Reduced anxiety‐like behavior and central neurochemical change in germ‐free mice

Background There is increasing interest in the gut‐brain axis and the role intestinal microbiota may play in communication between these two systems. Acquisition of intestinal microbiota in the immediate postnatal period has a defining impact on the development and function of the gastrointestinal, immune, neuroendocrine and metabolic systems. For example, the presence of gut microbiota regulates the set point for hypothalamic‐pituitary‐adrenal (HPA) axis activity. Methods We investigated basal behavior of adult germ‐free (GF), Swiss Webster female mice in the elevated plus maze (EPM) and compared this to conventionally reared specific pathogen free (SPF) mice. Additionally, we measured brain mRNA expression of genes implicated in anxiety and stress‐reactivity. Key Results Germ‐free mice, compared to SPF mice, exhibited basal behavior in the EPM that can be interpreted as anxiolytic. Altered GF behavior was accompanied by a decrease in the N‐methyl‐D‐aspartate receptor subunit NR2B mRNA expression in the central amygdala, increased brain‐derived neurotrophic factor expression and decreased serotonin receptor 1A (5HT1A) expression in the dentate granule layer of the hippocampus. Conclusions & Inferences We conclude that the presence or absence of conventional intestinal microbiota influences the development of behavior, and is accompanied by neurochemical changes in the brain.     

Amygdalar expression of the microRNA miR‐101a and its target Ezh2 contribute to rodent anxiety‐like behaviour

A greater understanding of neural mechanisms contributing to anxiety is needed in order to develop better therapeutic interventions. This study interrogates a novel molecular mechanism that shapes anxiety‐like behaviour, demonstrating that the microRNA miR‐101a‐3p and its target, enhancer of zeste homolog 2 (Ezh2) in the amygdala, contribute to rodent anxiety‐like behaviour. We utilized rats that were selectively bred for differences in emotionality and stress reactivity, showing that high‐novelty‐responding (HR) rats, which display low trait anxiety, have lower miR‐101a‐3p levels in the amygdala compared to low‐novelty‐responding (LR) rats that characteristically display high trait anxiety. To determine whether there is a causal relationship between amygdalar miR‐101a‐3p and anxiety behaviour, we used a viral approach to overexpress miR‐101a‐3p in the amygdala of HR rats and test whether it would increase their typically low levels of anxiety‐like behaviour. We found that increasing miR‐101a‐3p in the amygdala increased HRs' anxiety‐like behaviour in the open‐field test and elevated plus maze. Viral‐mediated miR‐101a‐3p overexpression also reduced expression of the histone methyltransferase Ezh2, which mediates gene silencing via trimethylation of histone 3 at lysine 27 (H3K27me3). Knockdown of Ezh2 with short‐interfering RNA (siRNA) also increased HRs' anxiety‐like behaviour, but to a lesser degree than miR‐101a‐3p overexpression. Overall, our data demonstrate that increasing miR‐101a‐3p expression in the amygdala increases anxiety‐like behaviour and that this effect is at least partially mediated via repression of Ezh2. This work adds to the growing body of evidence implicating miRNAs and epigenetic regulation as molecular mediators of anxiety behaviour.     

Overexpression of immediate early gene X‐1 (IEX‐1) enhances γ‐radiation‐induced apoptosis of human glioma cell line,U87‐MG

Malignant gliomas are usually incurable even if adjuvant therapy is delivered after neurosurgical treatment. Therefore, to enhance their radiation‐induced apoptosis, it is important to detect the mechanism(s) leading to the death of malignant glioma cells. We report that apoptosis was induced in a time‐dependent manner after γ‐radiation and that irradiated U87‐MG cells (human glioblastoma cell line) expressed immediate early gene X‐1 (IEX‐1) with p53. We also document that their apoptotic sensitivity to γ‐radiation was enhanced by the overexpression of IEX‐1. Our findings suggest that IEX‐1 may represent a new factor for the enhancement of radiation‐induced apoptosis of human glioma cells.     

