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 ß2 subunit (nAChRß2) 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 nAChRß2. 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 nAChRß2 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.