Liesl Duffy

Altered motor activity, exploration and anxiety in heterozygous neuregulin 1 mutant mice: implications for understanding schizophrenia

Human genetic studies have shown that neuregulin 1 (NRG1) is a potential susceptibility gene for schizophrenia. Nrg1 influences various neurodevelopmental processes, which are potentially related to schizophrenia. The neurodevelopmental theory of schizophrenia suggests that interactions between genetic and environmental factors are responsible for biochemical alterations leading to schizophrenia. To investigate these interactions and to match experimental design with the pathophysiology of schizophrenia, we applied a comprehensive behavioural phenotyping strategy for motor activity, exploration and anxiety in a heterozygous Nrg1 transmembrane domain mutant mouse model (Nrg1 HET) using different housing conditions and age groups. We observed a locomotion‐ and exploration‐related hyperactive phenotype in Nrg1 HETs. Increased age had a locomotion‐ and exploration‐inhibiting effect, which was significantly attenuated in mutant mice. Environmental enrichment (EE) had a stimulating influence on locomotion and exploration. The impact of EE was more pronounced in Nrg1 hypomorphs. Our study also showed a moderate task‐specific anxiolytic‐like phenotype for Nrg1 HETs, which was influenced by external factors. The behavioural phenotype detected in heterozygous Nrg1 mutant mice is not specific to schizophrenia per se, but the increased sensitivity of mutant mice to exogenous factors is consistent with the pathophysiology of schizophrenia and the neurodevelopmental theory. Our findings reinforce the importance of carefully controlling experimental designs for external factors and of comprehensive, integrative phenotyping strategies. Thus, Nrg1 HETs may, in combination with other genetic and drug models, help to clarify pathophysiological mechanisms behind schizophrenia.

Behavioural profile of a new mouse model for NPY deficiency

The abundantly expressed neuropeptide Y (NPY) plays an important role in anxiety and stress reactivity, as exogenous NPY administration reduces anxiety‐like behaviour in rodents. However, unlike the potent effects of NPY seen in pharmacological studies, two independent examinations of a genetic mouse model for NPY deficiency have shown only subtle, inconsistent and task‐dependent anxiety‐related phenotypes for male mutants. Here we present results of a newly developed germline NPY‐knockout model, which has been characterized behaviourally using a comprehensive multi‐tiered phenotyping strategy. Mice of both sexes were investigated in locomotion and exploration tasks, anxiety‐related paradigms, a hippocampus‐dependent memory test and a battery of basic tasks screening for sensory and motor functions. Male and female NPY‐deficient mice consistently demonstrated suppressed levels of locomotion and exploration. Furthermore, mutant mice exhibited a pronounced anxiogenic‐like phenotype when tested in spatiotemporal anxiety‐relevant paradigms (i.e. elevated‐plus maze, open field and light–dark task). Importantly, this phenotype was more pronounced in male NPY mutants, revealing a moderate sexually dimorphic impact of NPY deficiency on behaviour. Interestingly, lack of NPY did not result in impaired learning and memory in either sex. Our carefully selected comprehensive behavioural phenotyping strategy revealed a consistent hypolocomotive and sex‐dependent anxious‐like phenotype. This new NPY‐knockout mouse model reveals the importance of sex‐specific testing. It also offers a potent new model for research into anxiety‐related disorders and suggests potential treatment options for these conditions via the NPY system.

Cognition in transmembrane domain neuregulin 1 mutant mice

Neuregulin 1 (NRG1), which has been implicated in the development of schizophrenia, is expressed widely throughout the brain and influences key neurodevelopmental processes such as myelination and neuronal migration. The heterozygous transmembrane domain Nrg1 mutant mouse (Nrg1 TM HET) exhibits a neurobehavioural phenotype relevant for schizophrenia research, characterized by the development of locomotor hyperactivity, social withdrawal, increased sensitivity to environmental manipulation, and changes to the serotonergic system. As only limited data are available on the learning and memory performance of Nrg1 TM HET mice, we conducted a comprehensive examination of these mice and their wild type-like littermates in a variety of paradigms, including fear conditioning (FC), radial arm maze (RAM), Y maze, object exploration and passive avoidance (PA). Male neuregulin 1 hypomorphic mice displayed impairments in the novel object recognition and FC tasks, including reduced interest in the novel object and reduced FC to a context, but not a discrete cue. These cognitive deficits were task-specific, as no differences were seen between mutant and control mice in spatial learning (i.e. RAM and Y maze) for both working and reference memory measures, or in the PA paradigm. These findings indicate that neuregulin 1 plays a moderate role in cognition and present further behavioural validation of this genetic mouse model for the schizophrenia candidate gene neuregulin 1.

Behavioral profile of a heterozygous mutant mouse model for EGF-like domain neuregulin 1.

