Ventrolateral periaqueductal gray mediates rapid eye movement sleep regulation by melanin-concentrating hormone neurons

Abstract

Neurons containing melanin-concentrating hormone (MCH) in the lateral hypothalamic area (LH) have been shown to promote rapid eye movement sleep (REMs) in mice. However, the downstream neural pathways through which MCH neurons influence REMs remained unclear. Because MCH neurons are considered to be primarily inhibitory, we hypothesized that these neurons inhibit the midbrain 'REMs-suppressing' region consisting of the ventrolateral periaqueductal gray and the lateral pontine tegmentum (vlPAG/LPT) to promote REMs. To test this hypothesis, we optogenetically inhibited MCH terminals in the vlPAG/LPT under baseline conditions as well as with simultaneous chemogenetic activation of MCH soma. We found that inhibition of MCH terminals in the vlPAG/LPT significantly reduced transitions into REMs during spontaneous sleep-wake cycles and prevented the increase in REMs transitions observed after chemogenetic activation of MCH neurons. These results strongly suggest that the vlPAG/LPT may be an essential relay through which MCH neurons modulate REMs.

Keywords: REM sleep transitions; archearhodopsin T; chemogenetics; optogenetics; paradoxical sleep; sublaterodorsal nucleus.

Figure 1.. Anterograde tracing from lateral hypothalamic MCH neurons to the primary sites of REMs control.

MCH neurons (A– injection site in LH) innervate the REMs-suppressing vlPAG/LPT (B) as well as the REMs-generating SLD (C) in the brain stem, as indicated by the presence of ArchT-GFP labeled axon terminals (black color) in these regions. Black squares in A, B and C represent the regions magnified in A′, B′ and C′ and white squares in B′ and C′ represent the regions magnified in the Bʹʹ and Cʹʹ. Red arrowheads in Bʹʹ and Cʹʹ indicate individual boutons. Scale bars are: (A) 1mm; (B, C) 0.5 mm; (A′) 200 μm; (B′, C′) 100 μm; (Bʹʹ and Cʹʹ) 20 μm. 3V, 3^rd ventricle; 4V 4^th ventricle; Aq, aqueduct; f, fornix.

Figure 2.. Chemoactivation and optogenetic inhibition of MCH neurons.

(A) Triple-labeled neurons in the LH indicating the expression of hM3Dq-mCherry (red; native florescence) and ArchT-GFP (green; native florescence) in MCH neurons (blue; immunofluorescence labeling with rabbit anti-MCH and goat anti-rabbit antibodies). (B) Representative whole cell, current clamp recording from an ArchT-expressing MCH neuron (identified by GFP fluorescence) indicating hyperpolarization and complete cessation of action potentials induced by 565 nm light illumination (yellow/orange line above the trace). (C,D) c-Fos activity (black staining) in hM3Dq- and ArchT-expressing MCH neurons (mCherry labeling in brown) 2 h after saline (C) or CNO (0.3 mg/kg) application (D) indicating activation of MCH neurons by CNO. Scale bars are: (A) 20 μm; (C,D) overview images 100 μm; magnified images 50 μm.

Figure 3:. Map of injection sites and optical fiber tip locations in MCH-Cre mice.

(A) Outlines of AAV injection sites in three levels of the hypothalamus [AP −1.46, −1.70 and −1.94 (Franklin and Paxinos, 2007)]. (B) Location of optical fiber tips mapped onto two levels of the brainstem [AP −4.16 and −4.36 (Franklin and Paxinos, 2007)]. Animal IDs of all mice (n = 9) are listed in corresponding colors with injection site outlines and fiber placement markings. 3V, 3^rd ventricle; Aq, aqueduct; DMH, dorsomedial hypothalamic nucleus; DR, dorsal raphe nucleus; vlPAG/LPT, ventrolateral periaqueductual gray/lateral pontine tegmentum; PPT, pedunculopontine tegmental nucleus; VMH, ventromedial hypothalamic nucleus; xscp, decussation of the superior cerebellar peduncle.

