Nuclear factor kappa B signaling within the rat nucleus accumbens core sex-dependently regulates cue-induced cocaine seeking and matrix metalloproteinase-9 expression

Abstract

Chronic drug self-administration and withdrawal are associated with distinct neuroimmune adaptations that may increase drug craving and relapse vulnerability in humans. The nuclear factor kappa-B (NF-κB) pathway is a critical regulator of many immune- and addiction-related genes such as the extracellular matrix enzyme matrix metalloproteinase-9 (MMP-9), which is a known modulator of learning, memory, and synaptic plasticity. While some studies suggest striatal NF-κB signaling may regulate drug-conditioned behavior, no studies to date have examined whether NF-κB signaling within the nucleus accumbens core (NAc core) alters downstream neuroimmune function and cue-motivated cocaine seeking following a period of forced abstinence, whether any effects are specific to cocaine over other reinforcers, or whether sex differences exist. Here, we examined whether viral-mediated knockdown of the p65 subunit of NF-κB within the NAc core would alter MMP-9 expression and cue-induced cocaine- and sucrose-seeking behavior following a period of forced abstinence in male and female rats. We demonstrate that NAc core p65 knockdown results in a significant decrease in cue-induced cocaine seeking in males but not females. This effect was specific to cocaine, as p65 knockdown did not significantly affect cue-induced sucrose seeking in either males or females. Moreover, we demonstrate that males express higher levels of MMP-9 within the NAc core and nucleus accumbens shell (NAcSh) compared to females, and that p65 knockdown significantly decreases MMP-9 in the NAc core of males but not females among cocaine cue-exposed animals. Altogether, these results suggest that NAc core NF-κB signaling exerts modulatory control over cue-motivated drug-seeking behavior and downstream neuroimmune function in a sex-specific manner. These findings highlight the need to consider sex as an important biological variable when examining immunomodulatory mechanisms of cocaine seeking.

Keywords: Abstinence; Addiction; Cocaine seeking; Cue reactivity; Extracellular matrix; NF-κB; Neuroimmune signaling; Relapse; Sex differences.

Figure 1.. Lentiviral vector validation of p65 protein knockdown within the NAc core.

pLKO.1-puro-CMV-TurboGFP^™ positive control lentiviral particles (“Control” or “CTRL”) or pLKO.1-puro-CMV-TurboGFP^™-RELA shRNA lentiviral particles (“shRNA”) were administered into the NAc core of cocaine- and sucrose-naïve rats. Rats were sacrificed after 21 days for quantitative immunohistochemical staining of p65. (A) Timeline of experimental procedures. (B) Representative coronal section of lentiviral expression of tGFP (488 nm) within the dorsomedial NAc core. Cannula track is outlined by the red dotted line. (C) Representative coronal sections containing the NAc core that are immunostained for p65. The dorsomedial NAc core was imaged at 20x magnification and colocalization of p65 (568 nm) and tGFP (488 nm) was quantified. Scale bars = 50 μm. The shRNA-expressing lentivirus significantly reduced colocalization of p65 and tGFP relative to the control vector, indicating a significant reduction in p65 expression in successfully transduced cells. n = 5–6/group. *p < 0.05. (D) Anatomical map of NAc core viral infusions among animals tested for cue-induced cocaine or sucrose seeking (N = 47). Animals with misplaced viral infusions or GFP expression within the NAcSh or the dorsal striatum were excluded from the study (n = 7). AC = anterior commissure. Error bars = SEM.

Figure 1.. Lentiviral vector validation of p65 protein knockdown within the NAc core.

pLKO.1-puro-CMV-TurboGFP^™ positive control lentiviral particles (“Control” or “CTRL”) or pLKO.1-puro-CMV-TurboGFP^™-RELA shRNA lentiviral particles (“shRNA”) were administered into the NAc core of cocaine- and sucrose-naïve rats. Rats were sacrificed after 21 days for quantitative immunohistochemical staining of p65. (A) Timeline of experimental procedures. (B) Representative coronal section of lentiviral expression of tGFP (488 nm) within the dorsomedial NAc core. Cannula track is outlined by the red dotted line. (C) Representative coronal sections containing the NAc core that are immunostained for p65. The dorsomedial NAc core was imaged at 20x magnification and colocalization of p65 (568 nm) and tGFP (488 nm) was quantified. Scale bars = 50 μm. The shRNA-expressing lentivirus significantly reduced colocalization of p65 and tGFP relative to the control vector, indicating a significant reduction in p65 expression in successfully transduced cells. n = 5–6/group. *p < 0.05. (D) Anatomical map of NAc core viral infusions among animals tested for cue-induced cocaine or sucrose seeking (N = 47). Animals with misplaced viral infusions or GFP expression within the NAcSh or the dorsal striatum were excluded from the study (n = 7). AC = anterior commissure. Error bars = SEM.

Figure 2.. Cocaine and sucrose self-administration prior to lentivirus administration.

