Genetic lesioning of histamine neurons increases sleep-wake fragmentation and reveals their contribution to modafinil-induced wakefulness

Abstract

Acute chemogenetic inhibition of histamine (HA) neurons in adult mice induced nonrapid eye movement (NREM) sleep with an increased delta power. By contrast, selective genetic lesioning of HA neurons with caspase in adult mice exhibited a normal sleep-wake cycle overall, except at the diurnal start of the lights-off period, when they remained sleepier. The amount of time spent in NREM sleep and in the wake state in mice with lesioned HA neurons was unchanged over 24 hr, but the sleep-wake cycle was more fragmented. Both the delayed increase in wakefulness at the start of the night and the sleep-wake fragmentation are similar phenotypes to histidine decarboxylase knockout mice, which cannot synthesize HA. Chronic loss of HA neurons did not affect sleep homeostasis after sleep deprivation. However, the chronic loss of HA neurons or chemogenetic inhibition of HA neurons did notably reduce the ability of the wake-promoting compound modafinil to sustain wakefulness. Thus, part of modafinil's wake-promoting actions arise through the HA system.

Keywords: NREM sleep; caspase; chemogenetics; histamine; histidine decarboxylase; lesioning; modafinil; tuberomammillary nucleus; wakefulness.

Figure 1.

Chemogenetic inhibition of HA neurons induces sedation. (A) AAV-DIO-hM4Di-mCherry was injected bilaterally into the TMN area of HDC-ires-Cre mice to generate HDC-hM4Di mice. (B) Double-label immunohistochemistry from a series of coronal sections of the TMN area from an HDC-hM4Di mouse: mCherry (Red) and HDC (Green) confirm expression of the hM4Di-mCherry receptor in HA cells. Arrowheads indicate examples of double-labeled cells. The DAPI staining (purple) labels all the nuclei of cells in the section, indicating that most cells in the TMN are not HDC-positive. The hM4Di-mCherry receptor is extensively transported into the axons of HDC cells. Scale bars, 100 μm. VTM = the ventral part of the tuberomammillary nucleus; DTM = the dorsal part of the tuberomammillary nucleus; 3V = 3rd ventricle. (C) CNO given to HDC-hM4Di mice reduced locomotion. CNO was given midway through the “lights-off” active period. Distance traveled in total 30 min and locomotion speed of HDC-hM4Di mice that received saline (n = 6 mice) or 1 mg/kg CNO (n = 6 mice) i.p. injections. (Distance traveled: t(5) = 3.7, paired t-test, p = 0.013; locomotion speed: repeated measures two-way ANOVA and Bonferroni–Holm post hoc test. F(1, 5) = 13.496; 5 min: t(25) = 2.69, p = 0.01; 10 min: t(25) = 4.06, p = 0.0004; 15 min: t(25) = 2.71, p = 0.01; 20 min: t(25) = 3.15, p = 0.004; 25 min: t(25) = 2.78, p = 0.01; 30 min: t(25) = 2.11, p = 0.04. (D) CNO given to HDC-hM4Di mice evoked NREM sleep. CNO was given midway through the “lights-off” active period. An individual example of EMG, wake (W), NREM sleep (N), and REM (R) sleep, and EEG delta power spectrum of HDC-hM4Di mice that received saline or 1 mg/kg CNO i.p. injection. (E) CNO given to HDC-hM4Di mice evokes NREM sleep. The graph on the left shows the percentage and the graph on the right the total time (5 hr) of wake, NREM, and REM sleep of HDC-hM4Di mice that had received saline (n = 5 mice) or CNO (n = 5 mice) injections. [Paired t-test. Wake: t(4) = 7.28, p = 0.0018; NREM: t(4) = −6.84, p = 0.002; REM: t(4) = −1.74, p = 0.155]. Shading indicates “lights off.” (F) CNO given to HDC-hM4Di mice increases NREM delta power and decreases higher frequency powers. EEG power spectrum and power of different frequencies of NREM sleep of HDC-hM4Di mice that received saline or 1 mg/kg CNO i.p. injection. [Paired t-test. 0.5–4 Hz: t(4) = −7.61, p = 0.001; 4–8 Hz: t(4) = 0.45, p = 0.67; 8–14 Hz: t(4) = 2.92, p = 0.04; 14–30 Hz: t(4) = 3.22, p = 0.03]. (G) AAV-DIO-mCherry was injected bilaterally into the TMN area of HDC-Cre mice to generate HDC-hM4Di mice. CNO given to HDC-mCherry mice did not change total time (3, 5, or 12 hr) of wake, NREM, and REM sleep compared with saline injection. [Repeated measures two-way ANOVA and Bonferroni–Holm post hoc test. Wake: F(1, 4) = 0.066. 3 hr: t(8) = 0.04, p = 0.96; 5 hr: t(8) = 0.39, p = 0.7; 12 hr: t(8) = 2.71, p = 0.78; NREM: F(1, 4) = 0.007. 3 hr: t(8) = 0.08, p = 0.93; 5 hr: t(8) = 0.44, p = 0.66; 12 hr: t(8) = 0.58, p = 0.57; REM: F(1, 4) = 0.0006. 3 hr: t(8) = 0.15, p = 0.87; 5 hr: t(8) = 0.08, p = 0.93; 12 hr: t(8) = 0.19, p = 0.84.] All error bars represent the sem. (H) CNO given to HDC-mCherry mice did not affect NREM delta power and higher frequency powers. EEG power spectrum and power of different frequencies of NREM sleep of HDC-mCherry mice that received saline or 1 mg/kg CNO i.p. injection. [Paired t-test. 0.5–4 Hz: t(4) = 1.64, p = 0.17; 4–8 Hz: t(4) = −0.62, p = 0.56; 8–14 Hz: t(4) = −1.31, p = 0.25; 14–30 Hz: t(4) = −1.06, p = 0.34.]

