Individuals infected with human immunodeficiency virus-1 who abuse opiates can have a higher incidence of virus-associated neuropathology. Human immunodeficiency virus does not infect neurons, but viral proteins such as transactivator of transcription and glycoprotein 120, originating from infected glia, are neurotoxic. Moreover, functional changes in glial cells that enhance inflammation and reduce trophic support are increasingly implicated in human immunodeficiency virus neuropathology. In previous studies, co-exposure with morphine enhanced transactivator of transcription neurotoxicity towards cultured striatal neurons. Since those cultures contained µ-opioid receptor-expressing astroglia and microglia, and since glia are the principal site of infection in the central nervous system, we hypothesized that morphine synergy might be glially mediated. A 60 hour, repeated measures paradigm and multiple co-culture models were used to investigate the cellular basis for opiate-enhanced human immunodeficiency virus neurotoxicity. Morphine co-exposure significantly enhanced transactivator of transcription-induced neuron death when glia were present. Synergistic effects of morphine on transactivator of transcription neurotoxicity were greatest with neuron-glia contact, but also occurred to a lesser extent with glial conditioned medium. Importantly, synergy was lost if glia, but not neurons, lacked µ-opioid receptors, indicating that opiate interactions with human immunodeficiency virus converge at the level of µ-opioid receptor-expressing glia. Morphine enhanced transactivator of transcription-induced inflammatory effectors released by glia, elevating reactive oxygen species, increasing 3-nitrotyrosine production by microglia, and reducing the ability of glia to buffer glutamate. But neuron survival was reduced even more with glial contact than with exposure to conditioned medium, suggesting that noxious elements associated with cell contact augment the toxicity due to soluble factors. Similar morphine-transactivator of transcription synergy was also observed in studies with the clade C sequence of HIV-1 transactivator of transcription, which did not cause neuron death unless morphine was present. Several paradoxical observations related to opiate effects were noted when µ-opioid receptors were specifically ablated from either glia or neurons. This suggests that µ-opioid receptor loss in isolated cell types can fundamentally distort cell-to-cell signalling, revealing opponent processes that may exist in individual cell types. Our findings show the critical role of glia in orchestrating neurotoxic interactions of morphine and transactivator of transcription, and support the emerging concept that combined exposure to opiates and human immunodeficiency virus drives enhanced pathology within the central nervous system.

Background Human immunodeficiency virus (HIV) associated neurocognitive disorders (HAND), including memory dysfunction, continue to be a major clinical manifestation of HIV type-1 (HIV-1) infection. Viral proteins released by infected glia are thought to be the principal triggers of inflammation and bystander neuronal injury and death, thereby driving key symptomatology of HAND. Methods We used a GFAP-driven, doxycycline (DOX)-inducible HIV-1 Tat (transactivator of transcription) transgenic mouse model and examined structure-function relationships in hippocampal pyramidal CA1 neurons using morphologic (Golgi-silver impregnations, immunohistochemistry, TUNEL detection, synaptic protein markers, electron microscopy), electrophysiological (long-term potentiation (LTP)), and behavioral (Morris water maze, fear-conditioning) approaches. Results Tat induction caused a variety of different inclusions in astrocytes characteristic of lysosomes, autophagic vacuoles, and lamellar bodies, which were typically present within distal cytoplasmic processes. In pyramidal CA1 neurons, Tat induction reduced the number of apical dendritic spines, while disrupting the distribution of synaptic proteins (synaptotagmin 2 and gephyrin) associated with inhibitory transmission, but with minimal dendritic pathology and no evidence of pyramidal neuron death. Electrophysiological assessment of excitatory postsynaptic field potential (fEPSP) at Schaffer collateral/commissural fiber-CA1 synapses showed near total suppression of LTP in mice expressing Tat. The loss in LTP coincided with disruptions in learning and memory. Conclusion Tat expression in the brain results in profound functional changes in synaptic physiology and in behavior that are accompanied by only modest structural changes and minimal pathology. Tat likely contributes to HAND by causing molecular changes that disrupt synaptic organization, with inhibitory presynaptic terminals containing synaptotagmin 2 appearing especially vulnerable.