Human genetic studies have demonstrated that the neuregulin 1 gene (NRG1) is involved in the development of schizophrenia. Alternative splicing of NRG1 results in at least 15 distinct isoforms and all contain an extracellular epidermal growth factor (EGF)-like domain, which is sufficient for Nrg1s biological activity. Here, we characterize a heterozygous mutant model for mouse EGF-like domain neuregulin 1 (Nrg1) regarding schizophrenia-related behavioral domains. A comprehensive, multitiered phenotyping strategy was used to investigate locomotion, exploration, anxiety-related behaviors, and sensorimotor gating. Nrg1 mutant mice exhibited a hyper-locomotive phenotype and an improved ability to habituate to a new environment. Extensive analysis of anxiety-related behaviors revealed a wild type-like phenotype in this domain. However, a moderate impairment in sensorimotor gating was found after pharmacological challenge using psychoactive substances. Our study adds to the increasing behavioral data available from a variety of animal models for Nrg1 isoforms. We suggest a standardized and comprehensive behavioral phenotyping approach to distinguish between the different models and to clarify their relevance for schizophrenia research. Future behavioral investigations will focus on the negative and cognitive symptoms of schizophrenia.

Increased levels of serotonin2A receptors and serotonin transporter in the CNS of neuregulin 1 hypomorphic/mutant mice

Changes in neuregulin 1 expression have been reported in the CNS from subjects with schizophrenia. As neuregulin 1 is important in cortical development we postulated that changes in neuregulin 1 expression may contribute towards changes in cholinergic, glutamatergic and serotonergic markers that are well documented in the CNS of subjects with that disorder. To begin to test this hypothesis, we used in situ radioligand binding to measure levels of muscarinic M1/M4 receptors, the kainate receptor, the NMDA receptor, the serotonin 2A receptor, the serotonin 1A receptor and the serotonin transporter in the CNS from heterozygous transmembrane domain neuregulin 1 mutant mice. The major outcomes from these studies was the demonstration of an overall increase in levels of the serotonin 2A receptor (F=11.3, d.f.=3,1,72, p=0.0012) and serotonin transporter (F=5.00, d.f.=1,3,72, p<0.05) in the mutant mice. Levels of the other receptors did not vary in the mutant mice compared to their wild type-like litter mates. These data are the first evidence to suggest that NRG1 gene expression may be involved in regulating the development of the serotonergic system in the mammalian CNS.

Y1 receptors are critical for the proliferation of adult mouse precursor cells in the olfactory neuroepithelium

While the regenerative capacity of the olfactory neuroepithelium has been well studied less is known about the molecular events controlling precursor cell activity. Neuropeptide Y (NPY) is expressed at high levels in the olfactory system, and NPY has been shown to play a role in neuroregeneration of the brain. In this study, we show that the numbers of olfactory neurospheres derived from NPY, NPY/peptide YY, and Y1 receptor knockout mice are decreased compared with wild type (WT) controls. Furthermore, flow cytometric analysis of isolated horizontal basal cells, globose basal cells, and glandular cells showed that only glandular cells derived from WT mice, but not from NPY and Y1 receptor knockout mice, formed secondary neurospheres suggesting a critical role for NPY signaling in this process. Interestingly, olfactory function tests revealed that olfaction in Y1 knockout mice is impaired compared with those of WT mice, probably because of the reduced number of olfactory neurons formed. Together these results indicate that NPY and the Y1 receptor are required for the normal proliferation of adult olfactory precursors and olfactory function.

Distinct endocrine effects of chronic haloperidol or risperidone administration in male rats.

Antipsychotic drugs have been used effectively for the treatment of schizophrenia symptoms, but they are often associated with metabolic side effects such as weight gain and endocrine disruptions. To investigate the possible mechanisms of antipsychotic-induced metabolic effects, we studied the impact of chronic administration of a typical antipsychotic drug (haloperidol) and an atypical antipsychotic (risperidone) to male rats on food intake, body weight, adiposity, and the circulating concentrations of hormones and metabolites that can influence energy homeostasis. Chronic (28days) haloperidol administration had no effect on food intake, weight gain or adiposity in male rats, whereas risperidone treatment resulted in a transient reduction in food intake and significantly reduced body weight gain compared to vehicle-treated control rats. Whereas neither antipsychotic had any effect on serum lipid profiles, glucose tolerance or the circulating concentrations of hormones controlled by the hypothalamo-pituitary-thyroid (free T4), -adrenal (corticosterone), -somatotropic (IGF-1), or -gonadotropic axes (testosterone), haloperidol increased circulating insulin levels and risperidone increased serum glucagon levels. This finding suggests that haloperidol or risperidone induce distinct metabolic effects. Since metabolic disorders such as obesity and type 2 diabetes mellitus represent serious health issues, understanding antipsychotic-induced endocrine and metabolic effects may ultimately allow better control of these side effects.

