Chronic opioids induce synaptic plasticity, a major neuronal adaptation. Astrocyte activation in synaptogenesis may play a critical role in opioid tolerance, withdrawal, and dependence. Thrombospondins 1 and 2 (TSP1/2) are astrocyte-secreted matricellular glycoproteins that promote neurite outgrowth as well as dendritic spine and synapse formation, all of which are inhibited by chronic μ opioids. In prior studies, we discovered that the mechanism of TSP1 regulation by μ opioids in astrocytes involves crosstalk between three different classes of receptors, μ opioid receptor, EGFR and TGFβR. Moreover, TGFβ1 stimulated TSP1 expression via EGFR and ERK/MAPK activation, indicating that EGFR is a signaling hub for opioid and TGFβ1 actions. Using various selective antagonists, and inhibitors, here we compared the mechanisms of chronic opioid regulation of TSP1/2 isoform expression in vivo and in immortalized rat cortical astrocytes. TSP1/2 release from astrocytes was also monitored. Acute and chronic μ opioids, morphine, and the prototypic μ ligand, DAMGO, modulated TSP2 protein levels. TSP2 but not TSP1 protein content was up-regulated by acute (3 h) morphine or DAMGO by an ERK/MAPK dependent mechanism. Paradoxically, TSP2 protein levels were altered neither by TGFβ1 nor by astrocytic neurotrophic factors, EGF, CNTF, and BMP4. TSP1/2 immunofluorescence was increased in astrocytes subjected to scratch-wounding, suggesting TSPs may be useful markers for the "reactive" state of these cells and potentially for different types of injury. Previously, we determined that chronic morphine attenuated both neurite outgrowth and synapse formation in cocultures of primary astrocytes and neurons under similar temporal conditions that μ opioids reduced TSP1 protein levels in astrocytes. Here we found that, after the same 8 day treatment, morphine or DAMGO diminished TSP2 protein levels in astrocytes. Therefore, μ opioids may deter synaptogenesis via both TSP1/2 isoforms, but by distinct mechanisms. KEYWORDS: Opioids, morphine, opioid receptors, astrocytes, ERK/MAPK, growth factors A strocytes make integral contributions to neuronal signaling in the brain by releasing molecules that act at synapses during different stages of synaptogenesis. These phases include pre-and postsynaptic development, function, maintenance, and plasticity.1−7 One family of key astrocyte-secreting molecules includes thrombospondins (TSPs), oligomeric, multidomain glycoproteins that promote synapse formation in vivo and in vitro.8−14 Yet another molecule is TGFβ that induces excitatory synapses at least in part via a D-serine dependent mechanism. 15 Interestingly, TGFβ and TSPs maintain a reciprocal relationship in astrocytes. Early on, it was recognized that TSP1 activates TGFβ. 16 Alternatively, several independent research groups have reported that in astrocytes TGFβ increases TSP1 gene expression and/or protein levels as shown by immunoblotting, qRT-PCR, and in situ hybridization.17−20 Both of these processes appear to be initiated in the extrac...