Neurons containing orexin or melanin concentrating hormone reciprocally regulate wake and sleep

Abstract

Neurons containing orexin (hypocretin), or melanin concentrating hormone (MCH) are intermingled with each other in the perifornical and lateral hypothalamus. Each is a separate and distinct neuronal population, but they project to similar target areas in the brain. Orexin has been implicated in regulating arousal since loss of orexin neurons is associated with the sleep disorder narcolepsy. Microinjections of orexin into the brain or optogenetic stimulation of orexin neurons increase waking. Orexin neurons are active in waking and quiescent in sleep, which is consistent with their role in promoting waking. On the other hand, the MCH neurons are quiet in waking but active in sleep, suggesting that they could initiate sleep. Recently, for the first time the MCH neurons were stimulated optogenetically and it increased sleep. Indeed, optogenetic activation of MCH neurons induced sleep in both mice and rats at a circadian time when they should be awake, indicating the powerful effect that MCH neurons have in suppressing the wake-promoting effect of not only orexin but also of all of the other arousal neurotransmitters. Gamma-Aminobutyric acid (GABA) is coexpressed with MCH in the MCH neurons, although MCH is also inhibitory. The inhibitory tone of the MCH neurons is opposite to the excitatory tone of the orexin neurons. We hypothesize that strength in activity of each determines wake vs. sleep.

Keywords: hypothalamus; melanin concentrating hormone; optogenetics; sleep.

Figure 1

Distribution of MCH-immunoreactive neurons in wildtype C57Bl6/j mice (3 months old). Coronal sections (40 µm thick) were processed for immunohistochemical detection of MCH-immunoreactivity (rabbit anti-MCH; 1:5000 dilution; overnight incubation) and visualized using the avidin-biotin -diaminobenzidene method. The MCH-ir neurons were present only in the hypothalamus (boxed area in photo A). A major cluster is located in the zona incerta (photo B) and extends medially to the ventricle. Another major cluster is located laterally (arrowheads in D). Minor clusters are located around the fornix (photo C) and ventrally along the dorsal border of the VMH (arrow in photo D). The numbers in (B–D) represent distance (millimeters) caudal to bregma. The calibration bar in (D) = 250 µm. Abbreviations: 3v= third ventricle; f = fornix; mt = mammillothalamic tract; ot = optic tract; ZI = zona incerta.

Figure 2

Expression of ChR2-eYFP in MCH-immunoreactive neurons in the lateral hypothalamus of a representative wildtype C57BL/6J mouse. Compare with Figure 1. Panel (A) depicts the expression of ChR2-eYFP (green) in MCH neurons (red). Notice that the MCH neurons are diffusely distributed around the fornix and many MCH neurons located medial and ventral to the fornix did not contain the light-sensitive opsin. Panel (B) is a higher magnification view of a cluster of MCH neurons (arrow in panel A) that also contain ChR2-EYFP. Panel (C) depicts the close relationship between the orexin neurons (red) and MCH neurons (green ChR2-eYFP). The arrows in panel (C) show the encirclement of an orexin soma by MCH-ChR2-EYFP processes. Scale bar in (A) is 250 µm, (B) is 50 µm and (C) is 60 µm. Abbreviations: 3V = third ventricle; mt = mammillothalamic tract; mfb = medial forebrain bundle; f = fornix; VMH = ventromedial hypothalamus.

Figure 3

MCH/GABA neurons are antagonistic to the orexin neurons. We hypothesize that local interaction between these two opposing neurons regulate sleep and wake. Glia are also likely to interact with neurons along the lines hypothesized by Haydon’s studies. Abbreviations: Gamma-Aminobutyric acid (GABA); Norepinephrine (NE); Serotonin (5-HT); Acetylcholine (Ach); Dopamine (DA); Neuropeptide Y (NPY); Corticotropin releasing factor (CRF); Cocaine-Amphetamine-Regulated Transcript (CART); Neuronal activity regulated pentraxin (Narp).