Genetic depletion of brain 5HT reveals a common molecular pathway mediating compulsivity and impulsivity

Abstract

Neuropsychiatric disorders characterized by behavioral disinhibition, including disorders of compulsivity (e.g. obsessive-compulsive disorder; OCD) and impulse-control (e.g. impulsive aggression), are severe, highly prevalent and chronically disabling. Treatment options for these diseases are extremely limited. The pathophysiological bases of disorders of behavioral disinhibition are poorly understood but it has been suggested that serotonin dysfunction may play a role. Mice lacking the gene encoding brain tryptophan hydroxylase 2 (Tph2-/-), the initial and rate-limiting enzyme in the synthesis of serotonin, were tested in numerous behavioral assays that are well known for their utility in modeling human neuropsychiatric diseases. Mice lacking Tph2 (and brain 5HT) show intense compulsive and impulsive behaviors to include extreme aggression. The impulsivity is motor in form and not cognitive because Tph2-/- mice show normal acquisition and reversal learning on a spatial learning task. Restoration of 5HT levels by treatment of Tph2-/- mice with its immediate precursor 5-hydroxytryptophan attenuated compulsive and impulsive-aggressive behaviors. Surprisingly, in Tph2-/- mice, the lack of 5HT was not associated with anxiety-like behaviors. The results indicate that 5HT mediates behavioral disinhibition in the mammalian brain independent of anxiogenesis.

Fig. 1

Compulsive behaviors in Tph2−/− mice. (a) Tph2−/− (KO) mice (n = 6 males) shred significantly (Student’s t test, t₁₀ = 3.07, p < 0.05) more nestlet material than wild-type (WT) mice (n = 6 males); (b) Tph2−/− mice (n = 33; 22 males, 11 females) bury significantly (two-way ANOVA, F_1,66 = 35.61, p < 0.05 for genotype) more marbles than wild-type mice (n = 37; 26 males, 11 females); (c) Tph2−/− mice (n = 10; 5 males, 5 females) bury and displace significantly (two-way ANOVA, F_1,16 = 17.64, p < 0.005 for genotype) more food pellets than wild-type mice (n = 10; 5 males, 5 females); (d) Tph2−/− mice (n = 10; 5 males, 5 females) spend significantly (two-way ANOVA, F_1,15 = 22.47, p < 0.0005 for genotype) more time digging than wild-type mice (n = 9; 4 males, 5 females); (e) Total distance traveled or locomotor activity were not different (two-way ANOVA, p > 0.05) between Tph2−/− (n = 16; 9 males, 7 females) and wild-type mice (n = 25; 13 males, 12 females). Results are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 for Bonferroni post hoc comparisons between KO and WT groups. In tests where both males and females were used as subjects (Figs. 1b,d), the main effect of sex was not significant so data from both groups was combined for graphical purposes.

Fig. 2

Impulsive aggression in Tph2−/− mice. (a) Adult Tph2−/− male mice (n = 26) show significantly decreased attack latency (Student’s t test, t₄₁ = 3.66, p < 0.005) and increased number of attacks (Student’s t test, t₄₁ = 3.02, p < 0.005) compared to adult wild-type males in the classical resident-intruder test (n = 17); (b) Adult Tph2−/− male (M) mice (n = 12), show a significant increase in attack latency (Student’s t test, t₂₂ = 3.47, p < 0.005) and number of attacks (Student’s t test, t₂₂ = 2.85, p < 0.01) relative to adult male wild-type mice (n = 12) in the modified version of the resident-intruder test; (c) Adult Tph2−/− female (F) mice (n = 14) show a significant increase in attack latency (Student’s t test, t₂₄ = 3.26, p < 0.005) and number of attacks (Student’s t test, t₂₄ = 2.22, p < 0.05) relative to adult female wild-type mice (n = 12) in the modified resident-intruder test; (d) Tph2−/− weanlings (n = 30; 16 males, 14 females) show a significant increase in attack latency (Student’s t test, t₅₂ = 3.07, p < 0.005) and number of attacks (Student’s t test, t₅₂ = 2.54, p < 0.05) compared to wild-type weanling mice (n = 24; 12 males, 12 females); (d) Adult male Tph2−/− mice (n = 6) have significantly (two-way ANOVA, F_1,21 = 14.35, p < 0.005 for genotype and F_1,21 = 26.55, p < 0.0001 for sex) higher plasma testosterone levels compared to wild-type males (n = 6), while there is no difference in plasma testosterone levels in Tph2−/− females (n = 8) compared to wild-type females (n = 5). Results are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 for Bonferroni post hoc comparisons between KO and WT groups.

