The goal of the work proposed here is to directly relate cortical circuitry with the molecular components of neurotransmission: receptors and signal transduction proteins. We will study the relationship between the circuitry of primate prefrontal cortex and the D1 dopamine receptor and related molecules. This focus will allow us to relate our results to the circuitry of working memory, which is dysfunctional in schizophrenia. Working memory function depends on an optimal level of D1 receptor activation and is impaired by both over and under stimulation of D1 receptor. We hope that our results will lead to a better understanding of the mechanism of the D1 receptor's complex modulation of working memory, and ultimately to a better understanding of neuroleptic action. This work is comprised of four aims. 1) Identify the source of afferents that terminate onto D1-containing spines. This will suggest aspects of cortical circuitry that are critical for working memory function. We hypothesize that instrinsic axons target D1-containing spines, but not axons from distant sources. 2) Identify the source of afferents that terminate onto the spines that contain protein phosphatase 1 (PP1) isoforms. Pilot studies demonstrate that cortical spines can be subdivided based on their content of the isoforms PP1alpha and PP1gamma1. The results of this work will extend our understanding of the mechanisms by which cortical circuitry may be specialised. We hypothesize that afferents which do not terminate onto D1-containing spines still have access to the signal transduction pathway used by the D1 receptor. 3) Examine the subcellular distribution and relationship between D1, PP1alpha and PP1gamma1. The D1 receptor is found in spines that contain both PP1alpha and PP1gamma1. We hypothesize that the subcellular distribution of these isoforms differs within a single spine. If confirmed, this could suggest differential localization as the significance for multiple isoforms of a single protein within a spine. 4) Compare the distribution of group I metabotropic glutamate receptors (mGluRs) with that of the D1 receptor. Group I mGluRs potentiate D1 mediated effects in other brain areas. We hypothesize that one of these mGluRs will be found in the same spines as D1 receptors. This will add to our understanding of the interactions between G protein-coupled receptors. To accomplish these aims we will inject neuroanatomical tract tracers into the brains of young adult macaque monkeys to label parietal, thalamic and callosal afferents to prefrontal cortex, and within prefrontal cortex to label local horizontal and local intracolumnar axons. Double-labeling techniques appropriate for electron microscopy will be used to stain the labeled axons and either D1, PP1alpha or PP1gamma1. We will use serial section electron microscopy to determine if the postsynaptic targets of labeled axons contain these proteins. Post-embedding immunogold labeling will be used to examine the distribution of these proteins within a spine. Double-label experiments will directly compare their distribution and look for co-localization. Finally we will use single and pre-embedding double label methods to examine the subcellular localization of group I mGluRs and compare their distribution to that of the D1 receptor. The possibility of co-localization of the D1 receptor and the mGluR within a single spine will be examined.
