Differential stress induced c-Fos expression and identification of region-specific miRNA-mRNA networks in the dorsal raphe and amygdala of high-responder/low-responder rats.

Chronic stress triggers a variety of physical and mental health problems, and how individuals cope with stress influences risk for emotional disorders. To investigate molecular mechanisms underlying distinct stress coping styles, we utilized rats that were selectively-bred for differences in emotionality and stress reactivity. We show that high novelty responding (HR) rats readily bury a shock probe in the defensive burying test, a measure of proactive stress coping behavior, while low novelty responding (LR) rats exhibit enhanced immobility, a measure of reactive coping. Shock exposure in the defensive burying test elicited greater activation of HR rats' caudal dorsal raphe serotonergic cells compared to LRs, but lead to more pronounced activation throughout LRs' amygdala (lateral, basolateral, central, and basomedial nuclei) compared to HRs. RNA-sequencing revealed 271 mRNA transcripts and 33 microRNA species that were differentially expressed in HR/LR raphe and amygdala. We mapped potential microRNA-mRNA networks by correlating and clustering mRNA and microRNA expression and identified networks that differed in either the HR/LR dorsal raphe or amygdala. A dorsal raphe network linked three microRNAs which were down-regulated in LRs (miR-206-3p, miR-3559-5p, and miR-378a-3p) to repression of genes related to microglia and immune response (Cd74, Cyth4, Nckap1l, and Rac2), the genes themselves were up-regulated in LR dorsal raphe. In the amygdala, another network linked miR-124-5p, miR-146a-5p, miR-3068-3p, miR-380-5p, miR-539-3p, and miR-7a-1-3p with repression of chromatin remodeling-related genes (Cenpk, Cenpq, Itgb3bp, and Mis18a). Overall this work highlights potential drivers of gene-networks and downstream molecular pathways within the raphe and amygdala that contribute to individual differences in stress coping styles and stress vulnerabilities.

A2 noradrenergic neurons regulate forced swim test immobility.

The Wistar-Kyoto (WKY) rat is a widely used animal model of depression, which is characterized by dysregulation of noradrenergic signaling. We previously demonstrated that WKY rats show a unique behavioral profile on the forced swim test (FST), characterized by high levels of immobility upon initial exposure and a greater learning-like response by further increasing immobility upon re-exposure than the genetically related Wistar rats. In the current study we aimed to determine whether altered activation of brainstem noradrenergic cell groups contributes to this behavioral profile. We exposed WKY and Wistar rats, to either 5min of forced swim or to the standard two-day FST (i.e. 15min forced swim on Day 1, followed by 5min on Day 2). We then stained their brains for FOS/tyrosine hydroxylase double-immunocytochemistry to determine potential differences in the activation of the brainstem noradrenergic cell groups. We detected a relative hyperactivation in the locus coeruleus of WKY rats when compared to Wistars in response to both one- and two-day forced swim. In contrast, within the A2 noradrenergic cell group, WKY rats exhibited diminished levels of FOS across both days of the FST, suggesting their lesser activation. We followed up these observations by selectively lesioning the A2 neurons, using anti-dopamine-ß-hydroxylase-conjugated saporin, in Wistar rats, which resulted in increased FST immobility on both days of the test. Together these data indicate that the A2 noradrenergic cell group regulates FST behavior, and that its hypoactivation may contribute to the unique behavioral phenotype of WKY rats.

Novelty-seeking behavior predicts vulnerability in a rodent model of depression.

BACKGROUND: The onset of major depressive disorder is likely precipitated by a combination of heredity and life stress. The present study tested the hypothesis that rats selectively bred on a trait related to emotional reactivity would show differential susceptibility or resilience to the development of depression-like signs in response to chronic mild variable intermittent stress (CMS). METHODS: Male Sprague-Dawley rats that were bred based on the trait of either high or low locomotor activity in response to a novel environment were exposed to 4 weeks of CMS or control conditions. Changes in hedonic behavior were assessed using weekly sucrose preference tests and anxiety-like behavior was evaluated using the novelty-suppressed feeding test. RESULTS: During 4 weeks of CMS, bred low responder (bLR) rats became anhedonic at a faster rate and to a larger degree than bred high responder (bHR) rats, based on weekly sucrose preference tests. Measures of anxiety-like behavior in the novelty-suppressed feeding test were also significantly increased in the CMS-exposed bLR rats, though no differences were observed between CMS-exposed bHR rats and their unstressed controls. CONCLUSIONS: These findings present further evidence that increased emotional reactivity is an important factor in stress susceptibility and the etiology of mood disorders, and that bHR and bLR rats provide a model of resistance or vulnerability to stress-induced depression. Furthermore, exposing bHR and bLR rats to CMS provides an excellent way to study the interaction of genetic and environmental factors in the development of depression-like behavior.

