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, 29 (2), 91-102

Neuropeptide Y-induced Phase Shifts of PER2::LUC Rhythms Are Mediated by Long-Term Suppression of Neuronal Excitability in a Phase-Specific Manner

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Neuropeptide Y-induced Phase Shifts of PER2::LUC Rhythms Are Mediated by Long-Term Suppression of Neuronal Excitability in a Phase-Specific Manner

Rachel C Besing et al. Chronobiol Int.

Abstract

Endogenous circadian rhythms are entrained to the 24-h light/dark cycle by both light and nonphotic stimuli. During the day, nonphotic stimuli, such as novel wheel-induced exercise, produce large phase advances. Neuropeptide Y (NPY) release from the thalamus onto suprachiasmatic nucleus (SCN) neurons at least partially mediates this nonphotic signal. The authors examined the hypothesis that NPY-induced phase advances are accompanied by suppression of PER2 and are mediated by long-term depression of neuronal excitability in a phase-specific manner. First, it was found that NPY-induced phase advances in PER2::LUC SCN cultures are largest when NPY (2.35 µM) is given in the early part of the day (circadian time [CT] 0-6). In addition, PER2::LUC levels in NPY-treated (compared to vehicle-treated) samples were suppressed beginning 6-7 h after treatment. Similar NPY application to organotypic Per1::GFP SCN cultures resulted in long-term suppression of spike rate of green fluorescent protein-positive (GFP+) cells when slices were treated with NPY during the early or middle of the day (zeitgeber time [ZT] 2 or 6), but not during the late day (ZT 10). Furthermore, 1-h bath application of NPY to acute SCN brain slices decreased general neuronal activity measured through extracellular recordings. Finally, NPY-induced phase advances of PER2::LUC rhythms were blocked by latent depolarization with 34.5 mM K(+) 3 h after NPY application. These results suggest that NPY-induced phase advances may be mediated by long-term depression of neuronal excitability. This model is consistent with findings in other brain regions that NPY-induced persistent hyperpolarization underlies mechanisms of energy homeostasis, anxiety-related behavior, and thalamocortical synchronous firing.

Conflict of interest statement

DECLARATION OF INTEREST

The authors report no conflicts of interest. This work was supported by National Institutes of Health, Grants K99 GM086683 (KLG).

Figures

Figure 1
Figure 1. Phase specific NPY-induced phase advances of PER2 rhythms when applied during subjective day
(A) Scatterplot of 1-h NPY application to SCN slice cultures from PER2::LUC mice at CT 0–12 and corresponding fitted regression lines. (B) Means and SEMs for phase shifts resulting from NPY- and vehicle-treated samples pooled into 3-h time bins (N = 10–20/NPY or vehicle subgroup). (C–D) Representative bioluminescent traces recorded from SCN explants from PER2::LUC mice after NPY treatment at CT 2 (C) or CT 6 (D). The thin solid black lines represent the fitted sine-wave curves for the pretreatment traces and extended for four cycles. Gray lines indicate treatment. *Significantly different from control treatment at intervals CT 0–3 and CT 3–6, p < .05.
Figure 2
Figure 2. Temporal dynamics of PER2 expression following NPY application at ~CT 2 and 6
Normalized hourly means (± SEM) of baseline-subtracted PER2::LUC bioluminescence (see Methods) for 1.5 cycles following 1-h treatment with vehicle (CT 2: N = 4; CT 6: N = 5) or NPY (CT 2: N = 5; CT 6: N = 6) at CT 2 (panel A) or CT 6 (panel B). Note: the first two time points in each panel have fewer samples (Panel A, N = 1–4; Panel B, N = 3–6/group) due to transient increases in photon counts after relocation to the incubator/Lumicycle (see Methods).
Figure 3
Figure 3. Spike rate is positively correlated with Per1-driven fluorescence
(A) Representative photomicrograph of an SCN slice (N = 4) from a Per1::GFP mouse. Arrows indicate a dim cell (1) and a bright cell (2) within the same plane of view. (B) Representative traces from loose-patch electrophysiological recordings from cells (1) and (2) shown in (A). (C) Scatter plot depicting a significant correlation between fluorescence intensity and spike rate (Pearson correlation, R = .454, p < .05), as well as the fitted linear regression line and corresponding confidence intervals (95%).
Figure 4
Figure 4. Phase-specific effects of NPY-induced long-term suppression of spike frequency
(Left) Scatter plots of ZT and action potential frequency of cells treated with NPY at ZT 2 (A), ZT 6 (B), and ZT 10 (C). (Middle) Mean spike rate (± SEM) of NPY- and vehicle-treated groups in the first hour of recording compared to the second hour for NPY application at ZT 2 (A), ZT 6 (B), and ZT 10 (C). (Right) Representative traces for loose patch electrophysiological recordings of spontaneous action potentials from cells treated with NPY or vehicle (Control) at ZT 2 (A), ZT 6 (B), and ZT 10 (C).
Figure 5
Figure 5. NPY suppresses neuronal activity in acute SCN brain slices
Shown are the means ± SEM of hourly neuronal activity recorded in individual experiments from acute SCN brain slices. In slices treated with control medium, neuronal activity exhibited a clear peak in activity ~ZT 6, while in slices treated with NPY neuronal activity remained at relatively low levels throughout the recording period. Vertical bar indicates time of treatment: ZT 2–3 (Panel A) and ZT 6–7 (Panel B).
Figure 6
Figure 6. NPY-induced phase shifts and PER2 suppression are blocked by latent depolarization with high [K+]
(A) Means and SEMs of phase shifts resulting from NPY/vehicle (N = 6), NPY/high [K+] (N = 6), vehicle/high [K+] (N = 5), and vehicle/vehicle (N = 6) applied ~CT 6. (B) Group data illustrating the effects of all four treatment groups on PER2::LUC levels the first 12 h after treatment. Normalized and baseline-subtracted photon counts are expressed as mean ± SEM. (C) Representative bioluminescence trace from a PER2::LUC SCN explant after NPY treatment at CT6 and vehicle treatment at CT9. (D) Representative bioluminescence trace from a PER2::LUC SCN explant after NPY at CT6 and high [K+] at CT9. Gray lines indicate treatment. * Significantly different from all other treatment groups, p < .05.

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