Gene × Environment Interactions in Schizophrenia: Evidence from Genetic Mouse Models.

The study of gene × environment, as well as epistatic interactions in schizophrenia, has provided important insight into the complex etiopathologic basis of schizophrenia. It has also increased our understanding of the role of susceptibility genes in the disorder and is an important consideration as we seek to translate genetic advances into novel antipsychotic treatment targets. This review summarises data arising from research involving the modelling of gene × environment interactions in schizophrenia using preclinical genetic models. Evidence for synergistic effects on the expression of schizophrenia-relevant endophenotypes will be discussed. It is proposed that valid and multifactorial preclinical models are important tools for identifying critical areas, as well as underlying mechanisms, of convergence of genetic and environmental risk factors, and their interaction in schizophrenia.

Gut microbiota depletion from early adolescence in mice: Implications for brain and behaviour.

BACKGROUND: There is growing appreciation for the importance of bacteria in shaping brain development and behaviour. Adolescence and early adulthood are crucial developmental periods during which exposure to harmful environmental factors can have a permanent impact on brain function. Such environmental factors include perturbations of the gut bacteria that may affect gut-brain communication, altering the trajectory of brain development, and increasing vulnerability to psychiatric disorders. Here we assess the effects of gut bacterial depletion from weaning onwards on adult cognitive, social and emotional behaviours and markers of gut-brain axis dysfunction in mice. METHODS: Mice were treated with a combination of antibiotics from weaning onwards and effects on behaviours and potential gut-brain axis neuromodulators (tryptophan, monoamines, and neuropeptides) and BDNF expression were assessed in adulthood. RESULTS: Antibiotic-treatment depleted and restructured gut microbiota composition of caecal contents and decreased spleen weights in adulthood. Depletion of the gut microbiota from weaning onwards reduced anxiety, induced cognitive deficits, altered dynamics of the tryptophan metabolic pathway, and significantly reduced BDNF, oxytocin and vasopressin expression in the adult brain. CONCLUSIONS: Microbiota depletion from weaning onwards by means of chronic treatment with antibiotics in mice impacts on anxiety and cognitive behaviours as well as key neuromodulators of gut-brain communication in a manner that is similar to that reported in germ-free mice. This model may represent a more amenable alternative for germ-free mice in the assessment of microbiota modulation of behaviour. Finally, these data suggest that despite the presence of a normal gut microbiome in early postnatal life, reduced abundance and diversity of the gut microbiota from weaning influences adult behaviours and key neuromodulators of the microbiota-gut-brain axis suggesting that dysregulation of this axis in the post-weaning period may contribute to the pathogenesis of disorders associated with altered anxiety and cognition.

The microbiome: stress, health and disease.

Bacterial colonisation of the gut plays a major role in postnatal development and maturation of key systems that have the capacity to influence central nervous system (CNS) programming and signaling, including the immune and endocrine systems. Individually, these systems have been implicated in the neuropathology of many CNS disorders and collectively they form an important bidirectional pathway of communication between the microbiota and the brain in health and disease. Regulation of the microbiome-brain-gut axis is essential for maintaining homeostasis, including that of the CNS. Moreover, there is now expanding evidence for the view that commensal organisms within the gut play a role in early programming and later responsivity of the stress system. Research has focused on how the microbiota communicates with the CNS and thereby influences brain function. The routes of this communication are not fully elucidated but include neural, humoral, immune and metabolic pathways. This view is underpinned by studies in germ-free animals and in animals exposed to pathogenic bacterial infections, probiotic agents or antibiotics which indicate a role for the gut microbiota in the regulation of mood, cognition, pain and obesity. Thus, the concept of a microbiome-brain-gut axis is emerging which suggests that modulation of the gut microflora may be a tractable strategy for developing novel therapeutics for complex stress-related CNS disorders where there is a huge unmet medical need.

Social isolation-induced transcriptomic changes in mouse hippocampus impact the synapse and show convergence with human genetic risk for neurodevelopmental phenotypes.

