Morphine and gp120 toxic interactions in striatal neurons are dependent on HIV-1 strain

Abstract

A rigorously controlled, cell culture paradigm was used to assess the role of HIV-1 gp120 ± morphine in mediating opioid-HIV interactive toxicity in striatal neurons. Computerized time-lapse microscopy tracked the fate of individual neurons co-cultured with mixed-glia from mouse striata during opioid and gp120 exposure. Subpopulations of neurons and astroglia displayed μ-opioid receptor, CXCR4, and CCR5 immunoreactivity. While gp120 alone was or tended to be neurotoxic irrespective of whether X4-tropic gp120(IIIB), R5-tropic gp120(ADA), or dual-tropic gp120(MN) was administered, interactive toxicity with morphine differed depending on HIV-1 strain. For example, morphine only transiently exacerbated gp120(IIIB)-induced neuronal death; however, in combination with gp120(MN), morphine caused sustained increases in the rate of neuronal death compared to gp120(MN) alone that were prevented by naloxone. Alternatively, gp120(ADA) significantly increased the rate of neuron death, but gp120(ADA) toxicity was unaffected by morphine. The transient neurotoxic interactions between morphine and gp120(IIIB) were abrogated in the absence of glia suggesting that glia contribute significantly to the interactive pathology with chronic opiate abuse and neuroAIDS. To assess how mixed-glia might contribute to the neurotoxicity, the effects of morphine and/or gp120 on the production of reactive oxygen species (ROS) and on glutamate buffering were examined. All gp120 variants, and to a lesser extent morphine, increased ROS and/or decreased glutamate buffering, but together failed to show any interaction with morphine. Our findings indicate that HIV-1 strain-specific differences in gp120 are critical determinants in shaping both the timing and pattern of neurotoxic interactions with opioid drugs.

Figure 1

Cellular localization of CXCR4, CCR5, and/or MOR immunofluorescence in striatal neurons and astrocytes. Receptors were colocalized in neurons (a–c) or astrocytes (d–f), respectively, using antibodies against MAP2 or GFAP; cells were counterstained with Hoechst 33342 (blue). In neurons, CXCR4 (a), CCR5 (b), and MOR (c) immunoreactivity was associated with the cell body and dendrites, while CCR5 antigenicity appeared to extend into some axons (b). In GFAP-immunolabeled astrocytes, CXCR4 (d), CCR5 (e), and MOR (f) displayed a punctate pattern of labeling with some faint reactivity extending into the cell processes (d–f); scale bar = 10 µm.

Figure 2

Phase-contrast images showing time-lapse tracking of neuronal injury and death in the same neurons before and during exposure to vehicle-control medium (a), morphine (b), and/or gp120_IIIB (c). Although there are some small changes in the morphology of cells in (a) and (b), there is no neuron death over this 16 h time course. A neuron that dies over the same time course during gp120_IIIB treatment is followed in (c). Neuron death is often preceded by the systematic loss/degeneration of neurites (dendrite pruning; arrowheads) over a prolonged period. Death per se occurs more rapidly, as denoted here by a slight swelling and some loss of birefringence (arrow; 16 h) and DNA fragmentation thereafter (24 h and 60 h). Neuron death can be confirmed by annexin V reactivity, and the inability to exclude viability markers such as ethidium homodimer, ethidium monoazide, or trypan blue (see text). Some neurons remain viable despite morphine and gp120 co-exposure (open arrowheads “□” at 0 h and 60 h) (c); some surrounding cells, including astroglia, immature glial precursors and macrophages/microglia, are motile and can display sporadic movement, as indicated by the progenitor which moves to the top of the field between 7 h–8 h (hatched arrow) (c). Scale bar = 20 µm.

Figure 3

Time-dependent effects of morphine and/or HIV-1 gp120_IIIB on the survival of medium spiny striatal neurons cultured alone (a) or with a glial bedlayer (b). There were main effects of both time and treatment, as well as a significant interaction effect (time × treatment) (a), which were further examined using Duncan’s post-hoc analysis. Exposure to gp120_IIIB ± morphine was intrinsically neurotoxic, irrespective of whether neurons are cultured ± glia (a–b) (*P < 0.05 vs. control) (a). Morphine transiently and significantly accelerates gp120_IIIB neurotoxicity above control levels, but only when the neurons are co-cultured with glia (**P < 0.05 vs. control, but not morphine or gp120 treatment, at 12 h; n = 6 experiments) (b). Concurrent exposure to naloxone (Nal) negated the combined neurotoxic effects of morphine and gp120_IIIB at 12 h (b). Interestingly, morphine alone displayed modest, but significant neurotoxicity, but again, only in the presence of high-density glia (*P < 0.05 vs. control) (b). Neuron death is reported as the percentage dying relative to pretreatment numbers; data are the mean number of surviving neurons ± SEM from n = 4–6 experiments.

