Piotr Chmielarz

Inactivation of Glucocorticoid Receptor in Noradrenergic System Influences Anxiety- and Depressive-Like Behavior in Mice

The aim of this study was to investigate whether conditional inactivation of the glucocorticoid receptors (GRs) in noradrenergic neurons affects animal behavior in mice. Selective ablation of GRs in the noradrenergic system was achieved using the Cre/loxP approach. We crossed transgenic mice expressing the Cre recombinase under the dopamine beta-hydroxylase (DBH) promoter with animals harboring the floxed GR gene. The resulting GRDBHCre mutant mice exhibited no alterations in terms of normal cage behavior, weight gain, spatial memory or spontaneous locomotor activity, regardless of gender. To assess depressive- and anxiety-like behaviors we performed the Tail Suspension Test and the Light-Dark Box Test. While male mutant animals did not show any alternations in both tests, female GRDBHCre mutants displayed depressive- and anxiety-like behavior. Additionally, male GRDBHCre mice were exposed to chronic restraint stress but still exhibited immobility times and anxiety statuses similar to those of non-stressed animals while stressed control mice clearly revealed depressive- and anxiety-like phenotype. Thus, in males the effects of the mutation were precipitated only after chronic restraint stress procedure. Our data reveal a possible gender-dependent role of GRs in the noradrenergic system in anxiety- and depressive-like behavior in mice.

Acute and repeated treatment with the 5-HT7 receptor antagonist SB 269970 induces functional desensitization of 5-HT7 receptors in rat hippocampus

BACKGROUND SB 269970, a 5-HT(7) receptor antagonist may produce a faster antidepressant-like effect in animal models, than do antidepressant drugs, e.g., imipramine. The present work was aimed at examining the effect of single and repeated (14 days) administration of SB 269970 on the 5-HT(7) receptor in the hippocampus. METHODS The reactivity of 5-HT(7) receptors was determined using 5-carboxamidotryptamine (5-CT), which increased the bursting frequency of spontaneous epileptiform activity in hippocampal slices. Additionally, the effects of SB 269970 administration on the affinity and density of 5-HT(7) receptors were investigated using [(3)H]-SB 269970 and the influence of SB 269970 and imipramine on mRNA expression levels of Gα(s) and Gα(12) mRNA were studied using RT-qPCR. RESULTS Acute and repeated treatment with SB 269970 led to attenuation of the excitatory effects of activation of 5-HT(7) receptors. Neither single nor repeated administration of SB 269970 changed the mean affinity of 5-HT(7) receptors for [(3)H]-SB 269970. Repeated, but not single, administration of SB 269970 decreased the maximum density of [(3)H]-SB 269970 binding sites. While administration of imipramine did not change the expression of mRNAs for Gα(s) and Gα(12) proteins after both single and repeated administration of SB 269970, a reduction in Gα(s) and Gα(12) mRNA expression levels was evident. CONCLUSIONS These findings indicate that even single administration of SB269970 induces functional desensitization of the 5-HT(7) receptor system, which precedes changes in the receptor density. This mechanism may be responsible for the rapid antidepressant-like effect of the 5-HT(7) antagonist in animal models.

Gender differences in genetic mouse models evaluated for depressive-like and antidepressant behavior.

Depression is a mental disease that affects complex cognitive and emotional functions. It is believed that depression is twice as prevalent in women as in men. This phenomenon may influence the response to various antidepressant therapies, and these differences are still underestimated in clinical treatment. Nevertheless, most of the current findings are based on studies on male animal models, and relatively few of these studies take possible gender differences into consideration. Advancements in genetic engineering over the last two decades have introduced many transgenic lines that have been screened to study the pathomechanisms of depression. In this mini-review, we provide a compendious list of genetically altered mice that underwent tests for depressive-like or antidepressant behavior and determine if and how the gender factor was analyzed in their evaluation. Furthermore, we compile the gender differences in response to antidepressant treatment. On the basis of these analyses, we conclude that in many cases, gender variability is neglected or not taken into consideration in the presented results. We note the necessity of discussing this issue in the phenotypic characterization of transgenic mice, which seems to be particularly important while modeling mental diseases.

