Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section

Dopaminergic control of ADAMTS2 expression through cAMP/CREB and ERK: molecular effects of antipsychotics

A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at first episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we confirmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efficacy.Acknowledgements: We are highly indebted to the participants and their families for their cooperation in this study. We also thank IDIVAL biobank (Inés Santiuste and Jana Arozamena) for clinical samples and data as well as the PAFIP members (Marga Corredera) for the data collection. This work was supported by: SAF2016-76046-R and SAF2013-46292-R (MINECO and FEDER) to B.C.F., PI16/00156 (isciii and FEDER) to J.P.V., LUCHAMOS POR LA VIDA project to F.R.J. and J.P.V., SAF2017-83702-R (MINECO and FEDER), Red TERCEL RD12/0019/0024 (ISCIII) and GVA-PROMETEO 2018/041 (Generalitat Valenciana) to S.M. J.P.V. is supported by the RyC research programme (RYC-2013-14097) and F.R.J. by the predoctoral research programme (BES-2014-070615), from MINECO and FEDER

Metabotropic Glutamate Receptor 2 and Dopamine Receptor 2 Gene Expression Predict Sensorimotor Gating Response in the Genetically Heterogeneous NIH-HS Rat Strain

Altres ajuts: Foundation for the National Institutes of Health, R01MH084894Disruption of sensorimotor gating causes "flooding" of irrelevant sensory input and is considered a congenital trait in several neurodevelopmental disorders. Prepulse inhibition of acoustic startle response (PPI) is the operational measurement and has a high translational validity. Pharmacological studies in rodents have linked alterations in serotonin, dopamine and glutamate signalling to PPI disruption. How PPI response is associated with gene expression levels of these receptors is unknown. PPI response was assessed in 39 genetically heterogeneous National Institutes of Health-Heterogeneous Stock (NIH-HS) rats. Animals were classified as high, medium or low PPI. Expression levels of glutamate metabotropic receptor 2 (Grm2), dopamine receptor D2 (Drd2), dopamine receptor D1 (Drd1), serotonin receptor 1A (Htr1a), serotonin receptor 2A (Htr2a) and homer scaffolding protein 1 (Homer1) were investigated in prefrontal cortex (PFC) and striatum (STR). When comparing the two extreme phenotypes, only Drd2 in STR showed increased expression in the low PPI group. A multinomial model fitting all genes and all groups indicated that Grm2 in PFC, and Grm2 and Drd2 in the STR predicted PPI group. This was corroborated by a linear relationship of Grm2 with PPI in PFC, and Drd2 with PPI in STR. An interaction between levels of H3K27 trimethylation, associated with transcriptional repression, and PPI phenotype was observed for Drd2 in STR. Gene set enrichment analysis on a microarray dataset on Lewis rats confirmed enrichment of Drd2 in PFC in relation to PPI. These findings contribute to the understanding of the genetic substrate behind alterations in sensorimotor gating, relevant for its linkage to neurodevelopmental disorders

Increased thin-spine density in frontal cortex pyramidal neurons in a genetic rat model of schizophrenia-relevant features

The cellular mechanisms altered during brain wiring leading to cognitive disturbances in neurodevelopmental disorders remain unknown. We have previously reported altered cortical expression of neurodevelopmentally regulated synaptic markers in a genetic animal model of schizophrenia-relevant behavioral features, the Roman-High Avoidance rat strain (RHA-I). To further explore this phenotype, we looked at dendritic spines in cortical pyramidal neurons, as changes in spine density and morphology are one of the main processes taking place during adolescence. An HSV-viral vector carrying green fluorescent protein (GFP) was injected into the frontal cortex (FC) of a group of 11 RHA-I and 12 Roman-Low Avoidance (RLA-I) male rats. GFP labeled dendrites from pyramidal cells were 3D reconstructed and number and types of spines quantified. We observed an increased spine density in the RHA-I, corresponding to a larger fraction of immature thin spines, with no differences in stubby and mushroom spines. Glia cells, parvalbumin (PV) and somatostatin (SST) interneurons and surrounding perineuronal net (PNN) density are known to participate in FC and pyramidal neuron dendritic spine maturation. We determined by stereological-based quantification a significantly higher number of GFAP-positive astrocytes in the FC of the RHA-I strain, with no difference in microglia (Iba1-positive cells). The number of inhibitory PV, SST interneurons or PNN density, on the contrary, was unchanged. Results support our belief that the RHA-I strain presents a more immature FC, with some structural features like those observed during adolescence, adding construct validity to this strain as a genetic behavioral model of neurodevelopmental disorders