Evidence for neuronal desynchrony in the aged suprachiasmatic nucleus clock.

Aging is associated with a deterioration of daily (circadian) rhythms in physiology and behavior. Deficits in the function of the central circadian pacemaker in the suprachiasmatic nucleus (SCN) have been implicated, but the responsible mechanisms have not been clearly delineated. In this report, we characterize the progression of rhythm deterioration in mice to 900 d of age. Longitudinal behavioral and sleep-wake recordings in up to 30-month-old mice showed strong fragmentation of rhythms, starting at the age of 700 d. Patch-clamp recordings in this age group revealed deficits in membrane properties and GABAergic postsynaptic current amplitude. A selective loss of circadian modulation of fast delayed-rectifier and A-type K+ currents was observed. At the tissue level, phase synchrony of SCN neurons was grossly disturbed, with some subpopulations peaking in anti-phase and a reduction in amplitude of the overall multiunit activity rhythm. We propose that aberrant SCN rhythmicity in old animals--with electrophysiological arrhythmia at the single-cell level and phase desynchronization at the network level--can account for defective circadian function with aging.

Seasonal encoding by the circadian pacemaker of the SCN.

The circadian pacemaker of the suprachiasmatic nucleus (SCN) functions as a seasonal clock through its ability to encode day length [1-6]. To investigate the mechanism by which SCN neurons code for day length, we housed mice under long (LD 16:8) and short (LD 8:16) photoperiods. Electrophysiological recordings of multiunit activity (MUA) in the SCN of freely moving mice revealed broad activity profiles in long days and compressed activity profiles in short days. The patterns remained consistent after release of the mice in constant darkness. Recordings of MUA in acutely prepared hypothalamic slices showed similar differences between the SCN electrical activity patterns in vitro in long and short days. In vitro recordings of neuronal subpopulations revealed that the width of the MUA activity profiles was determined by the distribution of phases of contributing units within the SCN. The subpopulation patterns displayed a significantly broader distribution in long days than in short days. Long-term recordings of single-unit activity revealed short durations of elevated activity in both short and long days (3.48 and 3.85 hr, respectively). The data indicate that coding for day length involves plasticity within SCN neuronal networks in which the phase distribution of oscillating neurons carries information on the photoperiod's duration.

Amplitude of the SCN clock enhanced by the behavioral activity rhythm.

Circadian rhythms are regulated by the suprachiasmatic nucleus (SCN), a small structure at the base of the hypothalamus. While light effects on the SCN are well established, little is known of behavioral effects. This study elucidates direct modulating action of behavioral activity on the SCN by use of in vivo electrophysiology recordings, assessments of general locomotor behavior, and video-tracking of mice. The results show suppression of SCN neuronal activity by spontaneous behavior, the magnitude being dependent on the intensity, duration and type of behavioral activity. The suppression was moderate (32% of circadian amplitude) for low-intensity behavior and considerable (59%) for locomotor activity. Mild manipulation of the animals had reversed effects on the SCN indicating that different mechanisms are involved in the regulatory effect of spontaneous versus induced activity. The results indicate that exercise at the proper time of the cycle can boost the amplitude of the rhythm of the SCN clock itself. This has potentially beneficial effects for other rhythmic functions that are under the control of the SCN.

Fractal patterns of neural activity exist within the suprachiasmatic nucleus and require extrinsic network interactions.

The mammalian central circadian pacemaker (the suprachiasmatic nucleus, SCN) contains thousands of neurons that are coupled through a complex network of interactions. In addition to the established role of the SCN in generating rhythms of ~24 hours in many physiological functions, the SCN was recently shown to be necessary for normal self-similar/fractal organization of motor activity and heart rate over a wide range of time scales--from minutes to 24 hours. To test whether the neural network within the SCN is sufficient to generate such fractal patterns, we studied multi-unit neural activity of in vivo and in vitro SCNs in rodents. In vivo SCN-neural activity exhibited fractal patterns that are virtually identical in mice and rats and are similar to those in motor activity at time scales from minutes up to 10 hours. In addition, these patterns remained unchanged when the main afferent signal to the SCN, namely light, was removed. However, the fractal patterns of SCN-neural activity are not autonomous within the SCN as these patterns completely broke down in the isolated in vitro SCN despite persistence of circadian rhythmicity. Thus, SCN-neural activity is fractal in the intact organism and these fractal patterns require network interactions between the SCN and extra-SCN nodes. Such a fractal control network could underlie the fractal regulation observed in many physiological functions that involve the SCN, including motor control and heart rate regulation.

Ultraviolet light provides a major input to non-image-forming light detection in mice.

The change in irradiance at dawn and dusk provides the primary cue for the entrainment of the mammalian circadian pacemaker. Irradiance detection has been ascribed largely to melanopsin-based phototransduction [1-5]. Here we examine the role of ultraviolet-sensitive (UVS) cones in the modulation of circadian behavior, sleep, and suprachiasmatic nucleus (SCN) electrical activity. UV light exposure leads to phase-shifting responses comparable to those of white light. Moreover, UV light exposure induces sleep in wild-type and melanopsin-deficient (Opn4(-/-)) mice with equal efficacy. Electrical recordings from the SCN of wild-type mice show that UV light elicits irradiance-dependent sustained responses that are similar to those induced by white light, with characteristic fast transient components occurring at the light transitions. These responses are retained in Opn4(-/-) mice and preserved under saturating photopic conditions. The sensitivity of phase-shifting responses to UV light is unaffected by the loss of rods but is severely attenuated by the additional loss of cones. Our data show that UVS cones play an important role in circadian and sleep regulation in mice.

Phase resetting of the mammalian circadian clock relies on a rapid shift of a small population of pacemaker neurons.

