EcoHIV infection effects on cocaine seeking and neuroimmune responses in male mice depend on cocaine exposure pattern

Abstract

Cocaine use disorders (CUDs) and human immunodeficiency virus (HIV) remain persistent public health dilemmas throughout the world. Cocaine seeking increases over a protracted period of abstinence, an effect known as the incubation of craving. Little is known about how HIV may modulate this process. Thus, we sought to examine the impact of chronic HIV infection on the incubation of cocaine craving and associated changes in the expression levels of central neuroimmune and peripheral immune substrates. Here, male mice were inoculated with EcoHIV, which is a chimeric HIV-1 construct that produces chronic HIV infection in mice. Mice were conditioned with cocaine daily or intermittently in a conditioned place preference (CPP) paradigm, followed by 1 or 21 days of forced abstinence prior to assessing preference for the cocaine-paired chamber. Mice conditioned daily exhibited potentiated incubation of cocaine CPP after 21 days of abstinence, and EcoHIV increased cocaine CPP across the test session at both abstinence timepoints. Conversely, EcoHIV-infected mice conditioned intermittently showed higher cocaine seeking after 1 day of abstinence compared to 21 days. Analysis of corticolimbic CX3CL1-CX3CR1 and glutamate receptor expression revealed a positive relationship between cocaine seeking and medial prefrontal cortex (mPFC) CX3CL1 and GluA1, as well as a nucleus accumbens (NAc) GluN2A expression. Moreover, examination of peripheral immune markers showed that the effect of abstinence and EcoHIV infection on these measures depended on the cocaine exposure regimen. Altogether, these results highlight the importance of cocaine abstinence and exposure pattern as critical variables that modulate HIV-associated neuroimmune outcomes and relapse vulnerability.

Keywords: Cocaine craving; Cytokines; Fractalkine; HIV; NMDA.

Fig. 1.. Timeline of experimental procedures.

Male C57BL/6 J mice (N = 108) were inoculated with EcoHIV (300–400 ng p24, i.p.) or PBS, followed by blood collection on weeks 1, 3, and 5 post-inoculation. Beginning on Week 6 post-inoculation, mice were tested for their CPP chamber bias during a 20-min pretest session. Using a biased CPP design during cocaine conditioning, cocaine injections (10 mg/kg, i.p.) were paired with the chamber that was least preferred during the pretest while saline injections (10 mL/kg, i.p.) was paired with the opposite chamber. In Experiment 1, mice received a saline pairing in the morning (AM), followed by a cocaine pairing 6 h later (PM), for a total of 4 pairings each (i.e., “daily conditioning”). In Experiment 2, cocaine and saline pairings (4 each) occurred on alternating days (i.e., “intermittent conditioning”). After cocaine conditioning, mice were placed in forced abstinence for 1 or 21 days prior to a CPP test, where chamber preference was assessed in a drug-free state. Mice were sacrificed immediately after CPP testing for tissue collection.

Fig. 2.. Cocaine-induced locomotion during conditioning.

(A) In Experiment 1 (daily conditioning), EcoHIV-infected mice exhibited potentiated locomotor behavior on pairings 3 and 4 relative to sham mice (*p < 0.05 comparing EcoHIV-Cocaine vs. Sham-Cocaine; n = 23–24/group). Moreover, EcoHIV-infected mice exhibited a significant increase in horizontal locomotor activity at pairing 3 compared to pairing 1 (^#p < 0.05). (B) In Experiment 2 (intermittent conditioning, n = 23–24/group), cocaine-induced locomotor activity was significantly decreased at pairing 2, and then significantly increased by pairing 4, relative to pairing 1 regardless of infection status. Locomotor activity in response to saline was significantly reduced at pairings 2 and 3 relative to pairing 1 regardless of infection status. *p < 0.05 relative to pairing 1 collapsed across infection status. Error bars = ±SEM.

Fig. 3.. Cocaine conditioned place preference and associated locomotor behavior.