Leptin-receptor-expressing neurons in the dorsomedial hypothalamus and median preoptic area regulate sympathetic brown adipose tissue circuits.

Brown adipose tissue (BAT) thermogenesis is critical to maintain homoeothermia and is centrally controlled via sympathetic outputs. Body temperature and BAT activity also impact energy expenditure, and obesity is commonly associated with decreased BAT capacity and sympathetic tone. Severely obese mice that lack leptin or its receptor (LepRb) show decreased BAT capacity, sympathetic tone, and body temperature and thus are unable to adapt to acute cold exposure (Trayhurn et al., 1976). LepRb-expressing neurons are found in several hypothalamic sites, including the dorsomedial hypothalamus (DMH) and median preoptic area (mPOA), both critical sites to regulate sympathetic, thermoregulatory BAT circuits. Specifically, a subpopulation in the DMH/dorsal hypothalamic area (DHA) is stimulated by fever-inducing endotoxins or cold exposure (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Using the retrograde, transsynaptic tracer pseudorabies virus (PRV) injected into the BAT of mice, we identified PRV-labeled LepRb neurons in the DMH/DHA and mPOA (and other sites), thus indicating their involvement in the regulation of sympathetic BAT circuits. Indeed, acute cold exposure induced c-Fos (as a surrogate for neuronal activity) in DMH/DHA LepRb neurons, and a large number of mPOA LepRb neurons project to the DMH/DHA. Furthermore, DMH/DHA LepRb neurons (and a subpopulation of LepRb mPOA neurons) project and synaptically couple to rostral raphe pallidus neurons, consistent with the current understanding of BAT thermoregulatory circuits from the DMH/DHA and mPOA (Dimicco and Zaretsky, 2007; Morrison et al., 2008). Thus, these data present strong evidence that LepRb neurons in the DMH/DHA and mPOA mediate thermoregulatory leptin action.

Evolutionary sequence modeling for discovery of peptide hormones.

There are currently a large number of "orphan" G-protein-coupled receptors (GPCRs) whose endogenous ligands (peptide hormones) are unknown. Identification of these peptide hormones is a difficult and important problem. We describe a computational framework that models spatial structure along the genomic sequence simultaneously with the temporal evolutionary path structure across species and show how such models can be used to discover new functional molecules, in particular peptide hormones, via cross-genomic sequence comparisons. The computational framework incorporates a priori high-level knowledge of structural and evolutionary constraints into a hierarchical grammar of evolutionary probabilistic models. This computational method was used for identifying novel prohormones and the processed peptide sites by producing sequence alignments across many species at the functional-element level. Experimental results with an initial implementation of the algorithm were used to identify potential prohormones by comparing the human and non-human proteins in the Swiss-Prot database of known annotated proteins. In this proof of concept, we identified 45 out of 54 prohormones with only 44 false positives. The comparison of known and hypothetical human and mouse proteins resulted in the identification of a novel putative prohormone with at least four potential neuropeptides. Finally, in order to validate the computational methodology, we present the basic molecular biological characterization of the novel putative peptide hormone, including its identification and regional localization in the brain. This species comparison, HMM-based computational approach succeeded in identifying a previously undiscovered neuropeptide from whole genome protein sequences. This novel putative peptide hormone is found in discreet brain regions as well as other organs. The success of this approach will have a great impact on our understanding of GPCRs and associated pathways and help to identify new targets for drug development.

Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression.

Laser capture microdissection (LCM) is increasingly being used in quantitative gene expression studies of the nervous system. The current study aimed at determining the impact of various tissue manipulations on the integrity of extracted RNA in LCM studies. Our data indicate that various tissue preparation strategies prior to microdissection may decrease RNA quality by as much as 25%, thus affecting expression profiles of some genes. To circumvent this problem, we developed a strategy for reverse transcriptase real-time PCR that has considerable sensitivity and can be used to calculate relative changes in gene expression. This approach was validated in subregions of the rat cerebellum. Accordingly, expression of glial gene markers - myelin-associated glycoprotein and proteolipid protein 1 - was found 70-160-fold higher in the white matter layer of the cerebellar cortex as compared to the neuron-enriched granular layer. In contrast, expression of a specific neuronal maker, neuron-specific enolase, was found seven-fold higher in the granular layer, as compared to the white matter layer. Furthermore, this approach had high sensitivity and specificity as we were able to detect a 38% decrease in the expression of neuron-specific enolase without a change in the expression of glial markers following administration of the neurotoxin, ibotenic acid. These results demonstrate feasibility of performing accurate semi-quantitative gene expression analyses in LCM samples.