Inhibitory Neurotransmission Is Sex-Dependently Affected by Tat Expression in Transgenic Mice and Suppressed by the Fatty Acid Amide Hydrolase Enzyme Inhibitor PF3845 via Cannabinoid Type-1 Receptor Mechanisms

Abstract

(1) Background. The endocannabinoid (eCB) system, which regulates physiological and cognitive processes, presents a promising therapeutic target for treating HIV-associated neurocognitive disorders (HAND). Here we examine whether upregulating eCB tone has potential protective effects against HIV-1 Tat (a key HIV transactivator of transcription) protein-induced alterations in synaptic activity. (2) Methods. Whole-cell patch-clamp recordings were performed to assess inhibitory GABAergic neurotransmission in prefrontal cortex slices of Tat transgenic male and female mice, in the presence and absence of the fatty acid amide hydrolase (FAAH) enzyme inhibitor PF3845. Western blot and mass spectrometry analyses assessed alterations of cannabinoid receptor and enzyme protein expression as well as endogenous ligands, respectively, to determine the impact of Tat exposure on the eCB system. (3) Results. GABAergic activity was significantly altered upon Tat exposure based on sex, whereas the effectiveness of PF3845 to suppress GABAergic activity in Tat transgenic mice was not altered by Tat or sex and involved CB₁R-related mechanisms that depended on calcium signaling. Additionally, our data indicated sex-dependent changes for AEA and related non-eCB lipids based on Tat induction. (4) Conclusion. Results highlight sex- and/or Tat-dependent alterations of GABAergic activity and eCB signaling in the prefrontal cortex of Tat transgenic mice and further increase our understanding about the role of FAAH inhibition in neuroHIV.

Keywords: PF3845; Tat transgenic mice; anandamide; cannabinoid type 1 receptor; endocannabinoid enzyme inhibitor; endocannabinoids; fatty acid amide hydrolase; inhibitory postsynaptic potentials; neuroHIV; non-eCB lipids.

Figure 1

Tat induction alters the frequency and partially the amplitude of IPSCs in mPFC neurons in a sex-dependent manner. (A) Representative traces of sIPSCs in female and male Tat transgenic mPFC brain slices. (B) Tat induction significantly decreased mean sIPSC frequency for Tat(+) males compared to Tat(−) males, whereas a significant increase in sIPSC frequency was demonstrated for Tat(+) females compared to their respective Tat(−) counterparts. (C) No significant effects were noted on the mean amplitude of sIPSCs. (D) Similarly, for mIPSCs, Tat(+) males showed decreased mIPSC frequency compared to Tat(−) males and Tat(+) females showed increased mIPSC frequency compared to Tat(−) females. (E) For the mean amplitude of mIPSCs only Tat(+) males demonstrated decreased mIPSC amplitude compared to Tat(−) males. Female Tat transgenic mice did not significantly differ from each other. Data are raw data (mean ± SEM) separated by sex and genotype. Statistical significance was assessed by ANOVA followed by planned comparisons; * p < 0.05. In all panels, sample size is indicated as (cells/mice).

Figure 2

PF3845 decreases the frequency and partially the amplitude of IPSCs in mPFC neurons independent of Tat induction and sex. (A) Representative traces show sIPSCs in female Tat(−) and Tat(+) mPFC brain slices before and after PF3845 (1 µM) application. (B) Tat(+) males showed decreased sIPSC frequency compared to Tat(−) males, whereas Tat(+) females demonstrated increased sIPSC frequency compared to Tat(−) females. Further, PF3845 significantly decreased the mean sIPSC frequency in Tat(−) and Tat(+) brain slices for each sex with the downregulation being similar across all groups. (C) For the mean amplitude of sIPSCs, a significant downregulating effect by PF3845 was noted specifically for Tat(−) males. (D) For the mean mIPSC frequency, PF3845 significantly decreased mIPSC frequency in all groups with similar inhibitory effects across. (E) For the mean mIPSC amplitude, no significant effects were noted. Data are raw data (mean ± SEM) separated by sex and genotype. Statistical significance was assessed by ANOVA followed by Bonferroni’s post hoc test; * p < 0.05 vs. corresponding Tat(−) counterpart; ^# p < 0.05. In all panels, sample size is indicated as (cells/mice).

Figure 3

PF3845 effects on the frequency and amplitude of IPSCs (% of control) in mPFC neurons are blocked by CB₁R antagonist SR141716A but not CB₂R antagonist AM630. No significant effects were noted for sex and genotype on any sIPSC measure (% of control) and are therefore collapsed across. (A) Representative traces show sIPSCs before and after SR141716A (1 µM) ± PF3845 (1 µM) application. (B) No significant differences on sIPSC frequency and amplitude (% of control) were noted between the SR141716A alone and SR141716A in combination with PF3845 conditions, indicating the CB₁R antagonist SR141716A was able to block the downregulating effects of PF3845 on sIPSCs. Further, both conditions were not significantly different from control condition (0%). (C) Similarly, no significant effects were noted on the mIPSC frequency and amplitude (% of control). (D) Representative traces show sIPSCs before and after AM630 (1 µM) ± PF3845 (1 µM) application. (E) There was a significant difference between AM630 alone and AM630 in combination with PF3845 conditions on sIPSC frequency (% of control), indicating pretreatment of AM630 did not prevent the PF3845-induced decreases in the mean frequency of sIPSCs (% of control). This is supported by a significant downregulation of sIPSC frequency for the AM630 in combination with PF3845 condition compared to control (0%), which was not seen for the AM360 alone condition. No significant effects were noted on sIPSC amplitude (% of control). (F) Similarly, pretreatment of AM630 did not block the downregulating effects of PF3845 on mIPSC frequency and both conditions significantly decreased mean mIPSC frequency compared to control (0%). No significant effects were noted on mIPSC amplitude (% of control). Data are percent of control data (mean ± SEM) collapsed across sex and genotype. Statistical significance was assessed by ANOVA followed by Bonferroni’s post hoc test; ^# p < 0.05. One-sample t-tests with Bonferroni correction assessed significant changes from control condition (0%), ^a p < 0.05 vs. control (before treatment, 0%). In all panels, sample size is indicated as (cells/mice); please see Supplemental Table S1 for specific information on sample size for sex and genotype. SR1, SR141716A; PF, PF3845; AM, AM630.

