The P2X7 receptor is an adenosine triphosphate (ATP)-gated ion channel expressed in different cell types of the brain. Polymorphisms in the P2RX7 gene have repeatedly been associated with psychiatric disorders including major depression. Depression is a stress-related disorder in which a dysregulation of the immune system has attracted increasing attention as a potential disease mechanism. The well-documented role of P2X7 in inflammatory conditions advocates its involvement in immune system dysregulation and depression genesis. However, understanding its exact role requires further research using appropriate animal models. Unfortunately, some of the most widely used P2X7 knockout mouse models are limited in their utility by the continuous expression of certain P2rx7 splice variants or even activation of de novo transcripts. To overcome this limitation, we generated a novel constitutive and complete P2X7 KO mouse line. These KO mice lack all known murine splice variants and protein expression resulting in a loss-of-function as confirmed by calcium imaging and by the inability of P2X7-deficient peritoneal macrophages to mount an appropriate interleukin (IL)-1β response. Comprehensive characterization using a battery of tests assessing locomotion, anxiety- and depression-related as well as social behaviour revealed differences in locomotor and exploratory behaviours. P2X7 KO mice showed slightly increased locomotor activity and reduced anxiety-related behaviour at baseline. Under conditions of chronic stress exposure, genotype-dependent differences largely dissolved while P2X7 deficiency promoted enhanced stress resilience with regard to social behaviour. Taken together, our findings add further evidence for an involvement of the P2X7 in shaping different behavioural responses and their modulation by stressful environments. This novel loss-of-function model will contribute to a better understanding of P2X7 in stress-associated behaviours in basic and translational neuropsychiatric research.

The purinergic P2X7 receptor (P2X7R) has attracted considerable interest as a potential target for various central nervous system (CNS) pathologies including affective and neurodegenerative disorders. To date, the distribution and cellular localization of the P2X7R in the brain are not fully resolved and a matter of debate mainly due to the limitations of existing tools. However, this knowledge should be a prerequisite for understanding the contribution of the P2X7R to brain disease. Here, we generated a genetic mouse model by humanizing the P2X7R in the mouse as mammalian model organism. We demonstrated its functionality and revealed species-specific characteristics of the humanized receptor, compared to the murine ortholog, regarding its receptivity to activation and modulation by 2′,3′-O-(benzoyl-4-benzoyl)-adenosine 5′-triphosphate (BzATP) and trifluoperazine (TFP). This humanized P2rx7 allele is accessible to spatially and temporally controlled Cre recombinase-mediated inactivation. In contrast to previously generated knockout (KO) mice, none of the described P2rx7 splice variants evade this null allele. By selective disruption and assessment of human P2RX7 expression in different brain regions and cell types, we were able to demonstrate that the P2X7R is specifically expressed in glutamatergic pyramidal neurons of the hippocampus. Also, P2X7R is expressed in major non-neuronal lineages throughout the brain, i.e., astrocytes, oligodendrocytes, and microglia. In conclusion, this humanized mouse model provides the means for detailed assessment of human P2X7R function in vivo including evaluation of agonists or antagonists. In addition, this conditional allele will enable future loss-of-function studies in conjunction with mouse models for CNS disorders.

A single nucleotide polymorphism substitution from glutamine (Gln, Q) to arginine (Arg, R) at codon 460 of the purinergic P2X7 receptor (P2X7R) has repeatedly been associated with mood disorders. The P2X7R-Gln460Arg variant per se is not compromised in its function. However, heterologous expression of P2X7R-Gln460Arg together with wild-type P2X7R has recently been demonstrated to impair receptor function. Here we show that this also applies to humanized mice coexpressing both human P2X7R variants. Primary hippocampal cells derived from heterozygous mice showed an attenuated calcium uptake upon agonist stimulation. While humanized mice were unaffected in their behavioral repertoire under basal housing conditions, mice that harbor both P2X7R variants showed alterations in their sleep quality resembling signs of a prodromal disease stage. Also healthy heterozygous human subjects showed mild changes in sleep parameters. These results indicate that heterozygosity for the wild-type P2X7R and its mood disorder-associated variant P2X7R-Gln460Arg represents a genetic risk factor, which is potentially able to convey susceptibility to mood disorders.SIGNIFICANCE STATEMENTDepression and bipolar disorder are the most common mood disorders. The P2X7 receptor (P2X7R) regulates many cellular functions. Its polymorphic variant Gln460Arg has repeatedly been associated with mood disorders. Genetically engineered mice, with human P2X7R, revealed that heterozygous mice (i.e., they coexpress the disease-associated Gln460Arg variant together with its normal version) have impaired receptor function and showed sleep disturbances. Human participants with the heterozygote genotype also had subtle alterations in their sleep profile. Our findings suggest that altered P2X7R function in heterozygote individuals disturbs sleep and might increase the risk for developing mood disorders.