Figure 4:. Chemoactivation of MCH neurons specifically increases REMs.

Percentages of time spent in wake, NREMs and REMs (A, D, G), as well as number of bouts (B, E, H) and mean bout duration (C, F, I) during the 3 h recording period after saline (white bars) or CNO (black bars) injections in MCH-Cre mice (n = 9). Saline or CNO (0.3 mg/kg, i.p.) was administered at ZT6 and sleep-wake recordings were conducted between ZT7-ZT10. CNO injections specifically increased the amount of REMs (p=0.0021, 2-tailed paired t-test) and the number of REMs bouts (p=0.0106, 2-tailed paired t-test) without affecting other stages. Data are Mean ± SEM; * P < 0.05, ** P < 0.01.

Figure 5:. Optogenetic inhibition of MCH terminals in the vlPAG/LPT decreases NREMs-REMs transitions.

(A) Schematic illustration of the photo-inhibition paradigm during NREMs and REMs in MCH-Cre mice. Yellow/orange light (593.5 nm wave length) illuminations were applied after 30 seconds of stable NREMs and continued until the next wake bout irrespective of whether NREMs transitioned into REMs or wake. (B) Percentage of NREMs bouts transitioning to REMs. Repeated measures (RM) two-way ANOVA for ‘Drug treatment’ (F_(1,8)=32.95, p=0.0004) and ‘Laser treatment’ (F_(1,8)=49.73, p=0.0001), followed by Holm-Sidak’s multiple comparison test (saline + sham inhibition vs. saline + laser light, p=0.0068; saline + sham inhibition. vs. CNO + sham inhibition, p=0.0363; saline + sham inhibition vs. CNO + laser light, p=0.0309; saline + laser light vs. CNO + sham inhibition, p=0.0004; CNO + sham inhibition vs. CNO + laser light, p=0.0012). RM two-way ANOVAs for the duration of REMs bouts (C) and the latency to REMs onset (D) were not statistically significant (REMs bout duration ‘Drug treatment’ F_(1,8)=2.853, p=0.1297, ‘Laser treatment’ F_(1,8)=0.452, p=0.5203; REMs latency ‘Drug treatment’ F_(1,8)=1.797, p=0.2169, ‘Laser treatment’ F_(1,8)=0.5166, p=0.4928). (E) NREMs bout duration. RM two-way ANOVA for ‘Drug treatment’ (F_(1,8)=8.766, p=0.0181) and ‘Laser treatment’ F_(1,8)=19.3, p=0.0023, followed by Holm-Sidak’s multiple comparison test (saline + sham inhibition. vs. CNO + laser light, p=0.0282). (F) Percentage of NREMs during the 15 trials (starting with NREMs) within the 3h recording period. RM two-way ANOVA for ‘Drug treatment’ (F_(1,8)=6.879, p=0.0305) and ‘Laser treatment’ (F_(1,8)=61.61, p=0.≤0.0001), followed by Holm-Sidak’s multiple comparisons test (saline + sham inhibition vs. saline + laser light, p=0.05; saline + laser light vs. CNO + sham inhibition, p=0.0127; CNO + sham inhibition vs. CNO + laser light, p=0.0346). (G) Percentage of REMs during the 15 trials (starting with NREMs) within the 3h recording period. RM two-way ANOVA for ‘Drug treatment’ (F_(1,8)=6.879, p=0.0305) and ‘Laser treatment’ (F_(1,8)=61.61, p≤0.0001), followed by Holm-Sidak’s multiple comparisons test (saline + sham inhibition vs. saline + laser light, p=0.05; saline + laser light vs. CNO + sham inhibition, p=0.0127; CNO + sham inhibition vs. CNO + laser light, p=0.0346). Saline or CNO (0.3 mg/kg; i.p.) was injected at ZT6 and sham/laser light illumination was applied between ZT7-ZT10 (n = 9 mice). Data are Mean ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Scale bar in (A) is 20 sec.