Circadian regulation of dentate gyrus excitability mediated by G-protein signaling.

The central circadian regulator within the suprachiasmatic nucleus transmits time of day information by a diurnal spiking rhythm driven by molecular clock genes controlling membrane excitability. Most brain regions, including the hippocampus, harbor similar intrinsic circadian transcriptional machinery, but whether these molecular programs generate oscillations of membrane properties is unclear. Here, we show that intrinsic excitability of mouse dentate granule neurons exhibits a 24-h oscillation that controls spiking probability. Diurnal changes in excitability are mediated by antiphase G-protein regulation of potassium and sodium currents that reduce excitability during the Light phase. Disruption of the circadian transcriptional machinery by conditional deletion of Bmal1 enhances excitability selectively during the Light phase by removing G-protein regulation. These results reveal that circadian transcriptional machinery regulates intrinsic excitability by coordinated regulation of ion channels by G-protein signaling, highlighting a potential novel mechanism of cell-autonomous oscillations.

Examination of Diurnal Variation and Sex Differences in Hippocampal Neurophysiology and Spatial Memory.

Circadian rhythms are biological processes that cycle across 24 h and regulate many facets of neurophysiology, including learning and memory. Circadian variation in spatial memory task performance is well documented; however, the effect of sex across circadian time (CT) remains unclear. Additionally, little is known regarding the impact of time-of-day on hippocampal neuronal physiology. Here, we investigated the influence of both sex and time-of-day on hippocampal neurophysiology and memory in mice. Performance on the object location memory (OLM) task depended on both circadian time and sex, with memory enhanced at night in males but during the day in females. Long-term synaptic potentiation (LTP) magnitude at CA3-CA1 synapses was greater at night compared with day in both sexes. Next, we measured spontaneous synaptic excitation and inhibition onto CA1 pyramidal neurons. Frequency and amplitude of inhibition was greater during the day compared with night, regardless of sex. Frequency and amplitude of excitation was larger in females, compared with males, independent of time-of-day, although both time-of-day and sex influenced presynaptic release probability. At night, CA1 pyramidal neurons showed enhanced excitability (action potential firing and/or baseline potential) that was dependent on synaptic excitation and inhibition, regardless of sex. This study emphasizes the importance of sex and time-of-day in hippocampal physiology, especially given that many neurologic disorders impacting the hippocampus are linked to circadian disruption and present differently in men and women. Knowledge about how sex and circadian rhythms affect hippocampal physiology can improve the translational relevancy of therapeutics and inform the appropriate timing of existing treatments.

Circadian disruption of hippocampus in an early senescence male mouse model.

Age-related cognitive decline and disruptions in circadian rhythms are growing problems as the average human life span increases. Multiple strains of the senescence-accelerated mouse (SAM) show reduced life span, and the SAMP8 strain in particular has been well documented to show cognitive deficits in behavior as well as a bimodal pattern of circadian locomotor activity. However, little is known about circadian regulation within the hippocampus of these strains of mice. Here we test the hypothesis that in this early senescence model, disruption of the molecular circadian clock in SAMP8 animals drives disrupted behavior and physiology. We found normal rhythms in PER2 protein expression in the SCN of SAMP8 animals at 4 months, despite the presence of disrupted wheel-running activity rhythms at this age. Interestingly, a significant rhythm in PER2 expression was not observed in the hippocampus of SAMP8 animals, despite a significant 24-h rhythm in SAMR1 controls. We also examined time-restricted feeding as a potential strategy to rescue disrupted hippocampal plasticity. Time-restricted feeding increased long-term potentiation at Schaffer collateral-CA1 synapses in SAMP8 mice (compared to SAMR1 controls). Overall, we confirm disrupted circadian locomotor rhythms in this early senescence model (as early as 4 months) and discovered that this disruption is not due to arrhythmic PER2 levels in the SCN; however, other extra-SCN circadian oscillators (i.e., hippocampus) are likely impaired with accelerated aging.

Dysregulated clock gene expression and abnormal diurnal regulation of hippocampal inhibitory transmission and spatial memory in amyloid precursor protein transgenic mice.

