The optogenetic (r)evolution

Optogenetics is a rapidly evolving field of technology that allows optical control of genetically targeted biological systems at high temporal and spatial resolution. By heterologous expression of light-sensitive microbial membrane proteins, opsins, cell type-specific depolarization or silencing can be optically induced on a millisecond time scale. What started in a petri dish is applicable today to more complex systems, ranging from the dissection of brain circuitries in vitro to behavioral analyses in freely moving animals. Persistent technical improvement has focused on the identification of new opsins, suitable for optogenetic purposes and genetic engineering of existing ones. Optical stimulation can be combined with various readouts defined by the desired resolution of the experimental setup. Although recent developments in optogenetics have largely focused on neuroscience it has lately been extended to other targets, including stem cell research and regenerative medicine. Further development of optogenetic approaches will not only highly increase our insight into health and disease states but might also pave the way for a future use in therapeutic applications

Gene Expression Profiling Following Maternal Deprivation: Involvement of the Brain Renin-Angiotensin System

The postnatal development of the mouse is characterized by a stress hypo-responsive period (SHRP), where basal corticosterone levels are low and responsiveness to mild stressors is reduced. Maternal separation is able to disrupt the SHRP and is widely used to model early trauma. In this study we aimed at identifying of brain systems involved in acute and possible long-term effects of maternal separation. We conducted a microarray-based gene expression analysis in the hypothalamic paraventricular nucleus after maternal separation, which revealed 52 differentially regulated genes compared to undisturbed controls, among them are 37 up-regulated and 15 down-regulated genes. One of the prominently up-regulated genes, angiotensinogen, was validated using in-situ hybridization. Angiotensinogen is the precursor of angiotensin II, the main effector of the brain renin-angiotensin system (RAS), which is known to be involved in stress system modulation in adult animals. Using the selective angiotensin type I receptor [AT(1)] antagonist candesartan we found strong effects on CRH and GR mRNA expression in the brain and ACTH release following maternal separation. AT(1) receptor blockade appears to enhance central effects of maternal separation in the neonate, suggesting a suppressing function of brain RAS during the SHRP. Taken together, our results illustrate the molecular adaptations that occur in the paraventricular nucleus following maternal separation and contribute to identifying signaling cascades that control stress system activity in the neonate

Deducing corticotropin-releasing hormone receptor type 1 signaling networks from gene expression data by usage of genetic algorithms and graphical Gaussian models

<p>Abstract</p> <p>Background</p> <p>Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is a hallmark of complex and multifactorial psychiatric diseases such as anxiety and mood disorders. About 50-60% of patients with major depression show HPA axis dysfunction, i.e. hyperactivity and impaired negative feedback regulation. The neuropeptide corticotropin-releasing hormone (CRH) and its receptor type 1 (CRHR1) are key regulators of this neuroendocrine stress axis. Therefore, we analyzed CRH/CRHR1-dependent gene expression data obtained from the pituitary corticotrope cell line AtT-20, a well-established <it>in vitro </it>model for CRHR1-mediated signal transduction. To extract significantly regulated genes from a genome-wide microarray data set and to deduce underlying CRHR1-dependent signaling networks, we combined supervised and unsupervised algorithms.</p> <p>Results</p> <p>We present an efficient variable selection strategy by consecutively applying univariate as well as multivariate methods followed by graphical models. First, feature preselection was used to exclude genes not differentially regulated over time from the dataset. For multivariate variable selection a maximum likelihood (MLHD) discriminant function within GALGO, an R package based on a genetic algorithm (GA), was chosen. The topmost genes representing major nodes in the expression network were ranked to find highly separating candidate genes. By using groups of five genes (chromosome size) in the discriminant function and repeating the genetic algorithm separately four times we found eleven genes occurring at least in three of the top ranked result lists of the four repetitions. In addition, we compared the results of GA/MLHD with the alternative optimization algorithms greedy selection and simulated annealing as well as with the state-of-the-art method random forest. In every case we obtained a clear overlap of the selected genes independently confirming the results of MLHD in combination with a genetic algorithm.</p> <p>With two unsupervised algorithms, principal component analysis and graphical Gaussian models, putative interactions of the candidate genes were determined and reconstructed by literature mining. Differential regulation of six candidate genes was validated by qRT-PCR.</p> <p>Conclusions</p> <p>The combination of supervised and unsupervised algorithms in this study allowed extracting a small subset of meaningful candidate genes from the genome-wide expression data set. Thereby, variable selection using different optimization algorithms based on linear classifiers as well as the nonlinear random forest method resulted in congruent candidate genes. The calculated interacting network connecting these new target genes was bioinformatically mapped to known CRHR1-dependent signaling pathways. Additionally, the differential expression of the identified target genes was confirmed experimentally.</p

