This proposal focuses on developing technology for a new prototype optical diffusion fluorescence (ODF) probe to be used in vivo to study the role of astroglia cells in cocaine-induced effects on hemodynamics, metabolism and function in the brain. The optical probe in question is unique because it uses multiple wave- lengths to track cerebral blood volume (CBV), oxyhemoglobin (HbO2), deoxyhemoglobin (HbR) and intracellular calcium ([Ca++])i simultaneously at high temporal resolution in vivo. We have established proof-of- principle of the new probe design in the living rodent and recently obtained results demonstrating significant increases in cortical [Ca++]i following intravenous administration of cocaine and the local anesthetic lidocaine but not with methylphenidate indicating that cocaine's local anesthetic properties but not its catecholaminergic actions underlie this effect. We hypothesized that the cocaine-induced [Ca++]i increases are potentially harmful because the [Ca++]i increases would make the tissue more vulnerable to ischemia secondary to potential decreases in cerebral blood flow which would otherwise not induce ischemia and/or cell damage. We are proposing to further develop the ODF technology to isolate the cellular origin of the [Ca++]i increases induced by cocaine. Our main hypothesis states that the cocaine-induced [Ca++]I response originates from astroglia cells and not from neurons. We further hypothesize that glutamate in part mediates the increases in astrocytic [Ca++]i observed in cortex. To test our hypothesis we propose the following specific aims: i) To demonstrate that the ODF probe in combination with the calcium-sensitive fluorescence dye Rhod-2 (AM) and cell-specific tissue loading technology can be used to differentiate [Ca++]i dynamics in neuronal and astroglia cellular networks in vivo while simultaneously tracking changes in CBV, HbO2 and HbR; ii) To demonstrate that the cocaine-induced [Ca++]i response measured by the ODF probe originate in part from astroglia cells; iii) that the cocaine-induced [Ca++]i increases can be blocked by a selective metabotrobic glutamate receptor 1 (mGluR1) antagonist. The significance of this research to the field of drug addiction will be to 1) implement for the first time a new experimental methodology which is capable of tracking [Ca++]i transients from specific and large cell populations (astroglia cells) in the central nervous system in vivo, 2) directly address the role of astroglia cells in cocaine-induced changes in brain function in the living animal and 3) for the first time obtain information on [Ca++]i dynamics simultaneously with CBV, HbO2 and HbR in the normal and cocaine-exposed brain in vivo. Our long-term goal is to further solidify and simplify the ODF probe technology to assess brain function and oxygen content, thereby, allowing broad neuroscience community access to further studies in the area of drug addiction research. New information on the role of astroglia cells in the addiction process and/or neurotoxicity of drugs of abuse will potentially lead to identification of new treatment targets and new therapeutic strategies which will benefit public health. This proposal focuses on developing technology for a new prototype optical diffusion fluorescence (ODF) probe to be used in drug addiction research. The relevance of this research to public health is that the new probe in comparison to current technology (confocal and two-photon microscopes) is affordable and therefore can become widely accessible to researchers and thus help enhance our understanding of processes leading to addiction to drugs of abuse and ultimately to its treatment and prevention. ? ? ?