Fig. 3

Tph2−/− mice show motor but not cognitive impulsivity. (a) Tph2−/− knockout (KO) mice (n = 12; 5 males, 7 females) show significantly (two-way ANOVA, F_1,20 = 11.92, p < 0.005 for genotype) reduced hyponeophagia following an 18 hour fast relative to wild-type (WT) mice (n = 12; 8 males, 4 females); (b) Tph2−/− mice (n = 14; 5 males, 9 females) emerge from the dark significantly (two-way ANOVA, F_1,23 = 5.98, p < 0.05 for genotype) faster than wild-type mice (n = 13; 8 males, 5 females); (c) Tph2−/− mice (n = 6 males) show normal acquisition of the Barnes spatial learning task over 4 trial sessions as measured in duration to reach goal box (two-way ANOVA, p < 0.001 for trial, F_3,40 = 20.14) and (d) in distance traveled during the 4 learning trials (two-way ANOVA, p < 0.001 for trial, F_3,40 = 14.41) compared to wild-type mice (n = 6 males); (e) Tph2−/− mice (n = 6 males) also show normal reversal learning on the Barnes maze as measured in duration to reach the goal box (two-way ANOVA, F_3,40 = 21.45, p < 0.001 for trial) and (f) in distance traveled (two-way ANOVA, F_3,40 = 3.40, p < 0.05 for trial) during reversal learning trials. Results are mean ± SEM. *p < 0.05, **p < 0.01 for Bonferroni post hoc comparisons. KO mice did not differ from WT mice in acquisition of the Barnes maze task or in reversal learning.

Fig. 4

5HTP treatment attenuates compulsive and impulsive behaviors and restores 5HT levels in Tph2−/− mice. (a) Treatment with carbidopa (CD) (25mg kg⁻¹, ip) plus 5HTP (10mg kg⁻¹, ip) (n = 16; 7 males, 9 females), but not CD alone (n = 16; 7 males, 9 females), significantly (two-way ANOVA, F_1,28 = 31.0, p < 0.001 for genotype; F_1,28 = 7.323, p < 0.05 for sex) attenuates marble burying behavior in Tph2−/− knockout (KO) mice; (b) 5HTP treatment does not (two-way ANOVA, p > 0.05) affect locomotor activity in Tph2−/− mice (n = 12; 3 males, 9 females); (c) Treatment with CD + 5HTP (n=10), but not with vehicle alone (Con) (n = 15) or CD alone (n = 6), significantly attenuates latency to attack (one-way ANOVA, F_2,32 = 15.29, p < 0.0001) and number of attacks (one-way ANOVA, F_2,32 = 10.32, p < 0.0005) in adult male Tph2−/− mice. Impulsive aggressive behavior does not habituate over the course of repeated test sessions as seen in Tph2−/− mice not receiving any treatment. 5HTP treatment restores 5HT and 5HIAA levels in the brains of adult male Tph2−/− mice (n = 6 males for each genotype) 30 min after treatment in (d) hippocampus (one-way ANOVA, F_2,14 = 214, p < 0.0001 for 5HT; F_2,14 = 130.4, p < 0.0001 for 5HIAA), (e) frontal cortex (one-way ANOVA, F_2,14 = 52.63, p < 0.0001 for 5HT; F_2,14 = 99.79, p < 0.0001 for 5HIAA), (f) hypothalamus (one-way ANOVA, F_2,14 = 168.7, p < 0.0001 for 5HT; F_2,14 = 306.3, p < 0.0001 for 5HIAA), and (g) tegmentum (one-way ANOVA, F_2,14 = 196.8, p < 0.0001 for 5HT; F_2,14 = 77.54, p < 0.0001 for 5HIAA). Tryptophan (Trp) levels were the same in all groups and were not changed by any treatment. Note also that untreated Tph2−/− mice (n = 6 males) are devoid of 5HT and 5HIAA. Results are mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001 for Bonferroni post hoc comparisons between KO and WT mice (two-way ANOVAs) and ***p < 0.001 for Tukey’s post hoc comparisons (one-way ANOVAs). In tests where males and females were both used as subjects (Figs. 4a–c), the main effect of sex was not significant so data from both groups was combined for graphical purposes.

Fig. 5

Tph2−/− mice do not show anxiety-like behaviors. (a) Tph2−/− mice (n = 23; 11 males, 12 females) spend significantly (two-way ANOVA, F_1,44 = 6.55, p < 0.05 for genotype) more time in the lit compartment of the light/dark box and (b) take significantly (two-way ANOVA, F_1,44 = 6.09, p < 0.05 for genotype) longer to cross into the dark compartment while showing no differences in distance traveled compared to wild-type mice (n = 25; 12 males, 13 females); (c) KO (n = 23; 11 males, 12 females) and WT mice (n = 25; 12 males, 13 females) spend the same amount of time in either the open or closed arms of the elevated plus maze and (d) are not different in total distance traveled or resting time, indicating a lack of defect in exploratory behaviors or increased hyperactivity in Tph2−/− mice (two-way ANOVA, p > 0.05). Results are means ± SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 for Bonferroni post hoc comparisons between KO and WT groups. In tests where males and females were both used as subjects (Figs. 5a–d), the main effect of sex was not significant so data from males and females was combined for graphical purposes.