There is growing recognition that dorsal spinal cord gila (astrocytes & microglia) contribute to the creation & maintenance of enhanced pain, including pathological pain. In addition, they may play important roles in the development of morphine tolerance & tolerance-associated pain facilitation. While currently available methods have provided initial insights into the role of gila in pain regulation, all are severely limited. For example, in vivo pharmacology & analyses of homogenated spinal cord & CSF cannot identify which cell type(s) is/are responsible for the observed effects. In situ hybridization & immunohistochemistry cannot assess whether the end-product is actually released, so the physiological relevance of the changes are unclear. Furthermore, none of these techniques can address whether glial interactions are involved, such as microglial-derived products stimulating astrocytes to release the key painmodulating substance(s). Such glial synergies/interactions are well documented elsewhere in the CNS but have not yet been examined in spinal cord. New methodologies are needed to understand how dorsal spinal cord astrocytes & microglia, singly & interactively, modulate pain. New methodologies are needed to enable rapid isolation of pure astrocytes 8, pure microglia from the dorsal spinal cords of adult rats to allow assessment of how manipulations of interest (intrathecal HIV-1 gp120, chronic neuropathy, chronic morphine, & other clinically relevant pain models) alter these cells in terms of their receptor, mRNA & endproduct expression. New methods are also needed to enable manipulation of dorsal spinal cord astrocytes microglia in culture to test their responses (cellular mRNA, cellular proteins, & released endproducts) to pain-relevant transmitters & modulators. With rare exception, such studies are not possible in vivo since neurons, astrocytes &/or microglia likely express receptors for these pain-relevant substances & can release many of the same neuroactive compounds, thus confounding interpretations of the results. Thus this Small Grant proposal is focused on developing & refining the new methods described above. These new methods will then undergo initial assessments, in terms of functional characterization & receptor expression of the isolated astrocytes & microglia.