Auto-qPCR; a python-based web app for automated and reproducible analysis of qPCR data.

Quantifying changes in DNA and RNA levels is essential in numerous molecular biology protocols. Quantitative real time PCR (qPCR) techniques have evolved to become commonplace, however, data analysis includes many time-consuming and cumbersome steps, which can lead to mistakes and misinterpretation of data. To address these bottlenecks, we have developed an open-source Python software to automate processing of result spreadsheets from qPCR machines, employing calculations usually performed manually. Auto-qPCR is a tool that saves time when computing qPCR data, helping to ensure reproducibility of qPCR experiment analyses. Our web-based app ( https://auto-q-pcr.com/ ) is easy to use and does not require programming knowledge or software installation. Using Auto-qPCR, we provide examples of data treatment, display and statistical analyses for four different data processing modes within one program: (1) DNA quantification to identify genomic deletion or duplication events; (2) assessment of gene expression levels using an absolute model, and relative quantification (3) with or (4) without a reference sample. Our open access Auto-qPCR software saves the time of manual data analysis and provides a more systematic workflow, minimizing the risk of errors. Our program constitutes a new tool that can be incorporated into bioinformatic and molecular biology pipelines in clinical and research labs.

Netrin G1: its downregulation in the nucleus accumbens of cocaine-conditioned mice and genetic association in human cocaine dependence.

Netrin G1 is a presynaptic ligand involved in axonal projection. Although molecular mechanisms underlying cocaine addiction are still poorly understood, Netrin G1 might have a role as a regulator of anxiety, fear and spatial memory, behavioural traits impaired in the context of cocaine exposure. In this study, the Netrin G1 (Ntng1) expression was investigated in the nucleus accumbens of mice primarily conditioned to cocaine using a place preference paradigm. A genetic association study was then conducted on 146 multiplex families of the Collaborative study on Genetics of Alcoholism, in which seven single nucleotide polymorphisms located in the NTNG1 gene were genotyped. NTNG1 expression levels were also quantified in BA10, BA46 and the cerebellum of healthy controls (with no Axis 1 psychopathology). Decreased Ntng1 expression was initially observed in the nucleus accumbens of mice conditioned to cocaine. Significant genetic family-based associations were detected between NTNG1 polymorphisms and cocaine dependence. NTNG1 expression in BA10, BA46 and the cerebellum, however, were not significantly associated with any allele or haplotype of this gene. These results confirm that Ntng1 expression is disturbed in the nucleus accumbens of mice, after cocaine conditioning. A haplotype of NTNG1 was found to constitute a vulnerability factor for cocaine use disorder in patients, although none of its single nucleotide polymorphisms were associated with a differential expression pattern in healthy controls. The data suggest that change in the Ntng1 expression is a consequence of cocaine exposure, and that some of its genetic markers are associated with a greater risk for cocaine use disorder.

Chronic voluntary ethanol intake hypersensitizes 5-HT(1A) autoreceptors in C57BL/6J mice.

Alcoholism is a complex disorder involving, among others, the serotoninergic (5-HT) system, mainly regulated by 5-HT(1A) autoreceptors in the dorsal raphe nucleus. 5-HT(1A) autoreceptor desensitization induced by chronic 5-HT reuptake inactivation has been associated with a decrease in ethanol intake in mice. We investigated here whether, conversely, chronic ethanol intake could induce 5-HT(1A) autoreceptor supersensitivity, thereby contributing to the maintenance of high ethanol consumption. C57BL/6J mice were subjected to a progressive ethanol intake procedure in a free-choice paradigm (3-10% ethanol versus tap water; 21 days) and 5-HT(1A) autoreceptor functional state was assessed using different approaches. Acute administration of the 5-HT(1A) receptor agonist ipsapirone decreased the rate of tryptophan hydroxylation in striatum, and this effect was significantly larger (+75%) in mice that drank ethanol than in those drinking water. Furthermore, ethanol intake produced both an increased potency (+45%) of ipsapirone to inhibit the firing of 5-HT neurons, and a raise (+35%) in 5-HT(1A) autoreceptor-mediated stimulation of [(35)S]GTP-gamma-S binding in the dorsal raphe nucleus. These data showed that chronic voluntary ethanol intake in C57BL/6J mice induced 5-HT(1A) autoreceptor supersensitivity, at the origin of a 5-HT neurotransmission deficit, which might be causally related to the addictive effects of ethanol intake.

