Pharmacogenetic modulation of orexin neurons alters sleep/wakefulness states in mice

Abstract

Hypothalamic neurons expressing neuropeptide orexins are critically involved in the control of sleep and wakefulness. Although the activity of orexin neurons is thought to be influenced by various neuronal input as well as humoral factors, the direct consequences of changes in the activity of these neurons in an intact animal are largely unknown. We therefore examined the effects of orexin neuron-specific pharmacogenetic modulation in vivo by a new method called the Designer Receptors Exclusively Activated by Designer Drugs approach (DREADD). Using this system, we successfully activated and suppressed orexin neurons as measured by Fos staining. EEG and EMG recordings suggested that excitation of orexin neurons significantly increased the amount of time spent in wakefulness and decreased both non-rapid eye movement (NREM) and rapid eye movement (REM) sleep times. Inhibition of orexin neurons decreased wakefulness time and increased NREM sleep time. These findings clearly show that changes in the activity of orexin neurons can alter the behavioral state of animals and also validate this novel approach for manipulating neuronal activity in awake, freely-moving animals.

Figure 1. Specific expression of hM3D_q and hM4D_i in orexin neurons in mice.

A, Schematic representation of double-floxed Cre-dependent AAV vector expressing hM3D_q and hM4D_i under control of EF-1α promoter (rAAV-DIO-HAhM3Dq and rAAV-DIO-HAhM4Di). DIO, double-floxed inverted open reading frame; ITR, inverted terminal repeat; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. B, Coronal brain sections at the level of LHA (bregma-1.8 mm), prepared from orexin-cre mice expressing hM3D_q or hM4D_i following injection of Cre-dependent rAAV-DIO-HAhM3Dq or rAAV-DIO-HAhM4Di. Sections were stained with anti-HA antibody. Scale bars, 40 µm. C, Upper panels, Coronal section of LHA prepared from orexin-cre mice expressing hM3D_q and double stained with anti-orexin antibody (green) and anti-HA antibody (red), showing that most of orexin-ir neurons express HAhM3D_q. Lower panels, Coronal section of LHA prepared from orexin-cre mice expressing hM4D_i and double stained with anti-orexin antibody (green) and anti-HA antibody (red), showing that most of orexin-ir neurons express HAhM4D_i.

Figure 2. Activation or inhibition of orexin neurons by DREADD.

A–D, Representative images of Fos expression in orexin neurons, as shown by double staining of the LHA regions of wild type (A, C) and orexin-cre mice (B, D) injected with cre-activatable AAV carrying hM3D_q (A, B) or hM4Di (C, D) (rAAV-DIO-HAhM3Dq or rAAV-DIO-HAhM4Di) after injection of CNO. A, Only a small numbers of Fos-IR nuclei were observed in orexin-immunoreactive neurons from wild type mice administered CNO at 13:00 (ZT4) and killed at 15:00 (ZT6). Inset, high power view. B, Number of Fos- and orexin-double-positive neurons was higher in orexin-cre than in wild type (A) administered CNO at ZT4 and killed at ZT6. Inset, high power view. C, Many Fos- and orexin-double positive neurons were observed in wild type mice administered CNO at ZT12 and killed at ZT14. Inset, high power view. D, Fewer Fos- and orexin-double-positive neurons were observed in orexin-cre than in wild type mice administered CNO at ZT12 and killed at ZT14. Inset, high power view. E, Numbers of double-positive neurons at ZT6 in wild type and orexin-cre transgenic mice injected with hM3D_q virus and administered CNO (N = 5). F, Numbers of double-positive neurons at ZT14 in wild type and orexin-cre transgenic mice injected with hM4D_i virus and administered CNO (N = 5).

Figure 3. Effect of stimulation of orexinergic tone by hM3D_q on vigilance states of mice during light and dark periods (left and right panels, respectively).

A. Hourly analysis of sleep/wake states in transgenic and wild-type mice, both injected with rAAV-DIO-HAhM3Dq, after administration of CNO at ZT4 or ZT12. Amounts of wakefulness (Wake, upper panels), NREM sleep (middle panels), and REM sleep (lower panels) are shown. B, Latency to NREM sleep and REM sleep after CNO administration (N = 6 for transgenic mice, N = 5 for wild-type mice).

Figure 4. Effect of inhibition of orexinergic tone by hM4D_i on vigilance states of mice during light and dark periods (left and right panels, respectively).

A. Hourly analysis of sleep/wake states in transgenic and wild-type mice, both injected with rAAV-DIO-HAhM4Di, after administration of CNO at ZT4 or ZT12. Amounts of wakefulness (Wake, upper panels), NREM sleep (middle panels), and REM sleep (lower panels) are shown. B. Latency to NREM sleep and REM sleep after CNO administration (N = 5).