The role of Homer1c in metabotropic glutamate receptor‐dependent long‐term potentiation

Group I metabotropic glutamate receptors (mGluR1/5) play a role in synaptic plasticity and they demonstrate direct interactions with the neuronal Homer1c protein. We have previously shown that Homer1c can restore the plasticity deficits in Homer1 knockout mice (H1‐KO). Here, we investigated the role of Homer1c in mGluR‐dependent synaptic plasticity in wild‐type mice, H1‐KO, and H1‐KO mice overexpressing Homer1c (KO+H1c). We used a form of plasticity induced by activation of mGluR1/5 that transforms short‐term potentiaion (STP) induced by a subthreshold theta burst stimulation into long‐term potentiation (LTP). We have shown that although acute hippocampal slices from wild‐type animals can induce LTP using this stimulation protocol, H1‐KO only show STP. Gene delivery of Homer1c into the hippocampus of H1‐KO mice rescued LTP to wild‐type levels. This form of synaptic plasticity was dependent on mGluR5 but not mGluR1 activation both in wild‐type mice and in KO+H1c. mGluR1/5‐dependent LTP was blocked with inhibitors of the MEK‐ERK and PI3K‐mTOR pathways in KO+H1c mice. Moreover, blocking Homer1c–mGluR5 interactions prevented the maintenance of LTP in acute hippocampal slices from KO+H1c. These data indicate that Homer1c–mGluR5 interactions are necessary for mGluR‐dependent LTP, and that mGluR1/5‐dependent LTP involves PI3K and ERK activation. © 2013 Wiley Periodicals, Inc.     

Mice expressing the A53T mutant form of human alpha‐synuclein exhibit hyperactivity and reduced anxiety‐like behavior

Genetic mutations associated with α‐synuclein (α‐Syn) are implicated in the pathogenesis of Parkinson's disease (PD). PD is primarily a movement disorder, but patients are known to experience anxiety and other mood disorders. In this study, we examined the effect of the hA53T mutation during development by analyzing the protein expression of norepinephrine (NET), serotonin (SERT), and dopamine (DAT) transporters in addition to assessing locomotor and anxiety‐like behavior. We observed significant decreases in DAT expression at 8 months in transgenic animals compared with normal and younger mice. We used the elevated plus maze, open‐field test, and rotarod apparatus to evaluate wild‐type and hA53T hemizygous mice at 2, 8, and 12 months of age. Our results showed that 12‐month‐old transgenic mice spend more time in the open arms and display a greater number of open entries of the elevated plus maze compared with wild‐type controls and younger mice. Open‐field test results showed that 12‐month‐old mice travel a greater distance overall and travel more in the inner zone than either wild‐type or younger mice. Rotarod testing showed that 8‐ and 12‐month‐old transgenic mice perform better than either wild‐type controls or younger mice. Overall, 8–12‐month‐old transgenic mice showed a trend toward reduced anxiety‐like behavior and increased hyperactivity. These results indicate a possible role of the A53T α‐Syn mutation in anxiety‐like and hyperactive behaviors in a PD mouse model, suggesting that these behaviors might be comorbid with this disease. © 2010 Wiley‐Liss, Inc.     

Spatiotemporal expression of PSD‐95 in Fmr1 knockout mice brain

To investigate and compare the spatial and temporal expression of post‐synaptic density‐95 (PSD‐95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild‐type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD‐95 mRNA in the three brain areas was analyzed with quantitative RT‐PCR. PSD‐95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild‐type mice, the expression of PSD‐95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD‐95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P < 0.05, and P < 0.01, respectively). However, there was no significant difference of expression of PSD‐95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD‐95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD‐95 is possibly involved in hippocampal‐dependent learning defects, which are common in people with FXS.     

