Regulation of stress-provoked aggressive behavior using endocannabinoids

Abstract

Reactive impulsive aggression is characterized by outbursts of rage and violence when subjects encounter threatening stressful events. Although impulsive aggression and violence create a high-cost burden on health and society, relatively little is known about treatment. Early adolescent social isolation (SI) alters brain development and functions. It induces hyper-excitatory in the ventral hippocampus (vHip) to promote acute stress-provoked outbursts of aggression, referred to as impulsive aggression, in mouse models. Cannabinoid type 1 receptors (CB1Rs) act on presynaptic sites and suppress neurotransmitter release into synapses. Given that CB1R activation inhibits neurotransmitter releases and modulates excitatory network activity, we tested the hypothesis that CB1R activation reduces impulsive aggression in SI mice through decreasing excitatory activity in the vHip. Here, we report that CB1R agonists, WIN-552122 (WIN) or arachidonylcyclopropylamide (ACPA), ameliorated acute stress-provoked attack behavior in the resident-intruder test without affecting general locomotion activity. Increasing endocannabinoids (eCBs) by inhibiting degradation enzymes in the vHip reduced impulsive aggression, and the effect was blunted by administration of AM251, a CB1R antagonist. Acute stress in SI mice induced c-Fos expression, a marker of neuronal activation, on vHip neurons projecting to the ventromedial hypothalamus (VMH), a well-known brain area that controls attack behavior. eCB augmentation inhibited c-Fos expression in VMH-projecting vHip neurons surrounded by CB1Rs. These results suggest that enhancing eCB signaling in order to activate CB1Rs suppresses impulsive aggression via suppressing vHip→VMH neural activity and point to a role of CB1R activation in ameliorating impulsive aggression in adults who have had adverse experiences during early adolescence.

Keywords: Acute stress; Aggression; CB1 receptor; Endocannabinoid; Hippocampus; Social isolation.

Graphical abstract

Fig. 1

Administration of WIN 55212-2 inhibits impulsive aggression in post-weaning socially isolated mice, A. The experimental procedure. Mice at postnatal day 21 were randomly divided into group-housing (GH) or socially isolated (SI) mice (n = 8 per group). 5 weeks later, these mice underwent behavioral tests., B. In the no stress condition, there were no differences in the biting behavior between the SI and GH mice. Acute stress increased biting behavior in the SI mice but not in the GH mice. p < 0.01, acute stress vs. no stress in SI mice., C. The percentage of SI mice that engaged in more than the maximum level of biting of the non-stress-treated GH mice rose from 12.5% up to 62.5% after acute stress, D. The SI mice stayed less time in the central zone of the open field box compared to the GH mice (p < 0.01), suggesting that the SI mice were exhibiting anxiety-like behavior., E. There were no differences in the total distance traveled in the open field box between the SI and GH mice, suggesting no difference in general locomotor activity., F. The experimental procedure for the intraperitoneal injection of WIN 55212-2 (WIN), a CB1R agonist. Another three groups of SI mice received the vehicle, 0.1 and 0.5 mg/kg WIN, respectively (n = 5 per group). 30 min later, these mice received foot-shocks to induced impulsive aggression in the RI test., G. The administration of 0.1 and 0.5 mg/kg WIN decreased biting behavior compared to the vehicle dose. p < 0.001., H. 0.5 mg/kg WIN administration in SI mice decreased the level of biting toward vulnerable parts of intruders. p < 0.01. n = 5 per group., I. WIN treatment decreased the level of biting toward non-vulnerable parts of intruders. p < 0.01., J. WIN treatment decreased offensive behavior in the SI mice. p < 0.05., K. WIN treatment did not affect non-aggressive behavior in the SI mice. p > 0.05., L. There was no difference in the duration of ano-genital sniffing between the vehicle and 0.5 mg/kg WIN treatments. p > 0.05., M. 0.5 mg/kg WIN administration had no effect on the total distance the SI mice traveled in the open field box compared to the vehicle dose. p > 0.05. n = 5 per group., N and O. 0.5 mg/kg WIN treatment did not affect anxiety-like behavior. There were no differences in the distance traveled in the central zone (N, p > 0.05) or the frequency of entry into the central zone (O, p > 0.05)., P. Two groups of GH mice received the vehicle and 0.5 mg/kg WIN, respectively, and then received acute stress 30 min later (n = 10 per group). The WIN treatment had no effect on biting behavior compared to the vehicle. p > 0.05.Data represent as mean ± SEM.

