Colocalization of gamma-aminobutyric acid-like immunoreactivity and catecholamines in the feeding network of Aplysia californica.

Functional consequences of neurotransmitter coexistence and cotransmission can be readily studied in certain experimentally favorable invertebrate motor systems. In this study, whole-mount histochemical methods were used to identify neurons in which gamma-aminobutyric acid (GABA)-like immunoreactivity (GABAli) was colocalized with catecholamine histofluorescence (CAh; FaGlu method) and tyrosine hydroxylase (TH)-like immunoreactivity (THli) in the feeding motor circuitry (buccal and cerebral ganglia) of the marine mollusc Aplysia californica. In agreement with previous reports, five neurons in the buccal ganglia were found to exhibit CAh. These included the paired B20 buccal-cerebral interneurons (BCIs), the paired B65 buccal interneurons, and an unpaired cell with projections to both cerebral-buccal connectives (CBCs). Experiments in which the FaGlu method was combined with the immunohistochemical detection of GABA revealed double labeling of all five of these neurons. An antibody generated against TH, the rate-limiting enzyme in the biosynthesis of catecholamines, was used to obtain an independent determination of GABA-CA colocalization. Biocytin backfills of the CBC performed in conjunction with TH immunohistochemistry revealed labeling of the rostral B20 cell pair and the unpaired CBI near the caudal surface of the right hemiganglion. THli was also present in a prominent bilateral pair of caudal neurons that were not stained with CBC backfills. On the basis of their position, size, shape, and lack of CBC projections, the lateral THli neurons were identified as B65. Double-labeling immunohistochemical experiments revealed GABAli in all five buccal THli neurons. Finally, GABAli was observed in individual B20 and B65 neurons that were identified using electrophysiological criteria and injected with a marker (neurobiotin). Similar methods were used to demonstrate that a previously identified catecholaminergic cerebral-buccal interneuron (CBI) designated CBI-1 contained THli but did not contain GABAli. Although numerous THli and GABAli neurons and fibers were present in the cerebral and buccal ganglia, additional instances of their colocalization were not observed. These findings indicate that GABA and a catecholamine (probably dopamine) are colocalized in a limited number of interneurons within the central pattern generator circuits that control feeding-related behaviors in Aplysia.