After a weak, steady electric current of approximately 10 microamperes was imposed across the completely severed spinal cord of the larval lamprey Petromyzon marinus, enhanced regeneration was observed in the severed giant reticulospinal neurons. The current was applied with implanted wick electrodes for 5 to 6 days after transection (cathode distal to lesion). The spinal cords were examined 44 to 63 days after the operation by means of intracellular fluorescent dye injections and electrophysiology. Extracellular stimulation of whole cords showed that action potentials in most of the electrically treated preparations were conducted in both directions across the lesion, but they were not conducted in either direction in most of the sham-treated controls. In most of the electrically treated animals, processes from giant axons with swollen irregular tips, indicating active growth, were seen in or across the lesion. Only a few of the sham-treated controls showed these features. It is possible that these facilitated regenerative responses were mediated by the effects of the artificially applied electric fields on the natural steady current of injury entering the spinal lesion.
In the spinal cord of the lamprey, regeneration of giant reticulospinal axons occurs following transection. We show that partial degeneration of the proximal axonal segment, or "die-back," also occurs following spinal transection and it precedes regenerative outgrowth. The die-back during the first 5 days post-transection is reduced significantly by application of a 10-microA DC current across the site of transection, with the cathode distal to the lesion. Reversing the polarity of the applied current (cathode proximal to the lesion) increases the extent of axonal die-back relative to the sham-treated controls. Following spinal transection, saline-filled wick electrodes were implanted in the body musculature on either side of the lesion. Electrically treated animals received current across the lesion for 5 days, while the sham-treated controls received no current. After 5 days, several giant axons in each preparation were injected intracellularly in the spinal cord with the dye Lucifer Yellow. The extent of axonal die-back in the proximal cord stump was determined in the filled fibers by measuring the distance of the axon end from the site of lesion. The mean distances of axonal die-back were as follows: controls, 1750 microns +/- 45 SEM; cathode-distal, 740 microns +/- 33 SEM; cathode-proximal, 2820 microns +/- 60 SEM. These differences between the treatment groups proved to be significant using the Wilcoxon rank sum test. We propose that die-back is caused by the entry of cations driven into the cut surface of the cord by the endogenous injury current. The applied DC current interacts with the endogenous current of injury to either decrease or increase the flow of cations into the cord, depending on the direction of applied current flow across the lesion. This in turn causes a corresponding reduction or enhancement of the axonal die-back.
When the axon of the medial giant interneuron (MGI) of the cricket is axotomized close to the cell body, the normally stable, characteristic dendritic arborization is induced to sprout supernumerary neurites. The origin of the induced dendritic sprouts is not random; they emerge preferentially from the dendritic tips and branches close to the exit of the axon from the terminal ganglion. If any growth also occurred from the axon, there was a reciprocal relationship between the extent of dendritic and axonal sprouting. On the other hand, axotomy distant to the cell body induces sprouting only from the axon and does not alter the dendritic structure of the MGI.After crushing the ventral nerve cord distant to the cell body, the MGI sprouted neurites from the proximal axonal stump which crossed the site of lesion and continued growing within the distal cord. After a distant cut of the cord, however, the axonal neurites formed a neuroma in the proximal cord stump at the site of lesion and stopped elongating after 1 month. At this time, supernumerary sprouts first began to emerge from the normally smooth, rounded contours of the cell body.Based on these observations, we propose that axotomized neurons produce membrane at a constant rate. This newly synthesized membrane is preferentially inserted into neurites emerging from the proximal axonal stump. When axonal neurites stop growing in a neuroma following a distant cut, then this new membrane appears as supernumerary neurites from the soma. After a close cut, the axon often dies back into the ganglion and appears unable to receive the full complement of sprouting membrane. In such cases, the balance of the newly synthesized membrane is inserted into the dendrites and the characteristic structure of the arborization is significantly altered.Differential growth in discrete regions of a developing neuron eventually results in a fully differentiated nerve cell with three morphological compartments: the soma, the dendrites, and the axon. This structural specialization may arise during development from a selective distribution pattern of specific membrane components characteristic of each neuronal compartment. The distinct structural and functional properties that emerge in each compartment during development are stabilized and maintained to yield the mature neuron. ' Axotomy can be employed to study the insertion of newly synthesized membrane associated with the induced regenerative growth. In such an experimental framework, a mature, fully developed neuron is challenged by partial removal of a specific morphological compartment, namely, its axon. To restore its fully differentiated state, an injured neuron must specifically initiate axonal growth while retaining the mature properties of the undamaged dendrites and cell body. Sprouting induced by axotomy therefore provides a model system for studying the mechanisms controlling both regional growth and stabilization of structure and function in a single neuron.We have chosen to investigate regeneration of an injure...
The cell body of the medial giant interneuron (MGI) in the cricket normally does not spike in response to injected depolarizing currents. When axotomized 1 mm or more from the cell body (distant axotomy), the membrane properties of the soma remain unchanged. However, after axotomy close to the cell body (200 to 500 pm), the soma membrane becomes capable of generating action potentials by 6 hr after lesion. These regenerative spikes are 1 to 1.5 msec in duration and may reach 100 mV in peak amplitude. Ion substitutions indicate that these action potentials are primarily sodium dependent. A calcium-dependent component of soma membrane excitability that is normally present appears to be unaffected by axotomy. By 48 hr, the close axotomized MGI somata have lost the ability to generate action potentials and the membrane electrical properties return to normal.By 2 days after axotomy close to the soma, large, membrane-bound, electron-lucent vacuoles appear in the cytoplasm of the MGI cell body. Such vacuoles then disappear from axotomized MGI somata by 10 days. In addition, numerous arrays of densely packed, darkly staining microtubules are observed in the cell body, especially concentrated near the initial neurite. Neither of these specialized structures is observed in control, intact MGI somata. We propose that close axotomy disrupts the mechanisms which regulate the stability of the fully mature, differentiated neuron. The characteristic morphological and physiological stability of the MGI is lost: the dendritic arborization has been shown previously to be altered by extensive new outgrowth (Roederer, E., and M. J. Cohen (1983) J. Neurosci. 3: 1835-1847); there is a transient increase in soma membrane excitability, and new cytoplasmic organelles are induced.
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