Intracellular Ca2+ dynamics provide one of the primary means of signaling within astrocytes. The significance of astrocyte Ca2+ signaling includes bidirectional communication with neurons and governance of neural function through diverse mechanisms including gliotransmission and neurotransmitter transport. Reflecting the diverse roles for astrocyte Ca2+, there are distinct mechanisms and spatial domains of Ca2+ within the cell body and peripheral processes. However, despite the substantial evidence for fundamental roles for astrocyte Ca2+ in the cellular mechanisms of brain function and behavior, it remains largely unknown whether or how drugs of abuse affect astrocyte Ca2+ signaling. This is a particularly salient question, given the increasing evidence that astrocytes within the reward circuitry are chronically impaired both in structure and function. Further, limitations of existing methods of Ca2+ monitoring present significant hurdles toward assessment of astrocyte Ca2+ elevations associated with behavior in deep brain structures in rat. Toward that challenge, we have developed two novel AAVs which express the photoconvertible, ratiometric Ca2+ indicator CaMPARI2 under control of the astrocyte-specific GfaABC1D promoter, to allow irreversible marking of Ca2+-activated astrocytes. One variant is cytosolic and expressed primarily in the cell body, localized to primarily report somatic Ca2+ elevations; in contrast, the Lck-fusion variant can report Ca2+ elevations throughout the astrocyte and within the fine peripheral processes. Both variants demonstrate reliable, astrocyte-restricted expression in the nucleus accumbens, hippocampus and prefrontal cortex, and we have confirmed photoconversion upon Ca2+ stimulation in live slice. The goal of this proposal is to validate use of these newly developed tools to quantify astrocyte Ca2+ responses to stimuli in slice and in vivo, and to optimize parameters for which future studies can be designed to assess astroglial Ca2+ dynamics associated with drug self-administration and related behaviors.
Aim 1 will optimize conditions for astrocyte Ca2+ monitoring in live slice in response to both ATP and dopamine receptor agonism.
Aim 2 will validate and optimize conditions for Ca2+ monitoring following optical stimulation in vivo coupled with cocaine administration in rat, using an established experimental design for CaMPARI2 activation in vivo. These studies will inform how both acute and chronic cocaine exposure regulate Ca2+ dynamics in astrocytes. In both cases, CaMPARI2 photoconversion will be assessed by red:green fluorescence ratios as well as immunohistochemistry using a custom anti-CaMPARI2-red antibody. Results will allow for applications toward investigation of astrocyte Ca2+ across a broad field of cells, imaged at high resolution and with multiplexed assessment of gene and protein expression, and for interrogation of functional responsiveness of astrocytes across varied drugs of abuse and drug abuse paradigms.
Drug dependence and relapse to drug abuse represents a major public health crisis; accordingly, it is critical to understand what cellular mechanisms contribute to protracted drug abuse. However, little is known about astrocyte signaling during the stages of drug use. This proposal will develop, validate, and optimize astro- CaMPARI2 for assessment of astrocyte Ca2+ signaling following both acute and chronic cocaine exposure.
