Reference gene expression stability within the rat brain under mild intermittent ketosis induced by supplementation with medium-chain triglycerides.

Reverse transcription followed by quantitative (real-time) polymerase chain reaction (RT-qPCR) has become the gold standard in mRNA expression analysis. However, it requires an accurate choice of reference genes for adequate normalization. The aim of this study was to validate the reference genes for qPCR experiments in the brain of rats in the model of mild ketosis established through supplementation with medium-chain triglycerides (MCT) and intermittent fasting. This approach allows to reproduce certain neuroprotective effects of the classical ketogenic diet while avoiding its adverse effects. Ketogenic treatment targets multiple metabolic pathways, which may affect the reference gene expression. The standard chow of adult Wistar rats was supplemented with MCT (2 ml/kg orogastrically, during 6 h of fasting) or water (equivolume) for 1 month. The mRNA expression of 9 housekeeping genes (Actb, B2m, Gapdh, Hprt1, Pgk1, Ppia, Rpl13a, Sdha, Ywhaz) in the medial prefrontal cortex, dorsal and ventral hippocampus was measured by RT-qPCR. Using the RefFinder® online tool, we have found that the reference gene stability ranking strongly depended on the analyzed brain region. The most stably expressed reference genes were found to be Ppia, Actb, and Rpl13a in the medial prefrontal cortex; Rpl13a, Ywhaz, and Pgk1 in the dorsal hippocampus; Ywhaz, Sdha, and Ppia in the ventral hippocampus. The B2m was identified as an invalid reference gene in the ventral hippocampus, while Sdha, Actb, and Gapdh were unstable in the dorsal hippocampus. The stabilities of the examined reference genes were lower in the dorsal hippocampus compared to the ventral hippocampus and the medial prefrontal cortex. When normalized to the three most stably expressed reference genes, the Gapdh mRNA was upregulated, while the Sdha mRNA was downregulated in the medial prefrontal cortex of MCT-fed animals. Thus, the expression stability of reference genes strongly depends on the examined brain regions. The dorsal and ventral hippocampal areas differ in reference genes stability rankings, which should be taken into account in the RT-qPCR experimental design.

Neonatal Exposure to Bacterial Lipopolysaccharide Affects Behavior and Expression of Ionotropic Glutamate Receptors in the Hippocampus of Adult Rats after Psychogenic Trauma.

According to the two-hit hypothesis of psychoneuropathology formation, infectious diseases and other pathological conditions occurring during the critical periods of early ontogenesis disrupt normal brain development and increase its susceptibility to stress experienced in adolescence and adulthood. It is believed that these disorders are associated with changes in the functional activity of the glutamatergic system in the hippocampus. Here, we studied expression of NMDA (GluN1, GluN2a, GluN2b) and AMPA (GluA1, GluA2) glutamate receptor subunits, as well as glutamate transporter EAAT2, in the ventral and dorsal regions of the hippocampus of rats injected with LPS during the third postnatal week and then subjected to predator stress (contact with a python) in adulthood. The tests were performed 25 days after the stress. It was found that stress altered protein expression in the ventral, but not in the dorsal hippocampus. Non-stressed LPS-treated rats displayed lower levels of the GluN2b protein in the ventral hippocampus vs. control animals. Stress significantly increased the content of GluN2b in the LPS-treated rats, but not in the control animals. Stress also affected differently the exploratory behavior of LPS-injected and control rats. Compared to the non-stressed animals, stressed control rats demonstrated a higher locomotor activity during the 1st min of the open field test, while the stressed LPS-injected rats displayed lower locomotor activity than the non-stressed rats. In addition, LPS-treated stressed and non-stressed rats spent more time in the open arms of the elevated plus maze and demonstrated reduced blood levels of corticosterone. To summarize the results of our study, exposure to bacterial LPS in the early postnatal ontogenesis affects the pattern of stress-induced changes in the behavior and hippocampal expression of genes coding for ionotropic glutamate receptor subunits after psychogenic trauma suffered in adulthood.

Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples.