Forebrain CRF₁ modulates early-life stress-programmed cognitive deficits

Childhood traumatic events hamper the development of the hippocampus and impair declarative memory in susceptible individuals. Persistent elevations of hippocampal corticotropin-releasing factor (CRF), acting through CRF receptor 1 (CRF1), in experimental models of early-life stress have suggested a role for this endogenous stress hormone in the resulting structural modifications and cognitive dysfunction. However, direct testing of this possibility has been difficult. In the current study, we subjected conditional forebrain CRF1 knock-out (CRF1-CKO) mice to an impoverished postnatal environment and examined the role of forebrain CRF1 in the long-lasting effects of early-life stress on learning and memory. Early-life stress impaired spatial learning and memory in wild-type mice, and postnatal forebrain CRF overexpression reproduced these deleterious effects. Cognitive deficits in stressed wild-type mice were associated with disrupted long-term potentiation (LTP) and a reduced number of dendritic spines in area CA3 but not in CA1. Forebrain CRF1 deficiency restored cognitive function, LTP and spine density in area CA3, and augmented CA1 LTP and spine density in stressed mice. In addition, early-life stress differentially regulated the amount of hippocampal excitatory and inhibitory synapses in wild-type and CRF1-CKO mice, accompanied by alterations in the neurexin-neuroligin complex. These data suggest that the functional, structural and molecular changes evoked by early-life stress are at least partly dependent on persistent forebrain CRF1 signaling, providing a molecular target for the prevention of cognitive deficits in adults with a history of early-life adversity

Co-Expression of Wild-Type P2X7R with Gln460Arg Variant Alters Receptor Function

The P2X7 receptor is a member of the P2X family of ligand-gated ion channels. A single-nucleotide polymorphism leading to a glutamine (Gln) by arginine (Arg) substitution at codon 460 of the purinergic P2X7 receptor (P2X7R) has been associated with mood disorders. No change in function (loss or gain) has been described for this SNP so far. Here we show that although the P2X7R-Gln460Arg variant per se is not compromised in its function, co-expression of wild-type P2X7R with P2X7R-Gln460Arg impairs receptor function with respect to calcium influx, channel currents and intracellular signaling in vitro. Moreover, co-immunoprecipitation and FRET studies show that the P2X7R-Gln460Arg variant physically interacts with P2X7R-WT. Specific silencing of either the normal or polymorphic variant rescues the heterozygous loss of function phenotype and restores normal function. The described loss of function due to co-expression, unique for mutations in the P2RX7 gene so far, explains the mechanism by which the P2X7R-Gln460Arg variant affects the normal function of the channel and may represent a mechanism of action for other mutations.Fil: Aprile García, Fernando. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Metzger, Michael W.. Max Planck Institute of Psychiatry; AlemaniaFil: Paez Pereda, Marcelo. Max Planck Institute of Psychiatry; AlemaniaFil: Stadler, Herbert. Affectis Pharmaceuticals; AlemaniaFil: Acuña, Matias. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Liberman, Ana Clara. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Senin, Sergio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Gerez, Juan Atilio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; ArgentinaFil: Hoijman, Esteban. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Centro de Microscopías Avanzadas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Refojo, Damian. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mitkovski, Mišo. Max Planck Institute of Experimental Medicine; AlemaniaFil: Panhuysen, Markus. Affectis Pharmaceuticals; AlemaniaFil: Stühmer, Walter. Max Planck Institute of Experimental Medicine; AlemaniaFil: Holsboer, Florian. Max Planck Institute of Psychiatry; Alemania. HMNC Brain Health; AlemaniaFil: Deussing, Jan M.. Max Planck Institute of Psychiatry; AlemaniaFil: Arzt, Eduardo Simon. Max Planck Institute of Psychiatry; Alemania. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigación en Biomedicina de Buenos Aires - Instituto Partner de la Sociedad Max Planck; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

Differential CRH expression level determines efficiency of Cre- and Flp-dependent recombination