Dicer and microRNAs protect adult dopamine neurons

MicroRNAs (miRs) are important post-transcriptional regulators of gene expression implicated in neuronal development, differentiation, aging and neurodegenerative diseases, including Parkinson’s disease (PD). Several miRs have been linked to PD-associated genes, apoptosis and stress response pathways, suggesting that deregulation of miRs may contribute to the development of the neurodegenerative phenotype. Here, we investigate the cell-autonomous role of miR processing RNAse Dicer in the functional maintenance of adult dopamine (DA) neurons. We demonstrate a reduction of Dicer in the ventral midbrain and altered miR expression profiles in laser-microdissected DA neurons of aged mice. Using a mouse line expressing tamoxifen-inducible CreERT2 recombinase under control of the DA transporter promoter, we show that a tissue-specific conditional ablation of Dicer in DA neurons of adult mice led to decreased levels of striatal DA and its metabolites without a reduction in neuronal body numbers in hemizygous mice (DicerHET) and to progressive loss of DA neurons with severe locomotor deficits in nullizygous mice (DicerCKO). Moreover, we show that pharmacological stimulation of miR biosynthesis promoted survival of cultured DA neurons and reduced their vulnerability to thapsigargin-induced endoplasmic reticulum stress. Our data demonstrate that Dicer is crucial for maintenance of adult DA neurons, whereas a stimulation of miR production can promote neuronal survival, which may have direct implications for PD treatment.

Disruption of glucocorticoid receptors in the noradrenergic system leads to BDNF up-regulation and altered serotonergic transmission associated with a depressive-like phenotype in female GRDBHCre mice

Recently, we have demonstrated that conditional inactivation of glucocorticoid receptors (GRs) in the noradrenergic system, may evoke depressive-like behavior in female but not male mutant mice (GR(DBHCre) mice). The aim of the current study was to dissect how selective ablation of glucocorticoid signaling in the noradrenergic system influences the previously reported depressive-like phenotype and whether it might be linked to neurotrophic alterations or secondary changes in the serotonergic system. We demonstrated that selective depletion of GRs enhances brain derived neurotrophic factor (BDNF) expression in female but not male GR(DBHCre) mice on both the mRNA and protein levels. The possible impact of the mutation on brain noradrenergic and serotonergic systems was addressed by investigating the tissue neurotransmitter levels under basal conditions and after acute restraint stress. The findings indicated a stress-provoked differential response in tissue noradrenaline content in the GR(DBHCre) female but not male mutant mice. An analogous gender-specific effect was identified in the diminished content of 5-hydroxyindoleacetic acid, the main metabolite of serotonin, in the prefrontal cortex, which suggests down-regulation of this monoamine system in female GR(DBHCre) mice. The lack of GR also resulted in an up-regulation of alpha2-adrenergic receptor (α2-AR) density in the female but not male mutants in the locus coeruleus. We have also confirmed the utility of the investigated model in pharmacological studies, which demonstrates that the depressive-like phenotype of GR(DBHCre) female mice can be reversed by antidepressant treatment with desipramine or fluoxetine, with the latter drug evoking more pronounced effects. Overall, our study validates the use of female GR(DBHCre) mice as an interesting and novel genetic tool for the investigation of the cross-connected mechanisms of depression that is not only based on behavioral phenotypes.

Depressive-like immobility behavior and genotype × stress interactions in male mice of selected strains

Abstract In this study, we investigated whether basal immobility time of C57BL/6J mice, which are commonly used in transgenesis, interferes with detection of depressive-like behavior in the tail suspension test (TST) after chronic restraint stress (CRS). We included in the study mice of the C57BL/6N strain, not previously compared with C57BL/6J for behavior in the TST, and contrasted both strains with NMRI mice which exhibit low basal immobility. NMRI, C57BL/6J, and C57BL/6N male mice (n = 20 per strain) were tested under basal conditions and after CRS (2 h daily for 14 d). NMRI and C57BL/6J mice were differentiated in the TST by low and high basal immobility times, respectively, while the C57BL/6N and NMRI mice showed similar levels of basal immobility. CRSextended the immobility time of NMRI mice in the TST, whereas both C57BL/6J and C57BL/6N mice were unaffected regardless of their initial phenotype. We explored whether detailed analysis of activity microstructure revealed effects of CRS in the TST, which are not apparent in the overall comparison of total immobility time. Interestingly, unlike C57BL/6J and/6N strains which showed no sensitivity to CRS, stressed NRMI mice displayed distinct activity microstructure. In contrast to behavioral differences, all stressed mice showed significant retardation in body weight gain, decreased thymus weight and increased adrenal cortex size. However, after CRS, enlargement of the adrenal medulla was observed in both C57BL/6J and C57BL/6N mice, suggesting similar sympatho-medullary activation and stress coping mechanism in these substrains.

