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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.

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.

In crayfish, the severed distal segment of single lateral giant axon (SLGA) often survives for at least 10 months after lesioning if this segme;t retains a septal region of apposition with an adjacent, intact SLGA. In control (unsevered) SLGAs, this septal 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.

The distal stumps of severed medial giant axons (MGAs) and of nongiant 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.

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.

We report anomalies of lateral giant neurons in field-caught animals. In one case, both lateral giant neurons arising from the fifth abdominal ganglion did not terminate in the next-most anterior ganglion, but rather continued to the third abdominal ganglion. In another case, an additional large neuron arising in the fifth abdominal ganglion coursed in a posterior direction. All othe CNS structures appeared normal in animals having either anomaly.