Emma L. Burrows

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.

Increased adult hippocampal neurogenesis and abnormal migration of adult-born granule neurons is associated with hippocampal-specific cognitive deficits in phospholipase C-β1 knockout mice

Schizophrenia is a devastating psychiatric illness with a complex pathophysiology. We have recently documented schizophrenia‐like endophenotypes in phospholipase C‐β1 knockout (PLC‐β1−/−) mice, including deficits in prepulse inhibition, hyperlocomotion, and cognitive impairments. PLC‐β1 signals via multiple G‐protein coupled receptor pathways implicated in neural cellular plasticity; however, adult neurogenesis has yet to be explored in this knockout model. In this study, weemployed PLC‐β1−/− mice to elucidate possible correlates between aberrant adult hippocampal neurogenesis (AHN) and schizophrenia‐like behaviors. Using stereology and bromodeoxyuridine (BrdU) immunohistochemistry we demonstrated a significant increase in the density of adult‐generated cells in the granule cell layer (GCL) of adult PLC‐β1−/− mice compared with wild‐type littermates. Cellular phenotype analysis using confocal microscopy revealed these cells to be mature granule neurons expressing NeuN and calbindin. Increased neuronal survival occurred concomitant with reduced caspase‐3(+) cells in the GCL of PLC‐β1−/− mice. Stereological analysis of Ki67(+) cells in the subgranular zone suggested that neural precursor proliferation is unchanged in PLC‐β1−/− mice. We further showed aberrant migration of mature granule neurons within the GCL of adult PLC‐β1−/− mice with excessive adult‐generated mature neurons residing in the middle and outer GCL. PLC‐β1−/− mice exhibited specific behavioral deficits in location recognition, a measure of hippocampal‐dependent memory, but not novel object recognition. Overall, we have shown that PLC‐β1−/− mice have a threefold increase in net AHN, and have provided further evidence to suggest a specific deficit in hippocampal‐dependent cognition. We propose that abnormal cellular plasticity in these mice may contribute to their schizophrenia‐like behavioral endophenotypes.

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.

A neuroligin-3 mutation implicated in autism causes abnormal aggression and increases repetitive behavior in mice

BackgroundAggression is common in patients with autism spectrum disorders (ASD) along with the core symptoms of impairments in social communication and repetitive behavior. Risperidone, an atypical antipsychotic, is widely used to treat aggression in ASD. In order to understand the neurobiological underpinnings of these challenging behaviors, a thorough characterisation of behavioral endophenotypes in animal models is required.MethodsWe investigated aggression in mice containing the ASD-associated R451C (arginine to cysteine residue 451 substitution) mutation in neuroligin-3 (NL3). Furthermore, we sought to verify social interaction impairments and assess olfaction, anxiety, and repetitive and restrictive behavior in NL3R451C mutant mice.ResultsWe show a pronounced elevation in aggressive behavior in NL3R451C mutant mice. Treatment with risperidone reduced this aggression to wild-type (WT) levels. Juvenile and adult social interactions were also investigated, and subtle differences in initiation of interaction were seen in juvenile NL3R451C mice. No genotype differences in olfactory discrimination or anxiety were observed indicating that aggression was not dependent on altered olfaction, stress response, or social preference. We also describe repetitive behavior in NL3R451C mice as assessed by a clinically relevant object exploration task.ConclusionsThe presence of aberrant aggression and other behavioral phenotypes in NL3R451C mice consistent with clinical traits strengthen face validity of this model of ASD. Furthermore, we demonstrate predictive validity in this model through the reversal of the aggressive phenotype with risperidone. This is the first demonstration that risperidone can ameliorate aggression in an animal model of ASD and will inform mechanistic and therapeutic research into the neurobiology underlying abnormal behaviors in ASD.

Cognitive endophenotypes, gene–environment interactions and experience-dependent plasticity in animal models of schizophrenia

Schizophrenia is a devastating brain disorder caused by a complex and heterogeneous combination of genetic and environmental factors. In order to develop effective new strategies to prevent and treat schizophrenia, valid animal models are required which accurately model the disorder, and ideally provide construct, face and predictive validity. The cognitive deficits in schizophrenia represent some of the most debilitating symptoms and are also currently the most poorly treated. Therefore it is crucial that animal models are able to capture the cognitive dysfunction that characterizes schizophrenia, as well as the negative and psychotic symptoms. The genomes of mice have, prior to the recent gene-editing revolution, proven the most easily manipulable of mammalian laboratory species, and hence most genetic targeting has been performed using mouse models. Importantly, when key environmental factors of relevance to schizophrenia are experimentally manipulated, dramatic changes in the phenotypes of these animal models are often observed. We will review recent studies in rodent models which provide insight into gene-environment interactions in schizophrenia. We will focus specifically on environmental factors which modulate levels of experience-dependent plasticity, including environmental enrichment, cognitive stimulation, physical activity and stress. The insights provided by this research will not only help refine the establishment of optimally valid animal models which facilitate development of novel therapeutics, but will also provide insight into the pathogenesis of schizophrenia, thus identifying molecular and cellular targets for future preclinical and clinical investigations.

