we show that EE significantly increased pyramidal cell dendritic branching and BDNF protein levels in the hippocampus of wild-type mice; however, mGlu5 KO mice were resistant to these alterations, suggesting that mGlu5 is critical to these responses. A selective effect of EE on the behavioral response to the NMDA receptor antagonist MK-801 in mGlu5 KO mice was seen. MK-801-induced hyperlocomotion was further potentiated in enriched mGlu5 KO mice and treatment with MK-801 reinstated PPI disruption in EE mGlu5 KO mice only, a response that is absent under standard housing conditions. Together, these results demonstrate an important role for mGlu5 in environmental modulation of schizophrenia-related behavioral impairments. Furthermore, this role of the mGlu5 receptor is mediated by interaction with NMDA receptor function, which may inform development of novel therapeutics.

Identifying novel interventional strategies for psychiatric disorders: integrating genomics, 'enviromics' and gene-environment interactions in valid preclinical models.

Psychiatric disorders affect a substantial proportion of the population worldwide. This high prevalence, combined with the chronicity of the disorders and the major social and economic impacts, creates a significant burden. As a result, an important priority is the development of novel and effective interventional strategies for reducing incidence rates and improving outcomes. This review explores the progress that has been made to date in establishing valid animal models of psychiatric disorders, while beginning to unravel the complex factors that may be contributing to the limitations of current methodological approaches. We propose some approaches for optimizing the validity of animal models and developing effective interventions. We use schizophrenia and autism spectrum disorders as examples of disorders for which development of valid preclinical models, and fully effective therapeutics, have proven particularly challenging. However, the conclusions have relevance to various other psychiatric conditions, including depression, anxiety and bipolar disorders. We address the key aspects of construct, face and predictive validity in animal models, incorporating genetic and environmental factors. Our understanding of psychiatric disorders is accelerating exponentially, revealing extraordinary levels of genetic complexity, heterogeneity and pleiotropy. The environmental factors contributing to individual, and multiple, disorders also exhibit breathtaking complexity, requiring systematic analysis to experimentally explore the environmental mediators and modulators which constitute the ‘envirome’ of each psychiatric disorder. Ultimately, genetic and environmental factors need to be integrated via animal models incorporating the spatiotemporal complexity of gene–environment interactions and experience‐dependent plasticity, thus better recapitulating the dynamic nature of brain development, function and dysfunction.

Regulator of G-protein signalling 4 expression is not altered in the prefrontal cortex in schizophrenia

Objectives: Regulator of G-protein signalling 4 (RGS4) modulates signal transduction through several neurotransmitter receptor systems associated with the pathology of schizophrenia. A reported decrease in RGS4 expression in the prefrontal cortex of schizophrenia patients followed by supporting evidence from association studies implicated RGS4 as a susceptibility gene for schizophrenia. Subsequent efforts to extend these findings in post-mortem brain tissue have produced conflicting results. The aim of the present study was to reconcile these discrepancies by examining RGS4 expression in the dorsolateral prefrontal and parietal cortices from subjects with schizophrenia. Methods: RGS4 mRNA and protein levels were measured in post-mortem Brodmann area (BA)9 and BA40 tissue from 19 schizophrenia patients subjects and 19 pair-matched controls using in situ hybridization and western blotting. Results: Levels of RGS4 mRNA (F1,73=1.845; p >0.05) or protein (F1,72=3.336×10−4, p >0.05) did not vary significantly with diagnosis in BA9 or BA40 from subjects with schizophrenia. Conclusions: Altered RGS4 expression is not universally present throughout the cortex of people with schizophrenia.

Lower [3H]LY341495 binding to mGlu2/3 receptors in the anterior cingulate of subjects with major depressive disorder but not bipolar disorder or schizophrenia.

INTRODUCTION The glutamatergic system has recently been implicated in the pathogenesis and treatment of major depressive disorders(MDD) and mGlu2/3 receptors play an important role in regulating glutamatergic tone. We therefore measured cortical levels of mGlu2/3 to determine if they were changed in MDD. METHODS Binding parameters for [(3)H]LY341495 (mGlu2/3 antagonist) were determined to allow optimized in situ binding with autoradiography to be completed using a number of CNS regions. Subsequently, density of [(3)H]LY341495 binding was measured in BA24(anterior cingulate cortex), BA17(visual cortex) and BA46(dorsolateral prefrontal cortex) from subjects with MDD, Bipolar Disorder(BPD), Schizophrenia(SCZ), and controls, as well as rats treated with imipramine (20mg/kg), fluoxetine (10mg/kg), or vehicle. RESULTS mGlu2/3 are widely expressed throughout the brain with high levels observed in cortex. [(3)H]LY341495 binding was significantly lower in BA24 from subjects with MDD (mean ± SEM=141.3 ± 14.65 fmol/ETE) relative to controls (184.9 ± 7.76 fmol/ETE; Cohens d=1.005, p<0.05). There were no other differences with diagnoses, and chronic antidepressant treatment in rats had minimal effect on binding. LIMITATIONS Using this approach we are unable to determine whether the change represents fluctuations in mGlu2, mGlu3, or both. Moreover, using postmortem tissue we are unable to dissociate the irrevocable confound of suicidality upon binding levels. CONCLUSION We have demonstrated lower [(3)H]LY341495 binding levels in MDD in BA24-a brain region implicated in depression. Moreover we show that the lower levels are unlikely to be the result of antidepressant treatment. These data suggest that levels of either mGlu2 and/or mGlu3 are affected in the aetiology of MDD.

