Regeneration of synaptic connections by sensory neurons in leech ganglia maintained in culture

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Abstract
Leech ganglia maintained in organ culture were used to follow regeneration and the formation of synaptic connections by individual neurons. From earlier physiological studies on operated animals it is known that the c.n.s. of the leech is able to regenerate and that specific connections can be reformed. The present experiments show that specific regeneration also occurs in vitro but that axons do not simply grow directly back to their targets. (1) Axons were severed by crushing the connectives linking pairs of ganglia at the time of removal from the animal. Light and electron microscopy indicated that the procedure of crushing severed all the axons within the connectives. For several days after the lesion had been made, conduction of impulses from one ganglion to the next was abolished. (2) After 5-10 days in culture, stimulation of the connectives with external electrodes gave rise to impulses that were once again conducted beyond the site of the lesion. Characteristic excitatory and inhibitory synaptic potentials were evoked in identified sensory and motor cells in both ganglia by this indiscriminate stimulation of axons. Electron micrographs of the crushed region showed not only regenerated axons traversing the site of the lesion but also synaptic profiles similar to those seen in the neuropile of normal ganglia. Thus, pre- and post-synaptic specializations had been formed during regeneration in a part of the c.n.s. where they are not normally present. (3) Individual sensory neurons were injected with horseradish peroxidase to reveal the course taken by their regenerating axons. At the site of the crush profuse branching occurred/by 7 days. The arborization of a single axon was highly complex, with many varicosities present on fine branches. After two weeks in culture, one or more of the processes had usually grown beyond the crush and in certain instances had reached the next ganglion. Other branches ran back towards the ganglion in which the cell body was situated. During the period of the experiments (up to 45 days) no retraction of the sprouted fibres or of the arborization at the crush was observed. In addition to sprouting at the site of the lesion considerable sprouting also occurred within the ganglion, close to the cell body. (4) Individual mechanosensory neurons regenerated and would once again evoke synaptic potentials in their original targets after two weeks in culture. Thus, intracellular stimulation of single sensory cells in one ganglion gave rise to synaptic potentials in the appropriate motor neuron of the neighbouring ganglion. Injection of such sensory cells with horse-radish peroxidase showed that their axons had extended beyond the lesion and ramified in the neuropile of the next ganglion. (5) It is concluded that neurons in leech ganglia are able to regenerate and reform appropriate synaptic connections in culture. The degree of precision is hard to assess because of novel synaptic interactions and numerous additional sprouts that develop during regeneration.