Figure 2.

Selective genetic lesioning of HA neurons. (A) AAV-DIO-taCasp3-TEV was bilaterally injected into the TMN area of HDC-ires-Cre mice to generate HDC-Casp3 mice. To generate the controls, AAV-DIO-taCasp3-TEV was injected bilaterally into the TMN area of HDC-Cre-negative mice. (B) Casp3 efficiently kills HDC neurons. Six weeks after the AAV-DIO-taCasp3-TEV injections, immunohistochemistry was undertaken for HDC. Illustrative examples of HDC immunohistochemistry from a control mouse and an HDC-Casp3 mouse coronal section for the TMN area (three representative coronal sections on the rostral–caudal axis for HDC-immunostaining in the TMN are shown). The green dots indicate neuronal cell bodies stained for HDC, 3V, third ventricle. Scale bar, 200 μm. (C) Mapping the extent of HDC cell lesioning. Line drawings of sections showing HDC-positive cells (green dots) from individual control mice (n = 4 mice, designated as “mouse1” through to “mouse 4”) and HDC-Casp3 mice (n = 4 mice, designated as “mouse 1” through to “mouse 4”) along most of the rostral–caudal axis of the TMN area (bregma −1.94 to bregma −3). Few HDC-positive cells remained in the sections from the HDC-Casp3 mice. (D) Counts of HDC cell numbers along the rostral–caudal axis per section (bregma −1.94 to bregma −3) (left-hand graph) and total HDC cell numbers of control mice (n = 6 mice) and HDC-Casp3 mice (n = 6 mice) [t(10) = 10.86, unpaired t-test, p = 7.4E-7] (right-hand graph). All error bars represent the sem. The shaded envelopes on left-hand graph indicate sem.

Figure 3.

Ablation of HA neurons does not affect the overt sleep–wake cycle but induces more fragmented wakefulness and NREM sleep. (A, B, C) Percentage and time of wake, NREM, and REM sleep of HDC-Casp3 mice (n = 6 mice) and control mice (n = 9 mice) over the 24 hr cycle. [Unpaired t-test. Lights on: wake t(13) = 0.12, p = 0.9; NREM t(13) = −0.3, p = 0.76; REM t(13) = 0.19, p = 0.84; lights off: wake t(13) = 1.76, p = 0.1; NREM t(13) = −1.85, p = 0.08; REM t(13) = −0.83, p = 0.41]. (D, E, F) Episode duration of wake, NREM, and REM sleep of HDC-Casp3 mice (n = 6 mice) and control mice (n = 9 mice) across the 24 hr cycle and “lights on” and “lights off” periods [Unpaired t-test. Lights on: wake t(13) = 1.74, p = 0.1; NREM t(13) = 1.51, p = 0.15; REM t(13) = −0.46, p = 0.64; lights off: wake t(13) = 2.57, p = 0.02; NREM t(13) = 2.91, p = 0.01; REM t(13) = 0.64, p = 0.52.] (G, H) Vigilance state transitions of HDC-Casp3 mice (n = 6 mice) and control mice (n = 9 mice) during the “lights on” and “lights off” periods [Unpaired t-test. Lights off: wake to NREM t(13) = −3.69, p = 0.002; NREM to wake t(13) = −4.26, p = 9.2E-4.] All error bars represent the sem.

Figure 4.