Plasticity‐related gene 1 is important for survival of neurons derived from rat neural stem cells

Plasticity‐related gene 1 (Prg1) is a membrane‐associated lipid phosphate phosphatase. In this study, we first investigated the role of Prg1 in the survival of neurons derived from rat neural stem cells (NSCs) using small interfering RNA (siRNA). Prg1 knock‐down decreased the cell number. Interestingly, Prg1 knock‐down increased genomic DNA fragmentation, suggesting the possible induction of apoptosis. Exogenously expressed Prg1 rescued the cells from death and restored the loss of 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) activity induced with Prg1 siRNA. However, exogenously expressed mutated‐Prg1 (the 253rd amino acid, histidine253, had been changed to alanine) did not rescue the cell death or restore the MTT activity. Histidine253 of Prg1 has been reported to be important for lipid phosphate phosphatase activity. These results suggest that Prg1 is important for survival of neurons through its dephosphorylation activity. © 2013 Wiley Periodicals, Inc.     

G protein‐coupled receptor 37‐like 1 modulates astrocyte glutamate transporters and neuronal NMDA receptors and is neuroprotective in ischemia

We show that the G protein‐coupled receptor GPR37‐like 1 (GPR37L1) is expressed in most astrocytes and some oligodendrocyte precursors in the mouse central nervous system. This contrasts with GPR37, which is mainly in mature oligodendrocytes. Comparison of wild type and Gpr37l1^–/– mice showed that loss of GPR37L1 did not affect the input resistance or resting potential of astrocytes or neurons in the hippocampus. However, GPR37L1‐mediated signalling inhibited astrocyte glutamate transporters and – surprisingly, given its lack of expression in neurons – reduced neuronal NMDA receptor (NMDAR) activity during prolonged activation of the receptors as occurs in ischemia. This effect on NMDAR signalling was not mediated by a change in the release of D‐serine or TNF‐α, two astrocyte‐derived agents known to modulate NMDAR function. After middle cerebral artery occlusion, Gpr37l1 expression was increased around the lesion. Neuronal death was increased by ～40% in Gpr37l1^–/– brain compared to wild type in an in vitro model of ischemia. Thus, GPR37L1 protects neurons during ischemia, presumably by modulating extracellular glutamate concentration and NMDAR activation.     

Effects of (R)‐(−)‐5‐methyl‐1‐nicotinoyl‐2‐pyrazoline on glutamate transporter 1 and cysteine/glutamate exchanger as well as ethanol drinking behavior in male,alcohol‐preferring rats

Alcohol consumption is largely associated with alterations in the extracellular glutamate concentrations in several brain reward regions. We recently showed that glutamate transporter 1 (GLT‐1) is downregulated following chronic exposure to ethanol for 5 weeks in alcohol‐preferring (P) rats and that upregulation of the GLT‐1 levels in nucleus accumbens and prefrontal cortex results, in part, in attenuating ethanol consumption. Cystine glutamate antiporter (xCT) is also downregulated after chronic ethanol exposure in P rats, and its upregulation could be valuable in attenuating ethanol drinking. This study examines the effect of a synthetic compound, (R)‐(?)‐5‐methyl‐1‐nicotinoyl‐2‐pyrazoline (MS‐153), on ethanol drinking and expressions of GLT‐1 and xCT in the amygdala and the hippocampus of P rats. P rats were exposed to continuous free‐choice access to water, 15% and 30% ethanol, and food for 5 weeks, after which they received treatments of MS‐153 or vehicle for 5 days. The results show that MS‐153 treatment significantly reduces ethanol consumption. It was revealed that GLT‐1 and xCT expressions were downregulated in both the amygdala and the hippocampus of ethanol–vehicle‐treated rats (ethanol–vehicle group) compared with water‐control animals. MS‐153 treatment upregulated GLT‐1 and xCT expressions in these brain regions. These findings demonstrate an important role for MS‐153 in these glutamate transporters for the attenuation of ethanol‐drinking behavior. © 2015 Wiley Periodicals, Inc.     

