Aging affects GABAergic function and calcium homeostasis in the mammalian central clock

IntroductionAging impairs the function of the central circadian clock in mammals, the suprachiasmatic nucleus (SCN), leading to a reduction in the output signal. The weaker timing signal from the SCN results in a decline in rhythm strength in many physiological functions, including sleep–wake patterns. Accumulating evidence suggests that the reduced amplitude of the SCN signal is caused by a decreased synchrony among the SCN neurons. The present study was aimed to investigate the hypothesis that the excitation/inhibition (E/I) balance plays a role in synchronization within the network.MethodsUsing calcium (Ca2+) imaging, the polarity of Ca2+ transients in response to GABA stimulation in SCN slices of old mice (20–24 months) and young controls was studied.ResultsWe found that the amount of GABAergic excitation was increased, and that concordantly the E/I balance was higher in SCN slices of old mice when compared to young controls. Moreover, we showed an effect of aging on the baseline intracellular Ca2+ concentration, with higher Ca2+ levels in SCN neurons of old mice, indicating an alteration in Ca2+ homeostasis in the aged SCN. We conclude that the change in GABAergic function, and possibly the Ca2+ homeostasis, in SCN neurons may contribute to the altered synchrony within the aged SCN network

Exposure to long photoperiod increases peak time distribution.

<p>(A) Representative histograms of peak times of two individual slices from the anterior SCN in long (LP, <i>n</i> = 177 cells) and short photoperiod (SP, <i>n</i> = 183 cells) plotted in external time (ExT). (B) Phase distribution is defined as the standard deviation (SD) of peak time, of the first cycle <i>in vitro</i> (top panel). Phase distribution was calculated per slice, and is shown for the anterior and posterior SCN (bottom panel), in LP (green squares) and SP (red circles). Black bars indicate mean ± SEM; *** <i>p</i> < 0.001.</p

Peak time of PER2::LUC expression is similar in anterior and posterior SCN in both long and short photoperiod.

<p>(A) Overlays of brightfield images of anterior SCN cultures from short (SP) and long photoperiod (LP), with the corresponding bioluminescence image. Scale bar: 200 μm. (B) Intensity traces of PER2::LUC expression from single cells. Raw traces of bioluminescence intensity from the anterior SCN from SP (<i>n</i> = 183 cells; top panel), with corresponding smoothed traces (middle panel), and smoothed traces from the anterior SCN from LP (<i>n</i> = 177 cells; bottom panel). (C) Average peak time of PER2::LUC rhythms per slice, of the anterior and posterior SCN in LP (green squares, <i>n</i> = 5) and SP (red circles, <i>n</i> = 4) are plotted as external time (ExT). Grey background indicates projected dark period of light regime preceding the experiment. Black bars indicate mean ± SEM; * <i>p</i> < 0.05.</p

Functional clusters show distinct spatial distribution and region specific rhythm characteristics.

<p>(A) Representative examples of the communities detected by an advanced, unsupervised method for correlation matrix analysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168954#pone.0168954.ref015" target="_blank">15</a>]. The community detection method automatically divided the SCN in two distinct regions, a ventromedial (VM) and dorsolateral (DL) region in the anterior SCN (middle panel), and a medial (M) and lateral (L) region in the posterior SCN, in both long (LP; left panel), and short photoperiod (SP, right panel). (B) Peak times in external time (ExT) were averaged per region, per slice. Darkness and light of the previous light regime are represented by grey and white background respectively. (C) Phase distribution, defined as peak time standard deviation (SD), was calculated per region, per slice. (D) Single-cell period variability was averaged per region, per slice. All data are shown for the VM and DL region in the anterior, and M and L region in the posterior SCN, in LP (green squares) and SP (red circles). Black bars indicate mean ± SEM; * <i>p</i> < 0.01.</p

Single-cell period variability (SD) shows regional differences, especially after exposure to long photoperiod.

