Inactivation of median preoptic nucleus causes c-Fos expression in hypocretin- and serotonin-containing neurons in anesthetized rat

Abstract

The median preoptic nucleus (MnPN) of the hypothalamus contains sleep-active neurons including sleep-active GABAergic neurons and is involved in the regulation of nonREM/REM sleep. The hypocretinergic (HCRT) neurons of the perifornical-lateral hypothalamic area (PF-LHA) and serotonergic (5-HT) neurons of the dorsal raphe nucleus (DRN) are mostly active during waking and have been implicated in the regulation of arousal. MnPN GABAergic neurons project to the PF-LHA and DRN. It is hypothesized that MnPN promotes sleep by inhibiting multiple arousal systems including HCRT and other wake-active neurons within the PF-LHA and 5-HT neurons in the DRN. We examined the effects of inactivation of MnPN neurons by locally microinjecting 0.2 microl of 1 mM or 10 mM solutions of a GABA(A) receptor agonist, muscimol, into the MnPN on Fos expression (Fos-IR) in the PF-LHA neurons including HCRT neurons and 5-HT neurons in the DRN in anesthetized rats. Compared to artificial cerebrospinal fluid control, microinjection of muscimol into the MnPN resulted in significantly higher percentages of HCRT and non-HCRT neurons in the PF-LHA and 5-HT neurons in the DRN that exhibited Fos-IR. The percentage of melanin-concentrating hormone (MCH)+/Fos+ neurons in the PF-LHA did not change after muscimol treatments. These results support a hypothesis that the activation of MnPN neurons contributes to the suppression of wake-promoting systems including HCRT and other unidentified neurons in the PF-LHA and 5-HT neurons in the DRN. These results also suggest that MCH neurons may not be under MnPN inhibitory control. These findings are consistent with a hypothesized role of MnPN in sleep regulation.

Figure 1. Locations of the microinjection sites

Photomicrograph of a coronal section (20X magnification) through the MnPN of a rat, which was injected with aCSF, showing the tract of the injector cannula and the site of microinjection (A). Reconstruction diagrams through MnPN showing the locations of aCSF (B) and muscimol microinjection sites (C). D. Photomicrographs (40X) through the PF-LHA showing the anatomical distributions of the HCRT and MCH neurons and the grid system that used for the analyses. ac, anterior commissure; fx, fornix; LPO, lateral preoptic area; MnPN, median preoptic nucleus; MPO, medial preoptic area; mt, mammillothalamic tract; oc, optic chiasm; 3V, third ventricle.

Figure 2. Effects of muscimol microinjection into the MnPN on Fos-IR in HCRT+ and MCH+ neurons

Photomicrographs of coronal sections (100X magnification) from individual anesthetized rats microinjected with aCSF and 10 mM muscimol on HCRT neurons (A&B) and MCH neurons (E&F). The magnified images (600X) of the marked sections in figures A&B and E&F are shown in C&D and G&H, respectively. In aCSF treated animals, fewer Fos+ and HCRT+/Fos+ neurons were observed. In muscimol microinjected rats, although the numbers of HCRT+/Fos+ and single Fos+ neurons were higher, the numbers of MCH+/Fos+ neurons were similar to aCSF treated animals. Filled arrow, HCRT+/Fos+ or MCH+/Fos+ neuron; blank arrow, HCRT⁺/Fos- or MCH+/Fos- neuron; star, single Fos+ neuron.

Figure 3. Effects of muscimol microinjection into the MnPN on Fos-IR in PF-LHA neurons

Mean (±SEM) percentages of HCRT+/Fos+ (A), MCH+/Fos+ neurons (B), and the number of single Fos+ neurons (C) in perifornical (grid-1), medial (grid-2) and lateral (grid-3) part of the HCRT and MCH neuronal fields after aCSF and 2 doses of muscimol microinjections into the MnPN. As compared to the aCSF control, the percentages of HCRT+/Fos+ and single Fos-IR neurons increased significantly after muscimol microinjection into the MnPN. As compared to the HCRT+ neurons, significantly lower percentages of MCH+ neurons expressed Fos-IR after aCSF or muscimol microinjections. *, as compared to the aCSF treatment; *, P < 0.05; **, P < 0.01 level of significance.

Figure 4. Effects of muscimol microinjection into the MnPN on Fos-IR in 5-HT neurons

Mean (±SEM) percentages of 5-HT+/Fos+ (A), and the number of single Fos+ neurons (B) in the DRN after aCSF and 2 doses of muscimol microinjections into the MnPN. As compared to aCSF, the percentages of 5-HT+/Fos+ increased significantly after muscimol microinjection into the MnPN. The number of single Fos+ neurons also increased but that was not to a significant level. C. Photomicrograph of a coronal section (40X magnification) through the DRN of a rat showing the anatomical distributions of the 5-HT neurons and the grid system that used for the analyses. Photomicrographs (600X magnification) of sections through DRN after aCSF and 10mM muscimol microinjection are shown in D and E, respectively. In aCSF treated animals, fewer Fos+ and 5HT+/Fos+ neurons were observed, whereas in muscimol microinjected rats the numbers of 5-HT+/Fos+ and single Fos+ neurons were higher. Filled arrow, 5-HT+/Fos+ neuron; blank arrow, 5-HT⁺/Fos- neuron; star, single Fos+ neuron; aq, aqueduct; DRD, dorsal raphe nucleus, dorsal part; DRV, dorsal raphe nucleus, ventral part; DRVL, dorsal raphe nucleus, ventrolateral part; PAG, periaqueductal gray.