(A) Locomotor behavior during Experiment 1 (daily conditioning) CPP testing did not differ across treatment groups. (B) Among shams, cocaine seeking was increased across the session following 21d ABS relative to 1d ABS. This was further potentiated by EcoHIV infection at each timepoint (*p < 0.05 for main effect of Abstinence). (C) Locomotor behavior during Experiment 2 (intermittent conditioning) CPP testing did not differ across treatment groups. (D) Among mice conditioned intermittently, sham mice showed a trend towards abstinence-induced potentiation of cocaine seeking, while EcoHIV-infection produced the reverse effect (p < 0.0001 for Bin x Abstinence x Infection interaction from three-way ANOVA; post hoc comparisons at each timepoint did not achieve statistical significance). *p < 0.05; ^‡p < 0.10. Error bars = ±SEM. CPP scores = posttest time in cocaine-paired chamber − pretest time in cocaine-paired chamber.

Fig. 4.. Splenic HIV-1 LTR DNA levels following daily or intermittent cocaine conditioning and 1 or 21 days of abstinence.

HIV-1 LTR DNA was evaluated from all EcoHIV-infected mice. Two mice that had undetectable levels of HIV-1 DNA, akin to all sham samples tested, were excluded from the study. Among mice exposed to cocaine daily, splenic HIV-1 LTR DNA levels were increased at 1 day (1d ABS) compared to the 21 days of abstinence (21d ABS). Conversely, no differences were observed in DNA levels between abstinence timepoints among mice exposed to cocaine intermittently. **p < 0.01 relative to all other groups. Error bars = ±SEM.

Fig. 5.. mPFC CX3CL1 expression and associated cocaine seeking following daily conditioning and abstinence.

(A) Among mice conditioned daily, no group mean differences in mPFC CX3CL1 expression were detected due to EcoHIV, abstinence, or their interaction. Error bars = ±SEM. (B) However, cocaine-seeking behavior was significantly correlated with CX3CL1 expression only among sham mice across abstinence time points. Black = sham; Green = EcoHIV.

Fig. 6.. mPFC GluA1 expression and associated cocaine seeking following daily conditioning and abstinence.

(A) Among mice conditioned daily, no group mean differences in mPFC GluA1 expression were detected due to EcoHIV, abstinence, or their interaction. Error bars = ±SEM. (B) The slopes of the regression lines correlating mPFC GluA1 and cocaine seeking differed significantly across abstinence timepoints among the shams. Here, a significant correlation between mPFC GluA1 and cocaine seeking among shams was observed only after 21d ABS. (C) The slopes of the regression lines correlating mPFC GluA1 and cocaine seeking did not differ significantly across abstinence timepoints among the EcoHIV-infected mice. Here, no significant correlation was observed between mPFC GluA1 and cocaine seeking across all EcoHIV-infected mice.

Fig. 7.. NAc GluN2A expression and associated cocaine seeking following daily conditioning and abstinence.

(A) Among mice conditioned daily, no group mean differences in NAc GluN2A expression were detected due to EcoHIV, abstinence, or their interaction. Error bars = ±SEM. (B) Cocaine seeking behavior was significantly correlated with GluN2A expression only among sham mice across abstinence time points. Black = sham; Green = EcoHIV.

Fig. 8.. Plasma G-CSF and VEGF expression following daily cocaine exposure.

Multiplex cytokine arrays were used to explore peripheral cytokine, chemokine, and growth factor expression in plasma following daily cocaine conditioning and abstinence. (A) Regardless of abstinence, EcoHIV infection increased plasma expression of granulocyte colony stimulating factor (G-CSF) (*p < 0.05, main effect of infection). (B) Regardless of abstinence, EcoHIV infection decreased vascular endothelial growth factor (VEGF) expression in plasma (*p < 0.05, main effect of infection). Error bars = ±SEM.

Fig. 9.. Plasma IL-5 expression following intermittent cocaine exposure.

Multiplex cytokine arrays were used to explore peripheral cytokine, chemokine, and growth factor expression in plasma following intermittent cocaine conditioning and abstinence. Regardless of infection status, protracted abstinence from cocaine was associated with a significant increase in plasma expression of interleukin (IL)-5 (*p < 0.05, main effect of abstinence). Error bars = ±SEM.