Early life stress (ELS) can impact brain development and is a risk factor for neurodevelopmental disorders such as schizophrenia. Post-weaning social isolation (SI) is used to model ELS in animals, using isolation stress to disrupt a normal developmental trajectory. We aimed to investigate how SI affects the expression of genes in mouse hippocampus and to investigate how these changes related to the genetic basis of neurodevelopmental phenotypes. BL/6J mice were exposed to post-weaning SI (PD21-25) or treated as group-housed controls (n = 7-8 per group). RNA sequencing was performed on tissue samples from the hippocampus of adult male and female mice. Four hundred and 1,215 differentially-expressed genes (DEGs) at a false discovery rate of < 0.05 were detected between SI and control samples for males and females respectively. DEGS for both males and females were significantly overrepresented in gene ontologies related to synaptic structure and function, especially the post-synapse. DEGs were enriched for common variant (SNP) heritability in humans that contributes to risk of neuropsychiatric disorders (schizophrenia, bipolar disorder) and to cognitive function. DEGs were also enriched for genes harbouring rare de novo variants that contribute to autism spectrum disorder and other developmental disorders. Finally, cell type analysis revealed populations of hippocampal astrocytes that were enriched for DEGs, indicating effects in these cell types as well as neurons. Overall, these data suggest a convergence between genes dysregulated by the SI stressor in the mouse and genes associated with neurodevelopmental disorders and cognitive phenotypes in humans.

Genetic vs. pharmacological inactivation of COMT influences cannabinoid-induced expression of schizophrenia-related phenotypes.

Catechol-O-methyltransferase (COMT) is an important enzyme in the metabolism of dopamine and disturbance in dopamine function is proposed to be central to the pathogenesis of schizophrenia. Clinical epidemiological studies have indicated cannabis use to confer a 2-fold increase in risk for subsequent onset of psychosis, with adolescent-onset use conveying even higher risk. There is evidence that a high activity COMT polymorphism moderates the effects of adolescent exposure to cannabis on risk for adult psychosis. In this paper we compared the effect of chronic adolescent exposure to the cannabinoid WIN 55212 on sensorimotor gating, behaviours related to the negative symptoms of schizophrenia, anxiety- and stress-related behaviours, as well as ex-vivo brain dopamine and serotonin levels, in COMT KO vs. wild-type (WT) mice. Additionally, we examined the effect of pretreatment with the COMT inhibitor tolcapone on acute effects of this cannabinoid on sensorimotor gating in C57BL/6 mice. COMT KO mice were shown to be more vulnerable than WT to the disruptive effects of adolescent cannabinoid treatment on prepulse inhibition (PPI). Acute pharmacological inhibition of COMT in C57BL/6 mice also modified acute cannabinoid effects on startle reactivity, as well as PPI, indicating that chronic and acute loss of COMT can produce dissociable effects on the behavioural effects of cannabinoids. COMT KO mice also demonstrated differential effects of adolescent cannabinoid administration on sociability and anxiety-related behaviour, both confirming and extending earlier reports of COMT×cannabinoid effects on the expression of schizophrenia-related endophenotypes.

Phenotypic effects of repeated psychosocial stress during adolescence in mice mutant for the schizophrenia risk gene neuregulin-1: a putative model of gene × environment interaction.

There is a paucity of animal models by which the contributions of environmental and genetic factors to the pathobiology of psychosis can be investigated. This study examined the individual and combined effects of chronic social stress during adolescence and deletion of the schizophrenia risk gene neuregulin-1 (NRG1) on adult mouse phenotype. Mice were exposed to repeated social defeat stress during adolescence and assessed for exploratory behaviour, working memory, sucrose preference, social behaviour and prepulse inhibition in adulthood. Thereafter, in vitro cytokine responses to mitogen stimulation and corticosterone inhibition were assayed in spleen cells, with measurement of cytokine and brain-derived neurotrophic factor (BDNF) mRNA in frontal cortex, hippocampus and striatum. NRG1 mutants exhibited hyperactivity, decreased anxiety, impaired sensorimotor gating and reduced preference for social novelty. The effects of stress on exploratory/anxiety-related parameters, spatial working memory, sucrose preference and basal cytokine levels were modified by NRG1 deletion. Stress also exerted varied effect on spleen cytokine response to concanavalin A and brain cytokine and BDNF mRNA expression in NRG1 mutants. The experience of psychosocial stress during adolescence may trigger further pathobiological features that contribute to the development of schizophrenia, particularly in those with underlying NRG1 gene abnormalities. This model elaborates the importance of gene × environment interactions in the etiology of schizophrenia.

