Suppression of serotonin neuron firing increases aggression in mice

Abstract

Numerous studies link decreased serotonin metabolites with increased impulsive and aggressive traits. However, although pharmacological depletion of serotonin is associated with increased aggression, interventions aimed at directly decreasing serotonin neuron activity have supported the opposite association. Furthermore, it is not clear if altered serotonin activity during development may contribute to some of the observed associations. Here, we used two pharmacogenetic approaches in transgenic mice to selectively and reversibly reduce the firing of serotonin neurons in behaving animals. Conditional overexpression of the serotonin 1A receptor (Htr1a) in serotonin neurons showed that a chronic reduction in serotonin neuron firing was associated with heightened aggression. Overexpression of Htr1a in adulthood, but not during development, was sufficient to increase aggression. Rapid suppression of serotonin neuron firing by agonist treatment of mice expressing Htr1a exclusively in serotonin neurons also led to increased aggression. These data confirm a role of serotonin activity in setting thresholds for aggressive behavior and support a direct association between low levels of serotonin homeostasis and increased aggression.

Figure 1.

Normal number of serotonin neurons in Htr1a^RO mice. A, Representative images of brain sections containing the dorsal raphe nucleus taken from Htr1a^RO and control littermates stained with anti-Tph antibodies. B, No significant effect of genotype on total number of Tph-positive cells in dorsal and median raphe was observed (mean ± SEM, N = 4). Scale bars, 200 μm.

Figure 2.

Normal anxiety behavior in Htr1a^RO mice. No differences were detected between Htr1a^RO and control littermates in total distance traveled (A), fractional distance in center (B), and time in center in the open field test (C), and time in closed arms (D), time in open arms (E), and time in center platform in the elevated-plus maze (F; open field—control: N = 12, Htr1a^RO: N = 11; elevated-plus maze—control: N = 12, Htr1a^RO: N = 15).

Figure 3.

Increased aggression in Htr1a^RO mice. Male mice were isolated for 4 weeks before testing during three trials of the resident-intruder assay at one-week intervals starting at P60. A, The proportion of Htr1a^RO mice attacking an intruder was significantly higher compared with controls. B–D, Htr1a^RO mice showed a significant decrease in latency to the first attack (B), increase in total number of attacks (C), increase in tail rattling episodes (D), but no difference in total social interaction time (E; ano-genital sniffing, crawling over, and social grooming) during each of the three trials when compared with control littermates. Htr1a^RO mice also showed a significantly increased number of attacks during the 5 min following the first biting attack (F; controls: N = 9, Htr1a^RO: N = 12; *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4.

Pattern of aggressive behavior in Htr1a^RO and control mice. A, B, Timeline (15 min) showing initiation and duration of attacks and tail rattling events for individual mice in control (A) and Hr1a^RO animals (B) during the third trial of the resident-intruder assay.

Figure 5.

Increased aggression in Htr1a^RO mice overexpressing Htr1a in adulthood. A, Htr1a^RO and control littermates were treated until P40 with doxycycline to block overexpression of Htr1a during development and tested during three trials of the resident intruder assay at one-week intervals starting at P60. B–D, Htr1a^RO mice showed a significant decrease in latency to the first attack (B), increase in total number of attacks (C), but no difference in total social interaction time (D; ano-genital sniffing, crawling over, and social grooming) when compared with control littermates (controls: N = 10, Htr1a^RO: N = 9). E, Htr1a^RO and control littermates were treated from P21 with doxycycline to block overexpression of Htr1a during adulthood and tested during three trials of the resident intruder assay at one-week intervals starting at P60. F–H, Htr1a^RO mice showed no significant differences in latency to the first attack (F), total number of attacks (G), or total social interaction time (H; ano-genital sniffing, crawling over, and social grooming) when compared with control littermates with the exception of the last trial where Htr1a^RO animals showed a significant decrease in social interaction behavior (controls: N = 7, Htr1a^RO: N = 8; *p < 0.05, **p < 0.01).

Figure 6.

