Twenty-Seven Tamoxifen-Inducible iCre-Driver Mouse Strains for Eye and Brain, Including Seventeen Carrying a New Inducible-First Constitutive-Ready Allele.

To understand gene function, the cre/loxP conditional system is the most powerful available for temporal and spatial control of expression in mouse. However, the research community requires more cre recombinase expressing transgenic mouse strains (cre-drivers) that restrict expression to specific cell types. To address these problems, a high-throughput method for large-scale production that produces high-quality results is necessary. Further, endogenous promoters need to be chosen that drive cell type specific expression, or we need to further focus the expression by manipulating the promoter. Here we test the suitability of using knock-ins at the docking site 5' of Hprt for rapid development of numerous cre-driver strains focused on expression in adulthood, using an improved cre tamoxifen inducible allele (icre/ERT2), and testing a novel inducible-first, constitutive-ready allele (icre/f3/ERT2/f3). In addition, we test two types of promoters either to capture an endogenous expression pattern (MaxiPromoters), or to restrict expression further using minimal promoter element(s) designed for expression in restricted cell types (MiniPromoters). We provide new cre-driver mouse strains with applicability for brain and eye research. In addition, we demonstrate the feasibility and applicability of using the locus 5' of Hprt for the rapid generation of substantial numbers of cre-driver strains. We also provide a new inducible-first constitutive-ready allele to further speed cre-driver generation. Finally, all these strains are available to the research community through The Jackson Laboratory.

Inferior olive CRF plays a role in motor performance under challenging conditions.

A well-coordinated stress response is pivotal for an organisms' survival. Corticotropin-releasing factor (CRF) is an essential component of the emotional and neuroendocrine stress response, however its role in cerebellar functions is poorly understood. Here, we explore the role of CRF in the inferior olive (IO) nucleus, which is a major source of input to the cerebellum. Using a CRF reporter line, in situ hybridization and immunohistochemistry, we demonstrate very high levels of the CRF neuropeptide expression throughout the IO sub-regions. By generating and characterizing IO-specific CRF knockdown and partial IO-CRF knockout, we demonstrate that reduction in IO-CRF levels is sufficient to induce motor deficiency under challenging conditions, irrespective of basal locomotion or anxiety-like behavior. Furthermore, we show that chronic social defeat stress induces a persistent decrease in IO-CRF levels, and that IO-CRF mRNA is upregulated shortly following stressful situations that demand a complex motor response. Taken together our results indicate a role for IO-CRF in challenge-induced motor responses.

Adrenal and Ovarian Phenotype of a Tissue-Specific Urocortin 2-Overexpressing Mouse Model.

Urocortin 2 (UCN2) is a neuropeptide of the CRH family, involved in homeostatic mechanisms, the stress response, and control of anxiety. To elucidate the effects of UCN2 on steroidogenesis, we developed a mouse model that allows a Cre recombinase-determined conditional overexpression of UCN2 (UCN2-COE). In these mice SF1-Cre-driven overexpression of UCN2 was restricted to the adrenal glands, gonads, and parts of the hypothalamus. UCN2-COE animals of both sexes revealed significantly higher plasma UCN2 levels and significantly higher UCN2 expression levels in the adrenals and ovaries. In contrast, the baseline expression of UCN2 was already high in the testes of control mice with no further increase achievable in UCN2-COE animals. Adrenal steroidogenesis of UCN2-COE animals was investigated under baseline conditions, upon an ACTH stimulation test, and following a restraint stress test. A tendency toward lower expression of steroidogenic enzymes was detectable in UCN2-COE animals of both sexes with slight differences between males and females. A similar reduction in the expression levels of the final steps of ovarian steroidogenesis, accompanied by reduced plasma estradiol levels, was observed in female UCN2-COE animals. Thus, adrenal UCN2 overexpression resulted in down-regulation of adrenal steroidogenesis, suggesting a reduction in the stress response in the mouse (stress coping behavior). Similarly, UCN2 overexpression in the ovaries caused a decrease in steroidogenesis and reduction of follicles that had undergone ovulation. Nevertheless, this finding was not associated with reduced fertility.

Corticotropin-releasing hormone regulates common target genes with divergent functions in corticotrope and neuronal cells.

As a key regulator of the neuroendocrine stress axis and as a neuromodulator in the brain, the neuropeptide corticotropin-releasing hormone (CRH) plays an important role in various diseases of the central nervous system. Its cognate receptor CRH receptor type 1 (CRHR1) is a potential novel target for the therapeutic intervention in major depressive disorder. Therefore, a more precise understanding of involved intracellular signaling mechanisms is essential. The objective of this project was to identify specific target genes of CRHR1-mediated signaling pathways in the corticotrope cell line AtT-20 and in the neuronal cell line HN9 using microarray technology and qRT-PCR, respectively. In addition, we assessed the capacity of validated target genes to directly impact on CRHR1-dependent signaling using reporter assays. Thereby, we identified a set of CRHR1 downstream targets with diverging and cell type-specific roles which strengthen the role of CRH and CRHR1 as dynamic modulators of a variety of signal transduction mechanisms and cellular processes.

Glutamatergic and dopaminergic neurons mediate anxiogenic and anxiolytic effects of CRHR1.

The corticotropin-releasing hormone receptor 1 (CRHR1) critically controls behavioral adaptation to stress and is causally linked to emotional disorders. Using neurochemical and genetic tools, we determined that CRHR1 is expressed in forebrain glutamatergic and γ-aminobutyric acid-containing (GABAergic) neurons as well as in midbrain dopaminergic neurons. Via specific CRHR1 deletions in glutamatergic, GABAergic, dopaminergic, and serotonergic cells, we found that the lack of CRHR1 in forebrain glutamatergic circuits reduces anxiety and impairs neurotransmission in the amygdala and hippocampus. Selective deletion of CRHR1 in midbrain dopaminergic neurons increases anxiety-like behavior and reduces dopamine release in the prefrontal cortex. These results define a bidirectional model for the role of CRHR1 in anxiety and suggest that an imbalance between CRHR1-controlled anxiogenic glutamatergic and anxiolytic dopaminergic systems might lead to emotional disorders.