<p>Regional averages of single-cell period variability binned from all recordings for six areas of the anterior (top panels) and posterior SCN (bottom panels), in long (LP, left panels) and short photoperiod (SP, right panels). As indicated by the color bar on the right, dark (blue-black) colors indicate small, and light (green-yellow) colors indicate larger period variability. Scale bar: 200 μm.</p

Single-cell period variability (SD) increases after exposure to long photoperiod.

<p>(A) Representative examples of the variability in single-cell cycle-to-cycle interval from anterior SCN slices in long (LP, <i>n</i> = 177; top panel) and in short photoperiod (SP, <i>n</i> = 183; bottom panel). Black traces show the cycle-to-cycle interval of individual cells for the first three cycles <i>in vitro</i>; colored traces show the average period per cycle for the presented slice. (B) Cycle interval is defined as the cycle-to-cycle time difference between the half-maximum of the rising edge of the PER2::LUC expression rhythm. The variability in period is defined as the standard deviation (SD) of the cycle interval of individual cells, calculated for the first three cycles <i>in vitro</i> (top panel). Single-cell period variability was averaged per slice and is shown for the anterior SCN, in LP (green squares) and SP (red circles; bottom panel). Black bars indicate mean ± SEM. (C) Linear regression of the relationship between single-cell period variability and peak time SD for all recordings (<i>n</i> = 18, <i>p</i> < 0.001).</p

Expression Profiling after Prolonged Experimental Febrile Seizures in Mice Suggests Structural Remodeling in the Hippocampus

Febrile seizures are the most prevalent type of seizures among children up to 5 years of age (2-4% of Western-European children). Complex febrile seizures are associated with an increased risk to develop temporal lobe epilepsy. To investigate short- and long-term effects of experimental febrile seizures (eFS), we induced eFS in highly febrile convulsion-susceptible C57BL/6J mice at post-natal day 10 by exposure to hyperthermia (HT) and compared them to normotherm-exposed (NT) mice. We detected structural re-organization in the hippocampus 14 days after eFS. To identify molecular candidates, which entrain this structural re-organization, we investigated temporal changes in mRNA expression profiles eFS 1 hour to 56 days after eFS. We identified 931 regulated genes and profiled several candidates using in situ hybridization and histology at 3 and 14 days after eFS. This is the first study to report genome-wide transcriptome analysis after eFS in mice. We identify temporal regulation of multiple processes, such as stress-, immune- and inflammatory responses, glia activation, glutamate-glutamine cycle and myelination. Identification of the short- and long-term changes after eFS is important to elucidate the mechanisms contributing to epileptogenesis

Hierarchical clustering analysis of mRNA expression after eFS.

<p>Hierarchical clustering analysis of differentially expressed genes (930) in all experimental littermate couples (1 hour, 3 and 14 days post-eFS). Rows depict differential expression levels per HT/NT littermate couple, per time-point after eFS. Clustering was performed based on gene and sample clustering using Pearson correlation on average linkage. Tree cluster, right from heat-map, shows hierarchical distance between samples from the different time points. Differential gene expression (log2(Fc) between HT/NT littermate couples is shown in the heatmap as upregulated (red), downregulated (green), or no change (white) according to colored-scale bar.</p

Timed regulation of biological processes after eFS.

<p>Bar blackness indicates the peak effect of the process. P10: postnatal day 10, 1H: 1 hour post-eFS, 3D: 3 days post-eFS, 14D: 14 days post-eFS, 56D: 56 days post-eFS, Glu/Gln cycle: glutamate/glutamine cycle.</p

Validation of microarray results by qPCR analysis.

<p>Gene expression levels as determined by qPCR (Rn) in normothermic (NT) and hyperthermic (HT) animals compared to fold-change in expression of 5 genes as detected by microarray analysis (Rm). <i>P</i> < 0.05 was considered significant (one-tailed Student’s t-test). Indicated are mean ± standard error of the mean.</p