Membrane properties and selective connexions of identified leech neurones in culture

Abstract

SUMMARY1. Individual, identified neurones, dissected from the central nervous system ofthe 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.

In VitroSynaptogenesis between the Somata of IdentifiedLymnaeaNeurons Requires Protein Synthesis But Not Extrinsic Growth Factors or Substrate Adhesion Molecules

“…Whether synaptogenesis is also contingent on these factors has not yet been fully determined. The answer is difficult to discover in most preparations, because synapse formation requires neurite extension, which in turn relies on the availability of various growth factors (Henderson, 1996) and substrate adhesion molecules (Doherty and Walsh, 1989) as well as on newly synthesized proteins (Steward et al, 1988).To obtain synapses in the absence of neurite outgrowth, Fuchs et al (1981Fuchs et al ( , 1992 developed soma-soma synapses between the identified leech neurons. Haydon (1988) subsequently adopted this approach to develop and exploit soma-soma synapses between the identified Helisoma neurons.…”

“…To obtain synapses in the absence of neurite outgrowth, Fuchs et al (1981Fuchs et al ( , 1992 developed soma-soma synapses between the identified leech neurons. Haydon (1988) subsequently adopted this approach to develop and exploit soma-soma synapses between the identified Helisoma neurons.…”

Steps in the formation of neurites and synapses studied in cultured leech neurons

“…What kind of synapse will a Retzius and a P cell form if plated together? Even though Retzius and P cells form electrical synapses with different targets in culture, when plated together they form purely chemical inhibitory synapses, with the Retzius cell always being presynaptic (11).…”

“…De-Miguel of variable length from which they will regrow and form synaptic connections with other neurons (4,10). Early experiments showed that cultured leech neurons survive for weeks, preserving their resting potentials and the distinctive shape and duration of their action potentials (11). Moreover, cells can regrow neurites displaying characteristic branching patterns that depend on their identities and on the substrate (7,8), and, when plated in contact with other cells, they form specific chemical or electrical synapses.…”

Intracellular Recording, Sensory Field Mapping, and Culturing Identified Neurons in the Leech, <i>Hirudo medicinalis</i>

“…P, N, T, Rz). Moreover neurons can be removed from the animal and cultured in a suitable medium in which the cells maintain their electrical characteristics for as long as 45 days 1,2 .…”