Control of intermale aggression by medial prefrontal cortex activation in the mouse

Abstract

Aggressive behavior is widely observed throughout the animal kingdom because of its adaptiveness for social animals. However, when aggressive behavior exceeds the species-typical level, it is no longer adaptive, so there should be a mechanism to control excessive aggression to keep it within the adaptive range. Using optogenetics, we demonstrate that activation of excitatory neurons in the medial prefrontal cortex (mPFC), but not the orbitofrontal cortex (OFC), inhibits inter-male aggression in mice. At the same time, optogenetic silencing of mPFC neurons causes an escalation of aggressive behavior both quantitatively and qualitatively. Activation of the mPFC suppresses aggressive bursts and reduces the intensity of aggressive behavior, but does not change the duration of the aggressive bursts. Our findings suggest that mPFC activity has an inhibitory role in the initiation and execution, but not the termination, of aggressive behavior, and maintains such behavior within the adaptive range.

Figure 1. Injection of ChETA-EYFP lentivirus leads to functional ChETA expression on the excitatory neurons in the mPFC.

In this study, the mPFC area includes both the prelimbic cortex and medial orbital cortex. (A) EYFP expression on one side of the mPFC. The yellow dotted line indicates the outline of the guide cannula, and white boxes indicate the areas of c-Fos analysis. (B) Increased c-Fos expression following 1-min of stimulation with blue light on the EYFP-expressing side of the mPFC. Green: EYFP, Red: cFos. (C) The contralateral side of the mPFC did not show an increase in c-Fos expression. (D) Schematic representations of the lentivirus construct and the site of mPFC activation. (E) Quantification of c-Fos-positive cells in the stimulated and contralateral unstimulated sides of the mPFC in ChETA-EYFP-expressing animals (n = 5) and control EYFP-expressing animals (n = 4). * p<0.05 by t-test. Error bars represent SEM.

Figure 2. Activation of the mPFC, but not the OFC, inhibited attack bites of male mice.

(A) Schematic representation of the test schedule. Lentivirus infection was carried out at least 3 weeks before the optical stimulation experiment. Trial 1 examined the resident male's basal aggressive behavior without light stimulation. In Trial 2, the light stimulus was delivered to the mPFC 5 sec before the introduction of the intruder, and continued during a 3-min aggressive encounter. The light stimulus was then removed, and aggressive behavior was observed for an additional 3 min. In Trial 3, 2-min light stimuli were delivered at intervals over a 12-min session. (B) Schematic diagram of the ChETA-EYFP expression and light stimulation in the mPFC. (C) During the lights-on period in Trial 2, there was a significant reduction of the number of attack bites compared with that in Trial 1. (D) Light stimuli presented at intervals inhibited the frequency of attack bites in Trial 3. (E) The total number of attack bites in Trial 3 was also significantly reduced during the lights-on period compared with that in the lights-off period. (F) Schematic diagram of the control EYFP expression and light stimulation in the mPFC. (G) There was no statistically significant difference between Trial 1 and Trial 2. (H) Light stimuli presented at intervals did not change the attack bite behavior in the control mice. (I) The total number of attack bites in Trial 3 did not differ between the lights-on and lights-off periods. (J) Schematic diagram of ChETA-EYFP expression and light stimulation in the OFC. In this study, the OFC area was defined as including both the ventral and lateral orbital cortex. (K) There was no statistically significant difference between Trial 1 and Trial 2. (L) Light stimuli presented at intervals did not affect attack bite behavior in the OFC-stimulated animals. (M) The total number of attack bites in Trial 3 did not differ between the lights-on and lights-off periods. * p<0.05 by t-test with Bonferroni correction. Error bars represent SEM.

Figure 3. The mPFC activation inhibited aggressive behaviors specifically, and did not change non-aggressive behaviors in Trial 3.

The images show the duration of sideways threat (A), walking (B), rearing (C), tail rattle (D), self-grooming (E) and social contact (F). Whereas (A) and (D) denote aggressive behavior, the other four behavior are non-aggressive behaviors. * p<0.05 by t-test with Bonferroni correction. Error bars represent SEM.

Figure 4. Aggressive burst analysis during activation and inhibition of the mPFC.

(A) Temporal pattern of the probability of aggressive behavior during Trial 3 in ChETA expressing animals. Aggression probability was decreased throughout the light stimulation period. (B) Burst structure of aggressive behavior in individual ChETA expressing animals in Trial 3. The green bar indicates the occurrence of either attack bites or sideways threats. The red bar indicates an aggressive burst that started before the light stimulation and continued for a while after the light stimulation was applied. (C) The mPFC activation by ChETA stimulation caused a reduction of the frequency of aggressive bursts during the lights-on period. (D) The number of attack bites within a burst was lower during the lights-on period than during the lights-off period. (E) The duration of aggressive bursts was not changed by light stimulation in ChETA-expressing animals. (F) Analysis of the distribution of durations of aggressive bursts also showed no difference between the lights-on and lights-off periods. (G) Inhibition of the mPFC by eArchT3.0 increased the number of aggressive bursts slightly, albeit not significantly. (H) Inhibition of the mPFC increased the number of attack bites within a burst. (I–J) The duration of aggressive bursts was not changed by light stimulation in eArchT3.0-expressing animals. Whereas (C–F) were calculated by using the data from trial 1–3 in mPFC–ChETA group, (G–J) were calculated by using the data from trial 1 and trial 2 (lights-on) in mPFC–eArchT3.0 group. * p<0.05 by t-test. Error bars represent SEM.

Figure 5. Inhibition of the mPFC activity by the expression of eArchT3.0 on the excitatory neurons of the mPFC.

(Left) Schematic diagram of eArchT3.0 expression and light stimulation in the mPFC. (Right) During the lights-on period in Trial 2, there was a significant increase in the number of attack bites compared with that in the lights-off period in Trial 1 and Trial 2. * p<0.05 by t-test with Bonferroni correction. Error bars represent SEM.

Figure 6. Effect of the mPFC activation and inhibition on the attack and threat ratio of aggressive behavior.

(A) All data from Trials 1–3 were combined to estimate the attack-threat ratio for the ChETA experiment. There were significant reductions of the number of attack bites (A) and the duration of sideways threats (B) after mPFC activation. (C) There was a significant reduction of the attack/threat ratio by light stimulation. Inhibition of the mPFC by eArchT3.0 stimulation increased the frequency of attack bites (D), but had no effect on the duration of sideways threats (E). (F) There was a significant increase in the attack/threat ratio caused by stimulation. * p<0.05 by t-test. Error bars represent SEM.