Fig. 2

ACPA administration inhibits impulsive aggression in SI mice but not in GH mice, A. Another three groups of SI mice received the vehicle, 5 and 10 mg/kg ACPA, another CB1R agonist, respectively (n = 5 per group). 30 min later, these mice received foot-shocks to induced impulsive aggression in the RI test. ACPA administration in the SI mice decreased impulsive aggression compared to the vehicle dose (p < 0.05 in 5 mg/kg ACPA; p < 0.001 in 10 mg/kg ACPA)., B. Another two groups of GH mice received the vehicle and 10 mg/kg ACPA, respectively (n = 8 per group)., C. The biting levels were 3.25 ± 1.63 and 1.50 ± 0.93 in the vehicle and ACPA, respectively. An unpaired Student t-test showed no differences between the vehicle and ACPA (p > 0.05). Data represented as mean ± SEM., D and E. Detailed analysis of biting behavior. The vehicle and 10 mg/kg ACPA in the GH mice did not induced biting toward vulnerable parts of intruders (D, mean ± SEM as 0.875 ± 0.52 and 0.875 ± 0.74, respectively, p > 0.05) and non-vulnerable parts of intruders (E, mean ± SEM as 2.37 ± 1.19 and 0.63 ± 0.38, respectively, p > 0.05)., F. 10 mg/kg ACPA in GH mice did not induced offensive behavior compared to the vehicle. p > 0.05., G. The level of non-aggressive behavior in ACPA was lower than in the vehicle. p < 0.01., H. There were no differences in the duration of ano-genital sniffing between the vehicle and ACPA. p > 0.05., I. ACPA in the GH mice did not affect the total distance traveled compared to the vehicle. p > 0.05., J and K. ACPA in the GH mice did not affect anxiety-like behavior in the open field test. The indicators of anxiety-like behavior in the distance traveled in the central zone (J, p > 0.05) and in the frequency of entry into the central zone (K, p > 0.05).Data represented as mean ± SEM.

Fig. 3

Intra-hippocampal infusion of CB1R agonists in SI mice inhibits impulsive aggression., A. The experimental procedure for WIN infusion into the vHip. The vehicle, 1, and 2 μg/μl WIN were infused into the vHip of three groups of SI mice, respectively. 30 min later, these mice received foot-shocks to induce impulsive aggression. n = 6 per group., B. 2 μg/μl WIN decreased biting levels compared to the vehicle. Post hoc test, p < 0.05 in 2 μg/μl WIN vs. the vehicle. There were no differences between 1 μg/μl WIN and the vehicle. Post hoc test, p > 0.05 in 1 μg/μl WIN vs. the vehicle., C. There were no differences in the total distance traveled among the dose-groups in the open field test. The one-way ANOVA, p > 0.05., D and E. The 2 μg/μl WIN infusion into the vHip decreased anxiety-like behavior in the open field test. 2 μg/μl WIN increased the distance traveled in the central zone (D) and the frequency of entry into the central zone (E). Post hoc test, p < 0.001 in 2 μg/μl WIN vs. the vehicle. The 1 μg/μl WIN had no effects on anxiety-like behavior compared to the vehicle. Post hoc test, p > 0.05., F. Another two groups of SI mice received the vehicle and 4 ng/μl ACPA infusion into the vHip, respectively. n = 6 per group., G. ACPA infusion in SI mice decreased impulsive aggression compared to the vehicle. p < 0.05., H. The ACPA infusion into the vHip had no effects on general locomotor activity in the open field test. p > 0.05., I. ACPA infusion in SI mice had no effects on the distance traveled in the central zone. p > 0.05., J. The ACPA infusion into the vHip increased the frequency of entry into the central zone. p < 0.05.Data represented as mean ± SEM.