Ablation of HA neurons does not affect the EEG power spectrum during the spontaneous sleep–wake cycle. (A, B) EEG delta (0.5–4 Hz) and theta (4–8 Hz) power of wakefulness, NREM sleep, or REM sleep of control and HDC-Casp3 mice during the 12 hr “lights on” period (A) or the 12 hr “lights off” period (B). Two-way ANOVA and Bonferroni–Holm post hoc test. (Lights on: wake t = 0.65, p = 0.51; NREM t = 1.06, p = 0.29; REM t = −0.08, p = 0.93; lights off: wake t = 0.84, p = 0.4; NREM t = 0.63, p = 0.52; REM t = −0.16, p = 0.87.)

Figure 5.

Chronic lesioning of HA neurons partially attenuates modafinil-induced wakefulness. (A) An individual example of EMG, wake (W), NREM sleep (N), and REM (R) sleep, and EEG delta power of a control mouse that received vehicle or modafinil by i.p. injection. (B) Percentages of wake, NREM, and REM sleep of control mice (n = 6 mice) that received vehicle or modafinil i.p. injection. (C) An individual example of EMG, wake (W), NREM sleep (N), and REM (R) sleep, and EEG delta power of an HDC-Casp3 mouse that received vehicle or modafinil by i.p. injection. (D) Percentages of wake, NREM, and REM sleep of HDC-Casp3 mice (n = 5 mice) that received vehicle or modafinil injections. (E) Time (8 hr) of wake, NREM, and REM sleep of control mice (n = 6 mice) and HDC-Casp3 mice (n = 5 mice) that received vehicle or modafinil injections (control modafinil vs. CASP3 modafinil: wake: F(1, 4) = 5.57, t(4) = 5.1, p = 0.006; NREM: F(1, 4) = 6.48, t(4) = 5.42, p = 0.005; REM: F(1, 4) = 1.11, t(4) = 2.3, p = 0.08. (F) Sleep latency to NREM sleep of control mice (n = 6 mice) and HDC-Casp3 mice (n = 5 mice) that received vehicle or modafinil injections. [F(1, 4) = 7.56, control modafinil vs. CASP3 modafinil: t(4) = 4.18, p = 0.01.] Repeated measures two-way ANOVA and Bonferroni–Holm post hoc test. All error bars represent the sem.

Figure 6.

Chemogenetic inhibition of HA neurons partially attenuates modafinil-induced wakefulness. (A) An individual example of EMG, wake (W), NREM sleep (N), and REM (R) sleep, and EEG delta power of a control mouse that received saline and vehicle or saline and modafinil by i.p. injection. (B) Percentages of wake, NREM, and REM sleep of control mice (n = 5 mice) that received saline and vehicle or saline and modafinil i.p. injection. (C) An individual example of EMG, wake (W), NREM sleep (N), and REM (R) sleep, and EEG delta power of an HDC-hM4Di mouse that received CNO and vehicle or CNO and modafinil by i.p. injection. (D) Percentages of wake, NREM and REM sleep of HDC-hM4Di mice (n = 5 mice) that received CNO and vehicle or CNO and modafinil injections. (E) Time (8 hr) of wake, NREM, and REM sleep of HDC-hM4Di saline-injected mice (n = 5 mice) and HDC-hM4Di CNO-injected mice (n = 5 mice) that received vehicle or modafinil injections [saline and modafinil vs. CNO and modafinil: wake: F(1, 4) = 11.52, t(4) = 5.5, p = 0.005; NREM: F(1, 4) = 8.89, t(4) = 5.59, p=0.005; REM: F(1, 4) = 44.08, t(4) = 5.1, p = 0.006.] (F) Sleep latency to NREM sleep of HDC-hM4Di saline-injected mice (n = 5 mice) and HDC-hM4Di CNO-injected mice (n = 5 mice) that received vehicle or modafinil injections. [F(1, 4) = 10.23, saline and modafinil vs. CNO and modafinil: t(4) = 3.56, p = 0.02.] Repeated measures two-way ANOVA and Bonferroni–Holm post hoc test. All error bars represent the sem.

Figure 7.

Ablation of HA neurons does not affect sleep homeostasis after sleep deprivation or modafinil-induced wakefulness. (A, B) EEG power spectrum of NREM sleep (A) and total delta power (0.5–4 Hz) of NREM sleep (B) of control and HDC-Casp3 mice during the first hour of recovery sleep after modafinil-induced wakefulness. (C, D) EEG power spectrum of NREM sleep (A) and total delta power (0.5–4 Hz) of NREM sleep (B) of control and HDC-Casp3 mice during the first hour of recovery sleep after sleep deprivation. [Paired t-test. (B) Control: t(5) = −17.52, p = 0.00001; HDC-Casp3: t(5) = −4.62, p = 0.009; (D) control: t(4) = −7.6, p = 0.001; HDC-Casp3: t(3) = −15.41, p = 0.0005.]