A Role for Prefrontal Cortical NMDA Receptors in Murine Alcohol-Heightened Aggression

Abstract

Alcohol is associated with nearly half of all violent crimes committed in the United States; yet, a potential neural basis for this type of pathological aggression remains elusive. Alcohol may act on N-methyl-d-aspartate receptors (NMDARs) within cortical circuits to impede processing and to promote aggression. Here, male mice were characterized as alcohol-heightened (AHAs) or alcohol non-heightened aggressors (ANAs) during resident-intruder confrontations after self-administering 1.0 g/kg alcohol (6% w/v) or water. Alcohol produced a pathological-like pattern of aggression in AHAs; these mice shifted their bites to more vulnerable locations on the body of a submissive animal, including the anterior back and ventrum after consuming alcohol. In addition, through immunoblotting, we found that AHAs overexpressed the NMDAR GluN2D subunit in the prefrontal cortex (PFC) as compared to ANAs while the two phenotypes expressed similar levels of GluN1, GluN2A and GluN2B. After identifying several behavioral and molecular characteristics that distinguish AHAs from ANAs, we tested additional mice for their aggression following preferential antagonism of GluN2D-containing NMDARs. In these experiments, groups of AHAs and ANAs self-administered 1.0 g/kg alcohol (6% w/v) or water before receiving intraperitoneal (i.p.) doses of ketamine or memantine, or infusions of memantine directly into the prelimbic (PLmPFC) or infralimbic medial PFC (ILmPFC). Moderate doses of IP ketamine, IP memantine, or intra-PLmPFC memantine increased aggression in AHAs, but only in the absence of alcohol. Prior alcohol intake blocked the pro-aggressive effects of ketamine or memantine. In contrast, only memantine, administered systemically or intra-PLmPFC, interacted with prior alcohol intake to escalate aggression in ANAs. Intra-ILmPFC memantine had no effect on aggression in either AHAs or ANAs. In sum, this work illustrates a potential role of GluN2D-containing NMDARs in the PLmPFC in alcohol-heightened aggression. GluN2D-containing NMDARs are highly expressed on cortical parvalbumin-containing interneurons, suggesting that, in a subset of individuals, alcohol may functionally alter signal integration within cortical microcircuits to dysregulate threat reactivity and promote aggression. This work suggests that targeting GluN2D-NMDARs may be of use in reducing the impact of alcohol-related violence in the human population.

Figure 1

Behavioral and molecular characterization of alcohol-heightened aggressors (AHAs). (a) Aggressive male residents were assessed for aggression toward submissive intruders and characterization began after attack bite frequencies stabilized. Residents that consumed 1.0 g/kg of 6% EtOH (w/v) and expressed an average bite frequency that was ⩾2 SD higher than their average baseline attack bite frequency were identified as alcohol-heightened aggressors (AHAs; n=7); males that were not sensitive to alcohol’s pro-aggressive effects were characterized as ANAs (n=8). Data are shown as group mean±SEM. Attack bite topography of (b) ANAs and (c) AHAs was assessed after water intake and after 1.0 g/kg EtOH intake. AHAs increased the number of attacks to the more vulnerable, anterior back after consuming alcohol as compared to ANAs. Alcohol increased the total number of bites comprising the initial bout and bites to the ventrum. The proportion of bites to the posterior back, anterior back, ventrum and head during the initial aggressive bout are illustrated in pie charts. The frequency of attacks to each location is tabulated below, showing average attack bites for each bite target±SEM for ANAs and AHAs; ^a indicates a main effect of alcohol; for interactions, *p<0.05 indicates statistical significance between AHAs and ANAs, and ^#p<0.05 indicates statistical significance between EtOH and H₂O. Western blot analysis of NMDA receptor subunits revealed an increase in (f) GluN2D expression in the medial prefrontal cortex (mPFC) of AHAs compared to ANAs (n=6–8/phenotype group; *p=0.03) while there was no difference in PFC (d) GluN1 or (e) GluN2B. Data are shown as group mean±SEM.

Figure 2

Histological verification of cannula placements in the right infralimbic medial prefrontal cortex (mPFC). Photomicrographs from a representative placement verified after cresyl violet staining (right) adjacent to atlas images with distance from bregma (Paxinos and Franklin, 2001). Solid lines depict verified placements in the infralimbic mPFC. One alcohol non-heightened aggressor was excluded due to a cannula placement outside of the mPFC (dashed line). Mice received infusions of memantine first into the right prelimbic mPFC via microinjectors projecting 1 mm past the end of the cannula, and subsequently, into the right infralimbic mPFC via microinjectors with a 2 mm projection.

Figure 3

Aggression after alcohol or water intake and systemic NMDAR antagonist treatments. (a–d) Bar graphs depict baseline attack bite frequencies after 1.0 g/kg alcohol or water self-administration and vehicle injections. Alcohol-heightened aggressors (AHAs; left panels) attacked a submissive intruder more frequently after receiving a vehicle injection and consuming alcohol compared to water. Scatter plots display the percent change from baseline attack bite frequencies. In AHAs, low doses of (a) ketamine (n=10) or (c) memantine (n=8) escalated aggression; prior alcohol self-administration blocked these pro-aggressive effects. In alcohol non-heightened aggressors (ANAs; right panels), (b) ketamine (n=12) reduced aggression while (d) memantine (n=9) had a pro-aggressive effect when administered after alcohol self-administration. Higher doses of memantine and ketamine reduced aggression in (a,c) AHAs and (b,d) ANAs. Data are shown as mean±SEM; *p<0.05, **p<0.01 indicate statistical significance between vehicle and drug dose; ^#p<0.05, ^##p<0.01 indicate statistical significance between H₂O and EtOH.

Figure 4

Aggression after alcohol or water intake and intra-infralimbic or prelimbic mPFC memantine. Mice received intra-prelimbic (PLmPFC) and intra-infralimbic (ILmPFC) medial prefrontal cortical infusions of memantine after water or 1.0 g/kg alcohol self-administration. (a–d) Bar graphs depict baseline attack bite frequencies after alcohol or water self-administration and vehicle injections; scatter plots display the percent change from these baseline frequencies. Alcohol-heightened aggressors (AHAs; n=7; left panels) attacked a submissive intruder more frequently after receiving aCSF and consuming alcohol compared to water. In AHAs, memantine infused into the (a) PLmPFC, but not the (c) ILmPFC, escalated aggression. The pro-aggressive effect of intra-PLmPFC memantine was prevented when infusions were preceded by alcohol self-administration. In alcohol non-heightened aggressors (ANAs; n=9; right panels), (b) intra-PLmPFC memantine increased aggression when administered after alcohol consumption while (d) intra-ILmPFC memantine reduced the number of attack bites compared to baseline. Data are shown as mean±SEM; *p<0.05, **p<0.01 indicate statistical significance between vehicle and drug dose; ^#p<0.05, ^##p<0.01 indicate statistical significance between H₂O and EtOH.