Maturation of peptide-positive synaptic arbors in the medicinal leech requires rhythmic target activity

The formation and refinement of synaptic connections are dependent on the activity that emerges from nascent synaptic connections. Such activity has the effect of regulating the production and release of specific neurotransmitters. To determine the role of activity in regulating the production of peptide-positive synapses, we used antibodies against Phe-Met-Arg-Phe-NH₂ and acetylated α-tubulin as well as intracellular injections of Neurobiotin to examine varicosities belonging to heart excitor (HE) neurons on the heart tubes of medicinal leeches, Hirudo spp. We found that the production of peptide-positive varicosities increased considerably during the last week of embryogenesis, which coincided with the emergence of rhythmic activity of the heart tube. When we compromised central input to HE neurons with bicuculline or by surgical ablation of the central pattern generator during early embryogenesis, we found that activity in the heart tubes and its rhythmicity were greatly diminished. Furthermore, the activity of HE neurons had also lost its rhythmicity and appeared tonic, and production of peptide-positive varicosities was substantially reduced as well. Partial surgical ablations that preserved rhythmic activity in the heart tube while disrupting heart tube innervation by some HE neurons still resulted in peptide-positive varicosity production. Taken together, our results suggest that postsynaptic rhythmic activity of the heart tube is necessary and sufficient for the development and maturation of peptide-positive synapses.