Membrane properties and selective connexions of identified leech neurones in culture

1. Individual, identified neurones, dissected from the central nervous system of the leech and maintained in culture for several weeks, sprouted processes and formed synaptic connexions.2. The action potentials of isolated touch (T), pressure (P), nociceptive (N) cells and Retzius cells resembled those of their counterparts in situ, enabling them to be recognized unambiguously. Their input resistances were approximately 4 times greater than those of corresponding cells within the animal. In T, P and N cells trains of impulses were followed by a pronounced after-hyperpolarization, as in the animal.3. In certain cells, notably the L motoneurones, membrane properties became altered in culture. The current-voltage relation showed novel rectification and action potentials became much larger.4. Numerous neurites often extended for hundreds of micrometres from isolated neurones and ended in typical growth cones. Electron micrographs revealed that many fine axons were braided together to form thicker fascicles. Frequently, the processes were orientated between two neighbouring cells rather than at random. The fine structure of the cytoplasm, nucleus and organelles in cultured cells resembled those of their counterparts in situ. The glial cell that normally surrounds the neurones was, however, absent.5. Pairs of Retzius cells in culture usually became coupled electrically after about 6 days. Similarly L motoneurones became coupled in vitro. These junctions allowed current to pass in both directions and resembled those seen in the animal.6. Selective connexions were made by certain types of cells. Thus, P sensory neurones did not become coupled with Retzius cells but did develop electrical connexions with L motoneurones, as in the animal.7. Novel synaptic interactions not obvious in the animal could appear in culture. Retzius and L cells became electrically coupled and, in some instances where electrical coupling between Retzius cells failed to develop, chemically mediated inhibitory potentials became apparent.8. Isolated, identified leech neurones not only survive but regenerate processes and are capable of forming selective connexions in culture. The ability to define interactions between isolated pairs of cells offers the opportunity to explore in detail problems relating to synapse formation and cell-cell recognition.