Corticotropin-releasing hormone expressing (CRH+) neurons are distributed throughout the brain and play a crucial role in shaping the stress responses. Mouse models expressing site-specific recombinases (SSRs) or reporter genes are important tools providing genetic access to defined cell types and have been widely used to address CRH+ neurons and connected brain circuits. Here, we investigated a recently generated CRH-FlpO driver line expanding the CRH system-related tool box. We directly compared it to a previously established and widely used CRH-Cre line with respect to the FlpO expression pattern and recombination efficiency. In the brain, FlpO mRNA distribution fully recapitulates the expression pattern of endogenous Crh. Combining both Crh locus driven SSRs driver lines with appropriate reporters revealed an overall coherence of respective spatial patterns of reporter gene activation validating CRH-FlpO mice as a valuable tool complementing existing CRH-Cre and reporter lines. However, a substantially lower number of reporter-expressing neurons was discerned in CRH-FlpO mice. Using an additional CRH reporter mouse line (CRH-Venus) and a mouse line allowing for conversion of Cre into FlpO activity (CAG-LSL-FlpO) in combination with intersectional and subtractive mouse genetic approaches, we were able to demonstrate that the reduced number of tdTomato reporter expressing CRH+ neurons can be ascribed to the lower recombination efficiency of FlpO compared to Cre recombinase. This discrepancy particularly manifests under conditions of low CRH expression and can be overcome by utilizing homozygous CRH-FlpO mice. These findings have direct experimental implications which have to be carefully considered when targeting CRH+ neurons using CRH-FlpO mice. However, the lower FlpO-dependent recombination efficiency also entails advantages as it provides a broader dynamic range of expression allowing for the visualization of cells showing stress-induced CRH expression which is not detectable in highly sensitive CRH-Cre mice as Cre-mediated recombination has largely been completed in all cells generally possessing the capacity to express CRH. These findings underscore the importance of a comprehensive evaluation of novel SSR driver lines prior to their application

MAPK Signaling Determines Anxiety in the Juvenile Mouse Brain but Depression-Like Behavior in Adults

MAP kinase signaling has been implicated in brain development, long-term memory, and the response to antidepressants. Inducible Braf knockout mice, which exhibit protein depletion in principle forebrain neurons, enabled us to unravel a new role of neuronal MAPK signaling for emotional behavior. Braf mice that were induced during adulthood showed normal anxiety but increased depression-like behavior, in accordance with pharmacological findings. In contrast, the inducible or constitutive inactivation of Braf in the juvenile brain leads to normal depression-like behavior but decreased anxiety in adults. In juvenile, constitutive mutants we found no alteration of GABAergic neurotransmission but reduced neuronal arborization in the dentate gyrus. Analysis of gene expression in the hippocampus revealed nine downregulated MAPK target genes that represent candidates to cause the mutant phenotype

Forebrain CRF₁ modulates early-life stress-programmed cognitive deficits.

Childhood traumatic events hamper the development of the hippocampus and impair declarative memory in susceptible individuals. Persistent elevations of hippocampal corticotropin-releasing factor (CRF), acting through CRF receptor 1 (CRF₁), in experimental models of early-life stress have suggested a role for this endogenous stress hormone in the resulting structural modifications and cognitive dysfunction. However, direct testing of this possibility has been difficult. In the current study, we subjected conditional forebrain CRF₁ knock-out (CRF₁-CKO) mice to an impoverished postnatal environment and examined the role of forebrain CRF₁ in the long-lasting effects of early-life stress on learning and memory. Early-life stress impaired spatial learning and memory in wild-type mice, and postnatal forebrain CRF overexpression reproduced these deleterious effects. Cognitive deficits in stressed wild-type mice were associated with disrupted long-term potentiation (LTP) and a reduced number of dendritic spines in area CA3 but not in CA1. Forebrain CRF₁ deficiency restored cognitive function, LTP and spine density in area CA3, and augmented CA1 LTP and spine density in stressed mice. In addition, early-life stress differentially regulated the amount of hippocampal excitatory and inhibitory synapses in wild-type and CRF₁-CKO mice, accompanied by alterations in the neurexin-neuroligin complex. These data suggest that the functional, structural and molecular changes evoked by early-life stress are at least partly dependent on persistent forebrain CRF₁ signaling, providing a molecular target for the prevention of cognitive deficits in adults with a history of early-life adversity