Functional rescue of Htr1a autoreceptor. A, Schematic representation of Tph2-Htr1a transgene showing insertion of the Htr1a coding sequence at the start codon of the Tph2 gene in a mouse BAC. B, Crossing of the Tph2-Htr1a transgene to Htr1a^KO mice produced Htr1a^RR mice in which Htr1a expression as assessed by ¹²⁵I-MPPI autoradiography was restricted to raphe nuclei. C, Htr1a^RR mice showed significant hypothermia in response to the Htr1a agonist 8-OH-DPAT (0.5 mg/kg, s.c.). Time course and maximum hypothermic response (D) following systemic administration of the Htr1a agonist or saline in Htr1a^KO and Htr1a^RR littermates (N = 8; **p < 0.01).

Figure 7.

Electrophysiological characterization of Htr1a^RR mice. A–D, Whole-cell recordings. A, Effect of the Htr1a agonist 8-OH-DPAT on the membrane potential of dorsal raphe nucleus serotonin neurons in Htr1a^RR mice and its reversal by treatment with the Htr1a antagonist WAY100635 (50 nm). Downward deflections are responses to current pulses (−10 pA) used to monitor cell membrane resistance. B, Current–voltage relationships at different time points (a–c) of cell shown in A obtained with hyperpolarizing ramps. C, Effect of 8-OH-DPAT (100 nm) and 5-CT (300 nm) on membrane potential (V_Cell) of cells from Htr1a^KO (KO) and Htr1a^RR mice and their reversal by WAY100635 (50 nm). Numbers indicate the number of cells in each group. D, Effect of 8-OH-DPAT (100 nm) and 5-CT (300 nm) on the slope conductance (between −120 and −100 mV) determined from current–voltage ramps in cells from Htr1a^KO and Htr1a^RR mice and their reversal by WAY100635 (50 nm). E–K, Loose-seal cell-attached recordings. Representative data of the suppression of firing of serotonin (E), but not nonserotonin, constant frequency (F) and nonserotonin, irregular frequency (G) firing cells from Htr1a^RR mice by treatment with increasing doses of 8-OH-DPAT. Action current waveforms used to distinguish serotonin from nonserotonin cells are shown in the insets (calibration: E, 1 ms, 100 pA; F, 1 ms, 50 pA; G: 1 ms, 20 pA). H, Effect of single supramaximal concentration of 8-OH-DPAT (100 nm) and 5-CT (100 nm) on firing rate of serotonin and nonserotonin (Non-Ser) neurons. I, EC₅₀ curves for suppression of firing in dorsal raphe serotonin neurons in response to 5-CT showing wild-type control (WT) and functional recovery of Htr1a in Htr1a^RR. No response to 5-CT was seen in Htr1a^KO mice. Numbers indicate the total number of cells used for each genotype. J, Representative and summary (K) of data for the suppression of dorsal raphe serotonin neuron firing in wild-type control (WT), Htr1a^RR (shown in J), but not Htr1a^KO mice following treatment with the serotonin precursor tryptophan (Trp). Inset shows action current waveform used to identify serotonin neurons (calibration: 1 ms, 25 pA).

Figure 8.

Increased aggression in Htr1a^RR mice treated with 8-OH-DPAT. Three groups of male Htr1a^RR mice were isolated for 4 weeks before testing during three trials of the resident-intruder test starting at P60. Before testing on trial three, resident mice were injected with either the Htr1a agonist 8-OH-DPAT (0.2 mg/kg, s.c.) or antagonist WAY100635 (0.2 mg/kg, s.c.). A third group of mice received no injection (control). A, B, Treatment with 8-OH-DPAT caused a nonsignificant decrease in latency to first attack (A) and a significant increase in total attacks (B) toward the intruder when compared with behavior on the second trial or to behavior of WAY100635-treated mice. C, No significant difference in total social interaction time (ano-genital sniffing, crawling over, and social grooming) was observed between the three groups. Notably, no significant effect of WAY100635 was seen for any measure. No differences in behavior were detected between the three groups on the first two trials (control: N = 16, 8-OH-DPAT: N = 20, WAY100635: N = 9; **p < 0.01 Wilcoxon matched pairs test).