Mutant mouse models in evaluating novel approaches to antipsychotic treatment.

In this review we consider the application of mutant mouse phenotypes to the study of psychotic illness in general and schizophrenia in particular, as they relate to behavioral, psychopharmacological, and cellular phenotypes of putative import for antipsychotic drug development. Mutant models appear to be heuristic at two main levels; firstly, by indicating the functional roles of neuronal components thought to be of relevance to the putative pathobiology of psychotic illness, they help resolve overt behavioral and underlying cellular processes regulated by those neuronal components; secondly, by indicating the functional roles of genes associated with risk for psychotic illness, they help resolve overt behavioral and underlying cellular processes regulated by those risk genes. We focus initially on models of dopaminergic and glutamatergic dysfunction. Then, we consider advances in the genetics of schizophrenia and mutant models relating to replicable risk genes. Lastly, we extend this discussion by exemplifying two new variant approaches in mutant mice that may serve as prototypes for advancing antipsychotic drug development. There is continuing need not only to address numerous technical challenges but also to develop more "real-world" paradigms that reflect the milieu of gene × environment and gene × gene interactions that characterize psychotic illness and its response to antipsychotic drugs.

Dual hit mouse model to examine the long-term effects of maternal immune activation and post-weaning social isolation on schizophrenia endophenotypes.

Evidence suggests that early life adversity, such as maternal immune activation (MIA), can alter brain development in the offspring and confer increased risk for psychopathology and psychiatric illness in later life. In this study, the long-term effects of MIA, post-weaning social isolation, and the combination were assessed on behavioural and immunological profiles in adult male and female offspring. On gestation day 12.5, pregnant mice were weighed and injected with either polyinosinic:polycytidylic acid (5 mg/kg) or saline and cytokines levels were assayed 3 hrs later to confirm immune activation. The behaviour and immunological profiles of male and female offspring were examined in adolescence (P34-36), and adulthood (P55-80). MIA induced an increase in the pro-inflammatory cytokine IL-6 in pregnant dams three hours after administration (p < 0.001) that correlated with a decrease in body temperature (p < 0.05). The effect of MIA on the immunological phenotype of the offspring was evident in adolescence, but not in adulthood. MIA selectively induced hypoactivity in adolescent males, a phenotype that persisted until adulthood, but had no effect on cognition in males or females. In contrast, social isolation stress from adolescence resulted in impaired sociability (p < 0.05) and increased anxiety (p < 0.05) particularly in adult females. There was no synergistic effect of the dual-hit on immune parameters, sociability, anxiety or cognitive behaviours. Given the negative impact and sex-dependent effects of SI stress on locomotor and anxiety-like behaviour, future investigations should examine whether the health risks of social isolation, such as that experience during the COVID-19 pandemic, are mediated through increased anxiety.

Meta-Analysis of Brain Gene Expression Data from Mouse Model Studies of Maternal Immune Activation Using Poly(I:C).

