Abstract: Smoking and tobacco use (STU) is a major global health problem and worldwide more than six million people die due to tobacco related diseases each year. Although majority of smokers try to quit smoking several times in their life, traditional therapeutic approaches, which focus only on neuronal cells, have a very low success rate. Understanding the effect of nicotine on glial cells, synaptic communication and blood vasculature in the brain can provide further insights on the neurobiology of substance abuse and can potentially help to design better therapeutic approaches. Glial cells are nonexcitable cells in the brain which do not generate action potentials. Recently many additional functions of glial cells have been discovered which have challenged the traditional neuro-centric view in neuroscience. Astrocytes are major glial cells which regulate synapse, maintain homeostasis, maintain blood brain barrier, and regulate blood flow. Processes from one astrocyte can interact with as many as 10,000 synapses. Astrocyte also couple with blood vasculature and play key role in supply of oxygen and nutrients to the brain. In these studies, we investigated the effect of nicotine on astrocyte morphology and functional activity. By using tissue clearing and time lapse fluorescence imaging approaches, we showed nicotine induces morphological rearrangement of astrocytic processes mediated via nicotinic receptors. We also studied the functional consequences of astrocytic rearrangement in the brain in terms of cytokine release, cell viability and calcium activity. To further characterize the subcellular astrocyte calcium activity in physiological and pathological conditions, we developed a GCaMP based genetic probe by targeting the endoplasmic reticulum (ER) in astrocytes. This probe was shown to measure astrocyte specific calcium activity in the periphery of the ER in cell culture and in vivo. As astrocytic processes form intricated networks with synapses, we also developed a single molecule fluorescence imaging approach to characterize synaptic nicotinic receptors and observed nicotine-induced stoichiometric shift in post synaptic nicotinic receptors in the brain. By using pH sensitive super ecliptic pHluorin (SEP) probe, we showed nicotine also alters the trafficking of nicotinic receptors from the ER to the plasma membrane. In summary, we developed and applied several fluorescence microscopy-based tools to study the astrocyte activity in various physiological conditions. Nicotine was shown to alter the morphological and functional activity of astrocytes through nicotinic receptor activation. We also developed a genetic probe to image calcium activity in astrocytic processes. Our studies of synaptic nicotinic receptors showed nicotine alters their stoichiometry from low affinity subtype to high affinity subtype. These studies highlight the importance of fluorescence-based approach to study tobacco use disorder and provide further insights on nicotinic receptor pharmacology and astrocyte biology.