Orexinergic lateral hypothalamus (LH) projections to medial septum (MS) modulate ethanol-induced sedation in male and female mice and binge-like ethanol drinking in male mice only

Abstract

Orexin in both the lateral hypothalamus (LH) and medial septum (MS) is involved in sleep- and consciousness-related conditions. Since orexin modulates the intoxicating as well as rewarding effects of ethanol, this study focused on the role of orexin-projecting neurons from the LH to the MS, and this neurocircuit's role in mediating the sedative effects of alcohol. Drinking-in-the-Dark (DID) behavior was also assessed as a measure of the role of the LH-MS pathway in modulating binge-like ethanol intake, with a particular focus on sex differences in both behavioral paradigms. Male and female Hcrt-ires-cre mice received cannulation in the MS, while the LH was injected bilaterally with cre-dependent excitatory (Gq) Designer Receptor Exclusively Activated by Designer Drug (DREADD), inhibitory (Gi) DREADD or control virus. All subjects received a 3.75 g/kg dose of 20 % ethanol intraperitoneally and the sedative effect was assessed by the loss of righting reflex (LORR). After behavioral testing, brains were used for c-Fos immunohistochemistry analyses. A separate cohort of mice was used for a 2-week DID protocol using excitatory (Gq) DREADD and control virus. Gq DREADD-induced activation of the orexin neurocircuitry from the LH to the MS significantly reduced sedation time in both female and male mice. Furthermore, CNO treatment failed to alter ethanol sedation times in both animals expressing Gi DREADDs and control virus. There were no significant differences in blood ethanol concentrations (BECs) in any experimental group, suggesting that changes in sedation were not due to treatment-induced alterations of ethanol metabolism. Interestingly, in the DID study, only male mice decreased their ethanol consumption when Gq DREADDs were activated. These results provide novel evidence on the role played by this orexinergic LH to MS circuit on the sedative effects of ethanol and ethanol consumption in a sex-dependent manner. Thus, the MS should be considered further as a novel sexually dimorphic target.

Keywords: DREADDs; Drinking-in-the-Dark (DID); ethanol-induced sedation; lateral hypothalamus; medial septum; orexin; sex differences.

Figure 1:

Representative images and placement maps of cannulation in the MS and DREADD expression in the LH. A) Diagram of bilateral viral placement in the LH. B) Representative figure of DREADD expression and spread in the LH. C) Diagram of cannula placement in the MS. D) cannula placement in the in the MS for individual animals.

Figure 2:

A) Representative image of mCherry fluorescent terminals in the MS of Hcrt-ires-cre animals (YFP/Tx Red double labeling in yellow shows autofluorescence from guide cannula; Red mCherry labeling shows labeled terminals in MS). B) Representative image of pAAV-hSyn-DIO-hM3D(Gq)-mCherry fluorescence in the LH. C) Representative image of pAAV-hSyn-DIO- mCherry (control) fluorescence in the LH. D) Representative image of pAAV-hSyn-DIO-hM4D(Gi)-mCherry fluorescence in the LH. B)-D) shows the LH left and right at 20x magnification, arrow heads point to representative fluorescing neurons.

Figure 3:

MS-LH DREADD manipulation changes the sedative effects of ethanol intoxication. A) There is a significant decrease in sedation of Hcrt-cre mice when Gq DREADD becomes activated, with no significant changes in sedation time when Gi or control DREADD are manipulated. B) Ethanol metabolism rates are similar among Gq and control DREADD test subjects. C) Similarly, ethanol metabolism rates are similar among Gi and control DREADD mice. Data are represented as Mean ± SEM. *P < 0.05.

Figure 4:

c-Fos expression in the LH validates DREADD function. A) c-Fos levels in the LH are significantly lower in animals with activated Gi DREADD than animals with activated Gq DREADD, with control DREADD expression being in the middle. B) to D) shows representative images of c-Fos activation in the LH of Gq, Control, and Gi DREADD animals respectively. Black circles outline the LH area considered for analyses. Data are represented as Mean ± SEM. *P < 0.05.

Figure 5:

Gq DREADD activation of orexinergic LH-MS projection neurons blunts ethanol consumption in males only. A) No significant changes in ethanol consumption are seen in either Gq or control DREADD females when treated with CNO (900pmol) or vehicle. B) In control males, there is no change in ethanol drinking based on treatment, while Gq DREADD males significantly decreased their ethanol consumption when treated with CNO. C) No significant differences in BECs across DREADD or compound injected for female and D) male mice. Data are represented as Mean ± SEM. **P < 0.01.

Figure 6:

Schematic illustrating the effects of manipulating LH to MS orexinergic projections on ethanol sedation and consumption. Chemogenetic activation of orexinergic projections reduces ethanol sedation times in both males and females, but only modulates ethanol binge-drinking in male mice.