Circadian (~24h) rhythms depend on intra-cellular transcription-translation negative feedback loops (TTFLs). How these self-sustained cellular clocks achieve multi-cellular integration and thereby direct daily rhythms of behavior in animals is largely obscure. The suprachiasmatic nucleus (SCN) is the fulcrum of this gene-to-cell-to-circuit-to-behavior pathway in mammals. We describe cell-type-specific, functionally distinct, TTFLs in neurons and astrocytes of the SCN and show that- in the absence of other cellular clocks- the cell autonomous astrocytic TTFL alone can drive molecular oscillations in the SCN and circadian behavior in mice. Astrocytic clocks achieve this by reinstating clock gene expression and circadian function of SCN neurons via glutamatergic signals. These results provide a first demonstration that astrocytes can autonomously initiate and sustain complex mammalian behavior.
The blue-light sensitive photoreceptor cryptochrome (CRY) may act as a magneto-receptor through formation of radical pairs involving a triad of tryptophans. Previous genetic analyses of behavioral responses of Drosophila to electromagnetic fields using conditioning, circadian and geotaxis assays have lent some support to the radical pair model (RPM). Here, we describe a new method that generates consistent and reliable circadian responses to electromagnetic fields that differ substantially from those already reported. We used the Schuderer apparatus to isolate Drosophila from local environmental variables, and observe extremely low frequency (3 to 50 Hz) field-induced changes in two locomotor phenotypes, circadian period and activity levels. These field-induced phenotypes are CRY- and blue-light dependent, and are correlated with enhanced CRY stability. Mutational analysis of the terminal tryptophan of the triad hypothesised to be indispensable to the electron transfer required by the RPM reveals that this residue is not necessary for field responses. We observe that deletion of the CRY C-terminus dramatically attenuates the EMF-induced period changes, whereas the N-terminus underlies the hyperactivity. Most strikingly, an isolated CRY C-terminus that does not encode the Tryptophan triad nor the FAD binding domain is nevertheless able to mediate a modest EMF-induced period change. Finally, we observe that hCRY2, but not hCRY1, transformants can detect EMFs, suggesting that hCRY2 is blue light-responsive. In contrast, when we examined circadian molecular cycles in wild-type mouse suprachiasmatic nuclei slices under blue light, there was no field effect. Our results are therefore not consistent with the classical Trp triad-mediated RPM and suggest that CRYs act as blue-light/EMF sensors depending on trans-acting factors that are present in particular cellular environments.
Circadian rhythms in mammals are coordinated by the suprachiasmatic nucleus (SCN). SCN neurons define circadian time using transcriptional/ posttranslational feedback loops (TTFL) in which expression of Cryptochrome (Cry) and Period (Per) genes is inhibited by their protein products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual deletions accelerate and decelerate it, respectively. At the circuit level, neuronal interactions synchronize cellular TTFLs, creating a spatiotemporal wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins required for circadian function, we expressed CRY in SCN of Cry-deficient mice using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2:: EGFP under a minimal Cry1 promoter was circadian and rapidly induced PER2-dependent bioluminescence rhythms in previously arrhythmic Cry1/2-deficient SCN, with periods appropriate to each isoform. CRY1::EGFP appropriately lengthened the behavioral period in Cry1-deficient mice. Thus, determination of specific circadian periods reflects properties of the respective proteins, independently of their phase of expression. Phase of CRY1::EGFP expression was critical, however, because constitutive or phase-delayed promoters failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP induced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine vasopressin (AVP) because it was prevented by pharmacological blockade of AVP receptors. Thus, our genetic complementation assay reveals acute, protein-specific induction of cell-autonomous and network-level circadian rhythmicity in SCN never previously exposed to CRY. Specifically, Cry expression must be circadian and appropriately phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function.he suprachiasmatic nucleus (SCN) is a precise biological clock that coordinates circadian (∼24 h) rhythms in mammals. Composed of ∼10,000 neurons, the SCN sits atop the optic chiasm, through which it receives retinal innervation that entrains its cellular clockwork to the light/dark cycle. In turn, the SCN entrains peripheral circadian clocks distributed across the organism through neural pathways incorporating neuropeptidergic and GABA-ergic signaling (1). At the molecular level, circadian timing in SCN neurons revolves around transcriptional/posttranslational feedback loops (TTFL), in which the positive components BMAL1 and CLOCK activate E/E′-box-mediated transcription of the negative components Period (Per1, Per2) and Cryptochrome (Cry1, Cry2). PER/CRY heterodimers then inhibit BMAL1/CLOCK activity and thus prevent their own transcription. Subsequent proteasomal degradation of PER and CRY proteins relieves this inhibition, allowing the molecular cycle to begin again. Genetic studies have established that CRY1 and CRY2 are essential for behavioral and molecular circadian rhythmicity, acting as core components of th...
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