Decanalization mediating gene-environment interactions in schizophrenia and other psychiatric disorders with neurodevelopmental etiology

Over the past several decades, significant advances in genetics and neuroscience have transformed our understanding of how the brain produces adaptive behavior and the ways in which normal functioning becomes disrupted in neurodevelopmental disorders, such as schizophrenia and ASD. Nevertheless, we have only begun to comprehend how particular combinations of genomes and environmental histories combine to produce a given set of clinical symptoms. A major challenge for translating these findings to specific, effective treatments has been the heterogeneity that exists both in clinical presentation and in the genetic associations that have been uncovered. Both schizophrenia and ASD exhibit high heritability and significant research efforts have been geared toward uncovering genetic variation in a bid to explain cause. Genome-wide association studies (GWAS) have identified hundreds of common variants associated with complex diseases, however, the overall genetic risk explained by these loci remains modest (Manolio et al., 2009; Eichler et al., 2010). There is evidence for both common genetic variation and rare DNA sequence variants contributing to genetic susceptibility in both disorders (State and Levitt, 2011; Mowry and Gratten, 2013). This contribution of common and rare alleles is thought to be variable among cases, making it difficult to reliably detect an association signal among the heterogeneity and genetic noise. Adding further complexity, the genetic architecture may include epistatic (gene-gene) effects among interacting loci (Phillips, 2008). Even with sophisticated approaches to resolve gene–gene interactions acting within whole genome contexts, we are still faced with the conundrum of “missing heritability” (Manolio et al., 2009; Hannan, 2010; McGrath et al., 2011). The problem of missing heritability may be at least partly addressed by improving the discovery rate of genetic variants via statistically well-powered cohorts of individuals that are better characterized for disease phenotypes, genetic background, and environmental exposure (Manolio et al., 2009; Eichler et al., 2010). This approach is dependent on the idea that a combination of common variants of small effect, rare variants of large effect and environmental factors will lead to disease (Manolio et al., 2009; Eichler et al., 2010; Gibson, 2011). A focus on genetic risk factors alone has limited heuristic value due to the interdependent interactions between genetic and environmental factors that play key roles in pathogenesis. An alternative view suggests that gene-environment (G × E) interactions can account for a substantial proportion of disease risk (Svrakic et al., 2013). There is strong evidence that G × E interactions are ubiquitous, accounting for the greater part of phenotypic variation seen across genotypes (Grishkevich and Yanai, 2013). Research utilizing model organisms has identified that not all genes are equally likely to exhibit G × E interactions; promoter architecture, expression level, regulatory complexity, and essentiality correlate with the differential regulation of a gene by the environment (Grishkevich et al., 2012). In fact, genes that exhibit G × E interactions may confer evolutionary advantage in that they facilitate phenotypic plasticity and provide an organism with the flexibility to adjust its phenotype with respect to the specific environmental conditions experienced (Via et al., 1995; Lande, 2009). This plasticity could represent a substantial advantage in unpredictable environments but could also, when combined with genetic susceptibility, underlie disruptions in normal brain function.

Translational Assays for Assessment of Cognition in Rodent Models of Alzheimer’s Disease and Dementia

Cognitive dysfunction appears as a core feature of dementia, which includes its most prevalent form, Alzheimer’s disease (AD), as well as vascular dementia, frontotemporal dementia, and other brain disorders. AD alone affects more than 45 million people worldwide, with growing prevalence in aging populations. There is no cure, and therapeutic options remain limited. Gene-edited and transgenic animal models, expressing disease-specific gene mutations, illuminate pathogenic mechanisms leading to cognitive decline in AD and other forms of dementia. To date, cognitive tests in AD mouse models have not been directly relevant to the clinical presentation of AD, providing challenges for translation of findings to the clinic. Touchscreen testing in mice has enabled the assessment of specific cognitive domains in mice that are directly relevant to impairments described in human AD patients. In this review, we provide context for how cognitive decline is measured in the clinic, describe traditional methods for assessing cognition in mice, and outline novel approaches, including the use of the touchscreen platform for cognitive testing. We highlight the limitations of traditional memory-testing paradigms in mice, particularly their capacity for direct translation into cognitive testing of patients. While it is not possible to expect direct translation in testing methodologies, we can aim to develop tests that engage similar neural substrates in both humans and mice. Ultimately, that would enable us to better predict efficacy across species and therefore improve the chances that a treatment that works in mice will also work in the clinic.

Decreased expression of mGluR5 within the dorsolateral prefrontal cortex in autism and increased microglial number in mGluR5 knockout mice: Pathophysiological and neurobehavioral implications

Metabotropic glutamate receptor 5 (mGluR5) and microglial abnormalities have been implicated in autism spectrum disorder (ASD). However, controversy exists as to whether the receptor is down or upregulated in functioning in ASD. In addition, whilst activation of mGluR5 has been shown to attenuate microglial activation, its role in maintaining microglial homeostasis during development has not been investigated. We utilised published microarray data from the dorsolateral prefrontal cortex (DLPFC) of control (n=30) and ASD (n=27) individuals to carry out regression analysis to assess gene expression of mGluR5 downstream signalling elements. We then conducted a post-mortem brain stereological investigation of the DLPFC, to estimate the proportion of mGluR5-positive neurons and glia. Finally, we carried out stereological investigation into numbers of microglia in mGluR5 knockout mice, relative to wildtype littermates, together with assessment of changes in microglial somal size, as an indicator of activation status. We found that gene expression of mGluR5 was significantly decreased in ASD versus controls (p=0.018) as well as downstream elements SHANK3 (p=0.005) and PLCB1 (p=0.009) but that the pro-inflammatory marker NOS2 was increased (p=0.047). Intensity of staining of mGluR5-positive neurons was also significantly decreased in ASD versus controls (p=0.016). Microglial density was significantly increased in mGluR5 knockout animals versus wildtype controls (p=0.011). Our findings provide evidence for decreased expression of mGluR5 and its signalling components representing a key pathophysiological hallmark in ASD with implications for the regulation of microglial number and activation during development. This is important in the context of microglia being considered to play key roles in synaptic pruning during development, with preservation of appropriate connectivity relevant for normal brain functioning.