Maternal immune activation (MIA) is a known risk factor for schizophrenia (SCZ) and autism spectrum disorder (ASD) and is often modelled in animal studies in order to study the effect of prenatal infection on brain function including behaviour and gene expression. Although the effect of MIA on gene expression are highly heterogeneous, combining data from multiple gene expression studies in a robust method may shed light on the true underlying biological effects caused by MIA and this could inform studies of SCZ and ASD. This study combined four RNA-seq and microarray datasets in an overlap analysis and ranked meta-analysis in order to investigate genes, pathways and cell types dysregulated in the MIA mouse models. Genes linked to SCZ and ASD and crucial in neurodevelopmental processes including neural tube folding, regulation of cellular stress and neuronal/glial cell differentiation were among the most consistently dysregulated in these ranked analyses. Gene ontologies including K+ ion channel function, neuron and glial cell differentiation, synaptic structure, axonal outgrowth, cilia function and lipid metabolism were also strongly implicated. Single-cell analysis identified excitatory and inhibitory cell types in the cortex, hippocampus and striatum that may be affected by MIA and are also enriched for genes associated with SCZ, ASD and cognitive phenotypes. This points to the cellular location of molecular mechanisms that may be consistent between the MIA model and neurodevelopmental disease, improving our understanding of its utility to study prenatal infection as an environmental stressor.

Ethologically based behavioural and neurochemical characterisation of mice with isoform-specific loss of dysbindin-1A in the context of schizophrenia.

Dysbindin-1 is implicated in several aspects of schizophrenia, including cognition and both glutamatergic and dopaminergic neurotransmission. Targeted knockout of dysbindin-1A (Dys-1A KO), the most abundant and widely expressed isoform in the brain, is associated with deficits in delay/interference-dependent working memory. Using an ethologically based approach, the following behavioural phenotypes were examined in Dys-1A KO mice: exploratory activity, social interaction, anxiety and problem-solving ability. Levels of monoamines and their metabolites were measured in striatum, hippocampus and prefrontal cortex using high-performance liquid chromatography with electrochemical detection. The ethogram of initial exploration in Dys-1A KO mice was characterised by increased rearing from a seated position; over subsequent habituation, stillness was decreased relative to wildtype. In a test of dyadic social interaction with an unfamiliar conspecific in a novel environment, female KO mice showed an increase in investigative social behaviours. Marble burying behaviour was unchanged. Using the puzzle-box test to measure general problem-solving performance, no effect of genotype was observed across nine trials of increasing complexity. Dys-1A KO demonstrated lower levels of 5-HT in ratio to its metabolite 5-HIAA in the prefrontal cortex. These studies elaborate the behavioural and neurochemical phenotype of Dys-1A KO mice, revealing subtle genotype-related differences in non-social and social exploratory behaviours and habituation of exploration in a novel environment, as well as changes in 5-HT activity in brain areas related to schizophrenia.

Mutant models for genes associated with schizophrenia.

Schizophrenia is a highly complex and heritable psychiatric disorder in which multiple genes and environmental factors interact to cause the schizophrenia phenotype. A new generation of molecular studies has yielded numerous candidate genes with a putative role in risk for schizophrenia, whereas other genes regulate putative pathophysiological mechanisms. Mutant mice having either deletion (knockout) or insertion (knockin/transgenesis) of schizophrenia risk genes now allow the functional role of these genes to be investigated. In the present mini-review, we outline the advantages and limitations of various approaches to phenotypic assessment of mutant mouse models, including ethologically based methods. Thereafter, we consider recent findings, with a particular focus on, first, dopaminergic and glutamatergic pathophysiological models and, secondly, putative roles for DISC1 (disrupted in schizophrenia 1) and NRG1 (neuregulin 1) as susceptibility genes for schizophrenia. Finally, we identify current challenges associated with the use of genetic mutant models and highlight their potential value for exploring gene-gene and gene-environment interactions in relation to schizophrenia.

Acute stress in adolescence vs early adulthood following selective deletion of dysbindin-1A: Effects on anxiety, cognition and other schizophrenia-related phenotypes.

