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University of Texas Medical Branch, Galveston, Texas 77555-0641
1 Texas Tech Medical School, Lubbock, TX.
2 The University of Texas, Austin, TX.
The identification and characterization of processes and proteins involved in restoring a plasma membrane seal after axonal injury have been advanced by studies of invertebrate giant axons (GAs) with techniques inapplicable to smaller cells (1–7). These studies showed that Ca2+-induced endocytotic vesicles mediate sealing by protein-facilitated interactions and fusions (2–5) among themselves and with the plasma membrane, which enabled restoration of electrical function (6). In contrast to GAs isolated from squid, which vesiculate but do not form a seal 1–2 h after severance (7), transected crayfish medial GAs vesiculate and do seal within 1 h after severance (2–4). We thought that structural manifestations of sealing might be evident in scanning electron microscope (SEM) images of severed GAs isolated from the two invertebrates and fixed at times posttransection (PT) that are relevant to the previous assessments of sealing. Here we compare such micrographs, which show unorganized vesicles at an open, cut end of a squid GA, but a plug-like aggregation of vesicles at the closed, cut end of a crayfish GA. These structural comparisons at axonal cut ends are consistent with previous electrical (3,6,7) and dye exclusion (2–4) assessments of sealing in these two preparations.
Axons were prepared for SEM (JEOL model JSM840) as follows. GAs dissected from live squid (Loligo pealeii) were placed in artificial seawater (ASW) consisting of (in mM): 430 NaCl, 5 KCl, 10 CaCl2, 50 MgCl2, 5 TrisCl, pH 7.4 at 22 °C. GAs were transected in ASW with fine, spring microscissors. At 1 h PT, fixative (2% glutaraldehyde in ASW) was added; the GAs remained in fixative for 1–4 h, after which they were washed 3 times for 20 min each with ASW without fixative. The samples were placed in 1% osmium tetroxide in ASW for 1 h on ice and then washed 3 times in ASW. Next, the samples were dehydrated on ice in an ethanol (EtOH) series: 50%, 70%, 85%, and 95% for 10 min each, and lastly in 100% EtOH, 3 times for 15 min each. To dry the samples without surface tension changes, we placed them in a critical point dryer and then glued them to an SEM stub. Finally, the samples were sputter-coated with Au/Pd. Medial GAs were dissected from the ventral nerve cord of live crayfish (Procambarus clarkii) as described previously (2). The crayfish GAs were prepared like those of squid, except that medial GAs were placed in van Harreveld’s (vanH) solution (22 °C) consisting of (in mM): 205 NaCl, 5.4 KCl, 13.5 CaCl2, 2.6 MgCl2, 10 HEPES pH 7.4, and fixative was added at 30 min PT. In Ca2+-free vanH solution, CaCl2 was omitted, and 1 mM EGTA added.
The cut end of a squid GA (Fig. 1A) after transection in ASW and fixation at 1 h PT was slightly constricted, but open, with the glial sheath folded back on itself. At higher magnifications (Fig. 1B and C), the axoplasmic core appeared layered and surrounded by a large collection of vesicles on its periphery (axoplasmic cortex). The highest magnification of the vesicles (Fig. 1C) showed a vesicle size distribution ranging from submicrometer to several micrometers in diameter. Structures that would constitute a barrier to the movement of ions or molecules into or out of the cut end were not evident. In contrast, the cut end of a crayfish medial GA (Fig. 1D)—after transection in vanH solution and fixation at 30 min PT—was closed, as indicated by a seam made by the close apposition of the flattened boundary at the severed end. Furthermore, a prominent vesicular mass filled and protruded from an apparent residual opening at the constricted cut end. Higher magnification (Fig. 1E) showed that this vesicular mass was very densely packed and had a distribution of vesicle sizes similar to that in the squid GA. To confirm the essential role of Ca2+ in vesiculation and in the restoration of an ionic seal (structural barrier) after transection (2–5), the cut end of another crayfish GA (Fig. 1F) was transected in Ca2+-free vanH solution and fixed at 30 min PT. The cut end was open, and vesicles were absent.
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We thank Mr. Louis Kerr for assistance. This work was supported by NIH grant NS31256 and a Texas, Advanced Technology Program grant.
Literature Cited
This article has been cited by other articles:
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  H. M. Fishman and G. D. Bittner Vesicle-Mediated Restoration of a Plasmalemmal Barrier in Severed Axons Physiology, June 1, 2003; 18(3): 115 - 118. [Abstract] [Full Text] [PDF]
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