Rapid identification of novel genes expressed in a circadian manner in rat suprachiasmatic nuclei

The circadian clock in mammals is located in the suprachiasmatic nuclei (SCN). There is evidence that changes in gene expression are central to its mechanism. We are engaged in identifying the genes involved. The small size of the SCN, the large number of mammalian genes and the need to identify those differentially expressed over 24 h have required novel experimental procedures. mRNA differential display and an improved tissue micropunching method have been used to examine temporal changes in gene expression in the SCN of rats maintained in constant darkness. Several of the displayed cDNA species were found to be differentially expressed; they show no homology with published sequences. Riboprobes of these cDNA species were used for in situ hybridization. Emulsion-dipped sections confirmed that at least two of these differentially displayed mRNAs are expressed in a circadian manner

Circadian variation of EAAC1 glutamate transporter messenger RNA in the rat suprachiasmatic nuclei

Using in situ hybridization, we examined temporal changes of the EAAC1 glutamate transporter mRNA within the suprachiasmatic nuclei (SCN) of rats in constant darkness. Film autoradiographs showed that the SCN and supraoptic nuclei (SON) contained a marked density of hybridization signal. Analysis of silver grains per cell in emulsion-dipped sections indicated that cellular expression of EEAC1 mRNA in the SCN was elevated during the latter part of the subjective night and at the beginning of the subjective day, with a peak at circadian time 23.1 as determined by cosinor analysis. The times at which EAAC1 mRNA is highest correspond to the time points at which extracellular glutamate, a neurotransmitter that putatively mediates photic entrainment, has been reported to be low within the SCN. The presence of EAAC1 mRNA in the SCN and SON may partially explain the resistance of these nuclei to glutamate receptor-mediated excitotoxins; furthermore, the raised level preceding subjective dawn in the SCN may ensure sub-toxic levels of extracellular glutamate at the onset of photic stimulation during the LD cycle. In contrast, cellular expression of EAAC1 mRNA in the cingulate cortex and reticular thalamus remained constant at all time points studied. These results suggest that there is circadian control of the EAAC1 mRNA by the clock intrinsic to the SCN

Elevated BNP expression in mouse offspring left ventricles after protein restriction in utero

Objectives: We have previously shown that cyclin G1expression is reduced in fetal hearts after in utero protein restriction (PR) suggesting reduced cardiac cell cycle. However no difference in cyclin G1 expression was seen in adult offspring hearts. We hypothesised that the hearts of adult PR group should be under greater stress to maintain cardiac output. We therefore measured brain natriuretic peptide (BNP) expression in fetal hearts and left ventricles of adult offspring in the control (C) and PR groups because BNP is a marker of left ventricular dysfunction during volume overload or cardiac fibrosis (Nishikimi et al. Cardiovasc Res. 2006).Methods and results: Pregnant CD1 mice were placed on C (18% casein) or PR (9% casein) diet. Fetal hearts were collected on day 12 of gestation (C, n =11, PR, n =10) and the left ventricles (LV) of adult offspring at 6 months (C,n =17, PR, n =17). Fetal heart BNP mRNA expression relative to unit total RNA as measured by real-time PCR was similar in C and PR (C, 0.858F0.104 vs. PR, 0.761F0.096, p =NS). However, BNP expression in adult LV was greater in the PR than C (C, 7.043F0.68 vs. PR, 11.012F1.54, p =0.04).Conclusion: These results indicate that protein restriction in pregnancy induces cellular changes (indicated by cyclin G1 changes) in the fetal heart which places it under stress in adulthood (elevated BNP production). Because BNP can suppress ventricular remodelling, we are presently investigating cardiac structural changes to assess whether these alterations are adaptive or maladaptive

Impact on the developmental profile of the murine heart by maternal protein restriction during preganancy

Aims: We hypothesised that intrauterine environmental factors such as undernutrition may have an impact on cardiac development in prenatal life, leading to cardiac hypertrophy. In this study, we examined the effects of maternal dietary protein restriction (PR) during pregnancy on fetal heart development in the mouse. Study design and Subjects: CD1 mice were placed on control C (18% casein) or protein restricted PR (9% casein) diet during pregnancy. Fetal hearts were collected on day 12 of gestation and the left ventricles (LV) of adult offspring at 6 months.Outcome Measures: p53, e2f1 mRNA expression was measured by real time RT PCR, fetal heart ventricular volumes and surface area of by MRI.Results: No differences were demonstrated in p53 and E2f1 expression in fetal heart or adult LV. MRI revealed smaller hearts in the PR group, both for surface area (PR, 13831±68 vs. C, 16356±37 mm2, p<0.01) and ventricular volume (PR, 22114±43 v C, 27404±10 mm3; p<0.001).Conclusion: These results indicate that protein restriction during pregnancy leads to a smaller fetal heart (MRI scan), perhaps due to its effect on cell allocation and division. Lack of difference in the expression of p53 and E2f1, genes involved in cell inhibition and apoptosis and cell proliferation, respectively in either group suggest that changes in fetal heart size are due to the reduction of cardiomyocyte growth (supported by lower cyclin g1 expression previously described) in the PR group