Destruction of a single cell in the central nervous system of the leech as a means of analysing its connexions and functional role

Abstract

A method has been devised for killing an individual neurone in the C.N.S. of the leech by injecting it with Pronase. The technique has been used to examine the role of individual sensory and motor cells involved in producing reflex movements.1. After a neurone was injected with Pronase, either in an intact animal or an isolated ganglion, its cell body lost its resting and action potentials. Some hours later the injected cell's axons in the periphery failed to conduct impulses. In the intact animal the cell body could no longer be discerned after a few weeks.2. To test for destruction of processes within the neuropile, cells were injected first with the enzyme horseradish peroxidase (HRP) and then several hours later with Pronase. Absence of the characteristic HRP reaction product indicated that Pronase had spread throughout the arborization of the cell.3. Injection of Pronase into one cell did not produce overt electrophysiological or anatomical changes in other cells in the ganglion including neurones that were originally electrically coupled to the killed cell.4. Evidence that an individual cell was the only motoneurone supplying particular muscles was provided by destruction of that cell in otherwise intact animals, which resulted in a characteristic motor deficit in the area supplied by the killed cell. Over a period of months, functional recovery of the affected muscles occurred by way of homologous cells in adjacent ganglia.5. A further application of the technique was to trace the connexion that a particular sensory neurone makes onto two motoneurones that are electrically coupled. Normally, the sensory neurone gives rise to excitatory potentials in both post-synaptic cells. Synaptic potentials could still be recorded in one motor cell after the other had been destroyed by Pronase, indicating that synapses were made directly onto both of the motoneurones.