Neural substrates of elevated striatal dopamine synthesis and release in schizophrenia

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7. PROJECT SUMMARY/ABSTRACT Psychosis is a core clinical feature of schizophrenia (SZ) associated with elevated dopamine (DA) synthesis and release in the associative striatum (AST), but not the limbic striatum (LST). Determining the molecular and subcellular substrates of this region-specific presynaptic pathophysiology of psychosis in the disease state requires studies at the site of DA release, DA axonal boutons. This level of resolution in humans can only be achieved in postmortem brain studies. Elevated presynaptic DA signaling in the AST in SZ could be due to molecular changes within DA boutons. Specifically, greater protein levels of tyrosine hydroxylase (TH), vesicular monoamine transporter 2 (VMAT2) or the DA transporter (DAT), which govern DA synthesis, packaging for vesicular release and reuptake into the bouton, respectively, could represent molecular substrates of this pathophysiology. These molecular changes would be accompanied by ultrastructural alterations reflecting greater DA vesicular release. Alternately, or additionally, a greater density of DA boutons could be the substrate for elevated presynaptic DA signaling. Thus, in Aim 1 we test each of these possibilities by using triple-label immunofluorescence and confocal microscopy in SZ and unaffected comparison (CON) subjects to simultaneously quantify the abundance of TH, VMAT2 and DAT within DA boutons and the density of DA boutons in the AST and LST (Exp 1.1), and DA vesicle density is quantified via serial section transmission electron microscopy (Exp 1.2). Studies are repeated in monkeys chronically exposed to antipsychotic drugs (Exp 1.3). The findings in Aim 1 may result from alterations to local striatal cholinergic interneurons (ChIs) that affect DA boutons. Indeed, because markers of presynaptic DA signaling are normally greater in the AST than LST, an exaggeration of the factors that contribute to this regional specificity might underlie the AST-specific finding in SZ. ChIs are a compelling candidate as they exhibit greater functional and anatomical measures in the AST than LST and potently regulate DA synthesis and release by providing direct axo-axonic inputs to DA boutons. Activation of nicotinic acetylcholine receptors containing the β2 subunit (nAChRβ2) located on DA boutons induces DA release independent of DA neuron firing in the midbrain. Measures reflecting elevated presynaptic DA signaling in the AST in SZ might be the consequence of a greater proportion of DA boutons receiving ChI inputs and/or higher levels of molecular determinants of ChI synaptic strength, such as levels of ACh transferase (ChAT) and/or nAChRβ2. Thus, in Aim 2 we utilize quadruple-label immunofluorescence and confocal microscopy to simultaneously quantify the proportion of DA boutons receiving a ChI axo-axonic input and the abundance of ChAT and nAChRβ2 at these inputs in the AST and LST. As in Aim 1, studies are repeated in monkeys chronically exposed to antipsychotic drugs. These subcellular, molecular and ultrastructural analyses will provide the first comprehensive, region-specific analysis of the neural substrates and upstream factors of elevated presynaptic DA signaling in the AST of SZ and form the foundation of a future R01 application.