Changes in aggressive behavior due to dysbiosis of host gut microbiota

Technical Summary: Changes in Aggressive Behavior Due to Dysbiosis of Host Gut Microbiota

Problem Statement
Aggressive behavior, while a component of normal social interaction, can manifest as a morbid condition with severe societal and individual consequences. While the neurobiological underpinnings of aggression (involving the hypothalamus, monoamines, and hormones) are partially understood, the essential causes remain elusive. Recent research has proposed the "microbiota-gut-brain axis" as a novel mechanism, suggesting that the gut microbiota influences host behavior. Previous studies by the authors and others indicated that germ-free (GF) mice exhibit heightened aggression compared to specific pathogen-free (SPF) mice, and that early-life colonization of GF mice (conventionalization) can reduce this aggression. However, it remains unclear whether disrupting an already established gut microbiota in adulthood via antibiotics (inducing dysbiosis) can subsequently alter aggressive behavior.

Methodology
The study utilized a gnotobiotic mouse model to investigate the causal link between antibiotic-induced dysbiosis and aggression in a stabilized gut environment.

• Animal Model: Male BALB/c mice were derived from germ-free (GF) ancestors. These were converted to "ex-germ-free" (Ex-GF) mice by orally administering fecal matter from SPF mice at 4 weeks of age, followed by breeding to establish a second generation with indigenous gut microbiota.
• Experimental Design: At 6 weeks of age, when the gut microbiota was considered stable, Ex-GF mice were divided into two groups:
  1. Control Group: Received standard drinking water.
  2. Treatment Group (Ex-GF SM): Received drinking water supplemented with streptomycin (1 mg/mL) for six days. Streptomycin was selected as an aminoglycoside antibiotic specifically targeting aerobic and gram-negative bacteria, leaving anaerobic bacteria largely intact.
• Behavioral Assessment: At 8 weeks of age, aggressive behavior was evaluated in a strictly aseptic open-field arena. Mice were paired with castrated opponents (to reduce opponent aggression and clarify assessment). Aggression was qualitatively assessed over 10 minutes based on specific behaviors: biting, wrestling, tail-rattling, aggressive grooming, and chasing.
• Microbiota Analysis: Immediately following behavioral testing, cecal contents were collected to quantify bacterial populations. Aerobic bacteria were cultured on Trypticase soy agar, and total bacteria on BL agar.
• Statistical Analysis: Data were analyzed using the Mann-Whitney U test, with significance set at P < 0.05.

Key Results

• Behavioral Outcomes: Neither the streptomycin-treated group (Ex-GF SM) nor the non-treated control group exhibited any aggressive behavioral characteristics during the 10-minute observation period. The study found no significant difference in aggression levels between the two groups.
• Microbiota Disruption: The administration of streptomycin successfully induced dysbiosis. The number of aerobic bacteria in the cecum of the treated group was significantly reduced to 1/1000th of the control group (P = 0.002). The primary reduction was observed in the Enterobacteriaceae family.
• Total Bacterial Load: Despite the drastic reduction in aerobic bacteria, the total number of bacteria (including anaerobes) did not differ significantly between the two groups (P = 0.699).

Significance and Claims
The authors conclude that disrupting the gut microbiota in mice with an already established microbial community via antibiotics targeting aerobic bacteria does not exacerbate aggressive behavior. The study posits several key implications:

1. Critical Period for Intervention: The findings suggest that the gut microbiota's influence on aggression is most critical during early development. Once the microbiota is stabilized (around 8 weeks of age in mice), subsequent antibiotic-induced dysbiosis (specifically that which spares anaerobes) does not appear to alter aggression.
2. Role of Anaerobes: The lack of behavioral change may be attributed to the preservation of anaerobic bacteria, such as Bifidobacteria, which are known to modulate stress responses and behavior. The study references prior work indicating that Bifidobacterium infantis can attenuate hyperactivity and stress responses in GF mice.
3. Clinical Implications: The paper suggests that for human applications, particularly in infants, the timing of antibiotic use is crucial. Stabilizing the gut microbiota early in development may be necessary to prevent the exacerbation of aggression. Conversely, the use of antibiotics that do not target anaerobic bacteria in individuals with a stable microbiome may not increase aggression risks.
4. Maternal Influence: The authors highlight that maternal gut microbiota stability is vital for the healthy development of offspring microbiota, which in turn influences future aggression levels.

Limitations
The authors acknowledge that the study was conducted in a sterile isolator environment, which may limit the generalizability of the results to non-sterile settings. Additionally, the use of castrated opponents was a methodological necessity to maintain sterility, though the authors argue this did not bias the results given the absence of aggression in the castrated mice. Finally, the study notes that the relationship between aggression and gut microbiota requires further verification through additional animal models to fully establish causality.