Structure of allotransplanted ganglia and regenerated neuromuscular connections in crayfish.

In adult crayfish, Procambarus clarkii, motoneurons to a denervated abdominal superficial flexor muscle regenerate long-lasting and highly specific synaptic connections as seen from recordings of excitatory postsynaptic potentials, even when they arise from the ganglion of another crayfish. To confirm the morphological origins of these physiological connections we examined the fine structure of the allotransplanted tissue that consisted of the third abdominal ganglion and the nerve to the superficial flexor muscle (the fourth ganglion and the connecting ventral nerve cord were also included). Although there is considerable degeneration, the allotransplanted ganglia display intact areas of axon tracts, neuropil, and somata. Thus in both short (6-8 weeks) and long (24-30 weeks) term transplants approximately 20 healthy somata are present and this is more than the five axons regenerated to the host muscle. The principal neurite and dendrites of these somata receive both excitatory and inhibitory synaptic inputs, and these types of synaptic contacts also occur among the dendritic profiles of the neuropil. Axon tracts in the allotransplanted ganglia and ventral nerve cord consist largely of small diameter axons; most of the large axons including the medial and lateral giant axons are lost. The transplanted ganglia have many blood vessels and blood lacunae ensuring long-term survival. The transplanted superficial flexor nerve regenerates from the ventral to the dorsal surface of the muscle where it has five axons, each consisting of many profiles rather than a single profile. This indicates sprouting of the individual axons and accounts for the enlarged size of the regenerated nerve. The regenerated axons give rise to normal-looking synaptic terminals with well-defined synaptic contacts and presynaptic dense bars or active zones. Some of these synaptic terminals lie in close proximity to degenerating terminals, suggesting that they may inhabit old sites and in this way ensure target specificity. The presence of intact somata, neuropil, and axon tracts are factors that would contribute to the spontaneous firing of the transplanted motoneurons.

Regeneration of neuromuscular connections in crayfish allotransplanted neurons.

Transplantation of whole ganglia was used to study the regeneration of four of the neurons that innervate the superficial flexor muscles of the crayfish Procambarus clarkii. The isolated ganglia containing the somas of these neurons were successfully transplanted from one crayfish to another. Reinnervation proceeded across the muscle surface and by 8 to 10 weeks connections were detected across the entire target field. At different time periods after the transplant, junction potentials (JPs) produced in phase with spontaneous neuronal spikes were recorded. The distribution of JP sizes and their decay times were examined. JPs from transplanted preparations were smaller than JPs from control or normal regeneration animals. These JPs also failed to facilitate when stimulated at 1 and 10 Hz. These are normal characteristics of immature terminals, but in the transplant preparations, once established, they remained stable for the duration of the study. Thus, synaptogenesis appears to be arrested at a stage before synaptic efficacy is established in the allotransplants. In addition, connectivity maps were plotted for each axon over the muscle surface. Some muscle fibers did not receive any contacts, and overall innervation leveled off at around 60% of the muscle fibers, remaining stable for the duration of this study. Despite the incomplete physiological innervation, however, three of the four neurons showed the same medial/lateral preferences observed in control animals, regenerating their original patterns of connectivity across the muscle surface.

Synaptic repression at crayfish neuromuscular junctions. II. Evidence for calcium involvement.

The inability of synaptic junctions to generate normalisized postsynaptic potentials under normal physiological conditions was studied at crayfish neuromuscular synapses. Synaptic repression in the superficial flexor muscle system of the crayfish was induced by surgery: the nerve was cut in the middle of the target field, and the lateral muscle fibers were removed. After this surgery, the remaining medial synapses were unable to generate normal-sized junction potentials (jp) over the medial muscle population. In an attempt to study the mechanisms underlying this response, we varied the extracellular calcium concentration of the Ringers solution bathing the preparation, in both repressed and control animals, while monitoring the size of the same junction potential. The junction potential generated by the spontaneous activity of the nerve increased in size with increasing calcium concentrations in control animals, but failed to do so in repressed animals, that is, changes in external calcium concentrations did not affect repressed synapses. However, in the presence of the calcium ionophore A23187, control and repressed synapses both show an increase in the junction potential sizes they generate. Our data suggest that calcium is involved in the mechanisms that underlie synaptic repression in this crustacean neuromuscular system.

Synaptic repression at crayfish neuromuscular junctions. I. Generation after partial target area removal.

Synaptic repression, the inability of synaptic junctions to generate normal-sized postsynaptic potentials under normal physiological conditions, is reported here for crayfish neuromuscular synapses. The synapses in the superficial flexor muscle system of the crayfish change their efficiency in generating a postsynaptic response as a result of a specific alteration in their immediate environment. When the superficial flexor nerve is cut halfway into the target muscle field and the lateral muscle fibers are removed, the intact medial synapses do not generate normal-sized junction potentials (JP) at the 17 degrees-19 degrees C temperature of the Ringers solution. JPs cannot be recorded in 83% of the muscle fibers at 2 weeks after the operation and of the few JPs that can be detected, 80% are smaller than 1 mV in size. By 8 weeks after the operation, JPs were detected in 55% of the muscle fibers, and now only 46% of these are smaller than 1 mV. When the lateral muscle fibers are left in place during the original operation, providing a target area for the cut nerve to grow into, JPs were then detected in 60%-80% of all medial fibers at all time periods after the operation; their size profile, with 10%-25% of the muscle fibers having JP's less than 1 mV, was similar to control values. These results suggest that the efficiency of these synaptic contacts become affected as a result of partial axotomy and removal of the target area of the cut branches of the axons.

