Martis L. Ballinger

Heat-shock proteins in axoplasm: High constitutive levels and transfer of inducible isoforms from glia

To characterize heat‐shock proteins (HSPs) of the 70‐kDa family in the crayfish medial giant axon (MGA), we analyzed axoplasmic proteins separately from proteins of the glial sheath. Several different molecular weight isoforms of constitutive HSP 70s that were detected on immunoblots were approximately 1–3% of the total protein in the axoplasm of MGAs. To investigate inducible HSPs, MGAs were heat shocked in vitro or in vivo, then the axon was bathed in radiolabeled amino acid for 4 hours. After either heat‐shock treatment, protein synthesis in the glial sheath was decreased compared with that of control axons, and newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa appeared in both the axoplasm and the sheath. Because these radiolabeled proteins were present in MGAs only after heat‐shock treatments, we interpreted the newly synthesized proteins of 72 kDa, 84 kDa, and 87 kDa to be inducible HSPs. Furthermore, the 72‐kDa radiolabeled band in heat‐shocked axoplasm and glial sheath samples comigrated with a band possessing HSP 70 immunoreactivity. The amount of heat‐induced proteins in axoplasm samples was greater after a 2‐hour heat shock than after a 1‐hour heat shock. These data indicate that MGA axoplasm contains relatively high levels of constitutive HSP 70s and that, after heat shock, MGA axoplasm obtains inducible HSPs of 72 kDa, 84 kDa, and 87 kDa from the glial sheath. These constitutive and inducible HSPs may help MGAs maintain essential structures and functions following acute heat shock. J. Comp. Neurol. 396:1–11, 1998.

Reconnection of severed nerve axons with polyethylene glycol.

Severed medial giant axons in crayfish can be rejoined in vitro with polyethylene glycol (PEG) to produce axoplasmic continuity and through transmission of action potentials. Severed axon-like processes of a mammalian neuroblastoma/glioma cell line seem to be rejoined to the cell body using PEG in tissue culture. Our data suggest that PEG might be used to rejoin severed axons in vivo in various organisms.

Ultrastructural studies of severed medial giant and other CNS axons in crayfish.

SummaryThe distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.

DELAMINATING MYELIN MEMBRANES HELP SEAL THE CUT ENDS OF SEVERED EARTHWORM GIANT AXONS

Transected axons are often assumed to seal by collapse and fusion of the axolemmal leaflets at their cut ends. Using photomicroscopy and electronmicroscopy of fixed tissues and differential interference contrast and confocal fluorescence imaging of living tissues, we examined the proximal and distal cut ends of the pseudomyelinated medial giant axon of the earthworm, Lumbricus terrestris, at 5-60 min post-transection in physiological salines and Ca2+-free salines. In physiological salines, the axolemmal leaflets at the cut ends do not completely collapse, much less fuse, for at least 60 min post-transection. In fact, the axolemma is disrupted for 20-100 microm from the cut end at 5-60 min post-transection. However, a barrier to dye diffusion is observed when hydrophilic or styryl dyes are placed in the bath at 15-30 min post-transection. At 30-60 min post-transection, this barrier to dye diffusion near the cut end is formed amid an accumulation of some single-layered and many multilayered vesicles and other membranous material, much of which resembles delaminated pseudomyelin of the glial sheath. In Ca2+-free salines, this single and multilayered membranous material does not accumulate, and a dye diffusion barrier is not observed. These and other data are consistent with the hypothesis that plasmalemmal damage in eukaryotic cells is repaired by Ca2+-induced vesicles arising from invaginations or evaginations of membranes of various origin which form junctional contacts or fuse with each other and/or the plasmalemma.

Cooling of Peripheral Myelinated Axons Retards Wallerian Degeneration

The histological and ultrastructural status of intact and severed axons was examined in the ventral tail nerve of rats whose tails were maintained at 32, 23, and 13 degrees C. Compared to contralateral intact nerves, distal (anucleate) portions of severed myelinated axons morphologically and ultrastructurally degenerated within 3 days at 32 degrees C and within 6 days at 23 degrees C. In contrast, anucleate myelinated axons in ventral tail nerves maintained at 13 degrees C did not degenerate for at least 10 days. These and other data suggest that rapid Wallerian degeneration of anucleate myelinated axons is not an inevitable result of axonal severance in mammals.

