Traumatic brain injury (TBI) of all severities can result in chronic disturbances of cognitive, behavioral, emotional, and physical functioning. Recovery of cognitive function after TBI is a dynamic process in which alterations in neurotransmitter systems do not likely occur in isolation. Numerous studies have demonstrated that the dopaminergic (DA) innervation of medial septum and diagonal band of broca (medial septal area [MSA]) regions that are dense with cholinergic neurons can affect hippocampal acetylcholine (ACh) release. The goal of this R21 grant is to evaluate mild repetitive TBI-induced changes in the integrity of dopaminergic/cholinergic connectivity using contemporary tools including diffusion MRI tractography with network analysis, transgenic human tyrosine hydroxylase GFP expressing rats, and Fluorescence activated synaptosomal sorting (FASS) analysis. The overall hypothesis is that TBI produces a loss of DA fiber connectivity, including DAergic innervation of the MSA, which contributes to cholinergic deficits. Aim 1 will determine the effects of repeated mild fluid percussion TBI (rmTBI) on the connectivity of the nigrostriatal pathway using high-definition fiber tracking in conjunction with network topology analysis. Complimentary immunohistochemistry in hTH-GFP rats will be compared to the imaging tractography to enhance specificity to DA circuits. Aim 2 will determine if rmTBI results in a loss of DAergic innervation in the MSA, which is a region of cholinergic nuclei that project to the hippocampus. Changes in DA innervation will be determined by a novel florescence-activated synaptosome sorting (FASS) process to quantitate hTH-GFP-positive synaptosomes in the MSA. To determine if the loss of DAergic innervation of cholinergic systems is functionally significant, we will use microdialysis to measure hippocampal ACh levels evoked by electrical stimulation of the medial forebrain bundle. If successful, this project will demonstrate the use of contemporary and novel methods to evaluate the degree and consequences of DAergic dysconnectivity after TBI.