Patients with Alzheimer's disease (AD) often have fragmentation of sleep/wake cycles and disrupted 24-h (circadian) activity. Despite this, little work has investigated the potential underlying day/night disruptions in cognition and neuronal physiology in the hippocampus. The molecular clock, an intrinsic transcription-translation feedback loop that regulates circadian behavior, may also regulate hippocampal neurophysiological activity. We hypothesized that disrupted diurnal variation in clock gene expression in the hippocampus corresponds with loss of normal day/night differences in membrane excitability, synaptic physiology, and cognition. We previously reported disrupted circadian locomotor rhythms and neurophysiological output of the suprachiasmatic nucleus (the primary circadian clock) in Tg-SwDI mice with human amyloid-beta precursor protein mutations. Here, we report that Tg-SwDI mice failed to show day/night differences in a spatial working memory task, unlike wild-type controls that exhibited enhanced spatial working memory at night. Moreover, Tg-SwDI mice had lower levels of Per2, one of the core components of the molecular clock, at both mRNA and protein levels when compared to age-matched controls. Interestingly, we discovered neurophysiological impairments in area CA1 of the Tg-SwDI hippocampus. In controls, spontaneous inhibitory post-synaptic currents (sIPSCs) in pyramidal cells showed greater amplitude and lower inter-event interval during the day than the night. However, the normal day/night differences in sIPSCs were absent (amplitude) or reversed (inter-event interval) in pyramidal cells from Tg-SwDI mice. In control mice, current injection into CA1 pyramidal cells produced more firing during the night than during the day, but no day/night difference in excitability was observed in Tg-SwDI mice. The normal day/night difference in excitability in controls was blocked by GABA receptor inhibition. Together, these results demonstrate that the normal diurnal regulation of inhibitory transmission in the hippocampus is diminished in a mouse model of AD, leading to decreased daytime inhibition onto hippocampal CA1 pyramidal cells. Uncovering disrupted day/night differences in circadian gene regulation, hippocampal physiology, and memory in AD mouse models may provide insight into possible chronotherapeutic strategies to ameliorate Alzheimer's disease symptoms or delay pathological onset.

Circadian regulation of membrane physiology in neural oscillators throughout the brain.

Twenty-four-hour rhythmicity in physiology and behavior are driven by changes in neurophysiological activity that vary across the light-dark and rest-activity cycle. Although this neural code is most prominent in neurons of the primary circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus, there are many other regions in the brain where region-specific function and behavioral rhythmicity may be encoded by changes in electrical properties of those neurons. In this review, we explore the existing evidence for molecular clocks and/or neurophysiological rhythms (i.e., 24 hr) in brain regions outside the SCN. In addition, we highlight the brain regions that are ripe for future investigation into the critical role of circadian rhythmicity for local oscillators. For example, the cerebellum expresses rhythmicity in over 2,000 gene transcripts, and yet we know very little about how circadian regulation drives 24-hr changes in the neural coding responsible for motor coordination. Finally, we conclude with a discussion of how our understanding of circadian regulation of electrical properties may yield insight into disease mechanisms which may lead to novel chronotherapeutic strategies in the future.

Translational effects and coding potential of an upstream open reading frame associated with DOPA Responsive Dystonia.

Upstream open reading frames (uORFs) have emerged as major post-transcriptional regulatory elements in eukaryotic species. In general, uORFs are initiated by a translation start codon within the 5' untranslated region of a gene (upstream ATG; uATG), and they are negatively correlated with translational efficiency. In addition to their translational regulatory role, some uORFs can code for biologically active short peptides. The importance of uATGs/uORFs is further underscored by human diseases associated with single nucleotide polymorphisms (SNPs), which disrupt existing uORFs or introduce novel uORFs. Although several functional proteins translated from naturally occurring uORFs have been described, the coding potential of uORFs created by SNPs has been ignored because of the a priori assumption that these proteins are short-lived with no likely impact on protein homeostasis. Thus, studies on SNP-created uORFs are limited to their translational effects, leaving unexplored the potential cellular consequences of a SNP/uORF-encoded protein. Here, we investigate functionality of a uATG/uORF introduced by a +142C>T SNP within the GCH1 gene and associated with a familial form of DOPA Responsive Dystonia. We report that the +142C>T SNP represses GCH1 translation, and introduces a short, frame shifted uORF that encodes a 73-amino acid peptide. This peptide is localized within the nucleus and compromises cell viability upon proteasome inhibition. Our work extends the list of uATG/uORF associated diseases and advances research on peptides translated from SNP-introduced uORFs, a neglected component of the proteome.