Characterization of orderly spatiotemporal patterns of clock gene activation in mammalian suprachiasmatic nucleus

Abstract

Because we can observe oscillation within individual cells and in the tissue as a whole, the suprachiasmatic nucleus (SCN) presents a unique system in the mammalian brain for the analysis of individual cells and the networks of which they are a part. While dispersed cells of the SCN sustain circadian oscillations in isolation, they are unstable oscillators that require network interactions for robust cycling. Using cluster analysis to assess bioluminescence in acute brain slices from PERIOD2∷Luciferase (PER2∷LUC) knockin mice, and immunochemistry of SCN from animals harvested at various circadian times, we assessed the spatiotemporal activation patterns of PER2 to explore the emergence of a coherent oscillation at the tissue level. The results indicate that circadian oscillation is characterized by a stable daily cycle of PER2 expression involving orderly serial activation of specific SCN subregions, followed by a silent interval, with substantial symmetry between the left and right side of the SCN. The biological significance of the clusters identified in living slices was confirmed by co-expression of LUC and PER2 in fixed, immunochemically stained brain sections, with the spatiotemporal pattern of LUC expression resembling that revealed in the cluster analysis of bioluminescent slices. We conclude that the precise timing of PER2 expression within individual neurons is dependent on their location within the nucleus, and that small groups of neurons within the SCN give rise to distinctive and identifiable subregions. We propose that serial activation of these subregions is the basis of robustness and resilience of the daily rhythm of the SCN.

Period Coding of Bmal1 Oscillators in the Suprachiasmatic Nucleus

“…The neurons were effectively selected by convolution with a grid kernel that finds a spatial average around a dominant area of bioluminescence activities (see Materials and Methods). Although this method oversampled the number of oscillators compared with the traditional ROI method, the amount of false positives due to internal light scatter (Foley et al, 2011) was limited because the SCN is dense with neurons (Klein et al, 1991) and the method provided general spatiotemporal information consistent with results from the ROI method (Fig. 2).…”

“…In the adult SCN samples we have used, there was little spatial jittering of neurons during culture (inset, second row) except for the first 24 h after preparation of culture. Heterogeneous phases of bioluminescence activities in SCN cultures have been observed using Per1 (Quintero et al, 2003;Yamaguchi et al, 2003) and Per2 reporter systems (Evans et al, 2011;Foley et al, 2011;Fukuda et al, 2011), which were previously interpreted as spatially propagating waves or tides that expand and recede back to their point of origin (Yan et al, 2007;Butler and Silver, 2009). …”

Topological specificity and hierarchical network of the circadian calcium rhythm in the suprachiasmatic nucleus

“…7C, and Tables S3 and S4). patterns of clock gene expression using in situ hybridization and bioluminescence imaging (2,(10)(11)(12)(13)(14). However, the autonomous expression of a clock gene is either absent or undetectable in certain regions, particularly those where neurons receive major light information from the retina (18).…”

“…Heterogeneous distribution of the circadian phase in SCN slices was shown by in situ hybridization and bioluminescence studies examining clock gene expression (e.g., Per1 or Per2) (10)(11)(12)(13)(14). Disruption of intercellular coupling by inhibiting neuronal firing with tetrodotoxin (TTX) or mechanical isolation of SCN subregions severely reduced clock gene expression and mutual synchronization (10,11).…”

Functional Contributions of Strong and Weak Cellular Oscillators to Synchrony and Light-shifted Phase Dynamics

“…Light is the most powerful cue for most organisms (Pittendrigh and Daan, 1976;Tauber and Kyriacou, 2001). Light evoked changes in mammalian cellular oscillator dynamics is still largely unexplored due to the technical difficulty of introducing a physiological light signal in vitro (Evans et al, 2011;Foley et al, 2011;Webb et al, 2012). For the mammalian circadian pacemaker, the suprachiasmatic nucleus (SCN), physiologically activating retina-mediated photic responses while monitoring large numbers of deep brain individual cellular oscillators by imaging or electrophysiology remains a formidable challenge, both ex vivo and in vivo.…”