Sex differences and Tat expression affect dopaminergic receptor expression and response to antioxidant treatment in methamphetamine-sensitized HIV Tat transgenic mice

Abstract

Methamphetamine (Meth) abuse is a common HIV comorbidity. Males and females differ in their patterns of Meth use, associated behaviors, and responses, but the underlying mechanisms and impact of HIV infection are unclear. Transgenic mice with inducible HIV-1 Tat protein in the brain (iTat) replicate many neurological aspects of HIV infection in humans. We previously showed that Tat induction enhances the Meth sensitization response associated with perturbation of the dopaminergic system, in male iTat mice. Here, we used the iTat mouse model to investigate sex differences in individual and interactive effects of Tat and Meth challenge on locomotor sensitization, brain expression of dopamine receptors (DRDs) and regulatory adenosine receptors (ADORAs). Because Meth administration increases the production of reactive oxygen species (ROS), we also determined whether the effects of Meth could be rescued by concomitant treatment with the ROS scavenger N-acetyl cysteine (NAC). After Meth sensitization and a 7-day abstinence period, groups of Tat+ and Tat-male and female mice were challenged with Meth in combination with NAC. We confirmed that Tat expression and Meth challenge suppressed DRD mRNA and protein in males and females' brains, and showed that females were particularly susceptible to the effects of Meth on D1-like and D2-like DRD subtypes and ADORAs. The expression of these markers differed strikingly between males and females, and between females in different phases of the estrous cycle, in a Tat -dependent manner. NAC attenuated Meth-induced locomotor sensitization and preserved DRD expression in all groups except for Tat + females. These data identify complex interactions between sex, Meth use, and HIV infection on addiction responses, with potential implications for the treatment of male and female Meth users in the context of HIV, especially those with cognitive disorders.

Keywords: Dopamine receptors; Human immunodeficiency virus; Methamphetamine; N-acetyl cysteine; NeuroHIV; Reactive oxygen species; Tat.

Figure 1.. Effect of sex and NAC treatment on locomotor activity of Meth-sensitized Tat− and Tat+ mice.

(A) Motor activity of male and female Tat− and Tat+ mice was recorded for 30 min starting 5 minutes after challenge with Meth or saline (Sal). NAC at 100 mg/kg or Sal vehicle were administered at the same time as the challenge. Data are presented as the mean ± SEM of n=10–19 mice/group. Main effects: Tat (F1, 129=90.16, P<0.0001), NAC (F1, 129=13.2, p<0.05), METH (F1, 129=304.8, P<0.0001), and NAC × Meth × Sex interaction (F1, 129=5.7, P<0.05). *p<0.05 by ANOVA followed by Bonferroni’s post hoc test. (B) Serum progesterone levels were determined by ELISA at 24 h after challenge. Mean ± SD of duplicate samples.

Figure 2.. Effect of Tat expression, sex, and estrous cycle phase on ADORA transcription in the brains of Meth-challenged and NAC-treated mice.

Groups of Tat− (A, C, E) and Tat+ (B, D, F) were Meth sensitized, underwent a 7-day washout period, and were then challenged with saline alone (Sal), Meth alone, Sal plus NAC (Sal/NAC), or Meth plus NAC (Meth/NAC). At 24 h after challenge, the mice were euthanized and samples of caudate-putamen were analyzed by qRT-PCR for the expression of (A, B) ADORA 1, (C, D) ADORA 2a, and (E, F) ADORA 2b mRNA. Receptor mRNA levels were normalized to the mean GAPDH mRNA and 18S rRNA levels. *p<0.05 in mixed models and Bonferroni’s multiple comparisons. Comparisons between Tat+ and Tat− mice are described in the text.

Figure 3.. Effect of Tat expression, sex, and estrous cycle phase type 1 DRD (DRD1 and DRD5) transcription in the brains of Meth-challenged and NAC-treated mice.

Groups of Tat− (A, C) and Tat+ (B, D) mice were challenged as described for Figure 2. At 24 h after challenge, the mice were euthanized and samples of caudate-putamen were analyzed by qRT-PCR for the expression of (A, B) DRD1 and (C, D) DRD5 mRNA. Receptor mRNA levels were normalized to the mean GAPDH mRNA and 18S rRNA levels. *p<0.05 in mixed models and Bonferroni’s multiple comparisons. Comparisons between Tat+ and Tat− mice are described in the text.

Figure 4.. Effect of Tat expression, sex, and estrous cycle phase on type 2 DRD (DRD2, DRD3, and DRD4) transcription in the brains of Meth-challenged and NAC- treated mice.

Groups of Tat− (A, C, E), and Tat+ (B, D, F) mice were challenged as described for Figure 2. At 24 h after challenge, the mice were euthanized and samples of caudate-putamen were analyzed by qRT-PCR for the expression of (A, B) DRD2, (C, D) DRD3, and (E, F) DRD4 mRNA. Receptor mRNA levels were normalized to the mean GAPDH mRNA and 18S rRNA levels. *p<0.05 in mixed models and Bonferroni’s multiple comparisons. Comparisons between Tat+ and Tat− mice are described in the text.

Figure 5.. Quantification of DRD1 protein expression in the brains of Meth-challenged and NAC-treated mice by immunohistochemistry.

Groups of male and female Tat− and Tat+ mice were challenged as described for Figure 2. (A) At 24 h after challenge, serial sagittal sections of one hemisphere were stained with anti-DRD1 antibody. Images show 20x magnification of the hippocampal region, and rectangles containing cells of interest are additionally shown in the smaller images. (B) Quantification of DRD1 protein expression. Stained serial sagittal sections of one hemisphere were imaged and digitized using ImageJ. DRD1 expression is presented as the percentage area of positive staining normalized to the total brain area. Mean ± SEM of n=7–14 males and females/group. *p<0.05 by 2-way ANOVA and mixed effects analysis with Bonferroni’s post hoc test. Females in estrus showed the same expression pattern as animals in other estrous cycle phases.

Figure 6.. Quantification of DRD2 protein expression in the brains of Meth-challenged and NAC-treated mice by immunohistochemistry.

Groups of male and female Tat− and Tat+ mice were challenged as described for Figure 2 (A) At 24 h after challenge, serial sagittal sections of one hemisphere were stained with anti-DRD2 antibody. Images show 20x magnification of the hippocampal region, and rectangles containing cells of interest are additionally shown in the smaller images. (B) Quantification of DRD2 staining. Stained serial sagittal sections were imaged and digitized using ImageJ. DRD2 expression is presented as the percentage area of positive staining normalized to the total brain area. Mean ± SEM of n=7–14 males and females/group. *p<0.05 by 2-way ANOVA and mixed effects analysis with Bonferroni’s post hoc test. Females in estrus showed the same expression pattern as animals in other estrous cycle phases.