Histamine H1 receptor on astrocytes and neurons controls distinct aspects of mouse behaviour

Abstract

Histamine is an important neurotransmitter that contributes to various processes, including the sleep-wake cycle, learning, memory, and stress responses. Its actions are mediated through histamine H₁-H₄ receptors. Gene knockout and pharmacological studies have revealed the importance of H₁ receptors in learning and memory, regulation of aggression, and wakefulness. H₁ receptors are abundantly expressed on neurons and astrocytes. However, to date, studies selectively investigating the roles of neuronal and astrocytic H₁ receptors in behaviour are lacking. We generated novel astrocyte- and neuron-specific conditional knockout (cKO) mice to address this gap in knowledge. cKO mice showed cell-specific reduction of H₁ receptor gene expression. Behavioural assessment revealed significant changes and highlighted the importance of H₁ receptors on both astrocytes and neurons. H₁ receptors on both cell types played a significant role in anxiety. Astrocytic H₁ receptors were involved in regulating aggressive behaviour, circadian rhythms, and quality of wakefulness, but not sleep behaviour. Our results emphasise the roles of neuronal H₁ receptors in recognition memory. In conclusion, this study highlights the novel roles of H₁ receptors on astrocytes and neurons in various brain functions.

Figure 1

Generation and validation of conditional Hrh1 knockout mice. (A) Schematic overview of the generation of novel conditional knockout (cKO) mice: GFAP-Cre Hrh1^f/f (left) and CaMKII-Cre Hrh1^f/f (right), were generated by crossing Cre recombinase-expressing mice with mice that carried loxP recognition sites at the Hrh1 coding sequence (CDS) of exon 3 (ex3). (B) Representative PCR genotyping results of CaMKII-Cre (upper) or GFAP-Cre (lower) transgenic mice (cropped agarose gels are shown, see Supplementary Fig. S1 for full length gels). (C) Body weight was assessed at 12 weeks of age (n = 10–12). Data were analysed with one-way ANOVA and Tukey’s post-hoc test. (D) Left: Hrh1 expression in primary astrocyte or neuron cell cultures was assessed with real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as an internal control (n = 3–6). Data were analysed with t-test (*p < 0.05 and **p < 0.01). Right: Visualisation of successful recombination at the Hrh1 loxP sites (cropped agarose gel is shown, see Supplementary Fig. S1 for full length gel). NR: no recombination (3244 bp), R: recombination (1106 bp). (E) Left: Hrh1 expression in whole brain homogenates was assessed by real-time PCR. Gapdh served as an internal control (n = 6–7). Data were analysed with one-way ANOVA and Tukey’s post-hoc test (*p < 0.05, **p < 0.01 and ***p < 0.001). Right: Visualisation of successful recombination at the Hrh1 loxP sites (cropped agarose gel is shown, see Supplementary Fig. S1 for full length gel).

Figure 2

Behavioural assessment of cKO mice. (A) Exploratory behaviour and locomotor activity were assessed using the open-field test. Assessment of travel time, travel distance, average travel speed, and time spent in the centre of mice that moved freely in the open field for 30 min (n = 13–15). Two-way ANOVA and Bonferroni post-test were used for statistical analysis. (B) Light-dark box test was used to assess anxiety-like behaviour. After a 10 min session, the time spent in the light compartment was calculated (n = 12–15). Data were analysed with one-way ANOVA and Tukey’s post-hoc test (*p < 0.05 and ***p < 0.001). (C) Elevated plus-maze test was performed to assess anxiety-like behaviour. Mice were allowed to move freely on a cross-shaped platform for 10 min. The time spent in the open arms and number of arm entries were analysed (n = 12–15). Statistical differences were assessed with one-way ANOVA and Tukey’s post-hoc test. (D) Spatial memory was tested with the Y-maze test. Alternations were calculated after an 8-min session (n = 10–15). Data were analysed with one-way ANOVA and Tukey’s post-hoc test. (E) Recognition memory was assessed using the novel object recognition test. Mice were allowed to freely explore the environment and objects for 5 min. Cut-off time for total exploration was 20 s. Mice exhibiting decreased exploratory behaviour (<5 s of total exploration) were excluded from the statistical analysis (n = 5–10). Data were analysed with two-way ANOVA and Bonferroni post-hoc test (**p < 0.01).

Figure 3

Astrocyte-specific knockout mice showed increased aggressive behaviour. Aggressive behaviour of male cKO and control mice was assessed using the resident-intruder test. Resident mice were confronted in their home cage with an unfamiliar mouse for 5 min. The number of attacks (A), latency until the first attack (B), and cumulative time of attacks (C) were analysed (n = 11–14). In case of no attack, the latency of first attack was set to 300 s and cumulative time of attacks was set to 0. Statistical significance was assessed with one-way ANOVA and Tukey’s post-hoc test (***p < 0.001).

Figure 4

Deletion of astrocytic H₁ receptors affected the onset of nocturnal activity. Total home cage locomotor activity (A) and activity during the dark period (B) in the home cages of cKO mice and controls. (A) Mice were placed in individual home cages, and their vigilance level was assessed by an infrared detector for 48 h. Mobility counts were averaged for 3-hour intervals (n = 9–10). Data were assessed with two-way ANOVA and Bonferroni post-hoc test (**p < 0.01 and ***p < 0.001). (B) Mobility counts for the entire dark period (ZT 12–24) (n = 9–10). Data were analysed with one-way ANOVA and Tukey’s post-hoc test.

Figure 5

Sleep-wake cycle was not affected by conditional Hrh1 deficiency. Results from 24 h EEG/EMG recordings of cKO and control mice. (A) Time spent in each stage averaged for 3-hour intervals (n = 6–7). (B) Graphs represent the number of bouts for each stage during the light (left) and dark (right) phase (n = 6–7). (C) Graphs represent the average duration of each stage during the light (left) and dark (right) phase (n = 6–7). All data were analysed with one-way ANOVA and Tukey’s post-hoc test.

Figure 6

Astrocytic H₁ receptor deficiency was associated with an increased amount of delta waves during wakefulness Spectral distribution of EEG power densities was analysed for stages scored as wake (A,B), NREM sleep (C), and REM sleep (D). Left graphs: The power spectra were analysed for 0–25 Hz of each vigilance state over a period of 12 h. Right graphs: The percentage of delta waves (1.5–4 Hz) across the groups was assessed for wakefulness (dark and light phase) and NREM sleep (light phase). The percentage of theta waves (6–9 Hz) was analysed for REM sleep (light phase) (n = 6). Statistical differences were assessed with one-way ANOVA and Tukey’s post-hoc test (*p < 0.05).