Increased Sensitivity of Mice Lacking Extrasynaptic δ-Containing GABAA Receptors to Histamine Receptor 3 Antagonists

Abstract

Histamine/gamma-aminobutyric acid (GABA) neurons of posterior hypothalamus send wide projections to many brain areas and participate in stabilizing the wake state. Recent research has suggested that GABA released from the histamine/GABA neurons acts on extrasynaptic GABA_A receptors and balances the excitatory effect of histamine. In the current study, we show the presence of vesicular GABA transporter mRNA in a majority of quantified hypothalamic histaminergic neurons, which suggest vesicular release of GABA. As histamine/GABA neurons form conventional synapses infrequently, it is possible that GABA released from these neurons diffuses to target areas by volume transmission and acts on extrasynaptic GABA receptors. To investigate this hypothesis, mice lacking extrasynaptic GABA_A receptor δ subunit (Gabrd KO) were used. A pharmacological approach was employed to activate histamine/GABA neurons and induce histamine and presumably, GABA, release. Control and Gabrd KO mice were treated with histamine receptor 3 (Hrh3) inverse agonists ciproxifan and pitolisant, which block Hrh3 autoreceptors on histamine/GABA neurons and histamine-dependently promote wakefulness. Low doses of ciproxifan (1 mg/kg) and pitolisant (5 mg/kg) reduced locomotion in Gabrd KO, but not in WT mice. EEG recording showed that Gabrd KO mice were also more sensitive to the wake-promoting effect of ciproxifan (3 mg/kg) than control mice. Low frequency delta waves, associated with NREM sleep, were significantly suppressed in Gabrd KO mice compared with the WT group. Ciproxifan-induced wakefulness was blocked by histamine synthesis inhibitor α-fluoromethylhistidine (αFMH). The findings indicate that both histamine and GABA, released from histamine/GABA neurons, are involved in regulation of brain arousal states and δ-containing subunit GABA_A receptors are involved in mediating GABA response.

Keywords: Electroencephalogram; GABA; GABAA δ subunit; Gabrd KO mice; ciproxifan; extrasynaptic GABAA receptor; histamine; pitolisant.

Figure 1

Co-localization of Hdc mRNA with Gad67 or Vgat mRNA on maximum intensity projection images. (A) A representative image of double fluorescence in situ hybridization of Hdc and Gad67 in a coronal section of mouse brain on level −2.30 mm from Bregma. Scale bar is 200 µm. (B) A representative image of double fluorescence in situ hybridization of Hdc and Vgat in a coronal section of mouse brain on level −2.30 mm from Bregma. Scale bar is 100 µm. Small arrows indicate double-stained neurons, arrowheads denote cells with only one marker.

Figure 2

Effect of Hrh3 antagonists/inverse agonists on locomotion during the lights-on period. (A) Distance travelled in the open field after treatment with saline or different doses of ciproxifan (1, 3 and 10 mg/kg i.p.). (B) Vertical counts (frequency of rearings) in the same open field test. Number of animals is the same as in A. panel. (C) Vertical time (time spent rearing) in the same open field test. Number of animals is the same as in A. panel. (D) Distance travelled in the open field after treatment with saline or different doses of pitolisant (5 and 15 mg/kg i.p.). ^#p ≤ .05, ^##p ≤ .01, ^###p ≤ .001 for differences between the treatments.

Figure 3

Baseline EEG properties of Gabrd KO mice. (A) Schematic representation of EEG electrode placement (red dots). (B) EEG power spectrum during inactive (lights-on) and active (lights-off) periods. The black line above shows significant differences between the genotypes (n = 9 and 8 for WT and KO, respectively). (C) Representative examples of the power spectrum from 24-h baseline EEG recordings of Gabrd WT and KO mice. Arrows indicate beginning of lights-off time. Lower panels show power of delta and sigma bands. ZT – zeitgeber time. (D) Normalized power of frequency bands in Gabrd WT and KO mice (n = 9 and 8 for WT and KO, respectively).

Figure 4

Effect of ciproxifan on vigilance states during the lights-on period. (A) Relative amount of Wake, NREM, and REM episodes in Gabrd WT and KO mice during the next 4 h after treatment with saline, ciproxifan 3 or 10 mg/kg. (B) Wake, NREM, and REM bout duration in Gabrd WT and KO mice treated with saline, ciproxifan 3 or 10 mg/kg (n = 7, 5, and 5 for WT saline, cip3, and cip10, respectively; n = 6, 4, and 5 for KO saline, cip3, and cip10, respectively). Red and pink bars above show significant differences between the saline and treatment groups for each time interval (red for ciproxifan 3 mg/kg and pink for 10 mg/kg). ***p ≤ .001 for differences between the genotypes. ^##p ≤ .01, ^###p ≤ .001 for differences between the treatments.

Figure 5

Effect of ciproxifan on EEG power spectrum. (A) Example power spectrum from EEG recordings of Gabrd WT and KO mice after treatment with saline or ciproxifan 10 mg/kg. Lower panels show power of delta, alpha, and gamma2 bands in Gabrd WT and KO mice after treatment with ciproxifan 10 mg/kg. (B) Normalized power of frequency bands in Gabrd WT and KO mice treated with saline, ciproxifan 3 or 10 mg/kg (n = 7, 5, and 5 for WT saline, cip3, and cip10, respectively; n = 6, 4, and 5 for KO saline, cip3, and cip10, respectively).

Figure 6

Effect of αFMH pre-treatment on ciproxifan-induced sustained wakefulness. Relative amount of Wake, NREM, and REM episodes in Gabrd WT and KO mice pre-treated with either saline or αFMH and 20 h later treated with ciproxifan 10 mg/kg (n = 4 for WT saline+ciproxifan and αFMH+ ciproxifan; n = 3 and n = 4 for KO saline+ciproxifan and αFMH+ ciproxifan, respectively). ^#p ≤ .05, ^###p ≤ .001 for differences between the treatments.