RT-qPCR requires an adequate choice of stably expressed reference genes for accurate normalization of mRNA expression. However, testing a panel of reference genes is often time-consuming and expensive. In this work, we aimed to develop a set of multiplex real-time PCR assays for RT-qPCR analysis of commonly used housekeeping genes in laboratory rats. Using Hydrolysis probe (TaqMan®) technology, we have designed and optimized three triplex qPCR assays (Actb + Gapdh + B2m; Rpl13a + Sdha + Ppia; Hprt1+Pgk1+Ywhaz) demonstrating optimal PCR amplification efficiencies (from 94.7 to 100.5%) and repeatability. Novel assays allow expression analysis of 9 reference genes in 3 reactions making possible a more time-efficient choice of reference genes in RT-qPCR experiments in Wistar rats in comparison with widespread singleplex assays.

Developmental prefrontal mRNA expression of D2 dopamine receptor splice variants and working memory impairments in rats after early life Interleukin-1ß elevation.

Long (D2L) and Short (D2S) isoforms of D2 dopamine receptor differ in their biochemical and physiological properties, which could affect functioning of prefrontal cortex. Contribution of distinct D2 dopamine receptor isoforms to cognitive dysfunctions and its developmental regulation are currently not fully elucidated. In the present study, we evaluated developmental mRNA expression of D2S/D2L dopamine receptor isoforms within the rat medial prefrontal cortex (mPFC) in the model of neurodevelopmental cognitive dysfunction. Working memory performance (Y-maze spontaneous alternations) and D2S/D2L mRNA expression in the mPFC (by qRT-PCR) were evaluated in juvenile (P27), adolescent (P42-47) and adult (P75-90) rats after chronic early life treatment with proinflammatory cytokine interleukin (IL)-1ß (1 µg/kg i.p. daily P15-21). It was shown that IL-1ß elevation during the 3rd week of life leads to working memory deficit originating in juvenile animals and persisting into adulthood. D2S mRNA expression was strongly downregulated during adolescence, and such downregulation was exaggerated in animals injected with IL-1ß during P15-21. Early life IL-1ß administrations influenced developmental changes in the D2S/D2L mRNA ratio. This measure was found to be decreased in adolescent and adult control (intact and vehicle-treated) rats compared to juvenile control, while in the case of IL-1ß-treated animals, the decrease in D2S/D2L ratio was observed only in adulthood but not in adolescence compared to juvenile rats. During the adolescence, D2S mRNA expression was downregulated and D2S/D2L ratio was upregulated in the mPFC of rats treated with IL-1ß during the 3rd week of life compared to controls. Based on these data we conclude that changes in the developmental expression of D2 dopamine receptor splice variants within mPFC may underlie long-lasting cognitive deficit associated with neonatal pathology.

Insulin downregulates C3 gene expression in human HepG2 cells through activation of PPARγ.

C3 is an acute phase protein, and thus its plasma concentration increases quickly and drastically during the onset of inflammation. Insulin plays a complex role in inflammation. Elevated level of plasma C3 was shown to correlate with heightened fasting insulin levels and insulin resistance and appears to be a risk factor for the cardiovascular disease and atherosclerosis. The main source of plasma C3 is liver. Nothing is known about effects of insulin on C3 gene expression and protein secretion by hepatocytes. In light of these data we asked if insulin is capable of regulating C3 production in hepatocytes. Here we show that insulin downregulates C3 gene expression in human hepatoma cells HepG2 through activation of PI3K, mTORC1, p38 and MEK1/2 signaling pathways. Transcription factors PPARα, PPARγ, HNF4α and NF-κB are important contributors to this process. Insulin activates PPARγ through PI3K/Akt/mTORC1 pathway, which results in PPARγ binding to DR4 and DR0 cis-acting elements within the C3 promoter and subsequent displacement of HNF4α and PPARα from these sites. As a result PPARα/NF-κB complex, which exists on C3 promoter, is broken down and C3 gene expression is downregulated. The data obtained can potentially be used to explain the molecular mechanism underlying the correlation between heightened level of plasma C3 and insulin resistance in humans.

Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1ß treatment.

Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1ß (1µg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1ß. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1ß-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge.