Astrocytic HIV-1 Nef Expression Decreases Glutamate Transporter Expression in the Nucleus Accumbens and Increases Cocaine-Seeking Behavior in Rats

Abstract

Background/objectives: Cocaine use disorder is an intersecting issue in populations with HIV-1, further exacerbating the clinical course of the disease and contributing to neurotoxicity and neuroinflammation. Cocaine and HIV neurotoxins play roles in neuronal damage during neuroHIV progression by disrupting glutamate homeostasis in the brain. Even with combined antiretroviral therapy (cART), HIV-1 Nef, an early viral protein expressed in approximately 1% of infected astrocytes, remains a key neurotoxin. This study investigates the relationship among Nef, glutamate homeostasis, and cocaine in the nucleus accumbens (NAc), a critical brain region associated with drug motivation and reward.

Methods: Male and female Sprague Dawley rats were used to compare the effects of astrocytic Nef and cocaine by molecular analysis of glutamate transporters, GLT-1 and the cysteine glutamate exchanger (xCT), in the NAc. Behavioral assessments for cocaine self-administration were used to evaluate cocaine-seeking behavior.

Results: The findings indicate that both cocaine and Nef independently decrease the expression of the glutamate transporter GLT-1 in the NAc. Additionally, rats with astrocytic Nef expression exhibited increased cocaine-seeking behavior but demonstrated sex-dependent molecular differences after the behavioral paradigm.

Conclusions: The results suggest that the expression of Nef intensifies cocaine-induced alterations in glutamate homeostasis in the NAc, potentially underlying increased cocaine-seeking behavior. Understanding these interactions better may inform therapeutic strategies for managing cocaine use disorder in HIV-infected individuals.

Keywords: GLT-1; HIV-1 Nef; cocaine; glutamate; nucleus accumbens; sex-differences; xCT.

Figure 1

Timeline and schematic representation of bilateral infusions in the NAc at 5 weeks. (A) Specific timeline used for molecular experiments with rats. (B) Schematic representation showing location of the bilateral infusion site on the corresponding coronal section (red dots). Viral expression spans from approximately bregma 0.70 mm to 1.60 mm. GFAP-specific promoter expression of mCherry in NAc of Sprague Dawley rats 5 weeks after infusion; 10× magnification (100 µm scale). Right image shows white arrows pointing at positive punctate cytoplasmic staining in the NAc (right hemisphere); 100× magnification (10 µm scale). (C) Representative images of immunohistochemical staining with Nef antibody (1:50) in NAc slice of Control (top) and Nef-treated (bottom) rat brain tissue. DAB staining in brown with hematoxylin for nuclear counterstain. Pictures of 30 µm thick tissues taken at 60× magnification (62.18 µm scale). Zoomed area at 100× magnification (15 µm scale) of Nef-positive NAc slice demonstrating DAB staining with an astrocyte morphology.

Figure 2

Nef expression in the NAc combined with cocaine causes an increase in GFAP. (A) Representative images of free-floating immunofluorescence to identify astrocyte expression using GFAP (green, 1:200) and GLT-1 (red, 1:400). Nuclear counterstaining was used DAPI (blue). Pictures were taken at 100× magnification (10 µm scale). (B) Integrated density of GFAP in the infusion site demonstrates increased expression in the Nef+cocaine treatment. (C) Integrated density of GLT-1 in the infusion site shows decreased expression after 24 h of cocaine injection (15 mg/kg, i.p.) in Nef and cocaine treatments compared to control. Each sample represents an average of 3–4 60×mag fields per rat in each treatment. (D) Representative protein bands of GLT-1 (65 kD) and β-actin (42 kD). From left to right: control, Nef, cocaine, Nef+cocaine. (E) Protein expression of GLT-1 shows decrease 24 h after cocaine injection (i.p.). Ordinary one-way ANOVA with Tukey post-hoc and multiple comparisons was used to compare data within groups (control: n = 10–14; Nef: n = 12–15; cocaine: n = 10–15; Nef+cocaine: n = 11–13). Data represent mean +/− SEM adjusted to β-actin and normalized to control. One-way ANOVA main effect between treatments p < 0.05.

Figure 3

Nef expression in the NAc causes no effect on xCT expression. (A) Representative images of free-floating immunofluorescence to identify astrocyte expression using GFAP (red, 1:200) and xCT (green, 1:200). Nuclear counterstaining was used DAPI (blue). Pictures were taken at 100× (10 um scale). Each sample represents an average of 3–4 60× mag fields per rat in each treatment. (B) Integrated density of xCT expression in the NAc shows no significant differences between treatments. (C) Representative protein bands of xCT (37 kD) and β-actin (42 kD) per treatment. From left to right: control, Nef, cocaine, Nef+cocaine. (D) Protein expression of xCT in nucleus accumbens shows a decreased trend in cocaine treatment compared to control. Ordinary one-way ANOVA with Tukey post-hoc and multiple comparisons was used to compare data within groups (control: n = 9–13; Nef: n = 10–13; cocaine: n = 10–15; Nef+cocaine: n = 10–15). Data represent mean +/− SEM adjusted to β-actin and normalized to control.