Rats self-administered either cocaine or sucrose for a minimum of 12 days beginning on a FR-1 schedule of reinforcement and ending on a VR-5. (A) A post hoc Dunnett’s test of the session main effect revealed a significant increase in cocaine infusions earned at sessions 7–12 compared to session 1 regardless of group (*p < 0.05). (B) A post hoc Tukey’s test of the group-by-session interaction effect revealed CTRL-males had greater active lever presses than shRNA-females on session 11 (*p < 0.05). (C) A post hoc Tukey’s test of the group-by-session interaction effect revealed shRNA-males earned more sucrose pellets on sessions 10 and 12 than shRNA-females (*p < 0.05). (D) Overall, females tended to exhibit greater active lever presses during early sucrose self-administration compared to males, whereas males exhibited greater active lever presses during late sucrose self-administration. A Tukey’s multiple comparisons test revealed the following specific group differences across test sessions: *p < 0.05 comparing CTRL-male and shRNA-male each to CTRL-female and shRNA-female. **p < 0.5 comparing CTRL-male and shRNA-male each to CTRL-female and shRNA-female, as well as CTRL-female to shRNA-female. ^†p < 0.05 comparing CTRL-male to CTRL-female. ^‡p < 0.05 comparing CTRL-male and CTRL-shRNA to CTRL-female. ^p < 0.05 comparing CTRL-male to CTRL-female and shRNA-female, as well as shRNA-male to CTRL-female. ^#p < 0.05 comparing shRNA-males to shRNA-females. n = 5–6/group. Error bars = SEM.

Figure 3.. Cue-induced cocaine and sucrose seeking.

After lentivirus administration and 21 days of forced abstinence, rats were tested in a 1-hr cue-induced cocaine or sucrose seeking test. (A) shRNA-males exhibited significantly lower active lever presses compared to all other group (^#p < 0.01). All groups of rats except shRNA-males exhibited significantly greater active lever presses relative to inactive lever presses (*p < 0.01; n=5–7/group). (B) Post hoc tests revealed no significant differences in cue-induced cocaine and sucrose seeking across sex and virus type, however all groups exhibited significantly greater active lever presses relative to inactive lever presses (*p < 0.01; n=5–6/group). “ALPs” = active lever presses. ILPs = inactive lever presses. Error bars = SEM.

Figure 4.. NAc core MMP-9 expression in virus-treated male and female cocaine rats.

Immediately after the cue-induced cocaine or sucrose seeking test, rats were sacrificed for immunohistochemical analysis of MMP-9 expression within the NAc core. (A) Representative images of MMP-9 (568 nm) and DAPI (405 nm) immunoreactivity (IR) within the NAc core of male and female cocaine rats treated with control or shRNA lentivirus, imaged at 20x magnification. AC = anterior commissure. Scale bars = 50 μm. (B) shRNA-males exhibited a significant reduction in MMP-9 IR within the NAc core compared to CTRL-males. As well, CTRL-males showed significantly greater MMP-9 IR relative to both CTRL-females and shRNA-females. n = 5–7/group. **Difference from all other groups, Tukey’s test, p < 0.01. Error bars = SEM.

Figure 5.. NAc core MMP-9 expression in virus-treated male and female sucrose rats.

Immediately after the cue-induced cocaine or sucrose seeking test, rats were sacrificed for immunohistochemical analysis of MMP-9 expression within the NAc core. (A) Representative images of MMP-9 (568nm) and DAPI (405nm) immunoreactivity (IR) within the NAc core of male and female sucrose rats treated with control or shRNA lentivirus, imaged at 20x magnification. AC = anterior commissure. Scale bars = 50 μm. (B) A two-way ANOVA revealed a significant main effect of sex but no significant main effect of virus or sex-by-virus interaction. n = 5–6/group. *p < 0.05 for main effect of sex. Error bars = SEM.

Figure 6.. Male rats exhibit greater MMP-9 expression within the NAcSh.

We examined MMP-9 expression within the NAcSh to 1) probe whether our observed sex difference within the NAc core is also observed within the NAcSh and 2) confirm our viral manipulation was not associated with changes in MMP-9 expression observed within the NAcSh. (A) Anatomical location of NAcSh MMP-9 quantification and representative images of NAcSh MMP-9 (568nm) and DAPI (405nm) expression across virus type and sex. Scale bars = 50 μm. (B) Similar to the NAc core, males exhibited greater overall expression of MMP-9 compared to females (ANOVA main effect of sex, *p < 0.05). (C) Among male cocaine rats, shRNA treatment significantly reduced MMP-9 expression levels in the NAc core only (Tukey’s test, **p < 0.01 relative to all other groups). (D) Female cocaine rats exhibited significantly greater MMP-9 expression within the NAc core compared to the NAcSh (ANOVA main effect of brain region, *p<0.05), but no main effect of virus or virus-by-brain region interaction. NAcSh MMP-9 expression was not quantified in sucrose animals because it was completely undetectable in all but 2 animals.

Figure 7.. Total NAc core MMP-9 expression does not significantly correlate with cue-induced cocaine or sucrose seeking.

Relative MMP-9 immunoreactivity within the NAc core did not significantly correlate with cue-induced cocaine seeking in either (A) males or (B) females. Likewise, relative MMP-9 immunoreactivity within the NAc core did not significantly correlate with cue-induced sucrose seeking in either (A) males or (B) females. Dotted lines = 95% confidence bands. AU = arbitrary units.

Figure 8.. Total NAcSh MMP-9 expression does not significantly correlate with cue-induced cocaine seeking.

Relative MMP-9 immunoreactivity within the NAcSh did not significantly correlate with cue-induced cocaine seeking in either (A) males or (B) females. Dotted lines = 95% confidence bands. AUs = arbitrary units.