BY MEASUREMENT OF THE TIME COURSE OF INJURY CURRENT FOR 2.5 HR AT THE CUT END OF SEVERED GIANT AXONS (GAS) OF SQUID, WE ESTABLISHED PREVIOUSLY THAT GAS DO NOT SEAL WHEREAS SEVERED MEDIAL GIANT AXONS (MGAS) FROM EARTHWORMS DO SEAL. THE MAJOR DIFFERENCE BETWEEN THESE TWO GIANT AXONS IS THE GLIAL SHEATH; I.E., GAS ARE UNMYELINATED AND MGAS ARE MYELINATED. CONSEQUENTLY, BECAUSE SQUID GAS DO NOT SEAL, WE WISHED TO DETERMINE WHETHER SEALING AFTER AXOTOMY COULD OCCUR IN ANY OTHER UNMYELINATED GIANT AXON. CRAYFISH MGAS ARE UNMYELINATED AND ARE KNOWN TO SURVIVE TRANSECTION WHICH IMPLIES THAT THEY POSSESS A REPAIR MECHANISM. WE USED THE VIBRATING WIRE PROBE TO FOLLOW THE TIME COURSE OF INJURY CURRENT (II) FLOW AT THE CUT END OF CRAYFISH MGAS. IN MGAS FROM 11 DIFFERENT ANIMALS, II DECAYED TO BACKGROUND CURRENT DENSITY PRIOR TO AXOTOMY WITHIN 60 MIN OF TRANSECTION, INDICATING THE FORMATION OF A SEAL. THESE RESULTS WERE INDEPENDENT OF THE STATE OF AXOLEMMAL ION-CHANNEL CONDUCTION BECAUSE THE SAME RESULT WAS OBTAINED WHEN II WAS MEASURED IN THE PRESENCE OF ION CHANNEL BLOCKERS (TTX, 4-AP AND TEA) IN THE EXTERNAL SALINE. CONFIRMATION OF THE ~ELECTRICAL~ SEAL WAS MADE IN SEPARATE FLUORESCENT DYE UPTAKE EXPERIMENTS USING A ZEISS LASER SCANNING CONFOCAL (LSM-410) MICROSCOPE. FLUORESCEIN DYE OR LUCIFER YELLOW ADDED TO THE EXTERNAL SALINE WITHIN 30 MIN OF TRANSECTION WAS TAKEN UP BY MGAS WHEREAS WHEN ADDED 45 MIN AFTER TRANSECTION, THESE DYES WERE EXCLUDED. THUS, A PHYSICAL BARRIER WAS ESTABLISHED IN THE SAME TIME INTERVAL AS THE ELECTRICAL BARRIER. THESE DATA INDICATE THAT: 1) SEAL FORMATION CAN OCCUR IN SEVERED UNMYELINATED CRAYFISH MGAS, 2) SEAL FORMATION IS UNAFFECTED BY AXOLEMMAL ION CONDUCTION AND 3) FLUORESCENT DYE UPTAKE/EXCLUSION PROVIDES A COMPLEMENTARY METHOD TO VIBRATING PROBE MEASURES OF INJURY CURRENT IN THE ASSESSMENT OF THE EXTENT OF AXONAL REPAIR AFTER INJURY. IN OTHER EXPERIMENTS ON SQUID GAS, WE DISCOVERED THAT ADDITION OF THE NEUROFILAMENT PROTEASE CALPAIN TO THE EXTERNAL SOLUTION (ARTIFICIAL SEA WATER) INDUCED SEALING AS DETERMINED BOTH BY THE DECAY OF II TO BACKGROUND AND THE EXCLUSION OF FLUORESCENT DYE. THESE RESULTS ARE VERY SIGNIFICANT BECAUSE THEY SUGGEST THAT REPAIR CAN BE EFFECTED IN AXONS THAT DO NOT SEAL WITHOUT INTERVENTION.
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