Dopamine neuron synapses — like most CNS synapses — release more than one neurotransmitter, with roles that extend from development, to trophic support to more nuanced signaling. Dopamine neurons release glutamate as a cotransmitter. Glutamate cotransmission confers striking heterogeneity to the synaptic actions of dopamine neurons across their principal target neurons in the striatum. Tempering dopamine neuron glutamate cotransmission by conditional reduction of the glutamate synthesizing enzyme glutaminase, encoded by Gls1, reduces dopamine neuron glutamate cotransmission at phasic firing frequencies. Mice with a global heterozygous reduction of Gls1 show potentiated latent inhibition, and are less prone to psychostimulant sensitization, two behavioral effects involving aberrant salience attribution. Strikingly, these two phenotypes are seen in mice with a Gls1 reduction restricted to their dopamine neurons. The impact of the Gls1 reduction on dopamine neuron dependent behaviors runs counter to symptoms of schizophrenia, arguing for therapeutic potential. The subtlety of the reduction, affecting dopamine neuron glutamate cotransmission at phasic firing frequencies in the nucleus accumbens, identifies key circuitry involved in salience attribution. The driving hypothesis of this proposal is that dopamine neuron glutamate cotransmission is involved in salience attribution and that reducing cotransmission has therapeutic potential for schizophrenia pharmacotherapy. In this project, we will focus first on the synaptic impact of the Gls1 reduction on dopamine neuron dopamine and glutamate release (Aim 1), recording in cholinergic interneurons that respond to both transmitters, and in striatal projection neurons made into dopamine biosensors by expression of the dopamine-gated chloride channel LGC-53, enabling comparison of the differential impact of the Gls1 reduction on DA and GLU signals. Then, using an INTRSECT strategy (Aim 2), we will identify striatal dopamine neuron projections capable of glutamate cotransmission, and use optogenetic stimulation to examine their role in salience attribution. Finally, we will test glutaminase inhibition pharmacotherapy (Aim 3) showing first in brain slices that genetic reduction of Gls1 and pharmacological inhibition of glutaminase similarly and preferentially impact dopamine neuron glutamate cotransmission, and then induce a global Gls1 reduction in adulthood modeling pharmacotherapy to show the behavioral impact. Finally, we will make a dopamine neuron-selective Gls1 reduction in adulthood to evaluate both the synaptic and behavioral impact. This research should increase understanding of the role of dopamine neuron glutamate cotransmission and advance therapeutics based on activity dependent modulation of glutamate release, specifically glutaminase inhibition as a novel pharmacotherapy for schizophrenia.