The circadian pacemaker of the suprachiasmatic nuclei (SCN) contains a major pacemaker for 24 h rhythms that is synchronized to the external light-dark cycle. In response to a shift in the external cycle, neurons of the SCN resynchronize with different pace. We performed electrical activity recordings of the SCN of rats in vitro following a 6 hour delay of the light-dark cycle and observed a bimodal electrical activity pattern with a shifted and an unshifted component. The shifted component was relatively narrow as compared to the unshifted component (2.2 h and 5.7 h, respectively). Curve fitting and simulations predicted that less than 30% of the neurons contribute to the shifted component and that their phase distribution is small. This prediction was confirmed by electrophysiological recordings of neuronal subpopulations. Only 25% of the neurons exhibited an immediate shift in the phase of the electrical activity rhythms, and the phases of the shifted subpopulations appeared significantly more synchronized as compared to the phases of the unshifted subpopulations (p<0.05). We also performed electrical activity recordings of the SCN following a 9 hour advance of the light-dark cycle. The phase advances induced a large desynchrony among the neurons, but consistent with the delays, only 19% of the neurons peaked at the mid of the new light phase. The data suggest that resetting of the central circadian pacemaker to both delays and advances is brought about by an initial shift of a relatively small group of neurons that becomes highly synchronized following a shift in the external cycle. The high degree of synchronization of the shifted neurons may add to the ability of this group to reset the pacemaker. The large desynchronization observed following advances may contribute to the relative difficulty of the circadian system to respond to advanced light cycles.

Phase shifting capacity of the circadian pacemaker determined by the SCN neuronal network organization.

BACKGROUND: In mammals, a major circadian pacemaker that drives daily rhythms is located in the suprachiasmatic nuclei (SCN), at the base of the hypothalamus. The SCN receive direct light input via the retino-hypothalamic tract. Light during the early night induces phase delays of circadian rhythms while during the late night it leads to phase advances. The effects of light on the circadian system are strongly dependent on the photoperiod to which animals are exposed. An explanation for this phenomenon is currently lacking. METHODOLOGY AND PRINCIPAL FINDINGS: We recorded running wheel activity in C57 mice and observed large amplitude phase shifts in short photoperiods and small shifts in long photoperiods. We investigated whether these different light responses under short and long days are expressed within the SCN by electrophysiological recordings of electrical impulse frequency in SCN slices. Application of N-methyl-D-aspartate (NMDA) induced sustained increments in electrical activity that were not significantly different in the slices from long and short photoperiods. These responses led to large phase shifts in slices from short days and small phase shifts in slices from long days. An analysis of neuronal subpopulation activity revealed that in short days the amplitude of the rhythm was larger than in long days. CONCLUSIONS: The data indicate that the photoperiodic dependent phase responses are intrinsic to the SCN. In contrast to earlier predictions from limit cycle theory, we observed large phase shifting responses in high amplitude rhythms in slices from short days, and small shifts in low amplitude rhythms in slices from long days. We conclude that the photoperiodic dependent phase responses are determined by the SCN and propose that synchronization among SCN neurons enhances the phase shifting capacity of the circadian system.

Phase of the electrical activity rhythm in the SCN in vitro not influenced by preparation time.

The mammalian circadian clock, located in the suprachiasmatic nucleus (SCN) of the hypothalamus, drives daily rhythms in behavioral, physiological, and endocrine functions. The SCN has a genetic basis for rhythm generation and remains rhythmic when it is isolated and kept in constant conditions. This allows for an in vitro analysis of circadian attributes, which is a powerful approach in the study of SCN cellular mechanisms. For studying the phase of the SCN rhythm in vitro, it is important to assess whether preparation of the tissue itself introduces phase shifts. In the present study, we investigated whether preparation of hypothalamic brain slices affects the phase and waveform of the rhythm in electrical impulse frequency of the mouse SCN. Mice were kept under a 12:12 h light-dark cycle, and slices were prepared at six timepoints distributed over the 24 h cycle. We used the peak time and the time of the half-maximum levels in electrical activity as markers for circadian phase. The peak time in electrical activity was observed during the mid-subjective day, irrespective of the time of preparation, at a mean ZT of 5.18+/-0.20 h (n = 39). After preparation in red light at the end of the subjective night, the circadian phase appeared slightly advanced. When slices were prepared in the dark, using infrared illumination, the ANOVA showed no significant differences in peak times and time of half-maximum values between preparation times. The results affirm the value of the slice preparation for studying the phase of the SCN in vitro. We conclude that the phase and waveform of the electrical activity in the SCN in vitro is unaffected by the time of slice preparation but may be influenced by short light presentation when preparation is performed during the subjective night.

Heterogeneity of rhythmic suprachiasmatic nucleus neurons: Implications for circadian waveform and photoperiodic encoding.

Circadian rhythms in neuronal ensemble, subpopulations, and single unit activity were recorded in the suprachiasmatic nuclei (SCN) of rat hypothalamic slices. Decomposition of the ensemble pattern revealed that neuronal subpopulations and single units within the SCN show surprisingly short periods of enhanced electrical activity of approximately 5 h and show maximal activity at different phases of the circadian cycle. The summed activity accounts for the neuronal ensemble pattern of the SCN, indicating that circadian waveform of electrical activity is a composed tissue property. The recorded single unit activity pattern was used to simulate the responsiveness of SCN neurons to different photoperiods. We inferred predictions on changes in peak width, amplitude, and peak time in the multiunit activity pattern and confirmed these predictions with hypothalamic slices from animals that had been kept in a short or long photoperiod. We propose that the animals' ability to code for day length derives from plasticity in the neuronal network of oscillating SCN neurons.