Selective ablation of glucocorticoid receptors in the noradrenergic system affects evening corticosterone levels in a sex-dependent manner

BACKGROUND The hypothalamic-pituitary-adrenal (HPA) axis, which is involved in the release of corticosterone in response to stress, exhibits large circadian variations in its activity that can also be regulated by the noradrenergic system, thereby contributing to the pathophysiology of depression. We have recently shown that mice in which glucocorticoid receptors (GR) are selectively ablated in the noradrenergic system (GR(DBHCre) mice) exhibit sex-dependent phenotype alterations, manifested as increased anxiety- and depressive-like behaviors in female but not male mutants. METHODS In this study, we investigated the regulation of circadian HPA axis activity in GR(DBHCre) transgenic mice by measuring plasma corticosterone levels. RESULTS We found that evening plasma corticosterone increase was profoundly higher in females than males, and this diversification was further augmented in mutant GR(DBHCre) mice. CONCLUSIONS Our results provide evidence of the involvement of the noradrenergic system in the regulation of the sexually dimorphic circadian activity of the HPA axis.

Gender-dependent activity of CYP3A is indirectly modified by GR in the noradrenergic system

BACKGROUND The noradrenergic system is involved in the regulation of cytochrome P450 activity in the liver. We investigated the effect of selective ablation of the glucocorticoid receptor in the noradrenergic systemon the activity of the CYP3A isoform in mouse liver. METHODS The activity of CYP3A was studied by measuring the rate of testosterone 6β-hydroxylation in liver microsomes. RESULTS In mutant mice, the activity of CYP3A was reduced to 68% of the control in females, but remained unchanged in males. Chronic restraint stress increased CYP3A activity in mutant mice only. CONCLUSIONS The total basal activity of mouse CYP3A may be indirectly modulated by the glucocorticoid receptor in the noradrenergic system during a pubertal period.

Lack of riluzole efficacy in the progression of the neurodegenerative phenotype in a new conditional mouse model of striatal degeneration

Background Huntington’s disease (HD) is a rare familial autosomal dominant neurodegenerative disorder characterized by progressive degeneration of medium spiny neurons (MSNs) located in the striatum. Currently available treatments of HD are only limited to alleviating symptoms; therefore, high expectations for an effective therapy are associated with potential replacement of lost neurons through stimulation of postnatal neurogenesis. One of the drugs of potential interest for the treatment of HD is riluzole, which may act as a positive modulator of adult neurogenesis, promoting replacement of damaged MSNs. The aim of this study was to evaluate the effects of chronic riluzole treatment on a novel HD-like transgenic mouse model, based on the genetic ablation of the transcription factor TIF-IA. This model is characterized by selective and progressive degeneration of MSNs. Methods Selective ablation of TIF-IA in MSNs (TIF-IAD1RCre mice) was achieved by Cre-based recombination driven by the dopamine 1 receptor (D1R) promoter in the C57Bl/6N mouse strain. Riluzole was administered for 14 consecutive days (5 mg/kg, i.p.; 1× daily) starting at six weeks of age. Behavioral analysis included a motor coordination test performed on 13-week-old animals on an accelerated rotarod (4–40 r.p.m.; 5 min). To visualize the potential effects of riluzole treatment, the striata of the animals were stained by immunohistochemistry (IHC) and/or immunofluorescence (IF) with Ki67 (marker of proliferating cells), neuronal markers (NeuN, MAP2, DCX), and markers associated with neurodegeneration (GFAP, 8OHdG, FluoroJade C). Additionally, the morphology of dendritic spines of neurons was assessed by a commercially available FD Rapid Golgi Stain™ Kit. Results A comparative analysis of IHC staining patterns with chosen markers for the neurodegeneration process in MSNs did not show an effect of riluzole on delaying the progression of MSN cell death despite an observed enhancement of cell proliferation as visualized by the Ki67 marker. A lack of a riluzole effect was also reflected by the behavioral phenotype associated with MSN degeneration. Moreover, the analysis of dendritic spine morphology did not show differences between mutant and control animals. Discussion Despite the observed increase in newborn cells in the subventricular zone (SVZ) after riluzole administration, our study did not show any differences between riluzole-treated and non-treated mutants, revealing a similar extent of the neurodegenerative phenotype evaluated in 13-week-old TIF-IAD1RCre animals. This could be due to either the treatment paradigm (relatively low dose of riluzole used for this study) or the possibility that the effects were simply too weak to have any functional meaning. Nevertheless, this study is in line with others that question the effectiveness of riluzole in animal models and raise concerns about the utility of this drug due to its rather modest clinical efficacy.