Hippocampal-dependent neural immune interactions regulate heroin withdrawal and future enhanced fear learning

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Project Summary Opioid use disorder is a national crisis, and multiple lines of evidence indicate that heroin use leads to increased incidence and severity of anxiety disorders, including post-traumatic stress disorder (PTSD). Our laboratory has established a preclinical model that effectively demonstrates heroin withdrawal-induced hyperreactivity to future stressors, one of the key components of human PTSD. We discovered that withdrawal from escalating heroin administration results in an increase in dorsal hippocampal (DH) interleukin-1β (IL-1β), and blockade of DH IL-1 with receptor antagonist (IL-1RA) prevents heroin withdrawal enhanced fear learning. This IL-1β is expressed predominantly in astrocytes, and heroin withdrawal also upregulates glial fibrillary acidic protein (GFAP) expression, a marker for astrocyte reactivity. This suggests that astrocyte signaling may be involved in heroin withdrawal and future enhanced fear learning, however it is presently unknown whether there is a causal link between the two. The current proposal is a targeted approach to test the overall hypothesis that heroin withdrawal enhances fear learning through DH astrocyte signaling and alterations in astrocyte physiology. Aim 1 tests the hypothesis that DH astrocytes mediate heroin withdrawal-induced enhanced fear learning. We have successfully implemented Gi-coupled designer receptors exclusively activated by designer drugs (Gi-DREADDs) into our research program to manipulate the DH astrocyte signaling in vivo. Aim 1 tests if astroglial GPCR signaling is capable of influencing the long-term effects of heroin withdrawal on enhanced fear learning. In Aim 1, I will be trained in chemogenetic techniques, acquire extensive knowledge of this cutting-edge tool, and become proficient in using glial DREADDs to investigate how astrocyte signaling relates to behavior. Results will establish that DH astroglial signaling during heroin withdrawal mediates changes in the development of long- term fear. Aim 2 tests the hypothesis that heroin withdrawal alters DH astrocyte morphology and astrocyte- neuron interactions. We have established a method to analyze the complexity of individual astrocytes using the AAV5-GFAP-Lck-GFP construct, high-resolution confocal microscopy, and advanced analysis of 3-dimensional cellular reconstructions. Using this innovative technique, we will examine the effect of heroin withdrawal on astrocyte morphology and colocalization with post synaptic markers, a proxy for astrocyte-neuron interactions. In Aim 2, I will refine and enhance my skills in IHC, confocal microscopy, and advanced cellular analysis using the Bitplane Imaris software to quantify withdrawal-induced changes in astrocyte morphology and synaptic interactions. Results will establish that heroin use and withdrawal directly alters DH astrocyte physiology that may lead to enhanced fear learning and anxiety. Collectively, this proposal examines the role of hippocampal astrocytes in mediating enhanced fear learning following heroin withdrawal. These studies make important contributions to understanding the effects of heroin withdrawal on astrocytes and provide a valuable learning experience to foster the independence of my research career in addiction neurobiology.