A riot of rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus

Abstract

Background: In mammals, the synchronized activity of cell autonomous clocks in the suprachiasmatic nuclei (SCN) enables this structure to function as the master circadian clock, coordinating daily rhythms in physiology and behavior. However, the dominance of this clock has been challenged by the observations that metabolic duress can over-ride SCN controlled rhythms, and that clock genes are expressed in many brain areas, including those implicated in the regulation of appetite and feeding. The recent development of mice in which clock gene/protein activity is reported by bioluminescent constructs (luciferase or luc) now enables us to track molecular oscillations in numerous tissues ex vivo. Consequently we determined both clock activities and responsiveness to metabolic perturbations of cells and tissues within the mediobasal hypothalamus (MBH), a site pivotal for optimal internal homeostatic regulation.

Results: Here we demonstrate endogenous circadian rhythms of PER2::LUC expression in discrete subdivisions of the arcuate (Arc) and dorsomedial nuclei (DMH). Rhythms resolved to single cells did not maintain long-term synchrony with one-another, leading to a damping of oscillations at both cell and tissue levels. Complementary electrophysiology recordings revealed rhythms in neuronal activity in the Arc and DMH. Further, PER2::LUC rhythms were detected in the ependymal layer of the third ventricle and in the median eminence/pars tuberalis (ME/PT). A high-fat diet had no effect on the molecular oscillations in the MBH, whereas food deprivation resulted in an altered phase in the ME/PT.

Conclusion: Our results provide the first single cell resolution of endogenous circadian rhythms in clock gene expression in any intact tissue outside the SCN, reveal the cellular basis for tissue level damping in extra-SCN oscillators and demonstrate that an oscillator in the ME/PT is responsive to changes in metabolism.

Figure 1

PER2::LUC expression in the mediobasal hypothalamus. EM-CCD image illustrating PER2::LUC bioluminescence in the dorsomedial hypothalamus (DMH), particularly in the pars compacta region (DMHc), in the lateral and dorsal arcuate (ArcL and ArcD), the median eminence/pars tuberalis (ME/PT) and the ependymal cell layer of the 3rd ventricle (Ep), but not in the ventromedial hypothalamus (VMH). Calibration bar 250 μM. Lower panel delineates nuclei of interest.

Figure 2

Circadian rhythms of PER2::LUC expression in the MBH. (A) EM-CCD images from an MBH slice (representative of 12 independent experiments) showing one and a half circadian cycles of PER2::LUC bioluminescence expression. Single cells can be discriminated in the DMH (inset) and Arc. Calibration bar 250 μm. (B) Plots of relative PER2::LUC expression integrated across delineated DMH, Arc, ependymal cell layer, ME/PT and VMH. Expression is circadian in all regions except the VMH. Note how tissue wide circadian expression damps at different rates in different tissues. (C) Plots of integrated bioluminescence for representative individual cells in the DMHc, ArcD and ArcL from the same slice. Individual cells are still rhythmic after 8 days in culture, yet the amplitude and synchrony of rhythms decreases over time. (D) Rayleigh vector plots showing phase clustering of cells in the DMHc, ArcD and ArcL at 2 days and 5 days following culture (days indicated in panel C). Circadian rhythms are initially synchronized in all areas (day 2: DMHc, p < 0.005; ArcD, p < 0.005; ArcL p < 0.05) but become desynchronized over the course of the experiment (day 5: all p > 0.05). Filled circles indicate the phase of individual cells. Direction of arrow indicates the mean phase vector, its length indicates the significance of phase clustering, with the surrounding box indicating the variance of phase. The inner broken line indicates the significance threshold of p = 0.05.

Figure 3

Temporal patterns of electrical activity in mediobasal hypothalamic nuclei. Mediobasal hypothalamic nuclei exhibited a continuum of temporal profiles in electrical activity. Recordings of ArcD exhibited robust multiunit (MUA; A) and single unit activity (SUA; D) rhythms. MUA rhythms recorded from the DMHc (B) damped more rapidly than in the ArcD, while SUA profiles typically exhibited a single peak (E). In contrast, the ventromedial hypothalamus rarely sustained detectable MUA (C) or SUA (F) for longer than 18 h. Inset traces in D-F indicate the average spike waveforms for each cell (thick black line) and 8 consecutive matching spikes (thin grey lines); scale bars represent 15 μV (vertical) and 1 ms (horizontal). All figures are representative examples from single slices of MBH. Abbreviations as in text.

Figure 4

Circadian rhythms of PER2::LUC expression in the SCN. (A) Plot of relative PER2::LUC bioluminescence integrated across the whole SCN, imaged with an EM-CCD camera. (B) Plots showing circadian rhythms of PER2::LUC bioluminescence for representative individual cells from the slice in (A). (C) Detrended PMT recording of total PER2::LUC bioluminescence from an SCN slice.