Regeneration of an identifiable motoneuron in the crayfish. I. Patterns of reconnection and synaptic strength established in normal and altered target areas.

The superficial flexor muscles of the crayfish are innervated in a position-dependent connectivity pattern, which can be reestablished when the nerve to the muscle is cut. This article deals with the regeneration of the largest excitor motoneuron under three different target scenarios: (1) a normal target with all the muscle fibers present, (2) a reduced target lacking the medial or the lateral muscle fiber population, and (3) when the nerve enters the target in the middle of the muscle field. In scenario 1 the neuron is able to regenerate the normal connectivity pattern within 10 weeks after surgery: all the lateral fibers become innervated, with a linear decline in the probability of connections over the medial fibers. The medial fibers become transiently hyperinnervated before the normal pattern of connections is established. In scenario 2 the normal pattern of connections is established only when the lateral fibers were present; with only medial cells as a target, the transient hyperinnervation stage is stable and no decline in connections was observed. Analysis of regenerated junction potential sizes during the stable hyperinnervation stage show abnormal patterns, suggesting that some aspects of the regeneration program of this neuron can be affected when signals from its prime target cells are missing. In scenario 3 growth begins in both directions until the entire muscle becomes innervated. The normal pattern of connectivity finally emerges after continued lateral growth and diminished medial growth, suggesting that the position of the muscle fibers influences connectivity patterns during the final stages of regeneration.

Regeneration of an identifiable motoneuron in the crayfish. II. Patterns of reconnection and synaptic strength established in the presence of an extra nerve.

The regeneration of neuromuscular connections to the superficial flexor muscle system in the crayfish has been studied under a variety of experimental manipulations. These have provided insight into the factors that can influence the regeneration program of neurons. In this work the regeneration of the largest excitor motoneuron was studied under two different conditions: (1) when the original neuron and a transplanted neuron were growing simultaneously into a denervated target, and (2) when a transplanted neuron was growing into a target that had its original nerve supply intact. In condition 1 both the transplanted and the original neuron formed normal patterns of connectivity and synaptic strength in comparable periods of time. In condition 2 the rate of growth of the transplanted neuron is significantly reduced and does not extend into the lateral fibers of the muscle. It is concluded that the regeneration program of this neuron is not affected by the presence of other neurons growing at the same time into a denervated muscle. Since regeneration is seriously affected if growth occurs into a fully innervated target area, it is suggested that lack of growth stimuli from the target or competitive interactions between established and growing synaptic terminals could influence the regeneration program of this neuron.

Regeneration studies on a crayfish neuromuscular system. I. Connectivity changes after intersegmental nerve transplants.

The superficial flexor muscles of the crayfish are a neuromuscular system of a few muscle cells innervated by six neurons in a precise position-dependent pattern. The neurons are capable of regenerating their normal connectivity patterns within a short span of time when conditions are favorable. The superficial flexor muscles of the second and third segments, despite their similarities in neuronal and muscle cell size and number, have distinctive connectivity patterns; some homologous neurons form similar patterns but other homologous neurons form patterns that are reversed between segments. We transplanted each segment's nerve into each other's muscle in order to observe regeneration of the nerves into a target area that differed in connectivity patterns from their original muscle. During the first weeks of regeneration all neurons formed a connectivity pattern with more connections medially and declining connections laterally, a pattern determined by the medial location of the nerve transplant. This pattern is maintained for most of the neurons, but for some there is an eventual reduction in medial connections as maximum synapse formation shifts to the lateral muscle fibers. Three of the eight neurons studied were able to regenerate connectivity patterns that corresponded to their segment of origin and not to their host muscle. This suggests that intersegmental muscle differences are not influencing the formation of these connectivity patterns, so the neurons will follow their inherent synaptogenesis program.

Regeneration studies on a crayfish neuromuscular system. II. Effect of changing the nerve entry point into the muscle field on the gradient of innervation.

The superficial flexor muscle of the crayfish is a neuromuscular system in which the neurons form position-dependent connectivity patterns with the muscle fibers. This system could be formed with the help of a single medial-to-lateral gradient during development that embodies positional information. To test this gradient hypothesis we changed the nerve's normal medial entry point into the muscle by transplanting it to the middle of the muscle sheet. When all the muscle fibers were present in the target area, most of the neurons studied passed through a stage during regeneration in which they showed preference for either medial or lateral synapse formation. Those neurons that in normal animals innervated preferentially the medial fibers showed a medial preference for new contacts; the neuron that normally innervated the lateral fibers showed a lateral preference for new contacts; the neuron that normally innervated everywhere regenerated equally well into both medial and lateral fibers. Therefore, these neurons are able to detect information regarding their position within the muscle mass and respond to it by preferential synapse formation. The effect of a positional gradient could not be detected when half of the target field was removed prior to regeneration. In this instance, the neuron that innervated the missing target area now regenerated to almost all the available fibers. It is suggested that the interplay of positional cues with other factors at different points in time could determine the final connectivity patterns formed by these cells.