Serotonin transporter, 5‐HT1A receptor,and behavior in DBA/2J mice in comparison with four inbred mouse strains

Prepulse inhibition (PPI), the reduction in acoustic startle produced when it is preceded by a weak prepulse stimulus, is impaired in schizophrenic patients. The DBA/2J mouse strain displayed deficient PPI and is therefore suggested as an experimental animal model for the loss of sensorimotor gating in schizophrenia. Brain serotonin (5‐HT) has been implicated in the pathophysiology of several psychiatric disorders, including major depressive disorder and schizophrenia. In the present study, behavior, 5‐HT transporter (5‐HTT) mRNA level, 5‐HT_1A receptor mRNA level, and 5‐HT_1A receptor density in the brain regions were studied in DBA/2J mice in comparison with four inbred mouse strains (CBA/Lac, C57BL/6, BALB/c, and ICR). A decrease in 5‐HTT mRNA level in the midbrain and a reduced density of 5‐HT_1A receptors in the frontal cortex without significant changes in 5‐HT_1A receptor mRNA level in DBA/2J mice were found. It was shown that, along with decreased PPI, DBA/2J mice demonstrated considerably reduced immobility in the tail suspension test and in the forced swim test. No significant interstrain differences in intermale aggression, or in light‐dark box and elevated plus‐maze tests, were found. The results suggested the involvement of decreased 5‐HTT gene expression and 5‐HT_1A receptor density in genetically defined PPI deficiency and showed a lack of any association between PPI deficiency and predisposition to aggressive, anxiety, and depressive‐like behaviors. © 2009 Wiley‐Liss, Inc.     

The circadian gene Nr1d1 in the mouse nucleus accumbens modulates sociability and anxiety‐related behaviour

Nuclear receptor subfamily 1, group D, member 1 (Nr1d1) (also known as Rev‐erb alpha) has been linked to circadian rhythm regulation, mood‐related behaviour and disorders associated with social deficits. Recent work from our laboratory found striking decreases in Nr1d1 in the nucleus accumbens (NAc) in the maternal condition and indirect evidence that Nr1d1 was interacting with numerous addiction and reward‐related genes to modulate social reward. In this study, we applied our insights from the maternal state to nonparental adult mice to determine whether decreases in Nr1d1 expression in the NAc via adeno‐associated viral (AAV) vectors and short hairpin RNA (shRNA)‐mediated gene knockdown were sufficient to modulate social behaviours and mood‐related behaviours. Knockdown of Nr1d1 in the NAc enhanced sociability and reduced anxiety, but did not affect depressive‐like traits in female mice. In male mice, Nr1d1 knockdown had no significant behavioural effects. Microarray analysis of Nr1d1 knockdown in females identified changes in circadian rhythm and histone deacetylase genes and suggested possible drugs, including histone deacetylase inhibitors, that could mimic actions of Nr1d1 knockdown. Quantitative real‐time PCR (qPCR) analysis confirmed expression upregulation of gene period circadian clock 1 (Per1) and period circadian clock 2 (Per2) with Nr1d1 knockdown. The evidence for roles for opioid‐related genes opioid receptor, delta 1 (Oprd1) and preproenkephalin (Penk) was also found. Together, these results suggest that Nr1d1 in the NAc modulates sociability and anxiety‐related behaviour in a sex‐specific manner, and circadian, histone deacetylase and opioid‐related genes may be involved in the expression of these behavioural phenotypes.     

