Project Summary Cognitive deficits in several brain disorders are associated with altered dopamine (DA) levels in the cortex and abnormal cortical activity, and have a profound emotional and financial impact in our society. Imaging and post-mortem studies point to an overall decrease in cortical DA synthesis and release as an underlying pathology in brain disorders. However, due to our limited knowledge of normal connectivity, it has been challenging to establish links between changes in DA levels and alterations in neuronal circuits, which would significantly advance our mechanistic understanding of cortical dysfunctions. Progress is limited by the complexity of DA neuron synaptic actions in the cortex that involve the co-release of glutamate (GLU). Midbrain DA-GLU neurons are unique in their ability to increase neuronal firing through fast GLU-mediated signals and influence cortical activity, but it is not known how the function of these neurons is affected by hypodopaminergia. Here we will address this question by investigating, in mice, the molecular, synaptic, and circuit effects of decreasing DA synthesis in DA-GLU co-releasing neurons projecting to the cortex. These critical findings will guide the selection of future behavioral studies. Since DA neuron GLU co-transmission is preserved through phylogeny and is found in humans, our research will provide novel mechanistic insights into the DA dysregulation effects on cortical dysfunctions. It has been reported that decreased tyrosine hydroxylase (TH) expression (a key enzyme in DA synthesis) in hypothalamic DA-GLU neurons results in upregulation of vesicular glutamate transporter 2 (VGLUT2), suggesting increased GLU release. DA-GLU neurons projecting to the cortex make preferential connections to pyramidal output neurons in the lateral entorhinal cortex (LEC) and to GABA interneurons in the prefrontal cortex (PFC). Our hypothesis is that, when DA synthesis is decreased, both LEC- and PFC-projecting DA-GLU neurons will increase their release of GLU and use it as their main signaling molecule. This neurotransmitter switch, from DA-GLU to GLU-only, will differentially affect the activity of the LEC and PFC output neurons. We predict that, under hypodopaminergia, the firing of DA-GLU neurons will significantly increase LEC output activity due to strengthening of connections to pyramidal neurons, while significantly decreasing PFC output activity due to the strengthening of connections to GABAergic interneurons that inhibit PFC output neurons. To test this hypothesis, we will use intersectional viral strategies in TH-floxed mice to genetically inactive TH from LEC- and PFC-projecting DA neurons. We will then determine if this manipulation facilitates GLU co- transmission, by upregulating VGLUT2 expression at the transcript and protein levels in Aim 1; and by removing inhibitory effects mediated by DA on GLU co-transmission and increasing the DA-GLU neuron control over cortical activity in Aim 2, using ex vivo synaptic physiology and in vivo calcium imaging.

Project Narrative Cognitive deficits in several brain disorders are associated with decreased dopamine synthesis and release in the cortex, and have a profound emotional and financial impact in our society. This project will investigate how decreased dopamine synthesis affects the dopamine neuron control over cortical activity through the co- release of glutamate in mice. As dopamine neuron glutamate co-transmission is preserved through phylogeny and found in humans, our research will provide novel mechanistic insights into how dopamine dysregulation may affect cortical functions linked to cognitive deficits in brain disorders.