BACKGROUND: As exposure to stress has been linked to the onset and maintenance of psychotic illness, its pathogenesis may involve environmental stressors interacting with genetic vulnerability. AIM: To establish whether acute stress interacts with a targeted mutation of the gene encoding the neurodevelopmental factor dystrobrevin-binding protein 1 (DTNBP1), resulting in a specific loss of the isoform dysbindin-1A, to influence schizophrenia-relevant phenotypes in mice during adolescence and adulthood. METHODS: Male and female mice with a heterozygous or homozygous deletion of DTNBP1 were assessed in the open field test following acute restraint stress in adolescence (Day 35) and young adulthood (Day 60-70). Effects of acute restraint stress on memory retention in the novel object recognition test was also assessed in adulthood. Baseline corticosterone was measured in serum samples and, brain-derived neurotrophic factor (BDNF), glucocorticoid and mineralocorticoid receptor gene expression levels were measured in the hippocampus of adult mice. RESULTS: In the open field, deletion of dysbindin-1A induced hyperactivity and attenuated the action of stress to reduce hyperactivity in adolescence but not in adulthood; in females deletion of dysbindin-1A attenuated the effect of acute stress to increase anxiety-related behaviour in adolescence but not in adulthood. In the novel object recognition test, deletion of dysbindin-1A impaired memory and also revealed an increase in anxiety-related behaviour and a decrease in hippocampal BDNF gene expression in males. CONCLUSIONS: These data suggest that deletion of dysbindin-1A influences behaviours related to schizophrenia and anxiety more robustly in adolescence than in adulthood and that dysbindin-1A influences stress-related responses in a sex-dependent manner.

The probiotic Bifidobacteria infantis: An assessment of potential antidepressant properties in the rat.

It is becoming increasingly apparent that probiotics are important to the health of the host. The absence of probiotic bacteria in the gut can have adverse effects not only locally in the gut, but has also been shown to affect central HPA and monoaminergic activity, features that have been implicated in the aetiology of depression. To evaluate the potential antidepressant properties of probiotics, we tested rats chronically treated with Bifidobacteria infantis in the forced swim test, and also assessed the effects on immune, neuroendocrine and central monoaminergic activity. Sprague-Dawley rats were treated for 14 days with B. infantis. Probiotic administration in naive rats had no effect on swim behaviours on day 3 or day 14 following the commencement of treatment. However, there was a significant attenuation of IFN-gamma, TNF-alpha and IL-6 cytokines following mitogen stimulation (p<0.05) in probiotic-treated rats relative to controls. Furthermore, there was a marked increase in plasma concentrations of tryptophan (p<0.005) and kynurenic acid (p<0.05) in the bifidobacteria-treated rats when compared to controls. Bifidobacteria treatment also resulted in a reduced 5-HIAA concentration in the frontal cortex and a decrease in DOPAC in the amygdaloid cortex. The attenuation of pro-inflammatory immune responses, and the elevation of the serotonergic precursor, tryptophan by bifidobacteria treatment, provides encouraging evidence in support of the proposition that this probiotic may possess antidepressant properties. However, these findings are preliminary and further investigation into the precise mechanisms involved, is warranted.

Sexually dimorphic effects of maternal separation stress on corticotrophin-releasing factor and vasopressin systems in the adult rat brain.

Neonatal maternal separation has been widely used to model the well-established causal relationship between stress in early life and the later development of depression. As corticotrophin-releasing factor (CRF) and vasopressin (AVP) have been implicated in depression, we aimed to determine the long-term effects of maternal separation stress on these neuropeptide systems, and also to explore whether these effects are gender-dependent. Immunohistochemical staining of CRF, AVP and c-Fos was used to assess whether these neuropeptide systems were affected following an acute swim stress in male and female maternally separated rats. There was an increase in CRF-immunoreactivity (IR) (p<0.05), and an increased co-localisation of c-Fos and CRF (p<0.05) following stress, in the paraventricular nucleus of the hypothalamus (PVN) of maternally separated female rats only. We found no differences in CRF in the hypothalamus of maternally separated and control male rats. However, male maternally separated rats exhibited decreases in AVP-IR in both the non-stressed and stressed groups relative to controls (p<0.001). These data provide further evidence of the involvement of the neuropeptides CRF and AVP in the long-term maladaptive effects of maternal separation stress in early life. The enhanced CRF response to stress in MS females relative to males suggests that maternal separation stress results in a more reactive neuroendocrinological stress system in females, than in males. Furthermore, the sexually dimorphic effects of maternal separation on these neuropeptides indicate that gender is an important factor influencing the trajectory of early life stress effects on CRF and AVP systems in the brain.