From gene to brain to behavior: schizophrenia‐associated variation in AMBRA1 alters impulsivity‐related traits

Recently, genome‐wide association between schizophrenia and an intronic variant in AMBRA1 (rs11819869) was reported. Additionally, in a reverse genetic approach in adult healthy subjects, risk allele carriers showed a higher medial prefrontal cortex blood oxygen level‐dependent (BOLD) response during a flanker task examining motor inhibition as an aspect of impulsivity. To test whether this finding can be expanded to further aspects of impulsivity, we analysed the effects of the rs11819869 genotype on impulsivity‐related traits on a behavioral, temperament and neural level in a large sample of healthy adolescents. We consider this reverse genetic approach specifically suited for use in a healthy adolescent sample, as these individuals comprise those who will eventually develop mental disorders in which impulsivity is implicated. Healthy adolescents from the IMAGEN study were included in the neuropsychological analysis (n = 848) and a functional magnetic resonance imaging (fMRI) task (n = 512). Various aspects of impulsivity were assessed using the Temperament and Character Inventory‐Revised, the Substance Use Risk Profile Scale, the Cambridge Cognition Neuropsychological Test Automated Battery, and the Stop Signal Task (SST) in the fMRI paradigm. On a behavioral level, increased delay aversion was observed in risk allele carriers. Furthermore, risk allele carriers showed a higher BOLD response in an orbito‐frontal target region during the SST, which declined to trend status after Family Wise Error correction. Our findings support the hypothesis that the schizophrenia‐related risk variant of rs11819869 is involved in various aspects of impulsivity, and that this involvement occurs on a behavioral as well as an imaging genetics level.     

Disturbance of approach‐avoidance behaviors in non‐human primates by stimulation of the limbic territories of basal ganglia and anterior insula

The basal ganglia (BG) are involved in motivation and goal‐directed behavior. Recent studies suggest that limbic territories of BG not only support reward seeking (appetitive approach) but also the encoding of aversive conditioned stimuli (CS) and the production of aversive‐related behaviors (avoidance or escape). This study aimed to identify inside two BG nuclei, the striatum and pallidum, the territories involved in aversive behaviors and to compare the effects of stimulating these territories to those resulting from stimulation of the anterior Insula (aIns), a region that is well‐known to be involved in aversive encoding and associated behaviors. Two monkeys performed an approach/avoidance task in which they had to choose a behavior (approach or avoidance) in an appetitive (reward) or aversive (air‐puff) context. During this task, either one (single‐cue) or two (dual‐cue) CS provided essential information about which context‐adapted behavior should be selected. Microstimulation was applied during the CS presentation. Stimulation generally reduced approaches in the appetitive contexts and increased escape behaviors (premature responses) and/or passive avoidance (noninitiated action) in aversive context. These effects were more pronounced in ventral parts of all examined structures, with significant differences observed between stimulated structures. Thresholds to induce effects were lowest in the pallidum. Striatal stimulation led to the largest diversity of effects, with a subregion even leading to enhanced active avoidance. Finally, aIns stimulations produced stronger effects in the dual‐cue context. These results provide causal evidence that limbic territories of BG, like aIns, play crucial roles in the selection of context‐motivated behaviors.     

Maternal separation changes maternal care,anxiety‐like behaviour and expression of paraventricular oxytocin and corticotrophin‐releasing factor immunoreactivity in lactating rats

The early postnatal period is a time of tremendous change for the dam and her offspring. During this time, environmental insults such as repeated stress exposure can have detrimental effects. In research that has focused on the effect of postnatal stress exposure on the dams, conflicting changes in maternal care and anxiety‐like behaviour have been reported. Additionally, changes to hypothalamic neuropeptides that are crucially involved in the transition to motherhood and stress regulation, namely oxytocin and corticotrophin‐releasing factor (CRF), have not been examined. Accordingly, the present study aimed to determine (i) whether repeated postpartum stress increases engagement in maternal care behaviours and anxiety‐like behaviour and (ii) whether these behavioural changes correspond with changes to CRF‐ or oxytocin‐immunoreactive (‐IR) cells in the paraventricular nucleus (PVN) of the hypothalamus. A non‐lactating group was also included to control for the effects of lactation on anxiety and the hypothalamic neuroendocrine system. Following the birth of their litters, Long‐Evans dams were separated from their pups from postnatal day (PND) 1 to PND21 for either 15 minutes (maternal separation [MS]15) or 6 hours (MS360). Maternal behaviours were recorded for 30 minutes on select PNDs following the separation. On PND22, dams were exposed to the elevated plus maze, brains were collected, and immunofluorescence analysis of PVN oxytocin‐ and CRF‐IR cells was conducted. Our findings demonstrate that prolonged maternal separation altered typical maternal behaviours and reduced anxiety relative to MS15 dams. At the cellular level, oxytocin‐IR cells in the caudal PVN were reduced in MS360 dams to a level similar to that in non‐lactating controls, and PVN CRF‐IR cells were reduced relative to both MS15 and non‐lactating controls. Taken together, these data reveal the behavioural and neuronal changes that occur in the mother dam following repeated postnatal stress exposure.     

