Histamine N-methyltransferase regulates aggression and the sleep-wake cycle

Abstract

Histamine is a neurotransmitter that regulates diverse physiological functions including the sleep-wake cycle. Recent studies have reported that histaminergic dysfunction in the brain is associated with neuropsychiatric disorders. Histamine N-methyltransferase (HNMT) is an enzyme expressed in the central nervous system that specifically metabolises histamine; yet, the exact physiological roles of HNMT are unknown. Accordingly, we phenotyped Hnmt knockout mice (KO) to determine the relevance of HNMT to various brain functions. First, we showed that HNMT deficiency enhanced brain histamine concentrations, confirming a role for HNMT in histamine inactivation. Next, we performed comprehensive behavioural testing and determined that KO mice exhibited high aggressive behaviours in the resident-intruder and aggressive biting behaviour tests. High aggression in KO mice was suppressed by treatment with zolantidine, a histamine H2 receptor (H2R) antagonist, indicating that abnormal H2R activation promoted aggression in KO mice. A sleep analysis revealed that KO mice exhibited prolonged bouts of awakening during the light (inactive) period and compensatory sleep during the dark (active) period. Abnormal sleep behaviour was suppressed by treatment with pyrilamine, a H1R antagonist, prior to light period, suggesting that excessive H1R activation led to the dysregulation of sleep-wake cycles in KO mice. These observations inform the physiological roles of HNMT.

Figure 1

Hnmt deficiency increases brain histamine levels. (A) Histamine content in cortex, diencephalon, brainstem, and cerebellum homogenates (n = 5). 1-mHA, 1-methylhistamine; HA, histamine; n.d., not detected. (B) Histamine content in whole brain homogenates at various ages (n = 5–9). White bars, wild type (WT); black bars, knockout (KO). Student’s t-test, *p < 0.05. (C) Extracellular histamine concentrations in the hypothalamic area of WT (white circles) and KO mice (black square) for 24 h. Light period (white horizontal bar): ZT0-12, Dark period (black horizontal bar): ZT12-24 (n = 8).

Figure 2

HNMT-deficient mice exhibit high aggressive behaviour and low locomotor activity. (A) Time spent in the open arms of the elevated plus-maze (n = 10–12). (B) Time spent in the open arms of the elevated zero-maze (n = 10–12). (C) Time spent in the light box in the light/dark box test (n = 5–8). (D) Immobility time in the forced swim test (n = 8). (E) Alternation ratio in the Y-maze test (n = 8). (F) Latencies at 1 day or 7 days after training in the passive avoidance test (n = 8). (G) Latency to fall in the first and second trials of the rotarod test. (H) Total distance in the treadmill test (n = 10). (I) Withdrawal latency in the hot plate test (n = 10–12). (J) Time spent in the empty and stranger chambers in the three-chamber test (n = 5–8). (K) The number of attacks in the resident-intruder test (n = 8–11). (L) Ratios of the average speed, distance, movement time, and central area duration in the open field test (n = 5–8) (Student’s t-test, *p < 0.05). The performance value of WT mice was set to 1. (M) Locomotor activities in the home-cage. Light period (white horizontal bar), ZT0-12; dark period (black horizontal bar), ZT12-24 (n = 10) (Student’s t-test, *p < 0.05). (N) Immobility time in the home-cage during the first 2 h of the dark period (ZT12-14; white bars, WT; black bars, KO) (n = 10) (Student’s t-test, *p < 0.05).

Figure 3

Increased aggression in Hnmt-deficient mice is driven by H2R activation. (A) Images of wild type (WT, left) and knockout (KO, right) mice in their home cages. Most KO mice had skin injures. (B) Ratios of wounded mice (n = 26–28). (C) Intensity of biting behaviours on the aggressive response meter (n = 8) (Student’s t-test, *p < 0.05). (D) Serum testosterone concentrations (pg/mL). (E) The number of attacks over 5 min in the resident-intruder test with histamine receptor antagonist pretreatment. White bar, saline; red bar, 10 mg/kg pyrilamine (H1R antagonist); blue bar, 10 mg/kg zolantidine (H2R antagonist) (two-way ANOVA with Bonferroni correction; *p < 0.05; n.s.: not significant).

Figure 4

Sleep-wake cycle abnormalities in Hnmt-deficient mice are driven by H1R activation. (A) The ratios of wakefulness, NREM sleep, and REM sleep on the indicated Zeitgeber time (ZT) periods (n = 8). Light period (white horizontal bar), ZT0-12; dark period (black horizontal bar), ZT12-0 (n = 8, Student’s t-test; *p < 0.05). White bars, wild type (WT); black bars, knockout (KO). (B) Average duration of bouts during ZT0–6 and ZT12–18 (n = 8) (Student’s t-test, *p < 0.05). (C) Number of bouts during ZT0-6 and ZT12-18 (n = 8). (D) The spectral distribution of cortical EEG power density during wakefulness in the light and dark periods. Grey line, WT; black line, KO. Horizontal bars indicate statistical differences between the WT and KO groups. Bar graphs show the total power of EEG between 3.0 and 5.5 Hz. White bar, WT; black bar KO. (n = 8) (Student’s t-test, *p < 0.05). (E) Effects of pyrilamine and zolantidine administration at ZT0 on wakefulness in WT and KO mice during ZT0-6 and ZT12-18. (F) Effects of pyrilamine and zolantidine administration at ZT12 on wakefulness in WT and KO mice during ZT12-18 and ZT0-6. White bar, saline; red bar, 10 mg/kg pyrilamine (H1R antagonist); blue bar, 10 mg/kg zolantidine (H2R antagonist) (n = 6–8) (two-way ANOVA with Bonferroni multiple comparisons test; *p < 0.05; n.s.: not significant).