Nrxn3 upregulation in the globus pallidus of mice developing cocaine addiction.

Dysfunctions affecting the connections of basal ganglia lead to major neurological and psychiatric disorders. We investigated levels of mRNA for three neurexins (Nrxn) and three neuroligins (Nlgn) in the globus pallidus, subthalamic nucleus, and substantia nigra, in control conditions and after short-term exposure to cocaine. The expression of Nrxn2beta and Nlgn3 in the substantia nigra and Nlgn1 in the subthalamic nucleus depended on genetic background. The development of short-term cocaine appetence induced an increase in Nrxn3beta expression in the globus pallidus. Human NRXN3 has recently been linked to several addictions. Thus, NRXN3 adhesion molecules may play an important role in the synaptic plasticity of neurons involved in the indirect pathways of basal ganglia, in which they regulate reward-related learning.

Life-long hippocampal neurogenesis: environmental, pharmacological and neurochemical modulations.

It is now well documented that active neurogenesis does exist throughout the life span in the brain of various species including human. Two discrete brain regions contain progenitor cells that are capable of differentiating into neurons or glia, the subventricular zone and the dentate gyrus of the hippocampal formation. Recent studies have shown that neurogenesis can be modulated by a variety of factors, including stress and neurohormones, growth factors, neurotransmitters, drugs of abuse, and also strokes and traumatic brain injuries. In particular, the hippocampal neurogenesis may play a role in neuroadaptation associated with pathologies, such as cognitive disorders and depression. The increased neurogenesis at sites of injury may represent an attempt by the central nervous system to regenerate after damage. We herein review the most significant data on hippocampal neurogenesis in brain under various pathological conditions, with a special attention to mood disorders including depression and addiction.

Cannabinoid-serotonin interactions in alcohol-preferring vs. alcohol-avoiding mice.

Because cannabinoid and serotonin (5-HT) systems have been proposed to play an important role in drug craving, we investigated whether cannabinoid 1 (CB1) and 5-HT(1A) receptor ligands could affect voluntary alcohol intake in two mouse strains, C57BL/6 J and DBA/2 J, with marked differences in native alcohol preference. When offered progressively (3-10% ethanol) in drinking water, in a free-choice procedure, alcohol intake was markedly lower (approximately 70%) in DBA/2 J than in C57BL/6 J mice. In DBA/2 J mice, chronic treatment with the cannabinoid receptor agonist WIN 55,212-2 increased alcohol intake. WIN 55,212-2 effect was prevented by concomitant, chronic CB1 receptor blockade by rimonabant or chronic 5-HT(1A) receptor stimulation by 8-hydroxy-2-(di-n-propylamino)-tetralin, which, on their own, did not affect alcohol intake. In C57BL/6 J mice, chronic treatment with WIN 55,212-2 had no effect but chronic CB1 receptor blockade or chronic 5-HT(1A) receptor stimulation significantly decreased alcohol intake. Parallel autoradiographic investigations showed that chronic treatment with WIN 55,212-2 significantly decreased 5-HT(1A)-mediated [35S]guanosine triphosphate-gamma-S binding in the hippocampus of both mouse strains. Conversely, chronic rimonabant increased this binding in C57BL/6 J mice. These results show that cannabinoid neurotransmission can exert a permissive control on alcohol intake, possibly through CB1-5-HT(1A) interactions. However, the differences between C57BL/6 J and DBA/2 J mice indicate that such modulations of alcohol intake are under genetic control.

Alcohol intake after serotonin transporter inactivation in mice.

Knock-out mice lacking the serotonin transporter [5-hydroxytryptamine transporter (5-HTT)] were used to assess the influence of 5-HT re-uptake on ethanol consumption. Under a free-choice paradigm, alcohol intake was lower in mutant than in wild-type mice, and pharmacological blockade of 5-HTT by fluoxetine reduced alcohol intake in wild-type mice only. These data confirm the inhibitory effect of 5-HTT inactivation on ethanol intake.