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

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. We 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, we 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 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.

Regulation of persistent sodium currents by glycogen synthase kinase 3 encodes daily rhythms of neuronal excitability

“…For recordings in which the primary outcome was AP firing rate, the loose-patch recording technique was chosen, given that it does not affect endogenous membrane properties either by formation of a tight seal or by dialysis of intracellular milieu135455. Sample sizes were based on previous work with similar effect sizes and significant differences between groups, given the same methodologies for both loose patch and whole-cell electrophysiology565758. Fresh brain slices were prepared from mice killed between ZT 2–3 or ZT 11–12 for day and night recordings, respectively, using previously published methods (as in ref.…”

Disruption of circadian rhythmicity and suprachiasmatic action potential frequency in a mouse model with constitutive activation of glycogen synthase kinase 3

“…Electrophysiological signals were processed and controlled by a Multiclamp 700B amplifier, and pClamp 10.02 software (Axon Instruments, Union City, CA) in gap-free mode. Recordings were sampled at 20 kHz and filtered at 10 kHz (Besing et al, 2012). …”

The dynamics of GABA signaling: Revelations from the circadian pacemaker in the suprachiasmatic nucleus

“…Suppression of the firing of SCN neurons for 1 h with optogenetic manipulation has no effect on circadian phase during the night, but produces large phase delays at circadian time (CT) 0–6 and advances at CT 6–12 (Jones et al, 2015). Although acute suppression of firing rate by inhibitory neurotransmitters (e.g., NPY) does not appear to be correlated with the size of the phase shift (Gribkoff et al, 1998), the induction of persistent inhibition (lasting for 2–4 h after washout) is phase-dependent (Besing et al, 2012). Moreover, potassium induced depolarization several hours after NPY application completely blocks the NPY-induced phase shifts, suggesting that the resting membrane potential is critically important for phase resetting (Besing et al, 2012).…”

“…Although acute suppression of firing rate by inhibitory neurotransmitters (e.g., NPY) does not appear to be correlated with the size of the phase shift (Gribkoff et al, 1998), the induction of persistent inhibition (lasting for 2–4 h after washout) is phase-dependent (Besing et al, 2012). Moreover, potassium induced depolarization several hours after NPY application completely blocks the NPY-induced phase shifts, suggesting that the resting membrane potential is critically important for phase resetting (Besing et al, 2012). NPY and other neurotransmitters that typically produce phase advances during the day, hyperpolarize the resting membrane potential and reduce firing rate through activation of G-protein coupled inwardly rectifying K + (GIRK) channels (Scott et al, 2010; Hablitz et al, 2014, 2015).…”