Rapid artificial restoration of electrical continuity across a crush lesion of a giant axon

Action potentials never conducted through a crush lesion to the medial giant axon in the earthworm (Lumbricus terrestris) if the axon was exposed to normal or hypotonic salines that did not contain polyethylene glycol. However, action potentials, as well as electrotonic potentials, often conducted through a crush lesion exposed for 1 min to polyethylene glycol in hypotonic saline.

Calcium entry initiates processes that restore a barrier to dye entry in severed earthworm giant axons

After severance, axons can restore structural barriers that are necessary for recovery of their electrical function. In earthworm myelinated axons, such a barrier to dye entry is mediated by many vesicles and myelin-derived membranous structures. From time-lapse confocal fluorescence and DIC images, we now report that Ca2+ entry and not axonal injury per se initiates the processes that form a dye barrier, as well as the subsequent structural changes in this barrier and associated membranous structures. The time required to restore a dye barrier after transection also depends only on the time of Ca2+ entry.

Structural changes at cut ends of earthworm giant axons in the interval between dye barrier formation and neuritic outgrowth

We describe structural changes at the cut ends of invertebrate myelinated earthworm giant axons beginning with the formation of a dye barrier (15 minutes posttransection or postcalcium addition) and ending with the formation of a neuritic outgrowth (2–10 days posttransection). The morphology of the cut end, and the location and morphological configuration of the dye barrier, were assessed by time‐lapse confocal, fluorescence microscopy and by electron microscopy. During the interval from 15 to 35 minutes postcalcium addition, the dye barrier continuously migrated away from a cut axonal end; the dye barrier then remained stable for up to 5 hours. The size, packing density, and arrangement of membranous structures were correlated with changes in the dye barrier from 15 to 35 minutes postcalcium addition. During this interval, uptake of an externally placed hydrophilic dye by these membranous structures was also variable. After 35 minutes postcalcium addition, the membranous structures remained stable until they completely disappeared between 1 and 2 days posttransection. The disappearance of membranous structures always preceded neuritic outgrowth, which only arose from cut axonal ends. These results demonstrate that the dye barrier and associated membranous structures, which form after transection of earthworm giant axons, are very dynamic in the short term (35 minutes) with respect to their location and morphological configuration and suggest that axolemmal repair must be completed before neuritic outgrowth can occur. J. Comp. Neurol. 416:143–157, 2000.

Ultrastructural changes at gap junctions between lesioned crayfish axons

SummaryIn crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segment retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal region usually contains gap junctions and 50–60 nm vesicles near the axolemma of both SLGAs. From 1–14 days after lesioning, the distal segment of a severed SLGA undergoes obvious ultrastructural changes in mitochondria and neurotubular organization compared to control SLGAs or to adjacent, intact SLGAs in the same animal. Gap junctions are very difficult to locate in severed SLGAs within 24 h after lesioning. From two weeks to ten months after lesioning, the surviving stumps of severed SLGAs often appear remarkably normal except that structures normally associated with the presence of gap junctions remain very difficult to find.These and other data suggest that SLGA distal segments receive trophic support from adjacent, intact SLGAs. The mechanism of this support probably could not be via diffusion across gap junctions between intact and severed SLGAs since gap junctions largely disappear after lesioning. However, trophic maintenance could occur via the exocytotic — pinocytotic action of 50–60 nm vesicles which are always present on both sides of the septum between an intact SLGA and a severed SLGA distal segment.

Long-term survival of severed crayfish giant axons is not associated with an incorporation of glial nuclei into axoplasm

Glial nuclei have been reported to be incorporated into the axoplasm of surviving distal stumps (anucleate axons) weeks to months after lesioning abdominal motor axons in rock lobsters. We have not observed this phenomenon in crayfish medial giant axons (MGAs) which also survive for weeks to months after lesioning. Glial nuclei were not observed within MGAs perfused with a physiological intracellular saline. However, incorporation of glial nuclei was observed after MGAs were perfused with intracellular salines containing Fast green. From these and previously published data, we confirm that glial incorporation into axoplasm can occur, but we suggest that is is not a common mechanism used by crustaceans to provide for long-term survival of anucleate axons.