Effects of chronic risperidone treatment on the striatal protein profiles in rats

Extrapyramidal symptoms (EPS) commonly occur as side effects of antipsychotic drugs (APDs) and are most likely to arise when the occupancy of dopamine D(2) receptors in the striatum by these drugs exceeds 80%. We aimed to characterize changes in the protein expression profile in the striatum of rats after chronic (4 week) supra-therapeutic (EPS-inducing) treatment with risperidone (RIS), an atypical antipsychotic drug. Administration of RIS (2.1 mg/kg/day, via subcutaneous osmotic minipumps) induced significant vacuous chewing movements and catalepsy in male Sprague-Dawley rats over a 28-day treatment period compared with a vehicle (VEH) control group (n=12) (Karl et al., unpublished observation). Using two-dimensional gel electrophoresis (2DE), total protein extracts from the rat brain striatum were separated and protein expression was analyzed by Phoretix 2D Expression and Image Beta V4.02 software followed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). 2DE gels resolved up to 450 protein spots, presumably different proteins and/or their isoforms. There were 30 protein spots showing statistically significant different densities between the RIS- and VEH-treated groups. All 30 proteins were successfully identified by MALDI-TOF MS, 28 of these were divided into groups based on their known functions. These included metabolic, signaling, transport, protein metabolism, chaperone, DNA binding and cell cycle categories. We conclude that chronic risperidone treatment accompanied by an EPS-like behavioral phenotype results in alterations in the striatal protein profile possibly subsequent to blockade of dopaminergic systems. These results suggest that possible mechanisms involved in APD-induced EPS include metabolic dysfunction and oxidative stress.

Schizophrenia-relevant behaviours in a genetic mouse model for Y2 deficiency.

Expression levels of neuropeptide Y (NPY) are changed in schizophrenia patients. However, the direction of changes to NPY expression and the mechanisms behind NPYs impact on the development of the illness is not understood in detail. Here we investigated whether alterations in Y2 activity may be involved in the development of schizophrenia-related behaviours. We examined NPY Y2 receptor deficient male mice in behavioural domains relevant for the illness: locomotion, learning and memory, social interaction and sensorimotor gating (baseline and after acute challenge with psychotropic drugs) and the most relevant tasks were also completed in female Y2 mutants. Our investigations confirmed a hyper-locomotive phenotype for Y2 deficient male mice and no alterations in working and reference memory performance. Mutant males exhibited an increase in social interaction and moderately improved sensorimotor gating. The psychotropic drugs dexamphetamine and MK-801 affected prepulse inhibition similarly, whereas MK-801 appeared to be a slightly more potent stimulant for the acoustic startle response (ASR). Female Y2 deficient mice showed wild type-like performances in social interaction, working memory and prepulse inhibition. However, Y2 mutant females exhibited a moderately increased ASR compared to control mice. Taken together, lack of Y2 signalling in mice not only leads to altered locomotion but also changes social behaviours and affects sensorimotor gating. Thus, Y2 depletion influences a range of behaviours, which are potentially relevant for schizophrenia-related research.

Acoustic startle response and sensorimotor gating in a genetic mouse model for the Y1 receptor.

Recent research has highlighted a potential role for neuropeptide Y (NPY) and its Y(1) receptor in the development of schizophrenia. Genetic as well as molecular biological studies have demonstrated reduced levels of NPY in schizophrenia patients. Importantly, Y(1) receptors may mediate some of the potential effects of NPY on schizophrenia, as decreased Y(1) receptor expression has been found in the lymphocytes of schizophrenia patients. To clarify NPYs role in schizophrenia, we investigated a genetic animal model for Y(1) deficiency in regard to (i) acoustic startle response (ASR), (ii) habituation to ASR and (iii) sensorimotor gating [i.e. prepulse inhibition (PPI)] using two different PPI protocols. Mutant and wild type-like mice were screened for baseline behaviours and after pharmacological challenge with the psychotropic drugs dexamphetamine (DEX) and MK-801. Y(1) knockout mice (Y(1)(-/-)) showed a moderate reduction of the ASR and an impaired ASR habituation at baseline and after DEX treatment. The baseline PPI performance of Y(1) mutant mice was unaltered their response to DEX and MK-801 challenge was moderately different compared to control mice, which was dependent on the PPI protocol used. MK-801 challenge had a protocol-dependent differential effect in Y(1)(-/-) mice and DEX a more pronounced impact at the highest prepulse intensities. In conclusion, it appears that the Y(1) receptor influences the acoustic startle response and its habituation but does not play a major role in sensorimotor gating. Further explorations into the effects of Y(1) deficiency seem valid.