Verifying, Challenging, and Discovering New Synapses among Fully EM-Reconstructed Neurons in the Leech Ganglion
Neural circuits underpin the production of animal behavior, largely based upon the precise pattern of synaptic connectivity among the neurons involved. For large numbers of neurons, determining such \"connectomes\" by direct physiological means is difficult, as physiological accessibility is ultimately required to verify and characterize the function of synapses. We collected a volume of images spanning an entire ganglion of the juvenile leech nervous system via serial blockface electron microscopy (SBEM). We validated this approach by reconstructing a well-characterized circuit of motor neurons involved in the swimming behavior of the leech by locating the synapses among them. We confirm that there are multiple synaptic contacts between connected pairs of neurons in the leech, and that these synapses are widely distributed across the region of neuropil in which the neurons arbors overlap. We verified the anatomical existence of connections that had been described physiologically among longitudinal muscle motor neurons. We also found that some physiological connections were not present anatomically. We then drew upon the SBEM dataset to design additional physiological experiments. We reconstructed an uncharacterized neuron and one of its presynaptic partners identified from the SBEM dataset. We subsequently interrogated this cell pair via intracellular electrophysiology in an adult ganglion and found that the anatomically-discovered synapse was also functional physiologically. Our findings demonstrate the value of combining a connectomics approach with electrophysiology in the leech nervous system.\n\nSignificance StatementThe function of any nervous system depends on the arrangement of its component neurons into circuits. Determining this precise pattern requires an account of which neurons are linked by synapses, and where. Here we use serial electron microscopy to confirm, challenge, and discover synapses in the neuropil of one ganglion from a juvenile leech. Relying on the homology of the ganglion from animal to animal, we demonstrate that we can identify synapses we knew existed from previous physiological work, and that we can confirm a new anatomically-discovered synapse by subsequently recording from the same neurons in a different animal. Here we show how analyses of anatomical detail and physiologically determined interactions complementarily yield insight into how neural circuits produce behavior.