Fig. 4

Decreasing CB1R expression in the vHip exaggerates impulsive aggression in SI mice but not in GH mice., A and B. The experimental schema for CB1R knockdown (KD) (A). Lentiviruses carrying CB1R shRNA or the copGFP control were infused into the vHip (B). After 2 weeks of recovery, these mice received the RI test under no stress and then in a state of acute stress one day later (A)., C. Representative image showing the injection location of the lentivirus carrying the copGFP control. Green, GFP; blue, DAPI. D. Western blotting confirmed that the transduction of CB1R shRNA significantly decreased CB1R protein expression in the vHip. n = 7., E. CB1R knockdown in the SI mice further exaggerated impulsive aggression. There was a significant interaction effect of CB1R-KD and stress (p < 0.05). Acute stress increased biting behavior both in the control- and CB1R-KD-treated mice (no stress vs. acute stress, **p < 0.01 and ***p < 0.001 in the control and CB1R-KD mice, respectively). Importantly, CB1R-KD mice under acute stress exhibited a higher level of biting behavior compared to control mice under acute stress. Post hoc test, ###p < 0.001. n = 10 and 12 in copGFP control and CB1R shRNA, respectively., F. CB1R knockdown in the GH mice had no effects on impulsive aggression. The interaction effects of CB1R-KD and stress were not significant in the biting behavior of the GH mice. p > 0.05. n = 10 both in the control and shRNA.Data represented as mean ± SEM. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Increasing endocannabinoids by inhibiting degradation enzymes reduces impulsive aggression in a CB1R-dependent manner., A. The experimental schema for eCB augmentation in the vHip (left). Four groups of SI mice received the intra-vHip infusion of the vehicle (n = 7), UBR597+JZL184 (n = 6), URB597+JZL184+AM251 (n = 6), and AM251 only (n = 8), respectively. 5 ng/μl URB597 and 2 μg/μl JZL184 inhibited FAAH and MAGL degradation enzymes to increase AEA and 2-AG, respectively. AM251 (2.5 ng/μl) blocked CB1R activation. These mice encountered acute stress 30 min after drugs were infused into the vHip. The right panel shows that pharmacological eCB augmentation via the combination of URB597 and JZL184 treatment decreased impulsive aggression compared to the vehicle treatment. Post hoc test, p < 0.01. AM251 infusion blocked the anti-aggressive effects of eCB augmentation. Post hoc test, p > 0.05 in vehicle vs URB597+JZL184+AM251. Inhibiting CB1Rs via AM251 treatment increased impulsive aggression compared to the vehicle. Post hoc test, p < 0.01 in vehicle vs AM251., B. Another three groups of SI mice received the intra-vHip infusion of the vehicle, URB597, and URB597+AM251, respectively. n = 5 per group. AEA augmentation reduced impulsive aggression compared to the vehicle, the effects of which were blocked by AM251 infusion. Post hoc test, p < 0.05 in URB597 vs. the vehicle; p > 0.05 in URB597+AM251 vs. the vehicle., C. Another three groups of SI mice received the intra-vHip infusion of the vehicle (n = 7), JZL184 (n = 8), and JZL184+AM251 (n = 5), respectively. 2-AG augmentation reduced impulsive aggression compared to the vehicle, the effect of which was blocked by AM251 infusion. Post hoc test, p < 0.05 in JZL184 vs. the vehicle; p > 0.05 in JZL184+AM251 vs. the vehicle., D and E. 2-AG augmentation in the vHip reduced the level of biting toward the vulnerable parts (D, p < 0.05) and non-vulnerable parts of intruders (E, p < 0.01). n = 7 and 8 in vehicle and JZL184, respectively. F ~ H. 2-AG augmentation via JZL184 did not affect offensive behavior (F, p > 0.05), non-aggressive behavior (G, p > 0.05), or the duration of ano-genital sniffing (H, p > 0.05). n = 7 and 8 in the vehicle and JZL184, respectively.Data represented as mean ± SEM.