Figure 4

Behavioral timeline and schematic representation of lentiviral infusions in NAc core. (A) Behavioral timeline used for cocaine self-administration experiments with rats. (B) Schematic representation showing placement of the infusion site of each rat in the corresponding coronal section of the NAc ranging from 0.70 mm to 1.60 mm (male control vectors—black dots; male Nef vectors—red dots; female control vectors—red triangles; female Nef vectors—gray triangles). (C) GFAP-specific promoter expression of mCherry in NAc of Sprague Dawley rats 10 weeks after infusion; 10× magnification (100 µm scale) with zoomed-in area at 15 µm scale showing morphology of cells infected. (D) Representative images of Nef immunohistochemistry at infusion site after behavioral paradigm. DAB staining in brown with hematoxylin for nuclear counterstain. Red arrows represent astrocytes positive for Nef. Pictures of 30 µm thick tissues taken at 20× magnification (100 µm scale).

Figure 5

Nef-expressing male rats show more cocaine-seeking behavior than cocaine male rats. (A) Active lever presses of control, Nef, cocaine, and Nef+cocaine male rats during self-administration phase and extinction. (B) Cocaine infusions of cocaine vs. Nef+cocaine rats and saline infusions for control and Nef rats during self-administration. *, **, ***, **** represent significant differences between cocaine and Nef+cocaine groups during self-administration at p < 0.05, 0.01, 0.001, 0.0001, respectively. (C) Average right lever presses (correct) during extinction of cocaine vs. Nef+cocaine rats compared to right active lever presses during cue-primed reinstatement. (D) Average right lever presses of cocaine vs. Nef+cocaine animals compared to right active lever presses during cocaine-primed reinstatement. Nef+cocaine rats significantly increased active lever presses in cocaine-primed reinstatement compared to cocaine group. Two-way, repeated measures ANOVA with Fisher’s LSD post-hoc analysis indicated all rats reinstated cocaine seeking compared with extinction responding. Data represent mean +/− SEM.

Figure 6

Nef-expressing female rats show more cocaine-seeking behavior than cocaine female rats. (A) Active lever presses of control, Nef, cocaine, and Nef+cocaine female rats during self-administration phase and extinction. (B) Cocaine infusions of cocaine vs. Nef+cocaine rats and saline infusions for control and Nef rats during self-administration. No differences were observed between the cocaine and Nef+cocaine female groups. (C) Average right lever presses (correct) during extinction of cocaine vs. Nef+cocaine rats compared to right active lever presses during cue-primed reinstatement. (D) Average right lever presses of cocaine vs. Nef+cocaine animals compared to right active lever presses during cocaine-primed reinstatement. Nef+cocaine rats significantly increased active lever presses in cocaine-primed reinstatement compared to cocaine group. Two-way, repeated measures ANOVA indicated that all rats reinstated cocaine seeking compared with extinction responding. Data represent mean +/− SEM.

Figure 7

Nef alters glutamate transporter expression after cocaine self-administration paradigm in male and female rats. (A) Representative protein bands of GLT-1 (kDa) with analysis of Western blots per group. Protein expression of GLT-1 shows a decrease in all treatments compared to cocaine after cocaine-primed reinstatement of male rats. (B) Representative protein bands of xCT (37 kDa) in NAc of male rats with analysis per group. Protein expression of xCT in the NAc shows a decrease in cocaine and Nef+cocaine groups after behavioral paradigm of male rats. (C) Representative protein bands of GFAP (50 kDa) in the NAc of male rats with analysis per group. GFAP expression shows no difference between groups of male rats after behavioral paradigm. (male groups, control: n = 8; Nef: n = 8; cocaine: n = 9; Nef+cocaine: n = 10). (D) Representative protein blots of GLT-1 in the NAc of female rats with analysis per group. Protein expression of GLT-1 shows decrease in Nef and cocaine groups but increase in Nef+cocaine group after cocaine-primed reinstatement in female rats. (E) Representative blots of xCT in the NAc of female rats and analysis after behavioral paradigm per group. Protein expression of xCT in the NAc shows decrease in cocaine and Nef+cocaine after behavioral paradigm in female rats. (F) Representative images of GFAP protein expression with analysis per group of female rats. GFAP trends toward an increase in the Nef+cocaine female group compared to control 24 h after cocaine-primed reinstatement. One-way ANOVA with Tukey post-hoc and multiple comparisons was used to compare data within groups. (Female groups, control: n = 8; Nef: n = 9; cocaine: n = 9; Nef+cocaine: n = 11). Data represent mean +/− SEM adjusted to β-actin and normalized to control.