Altered cytokine profile, pain sensitivity, and stress responsivity in mice with co-disruption of the developmental genes Neuregulin-1×DISC1.

The complex genetic origins of many human disorders suggest that epistatic (gene×gene) interactions may contribute to a significant proportion of their heritability estimates and phenotypic heterogeneity. Simultaneous disruption of the developmental genes and schizophrenia risk factors Neuregulin-1 (NRG1) and Disrupted-in-schizophrenia 1 (DISC1) in mice has been shown to produce disease-relevant and domain-specific phenotypic profiles different from that observed following disruption of either gene alone. In the current study, anxiety and stress responsivity phenotypes in male and female mutant mice with simultaneous disruption of DISC1 and NRG1 were examined. NRG1×DISC1 mutant mice were generated and adult mice from each genotype were assessed for pain sensitivity (hot plate and tail flick tests), anxiety (light-dark box), and stress-induced hypothermia. Serum samples were assayed to measure circulating levels of pro-inflammatory cytokines. Mice with the NRG1 mutation, irrespective of DISC1 mutation, spent significantly more time in the light chamber, displayed increased core body temperature following acute stress, and decreased pain sensitivity. Basal serum levels of cytokines IL8, IL1ß and IL10 were decreased in NRG1 mutants. Mutation of DISC1, in the absence of epistatic interaction with NRG1, was associated with increased serum levels of IL1ß. Epistatic effects were evident for IL6, IL12 and TNFα. NRG1 mutation alters stress and pain responsivity, anxiety, and is associated with changes in basal cytokine levels. Epistasis resulting from synergistic NRG1 and DISC1 gene mutations altered pro-inflammatory cytokine levels relative to the effects of each of these genes individually, highlighting the importance of epistatic mechanisms in immune-related pathology.

Microbial regulation of hippocampal miRNA expression: Implications for transcription of kynurenine pathway enzymes.

Increasing evidence points to a functional role of the enteric microbiota in brain development, function and behaviour including the regulation of transcriptional activity in the hippocampus. Changes in CNS miRNA expression may reflect the colonisation status of the gut. Given the pivotal impact of miRNAs on gene expression, our study was based on the hypothesis that gene expression would also be altered in the germ-free state in the hippocampus. We measured miRNAs in the hippocampus of Germ free (GF), conventional (C) and Germ free colonised (exGF) Swiss Webster mice. miRNAs were selected for follow up based on significant differences in expression between groups according to sex and colonisation status. The expression of miR-294-5p was increased in male germ free animals and was normalised following colonisation. Targets of the differentially expressed miRNAs were over-represented in the kynurenine pathway. We show that the microbiota modulates the expression of miRNAs associated with kynurenine pathway metabolism and, demonstrate that the gut microbiota regulates the expression of kynurenine pathway genes in the hippocampus. We also show a sex-specific role for the microbiota in the regulation of miR-294-5p expression in the hippocampus. The gut microbiota plays an important role in modulating small RNAs that influence hippocampal gene expression, a process critical to hippocampal development.

Epistatic and Independent Effects on Schizophrenia-Related Phenotypes Following Co-disruption of the Risk Factors Neuregulin-1 × DISC1.