Figure 4

Time-dependent effects of exposure to morphine (Morph) and/or R5-tropic HIV-1 gp120_ADA in neuron and mixed-glial co-cultures for 72 h. Analyses for R5-tropic gp120_ADA showed a main effect for time, but not for treatment (a). Although there was a significant interaction effect (time × treatment), Duncan’s post-hoc testing did not reveal significance at particular times. When the analysis was simplified by eliminating the morphine group, there was decline in survival for R5-tropic gp120_ADA exposed neurons at the last time point of assessment (*P < 0.05 vs. control at 72 h) (a). Otherwise, values for gp120_ADA treatment alone vs. control (e.g., P = 0.056 at 68 h) or gp120_ADA + morphine (e.g., P = 0.054 at 56 h) were close, but not significant; data are the mean number of surviving neurons ± SEM from n = 4–6 experiments.

Figure 5

Time-dependent effects of exposure to morphine (Morph), X4/R5-tropic HIV-1 gp120_MN and/or naloxone (Nal) in neuron and mixed-glial co-cultures for 72 h (a). There were significant time and treatment effects for dual-tropic gp120_MN, as well as a significant interaction effect. Post-hoc analyses showed that all treatment groups were different from control values starting at 20 h following continuous exposure, except for morphine whose effects began at 40 h (*P < 0.05 vs. control). Additionally, morphine co-treatment enhanced the effects of gp120_MN at both early time points (20–28 h), and at 56 h and thereafter (^¶P ≤ 0.05), and the effects of morphine were prevented by naloxone (Nal) (^#P ≤ 0.05 vs. morphine + gp120_MN; *P < 0.05 vs. control). Unlike gp120_ADA, co-administering morphine exacerbated the neurotoxicity of gp120_MN suggesting that morphine interacts differently with gp120 variants from different HIV-1 strains. Data are the mean number of surviving neurons ± SEM from n = 4–6 experiments (a). Effects of morphine (500 nM) and/or dual-tropic gp120_MN (500 pM) on glutamate buffering (b). Mixed-glial cultures were challenged with 1 mM glutamate and the levels were assessed at 0, 15, 30, 45, 60, 120 and 240 min. Exogenously administered glutamate was rapidly depleted during the first 60 min and continued to slowly decrease thereafter in untreated controls. gp120_MN treated cultures ± morphine, demonstrated a significant deficit in the ability to uptake glutamate beginning at 45 min which persisted throughout the experiment. Values indicate the amount of glutamate (mM) remaining in the media over the 240 min time course ± SEM of n = 3 experiments (*P < 0.05 vs. control; ^§P < 0.05 control vs. gp120 alone or morphine + gp120; ^¶P < 0.05 gp120 vs. morphine + gp120; ^‡P < 0.05 control vs. gp120 alone) (b).

Figure 6

Effects of gp120 and morphine on ROS production in mixed-glia assessed by DCF fluorescence. A concentration-effect curve revealed significant increases in DCF relative fluorescence following 45 min exposure to gp120_IIIB with an EC₅₀ = 8.39 × 10⁻⁹ (*P < 0.05) (a). Similar concentration-effect curves for gp120_ADA (b) and gp120_MN (c) also show ROS elevation, but with somewhat different kinetics. Gp120_IIIB ± morphine significantly increased ROS formation in mixed-glial cultures but without any additive or synergistic effect (d). Cells were pretreated with vehicle, NAC or DPI for 20 min before incubation with DCF-DA and subsequently treated with gp120_IIIB (500 pM) and/or morphine (500 nM) for 40 min prior to assay. Mean DCF relative fluorescence units (MFI) were used as an estimate of ROS and were compared for each treatment. Gp120_IIIB and morphine both markedly increased ROS production (*P < 0.05 vs. control). NAC and/or DPI significantly decreased gp120_IIIB and/or morphine-induced ROS production (^#P < 0.05 vs. treatment with gp120 and/or morphine alone); data are the mean ± SEM of n = 3 experiments (d).