In this study, regression analysis revealed that TOC is the principal factor in controlling the fate of organo-halogenated contaminants (OHCs: PCBs, PBDEs, OCPs) in Soan River, Pakistan. The OHCs(adsorbed TOC) burial flux (OHCs(adsorbed TOC)Bf; mg/cm2·yr) was calculated in the following ranges: ∑ PCBs (0.07–0.31), ∑ PBDEs (0.005–0.029), ∑ HCHs (0.015–0.046) and ∑ DDTs (0.007–0.039). Apart from OHCs(adsorbed TOC)Bf, the levels of OHCs were in the following order: PCBs > DDTs > PBDEs > HCH > Chlordane > HCB. PBDEs and PCB congener patterns showed following order respectively: BDE-149 > -153 > -18 > -138 > -44 and PCB-149 > -153 > -18 > -138 > -44. DDT isomers and metabolites' pattern were p,p′-DDT > p,p′-DDD > p,p′-DDE > o,p′-DDT > o,p′-DDD > o,p′-DDE and HCHs were β-HCH > α-HCH > γ-HCH > δ-HCH. PBDE composition had similarities to penta-BDE and DE-71 mixtures and PCBs with commercial products Aroclor-1254 and -1260. (DDE + DDD)/∑ DDTs and p,p′-DDT/p,p′-DDE suggested the recent input of DDTs in sediments while α/γ-HCH indicated past usage of lindane and technical mixtures. Risk assessment suggested that Soan River and its tributaries are potentially at risk against most of the OHCs.

A recent association study analyzing panic disorder identified single nucleotide polymorphisms (SNPs) in TMEM132D. In a mouse model of extremes in trait anxiety, anxiety-related behaviour was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex. Screening for sites and motifs suggests a role of TMEM132D in cell adhesion but until now its function is unclear. The aim of this study is to investigate the function of TMEM132D as a new candidate gene using in vitro and in vivo approaches. Expression of a TMEM132D-GFP fusion protein in HEK cells confirmed its predicted localization in the cellular membrane. Overexpression significantly increased the number of filopodia-like structures. Additionally, we observed a strong colocalization of TMEM132D with actin filaments as shown by phalloidin staining. The expression pattern of Tmem132d in the brain of wild-type animals at different postnatal stages and in adult mice was analyzed by in situ hybridization (ISH). Conditional knock-out (KO) mice are currently established using embryonic stem cell clones received from the German Genetrap Consortium (GGTC) based on a conditional gene trapping approach. Specific expression of the LacZ reporter gene was analyzed by X-Gal staining confirming the results obtained by ISH. Colocalization studies in these animals revealed expression of the LacZ protein with different neuronal markers. The primary characterization and behavioural analysis of KO mice is in progress.

Recent linkage and association studies suggest P2RX7 as a novel susceptibility gene for major depressive disorder (MDD) and bipolar disorder (BP). A non-synonymous SNP has been shown to be associated with reduced calcium influx and is likely to affect P2RX7 oligomerization and interaction. To study the functional relevance of the polymorphism in an appropriate in vivo model, we generated humanized mouse mutants in which the murine P2RX7 gene was substituted by the wild-type or the disease-associated isoform of human P2RX7. A knock-in approach based on homologous recombination in embryonic stem (ES) cells was used. ES cell clones were screened by Southern blot analysis. Positive clones were injected into blastocysts and chimeric animals were screened for germline transmission by PCR. In case of the mutant variant of human P2RX7 we obtained two correctly recombined ES cell clones and 24 chimeras in total. In terms of the wild-type variant we identified four positive clones which resulted in a total of 23 chimeras. In both cases the humanized allele was transmitted through the germline. The correct expression of the human P2RX7 variants in the brain of mutant mice was confirmed by ISH using a human-specific probe. Using these mouse lines, we will be able to functionally validate the human association data. Future analyses will involve molecular, endocrinological, electrophysiological and behavioural paradigms.

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

Conditional mouse mutants overexpressing corticototropin-releasing hormone (CRH) restricted to the central nervous system exhibit enhanced active stress-coping behavior. A highly flexible gain-of-function mouse model was created by combining the properties of the ubiquitously expressed ROSA26 locus with those of the Cre/loxP system. The knock-in of a Crh-LacZ expression unit, which is sensitive to activation by Cre recombinase, allows the spatio-temporally controlled overexpression of CRH at different dosages. In control mice (left), only endogenous CRH expression was detectable in the brain, whereas heterozygous (middle) and homozygous (right) CRH-COE-Nes mice expressed increasing levels of exogenous CRH throughout the brain. The pattern of CRH induction paralleled the activation of the simultaneously introduced LacZ reporter gene (left brain half). CRH-COE-Nes mice exhibited a marked gene-dosage-dependent increase in active stress-coping behavior as reflected by reduced immobility in the forced swim test (bottom), which depends on catecholaminergic transmission and enhanced activation of the locus coeruleus. For more information on this topic, please refer to article by Deussing et al. on pages 1028–1042.

The corticotropin-releasing hormone (CRH) plays a central role in the stress response. Besides its function in modulating a wide range of behaviors CRH is the major regulator of the hypothalamic-pituitary-adrenocortical (HPA) system. Chronically elevated CRH levels are implicated in the pathogenesis and maintenance of depression. To study the effects of central CRH hyperdrive we generated a mouse line overexpressing CRH in a spatio-temporally regulated fashion. Restricting CRH overexpression to the CNS enabled us to investigate the CNS effects of CRH without affecting the peripheral CRH system or the circadian HPA axis regulation under basal conditions. In contrast, under stress these mice showed a hyperactive HPA axis and an antidepressant-like behavior in respective screening paradigms, which is mediated via catecholamines. To investigate the molecular mechanisms underlying this phenotype we studied mRNA and protein expression levels. In situ hybridization was performed to reveal changes in transcription of genes related to the CRH system and the immediate early genes c-fos and zif-268, and protein expression of CRHR1 and CRHR2 was monitored via ligand binding autoradiography. Altogether, we found profound and brain region-specific alterations in gene expression of the analyzed targets in response to chronic CRH exposure as well as in response to stress. In conclusion our conditional CRH overexpressing mice have proven as a valuable tool for testing effects of CRH excess.