Few studies have addressed likely gene × gene (ie, epistatic) interactions in mediating risk for schizophrenia. Using a preclinical genetic approach, we investigated whether simultaneous disruption of the risk factors Neuregulin-1 (NRG1) and Disrupted-in-schizophrenia 1 (DISC1) would produce a disease-relevant phenotypic profile different from that observed following disruption to either gene alone. NRG1 heterozygotes exhibited hyperactivity and disruption to prepulse inhibition, both reversed by antipsychotic treatment, and accompanied by reduced striatal dopamine D2 receptor protein expression, impaired social cognition, and altered glutamatergic synaptic protein expression in selected brain areas. Single gene DISC1 mutants demonstrated a disruption in social cognition and nest-building, altered brain 5-hydroxytryptamine levels and hippocampal ErbB4 expression, and decreased cortical expression of the schizophrenia-associated microRNA miR-29b. Co-disruption of DISC1 and NRG1, indicative of epistasis, evoked an impairment in sociability and enhanced self-grooming, accompanied by changes in hypothalamic oxytocin/vasopressin gene expression. The findings indicate specific behavioral correlates and underlying cellular pathways downstream of main effects of DNA variation in the schizophrenia-associated genes NRG1 and DISC1.

Prenatal stress-induced alterations in major physiological systems correlate with gut microbiota composition in adulthood.

Early-life adverse experiences, including prenatal stress (PNS), are associated with a higher prevalence of neurodevelopmental, cardiovascular and metabolic disorders in affected offspring. Here, in a rat model of chronic PNS, we investigate the impact of late gestational stress on physiological outcomes in adulthood. Sprague-Dawley pregnant dams were subjected to repeated restraint stress from embryonic day 14 to day 20, and their male offspring were assessed at 4 months of age. PNS induced an exaggeration of the hypothalamic-pituitary-adrenal (HPA) axis response to stress, as well as an elevation of blood pressure and impairment of cognitive function. Altered respiratory control was also observed, as demonstrated by increased variability in basal respiratory frequency and abnormal frequency responses to both hypoxic and hypercapnic challenges. PNS also affected gastrointestinal neurodevelopment and function, as measured by a decrease in the innervation density of distal colon and an increase in the colonic secretory response to catecholaminergic stimulation. Finally, PNS induced long lasting alterations in the intestinal microbiota composition. 16S rRNA gene 454 pyrosequencing revealed a strong trend towards decreased numbers of bacteria in the Lactobacillus genus, accompanied by elevated abundance of the Oscillibacter, Anaerotruncus and Peptococcus genera in PNS animals. Strikingly, relative abundance of distinct bacteria genera significantly correlated with certain respiratory parameters and the responsiveness of the HPA axis to stress. Together, these findings provide novel evidence that PNS induces long-term maladaptive alterations in the gastrointestinal and respiratory systems, accompanied by hyper-responsiveness to stress and alterations in the gut microbiota.

Premature responding following bilateral stimulation of the rat subthalamic nucleus is amplitude and frequency dependent.

The subthalamic nucleus (STN), a key component of the basal ganglia circuitry, functions as an internal clock that regulates the correct sequence of movements in a motor response. The importance of the STN in motor function is evidenced by its involvement in Parkinson disease (PD). This nucleus has also been associated with the attentional and emotional aspects of motor behavior through its connections with the limbic and prefrontal areas of the brain. As lesions of the STN have been shown to increase premature responding in a serial reaction time task in rats, indicative of its involvement in cognitive performance, the present study aimed to investigate whether bilateral deep brain stimulation (DBS) of the STN, in non-lesioned rats, affects cognitive functions and whether these are dependent on certain stimulation parameters. Rats were trained in a choice reaction time task and implanted bilaterally with electrodes. Stimulation parameters (amplitude, frequency and pulse width) were varied during the test procedure, after which rats were sacrificed and the brains processed for histochemical staining. Results show no change in reaction times or motor times during stimulation. However, a linear decrease in premature responses was observed with decreasing amplitudes and at high frequencies only. These results are the first to demonstrate that bilateral STN HFS has a positive effect on cognition in freely moving rats. This latter result is in contrast to findings following lesions of the STN, and suggests that current strength and frequency of stimulation are parameters that are integral to the mediation of stimulation effects. Furthermore, the overall effects of DBS on neuronal cells cannot be classified simply as being "inhibitory" and evidently mediates its effects by more complex mechanisms than lesions of the same brain area.