HIV-1 infection predisposes the central nervous system to damage by opportunistic infections and environmental insults. Such maladaptive plasticity may underlie the exaggerated comorbidity seen with HIV-1 infection and opioid abuse. Although morphine and HIV-1 Tat synergize at high concentrations to increase neuronal death in vitro, we questioned whether chronic low Tat exposure in vivo might contribute to the spectrum of neuropathology through sublethal neuronal injury. We used a doxycycline-driven, inducible, HIV-1 Tat transgenic mouse, in which striatal neuron death was previously shown to be absent, to examine effects of differential Tat expression, alone and combined with morphine. Low constitutive Tat expression caused neurodegeneration; higher levels induced by 7 days of doxycycline significantly reduced dendritic spine numbers. Moreover, Tat expression widely disrupted the endogenous opioid system, altering and , but not ␦, opioid receptor and proopiomelanocortin, proenkephalin, and prodynorphin transcript levels in cortex, hippocampus, and striatum. In addition to markedly reducing spine density by itself, morphine amplified the effect of higher levels of Tat on spines, and also potentiated Tat-mediated dendritic pathology, thus contributing to maladaptive neuroplasticity at multiple levels. The dendritic pathology and reductions in spine density suggest that sustained Tat ؎ morphine exposure un- Exposure to HIV results in neurodegenerative alterations in the central nervous system (CNS) of a substantial proportion of patients, even in the era of highly active anti-retroviral therapy. Highly active anti-retroviral therapy does not readily cross the blood-brain barrier, making the CNS a safe-haven for infection, and permitting ongoing degenerative changes even when viral titers are quite low in the periphery. [1][2][3][4][5][6] There is considerable evidence, both in patients and in experimental models, that coexposure to abused opiate drugs can hasten the onset and worsen the outcome of HIV encephalitis and other neurodegenerative changes.7-15 A more limited number of studies show that opioids increase viral loads, and hasten disease progression and/or neuropathology in simian immunodeficiency models, 16 -19 although this has been controversial. 20 -22 Our in vitro work has consistently shown evidence for interactions between Tat or gp120 and morphine that accelerate neurodegeneration. These interactive effects appear to be orchestrated by glial cells, 23 and likely involve synergistic upregulation of proinflammatory chemokine/cytokine release and production of reactive species. 24 -26 The present work was undertaken to extend previous results suggesting that HIV-1 Tat exposure might disrupt endogenous opioid and chemokine signaling.

Opiate abuse and HIV-1 have been described as interrelated epidemics, and even in the advent of combined anti-retroviral therapy, the additional abuse of opiates appears to result in greater neurologic and cognitive deficits. The central nervous system (CNS) is particularly vulnerable to interactive opiate-HIV-1 effects, in part because of the unique responses of microglia and astroglia. Although neurons are principally responsible for behavior and cognition, HIV-1 infection and replication in the brain is largely limited to microglia, while astroglia and perhaps glial progenitors can be latently infected. Thus, neuronal dysfunction and injury result from cellular and viral toxins originating from HIV-1 infected/exposed glia. Importantly, subsets of glial cells including oligodendrocytes, as well as neurons, express µ-opioid receptors and therefore can be direct targets for heroin and morphine (the major metabolite of heroin in the CNS), which preferentially activate µ-opioid receptors. This review highlights findings that neuroAIDS is a glially driven disease, and that opiate abuse may act at multiple glial-cell types to further compromise neuron function and survival. The ongoing, reactive cross-talk between opiate drug and HIV-1 co-exposed microglia and astroglia appears to exacerbate critical proinflammatory and excitotoxic events leading to neuron dysfunction, injury, and potentially death. Opiates enhance synaptodendritic damage and a loss of synaptic connectivity, which is viewed as the substrate of cognitive deficits. We especially emphasize that opioid signaling and interactions with HIV-1 are contextual, differing among cell types, and even within subsets of the same cell type. For example, astroglia even within a single brain region are heterogeneous in their expression of µ-, δ-, and κ-opioid receptors, as well as CXCR4 and CCR5, and Toll-like receptors. Thus, defining the distinct targets engaged by opiates in each cell type, and among brain regions, is critical to an understanding of how opiate abuse exacerbates neuroAIDS.

HIV-1 proteins, such as Tat and gp120 are believed to play a crucial role in the central nervous system (CNS) pathology of acquired immune deficiency syndrome (AIDS). The present study sought to determine the potential role of Tat and/or gp120 on behavioral development and the relationship with the assessed long-term effects of the HIV-1 proteins on the rat hippocampus. Male pups of 13 Sprague-Dawley litters were bilaterally injected on postnatal day (P)1. Every litter contributed an animal to each of four treatment condition: VEH (0.5µl sterile buffer), gp120 (100ng), Tat (25µg) or combined gp120+Tat (100ng+25µg). Body weight was not affected by either protein group. Tat revealed a transient effect on many of the behavioral assessments early in development as well as on preattentive processes and spatial memory in adulthood. Gp120 had more selective effects on negative geotaxis (P8-10) and on locomotor activity (P94-96). Combined gp120+Tat effects were noted for eye opening with potential interactive effects of gp120 and Tat on negative geotaxis. Anatomical assessment at ~7½ month of age was conducted by using design-based stereology to quantify the total cell number in five hippocampal subregions [granule layer (GL), hilus of the dentate gyrus (DGH), cornu ammonis fields (CA)2/3, CA1, and subiculum (SUB)] (Fitting et al., 2007a). A relationship between early reflex development and estimated cell number in the adult hippocampus was indicated by simple regression analyses. In addition, estimated number of neurons and astrocytes in the DGH explained 81% of the variance of the distribution of searching behavior in the probe test. Collectively, these data indicate that the DGH may participate in the spatial memory alterations observed in adulthood consequent to neonatal exposure to HIV-1 proteins. (Supported by DA013137, DA014401, HD043680).