Ultrastructural localization of Leu5-enkephalin immunoreactivity in mesocortical neurons and their input terminals in rat ventral tegmental area

Enkephalin (ENK) immunoreactivity is widely distributed in the ventral tegmental area (VTA), where endogenous ENK and dynorphin opioid peptides are known to have opposing actions in reward, stress, cognition, and fear-related behaviors. Many neurons in the VTA give rise to mesocortical projections terminating in the medial prefrontal cortex (mPFC), and these projections have been implicated to varying extents in all these functions. To determine whether there is a synaptic basis for ENK and/or dynorphin modulation of cortically projecting neurons within the VTA, we combined retrograde tract-tracing from the mPFC with dual immunocytochemical-labeling electron microscopy in the rat VTA. The retrograde tracer Fluorogold (FG) was microinjected into mPFC. At optimal survival periods, sections through the VTA were processed for immunolabeling of anti-FG and a Leu(5)-ENK antibody recognizing both ENK and dynorphin peptides. Over 26% of the retrogradely labeled neuronal somatodendritic profiles (n = 177) were contacted by ENK-immunoreactive axonal profiles including small axons and axon terminals. The axon terminals varied in their subcellular distribution of ENK immunoreactivity and also differed in forming either inhibitory-type (symmetric) or excitatory-type (asymmetric) synapses. Many of the axonal profiles also were apposed to FG-labeled somata or dendrites without forming recognizable synapses. Approximately one-third of the mesocortical neuronal perikarya also showed sparsely distributed somatodendritic ENK-immunoreactivity. Our results provide ultrastructural evidence that ENK and possibly dynorphin in the rat VTA have distributions consistent with involvement in diverse physiological actions affecting the output of mesocortical neurons, some of which also contain one or both peptides.