Figure 5

Micro-dissected Arc complex and DMH maintain circadian rhythms in PER2::LUC expression in normal and TTX containing media. EM-CCD images of micro-dissected Arc complex (Arc/ME/PT) (A) and DMH (B) captured before total bioluminescence was recorded in photomultiplier tubes (PMTs). (C; Arc complex) and (D; DMH) are detrended PMT recordings of relative PER2::LUC bioluminescence (counts per minute) emitted from the slices shown in (A) and (B). Inhibition of sodium channel dependent action potentials with TTX (0.5 μM) in micro-dissected Arc complex (F) and DMH (E) does not inhibit circadian rhythms in PER2::LUC expression.

Figure 6

Circadian rhythms of PER2::LUC expression continue in the presence of TTX. (A) Representative example of total bioluminescence integrated across delineated DMH, Arc, ependymal cell layer (Ep), ME/PT and VMH in an intact MBH slice culture imaged on an EM-CCD camera. All areas except the VMH are initially rhythmic in control medium. After 2 days, the slice was treated with 0.5 μM TTX for 3 days before return to control medium, grey shading represents TTX in the culture medium. All previously rhythmic areas sustained rhythmicity in the presence of TTX. (B) EM-CCD image showing PER2::LUC bioluminescence expression in an MBH cultured with 0.5 μM TTX. Single cells can be discriminated in the DMH (inset) and Arc. Calibration bar 250 μm. (C) Plots showing integrated bioluminescence for representative individual cells in DMHc, ArcD (dark blue) and ArcL (light blue). Individual cellular rhythms continue in the presence of TTX.

Figure 7

Circadian rhythms of PER2::LUC expression are revived by forskolin treatment. (A) EM-CCD images from the MBH slice depicted in Fig. 2 showing one and a half circadian cycles of PER2::LUC bioluminescence expression following addition of 10 μM forskolin to the culture medium. Single cells can be discriminated in the DMH (inset) and Arc. Calibration bar 250 μm. (B) Plots of relative PER2::LUC expression integrated across delineated DMH, Arc, ependymal cell layer, ME/PT and VMH. Circadian rhythms in all regions except the VMH are revived, and in the Arc, ME/PT and ependymal cell layer are potentiated with respect to initial amplitude. (C) Circadian rhythms in six representative individual cells in the DMHc, ArcD and ArcL are resynchronized. (D) Rayleigh vector plots showing phase clustering of cells in the DMHc, ArcD and ArcL at 2 days and 5 days following forskolin treatment (days indicated in panel C). Circadian rhythms are initially resynchronized in all areas (day 2: DMHc, p < 0.05; ArcD, p < 0.00001; ArcL p < 0.00001). Five days after forskolin treatment cells in the ArcD and ArcL are still synchronized (ArcD, p < 0.00001; ArcL p < 0.001). This continued synchrony is reflected in the sustained circadian rhythm in the Arc at day 5 after forskolin, when signal is integrated across the whole Arc (B). In contrast, individual cells in the DMHc become desynchronized by 5 days after forskolin (p > 0.05), which is reflected in the arrhythmicity in the whole DMH (B) at this time.

Figure 8

Forskolin synchronizes cellular oscillators in the MBH. Plots showing cross correlations between pairs of oscillating cells in the ArcD (left) and DMHc (right). Correlations were calculated from raw data using a moving window (duration 48 h) with the test cell shifted in time between -24 and +24 hours. The color scale indicates the strength of correlation at any given point with 1 (dark red) indicating perfect correlation and -1 (dark blue) perfect anti-correlation. Initially the cells shown were weakly synchronized (ArcD) or exhibited different circadian periods (DMHc). After addition of forskolin at time 150 h, the periods of cellular oscillators synchronized (indicated by stronger vertical banding) and adopted a stable phase relationship either in phase (DMHc) or with one cell phase leading the other by ~6 h (ArcD).

Figure 9

The phase of peak PER2::LUC expression is altered in the ME/PT of food deprived mice. (A) Rayleigh vector plots showing the phase of peak PER2::LUC expression in vitro, calculated as the time of peak bioluminescence after cull of animal, in the ME/PT of food deprived and control animals. In control animals the phase was significantly correlated with time of cull, peaking on average 31.3 ± 0.9 h after cull (n = 12) versus 42.4 ± 2.3 h in food deprived animals (n = 5, p < 0.001). (B) Lower panels highlight the differences in phase of PER2::LUC expression in neuronal tissue of the MBH in representative control and food deprived mice. Time of peak PER2::LUC is indicated by colored arrowheads; Arc (red), DMH (blue) and the ME/PT (purple). Note the altered peak phase of the ME/PT in the food deprived mouse.