To investigate autonomic substrates of brainstem-gut circuitry identified using trans-synaptic tracing with pseudorabies virus (PRV)-152, a strain that expresses enhanced green fluorescent protein, and PRV-614, a strain that expresses enhanced red fluorescent protein, injecting into the rat rectum wall. 3-7 days after PRV-152 injection, spinal cord and brainstem were removed and sectioned, and processed for PRV-152 visualization using immunofluorescence labeling against PRV-152. 6 days after PRV-614 injection, brainstem was sectioned and the neurochemical phenotype of PRV-614-positive neurons was identified using double immunocytochemical labeling against PRV-614 and TPH. We observed that the largest number of PRV-152- or PRV-614-positive neurons was located in the gigantocellular reticular nucleus (Gi), lateral paragigantocellular (LPGi), rostral ventrolateral reticular nucleus (RVL), solitary tract nucleus (Sol), locus coeruleus (LC), raphe magnus nucleus (RMg), subcoeruleus nucleus (SubCD). Double-labeled PRV-614/tryptophan hydroxylase (TPH) neurons were concentrated in the RMg, LPGi and Sol. These brainstem neurons are candidates for relaying autonomic command signals to the gut. The autonomic substrate of brainstem-gut circuitry likely plays an important role in mediating different aspects of stress behaviors.