Two successive phases in the threat‐related attentional response of anxious subjects: neural correlates

Background: It is well known that highly anxious individuals are characterized by the allocation of a greater amount of attentional resources to threatening stimulation. However, neural data in relation to the time course of attentional biases in anxiety are still surprisingly scarce and preliminary. The present research explored attentional biases in anxious subjects (grouped according to their scores in trait and state forms of the State–Trait Anxiety Inventory) over time by using event‐related potentials (ERPs) and through the application of source localization methodologies. Methods: Participants (n=27) were asked to perform an indirect visual matching task in a cue‐target paradigm. The targets consisted of three types of emotional pictures: positive arousing, negative arousing, and neutral. ERPs in response to target stimulation were submitted to temporal and spatial principal component analyses. Results: Statistical analyses revealed that negative targets elicited higher amplitudes than positive pictures in P200. Subsequently, greater amplitudes in response to positive than to negative pictures were observed in P500. Source analyses (standardized low resolution brain electro‐magnetic tomography: sLORETA) indicated an involvement of visual association cortical areas (i.e., precuneus and cuneus) both in P200 and P500. Conclusions: The results might be interpreted in line with the orienting‐avoidance hypothesis toward threatening events in anxious subjects. This attentional pattern was only manifested by individuals with high levels of both trait and state anxiety. Further investigation should be done in order to better understand the brain mechanisms underlying the attentional biases in anxiety and to apply this knowledge to the development of cognitive therapies. Depression and Anxiety, 2009. © 2009 Wiley‐Liss, Inc.     

Cannabinoid Modulation of Midbrain Urocortin 1 Neurones During Acute and Chronic Stress

Neurones in the centrally projecting Edinger–Westphal nucleus (EWcp) are the main site of urocortin 1 (Ucn1) synthesis in the mammalian brain, and are assumed to play a role in the stress response of the animal. Because endocannabinoid signalling has also been strongly implicated in stress, we hypothesised that endocannabinoids may modulate the functioning of the urocortinergic EWcp. First, using in situ hybridisation, we demonstrated cannabinoid receptor 1 (CB1R) mRNA expression in mouse EWcp‐neurones that were Ucn1‐negative. Dual‐ and triple‐label immunocytochemistry revealed the presence of CB1R in several GABA‐immunopositive fibres juxtaposed to EWcp‐Ucn1 neurones. To test functional aspects of such an anatomical constellation, we compared acute (1 h of restraint) and chronic (14 days of chronic mild stress) stress‐induced changes in wild‐type (WT) and CB1R knockout (CB1R‐KO) mice. Acute and especially chronic stress resulted in an increase in Ucn1 content of the EWcp, which was attenuated in CB1R‐KO mice. CB1R‐KO mice had higher basal and chronic stress‐induced adrenocorticotrophin and corticosterone levels and were more anxious on the elevated plus‐maze versus WT. Collectively, our results show for the first time EWcp‐Ucn1 neurones are putatively innervated by endocannabinoid sensitive, inhibitory, GABAergic afferents. In addition, we provide novel evidence that the absence of the CB1 receptor alters the Ucn1 mRNA and peptide levels in EWcp neurones, concomitant with an augmented stress response and increased anxiety‐like behaviour.