Fig. 6

eCB augmentation inhibits the activation of vHip neurons projecting to the VMH. A. Schema of the experimental design. Red retrobeads were infused into the VMH, and tips of drug cannulas were implanted in the vHip of SI mice. After two weeks of recovery, the three retrobead-treated groups of mice received an intra-vHip infusion of the vehicle, 5 ng/μl URB597, and 2 μg/μl JZL184, respectively. One hour after the drug infusion, these SI mice received foot-shocks to induce c-Fos expression and then were sacrificed 90 min later. n = 3 per group., B. Representative image showing the injected location of retrobead infusion in the VMH. Scale bar: 550 μm, C. Representative images showing signals of red retrobeads (red), CB1Rs (green), and c-Fos (blue) in the ventral CA1 of the vehicle (top), UBR597 (middle), and JZL184 (bottom) -infused mice. Acute stress induced c-Fos expression (blue) surrounded by CB1Rs (green). c-Fos signals were co-localized with red retrobeads. URB597 infusion and JZL184 infusion significantly decreased c-Fos expression, suggesting that AEA and 2-AG augmentation suppressed acute stress-provoked vHip activation. Scale bar: 40 μm. The right panels show the magnified insets as white dotted squares., D. Representative confocal image showing c-Fos-positive cells co-localized with red retrobeads. The CB1R signals surrounded the c-Fos-positive cells. Scale bar: 10 μm., E. Quantitative analysis of Fig. 6C. In the vHip, URB597 infusion and JZL184 infusion decreased c-Fos expression surrounded by CB1R signals in retrobead-positive cells compared to the vehicle. Post hoc test, p < 0.001; n = 12 brain slices from 3 mice per group. Data represented as mean ± SEM. . (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. S1

The infusion of CB1R agonists into the dorsal striatum has no effect on the SI mice., A. The experimental procedure for WIN infusion into the dorsal striatum. Two groups of SI mice received the vehicle and 2 μg/μl WIN infusion into the dorsal striatum, respectively. 30 min later, these mice received foot-shocks to induce impulsive aggression. n = 5 per group., B. The WIN infusion into the dorsal striatum had no effects on impulsive aggression compared to the vehicle. p > 0.05., C. There were no differences in the total distance traveled between the WIN and vehicle groups in the open field test. p > 0.05., D and E. The infusion into the dorsal striatum did not affect anxiety-like behavior in the open field test. There were no differences between the WIN and vehicle groups in the distance traveled in the central zone (D) and the frequency of entry into the central zone (E). p > 0.05., F. Another two groups of SI mice received the vehicle and 4 ng/μl ACPA infusion into the dorsal striatum, respectively. n = 6 per group., G. The ACPA infusion into the dorsal striatum had no effects on impulsive aggression. p > 0.05., H ~ J. In the open field, there were no differences between the vehicle and ACPA infusion groups in the total distance traveled (H, p > 0.05), the distance traveled in the central zone (I, p > 0.05), and the frequency of entry into the central zone (J, p > 0.05).Data represented as mean ± SEM.

Fig. S2

The AM251 infusion into the hippocampus has no effects in GH mice., A. The experimental procedure for AM251 infusion into the vHip. Two groups of GH mice received the infusion of vehicle and 2.5 ng/μl AM251, respectively. n = 9 per group., B and C. 2.5 ng/μl AM251 infusion did not affect biting behavior (B) or offensive behavior (C) as compared to the vehicle. p > 0.05., D. 2.5 ng/μl AM251 infusion decreased the level of non-aggressive behavior. p < 0.05., E. AM251 infusion did not affect the duration of ano-genital sniffing. p > 0.05., F ~ H. In the open field test, there were no differences between the AM251 and vehicle infusion groups in the total distance traveled (F, p > 0.05), the distance traveled in the central zone (G, p > 0.05), and the frequency of entry into the central zone (H, p > 0.05). Data represented as mean ± SEM.

Fig. S3

eCB augmentation in the ventral hippocampus decreased anxiety-like behavior in SI mice., A. The experimental schema. Four groups of SI mice received the intra-vHip infusion of the vehicle (n = 12), 5 ng/μl URB597 (n = 5), 2 μg/μl JZL184 (n = 4), and 2.5 ng/μl AM251 (n = 8), respectively., B. There were no differences in the total distance traveled among the four groups. p > 0.05., C. URB597 infusion in the SI mice increased the distance traveled in the central zone. Post hoc test, p < 0.01 in URB597 vs. the vehicle., D. URB597 infusion and JZL184 infusion increased the frequency of entry into the central zone compared to the vehicle. Post hoc test, p < 0.05. Data represented as mean ± SEM.