Figure 4

Effects of PF3845 on the frequency and amplitude of IPSCs (% of control) in mPFC neurons are blocked in the absence of extracellular and intracellular calcium. No significant effects were noted for sex and genotype on any sIPSC measure and are therefore collapsed across. (A) Representative traces show sIPSCs before and after 0 Ca²⁺ ± PF3845 (1 µM) bath application. (B) Removing extracellular calcium from the aCSF in the presence or absence of PF3845 (1 µM) significantly downregulated sIPSC frequency or amplitude (% of control; except for sIPSC amplitude at 0 Ca²⁺ condition) compared to control condition (before removal of 0 Ca²⁺, 0%). Importantly, the 0 Ca²⁺ ± PF3845 condition was not significantly different from 0 Ca²⁺, indicating PF3845 had no significant effect on sIPSC frequency or amplitude in the absence of extracellular calcium. (C) Similarly, mIPSC frequency or amplitude (% of control) was significantly downregulated by 0 Ca²⁺ ± PF3845 (except for mIPSC amplitude at 0 Ca²⁺) with PF3845 showing no further downregulation in the absence of extracellular calcium compared to the 0 Ca²⁺ condition. (D) Representative traces show sIPSCs before and after CdCl₂ (200 µM) ± PF3845 (1 µM) bath application. (E) Application of CdCl₂ significantly downregulated sIPSC frequency and amplitude (% of control) in the presence or absence of PF3845 compared to control condition (before CdCl₂ treatment, 0%). No differences were noted between CdCl₂ and CdCl₂ ± PF3845 treatment conditions. (F) For mIPSCs, application of CdCl₂ significantly downregulated mIPSC frequency (% of control) from control condition (0%), with CdCl₂ ± PF3845 showing no further downregulation compared to the CdCl₂ condition. No significant differences were noted for mIPSC amplitude (% of control). (G) Representative traces show sIPSCs before and after thapsigargin (1 µM) ± PF3845 (1 µM) bath application. (H) Application of thapsigargin significantly downregulated sIPSC frequency and amplitude (% of control) in the presence or absence of PF3845 compared to control condition (before thapsigargin treatment, 0%). No differences were noted between thapsigargin and thapsigargin + PF3845 treatment conditions, indicating that depletion of intracellular calcium stores blocked PF3845′s downregulating effects on sIPSC frequency and amplitude (% of control). (I) Similarly, mIPSC frequency was significantly downregulated by thapsigargin, with PF3845 showing no further downregulation compared to thapsigargin alone. No significant differences were noted for mIPSC amplitude (% of control). Data are percent of control data (mean ± SEM) collapsed across sex and genotype. Statistical significance was assessed by ANOVA followed by Bonferroni’s post hoc test and revealed no significant effects. One-sample t-tests with Bonferroni correction assessed significant changes from control condition (0%), ^a p < 0.05 vs. control (before treatment, 0%). In all panels, sample size is indicated as (cells/mice); please see Supplemental Table S1 for specific information on sample size for sex and genotype. 0 Ca²⁺, zero extracellular calcium; PF, PF3845; Thap, thapsigargin.

Figure 5

CB₁R, FAAH, and MAGL protein expression levels are not altered by Tat and sex. Western blot analyses of protein expression levels for CB₁R (A), FAAH (B), and MAGL (C) were assessed in the PFC of Tat transgenic mice. No significant effects were noted for any of the three measures. Data are eCB/β-Actin ratio data (mean ± SEM) separated by sex and genotype. Statistical significance was assessed by ANOVA. Sample size: n = 3 mice per group and sex.

Figure 6

AEA and seven related non-eCB lipids are significantly upregulated in the PFC of female Tat(+) mice. Concentrations of AEA, 2-AG and other lipid molecules were assessed in the PFC of Tat transgenic mice using LC/MS/MS. Lipid concentrations were normalized to pg/mg of tissue. (A) No significant effects were noted for 2-AG or its related non-eCB lipid 2-LG. (B) For AEA and eight related non-eCB lipids, a significant sex and/or sex x genotype interaction was noted (see Table 1 for ANOVA results) with female Tat(+) mice demonstrating significant increased lipid concentrations in the PFC compared to one or more of the other three groups, except for PEA. Data of (non-)eCB concentration are expressed in pg/mg (mean ± SEM) separated by sex and genotype. Statistical significance was assessed by ANOVA followed by Bonferroni post hoc tests; * p < 0.05 vs. Tat(−) female, ^# p < 0.05 vs. Tat(+) male, ^§ p < 0.05 vs. Tat(−) male. Sample size: n = 9, Tat(−) mice per sex; n = 8, Tat(+) mice per sex. Please see Supplemental Table S2 for the actual concentration values (mean ± SEM).