Sensitivity to MK-801 in phospholipase C-β1 knockout mice reveals a specific NMDA receptor deficit

Phospholipase C-β1 (PLC-β1) is a critical component of multiple signalling pathways downstream of neurotransmitter receptors. Mice lacking this enzyme display a striking behavioural phenotype with relevance to human psychiatric disease. Glutamatergic dysfunction is strongly associated with several abnormal behavioural states and may underlie part of the phenotype of the phospholipase C-β1 knockout (KO) mouse. A heightened response to glutamatergic psychotomimetic drugs is a critical psychosis-related endophenotype, and in this study it was employed as a correlate of glutamatergic dysfunction. Control (n=8) and PLC-β1 KO mice (n=6) were treated with MK-801, a NMDA receptor (NMDAR) antagonist, following either standard housing or environmental enrichment, and the motor function and locomotor activity thus evoked was assessed. In addition, MK-801 binding to the NMDAR was evaluated through radioligand autoradiography in post-mortem tissue (on a drug-naive cohort). We have demonstrated a significantly increased sensitivity to the effects of the NMDA antagonist MK-801 in the PLC-β1 KO mouse. In addition, we found that this mouse line displays reduced hippocampal NMDAR expression, as measured by radioligand binding. We previously documented a reversal of specific phenotypes in this mouse line following housing in an enriched environment. Enrichment did not alter this heightened MK-801 response, nor NMDAR expression, indicating that this therapeutic intervention works on specific pathways only. These findings demonstrate the critical role of the glutamatergic system in the phenotype of the PLC-β1 KO mouse and highlight the role of these interconnected signalling pathways in schizophrenia-like behavioural disruption. These results also shed further light on the capacity of environmental factors to modulate subsets of these phenotypes.

5-HT2 Receptor Regulation of Mitochondrial Genes: Unexpected Pharmacological Effects of Agonists and Antagonists

In acute organ injuries, mitochondria are often dysfunctional, and recent research has revealed that recovery of mitochondrial and renal functions is accelerated by induction of mitochondrial biogenesis (MB). We previously reported that the nonselective 5-HT2 receptor agonist DOI [1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine] induced MB in renal proximal tubular cells (RPTCs). The goal of this study was to determine the role of 5-HT2 receptors in the regulation of mitochondrial genes and oxidative metabolism in the kidney. The 5-HT2C receptor agonist CP-809,101 [2-[(3-chlorophenyl)methoxy]-6-(1-piperazinyl)pyrazine] and antagonist SB-242,084 [6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride] were used to examine the induction of renal mitochondrial genes and oxidative metabolism in RPTCs and in mouse kidneys in the presence and absence of the 5-HT2C receptor. Unexpectedly, both CP-809,101 and SB-242,084 increased RPTC respiration and peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) mRNA expression in RPTCs at 1–10 nM. In addition, CP-809,101 and SB-242,084 increased mRNA expression of PGC-1α and the mitochondrial proteins NADH dehydrogenase subunit 1 and NADH dehydrogenase (ubiquinone) β subcomplex 8 in mice. These compounds increased mitochondrial genes in RPTCs in which the 5-HT2C receptor was downregulated with small interfering RNA and in the renal cortex of mice lacking the 5-HT2C receptor. By contrast, the ability of these compounds to increase PGC-1α mRNA and respiration was blocked in RPTCs treated with 5-HT2A receptor small interfering RNA or the 5-HT2A receptor antagonist eplivanserin. In addition, the 5-HT2A receptor agonist NBOH-2C-CN [4-[2-[[(2-hydroxyphenyl)methyl]amino]ethyl]-2,5-dimethoxybenzonitrile] increased RPTC respiration at 1–100 nM. These results suggest that agonism of the 5-HT2A receptor induces MB and that the classic 5-HT2C receptor agonist CP-809,101 and antagonist SB-242,084 increase mitochondrial genes and oxidative metabolism through the 5-HT2A receptor. To our knowledge, this is the first report that links 5-HT2A receptor agonism to mitochondrial function.