Dopamine (DA) neurons in the ventral tegmental area (VTA) are thought to encode an error prediction signal, in that the firing of DA neurons and release of DA may encode errors in predictions about rewards. In order for this to occur, DA release must be temporally precise to ensure that the errors in reward prediction provide information about the reward and the associated events that had just occurred. This theory assumes the VTA DA signal is fast acting and brief. This simple assumption has not been adequately tested, and evidence for fast DA signaling is in fact lacking. Within the prefrontal cortex (PFC) at least, both pre-and postsynaptic indicies of DA function in vivo or in vitro, suggest that DA signaling is slow in onset and slow to recover, and is therefore inconsistent with the properties required for an error prediction signal. On the other hand, although DA receptors are typically not ionotropic, in vivo intracellular recordings show that PFC neurons respond to VTA stimulation with a fast EPSP. Preliminary data suggest this EPSP has properties similar to glutamate mediated EPSPs, is blocked by destruction of DA cells in the VTA and peripheral administration of a glutamate antagonist. These findings suggest that VTA DA cells may encode a fast signal in the PFC via co-release of another transmitter, possibly glutamate. This idea has a long and controversial history but co-release of glutamate by VTA DA neurons would remove the burden of concurrently encoding fast and slow signals by postsynaptic DA receptors. Moreover, glutamate release due to VTA firing could produce fast changes in the membrane potential of PFC neurons that would provide the temporally precise signal required for the error prediction theory. The first part of this proposal will test the hypothesis that the EPSP in the VTA-PFC pathway is mediated by glutamate. The second part will test the temporal limits of DA signaling in the PFC. Part 1 will involve in vivo intracellular recordings from PFC neurons in combination with administration of pharmacological agents (either applied peripherially or via a dialysis probe in PFC) that block DA, glutamate or GABA receptors in an attempt to block the EPSP in the VTA-PFC pathway. Additional experiments will be performed in VTA-PFC organotypic slice co-cultures to test the properties of the EPSP in a more rigorous manner. These experiments will also determine the validity of the organotypic slice co-culture as a means of studying the VTA-PFC pathway based on its similarities to the in vivo preparation. Part 2 will involve simultaneous measurements of DA release in PFC and the electrophysiological response of PFC neurons to VTA stimulation in an attempt to determine the parameters of this release and how quickly it can alter the properties of PFC neurons. A new theory will be proposed that incorporates fast and slow signaling from VTA neurons and that explains the existing literature while providing a new way to think about normal and pathological states involving PFC DA.