Transected axons are often assumed to seal at their cut ends by the formation of continuous membrane barriers that allow for the restoration of function in the axonal stumps. We have used several electrophysiological measures (membrane potential, input resistance, injury current density) and several morphological measures (phase- contrast, video-enhanced differential interference contrast, light, and electron microscopies) of living and fixed material to assess the extent and mechanism of sealing within hours after transecting giant axons of squid (Loligo pealei and Sepioteuthis lessoniana) and earthworms (Lumbricus terrestris). Our electrophysiological data suggest that the proximal and distal ends of transected squid giant axons do not completely seal within 2.5 hr in physiological saline. In contrast, the same set of measures suggest that proximal and distal ends of transected earthworm giant axons seal within 1 hr in physiological saline. Our morphological data show that the cut ends of both squid and earthworm axons constrict, but that a 20–70-microns- diameter opening always remains at the cut end that is filled with vesicles. Axonal transection induces the formation of vesicles that are observed in the axoplasm within minutes in standard salines and that rapidly migrate to the cut ends. These injury-induced vesicles are loosely packed near the cut ends of squid giant axons, which do not functionally seal within 2.5 hr of transection. In contrast, vesicles formed a tightly packed plug at the cut ends of earthworm medial giant axons, which do functionally seal within 1 hr of transection in physiological saline. Since we detect no single continuous membrane that spans the cut end, sealing does not appear to occur by the fusion of constricted axolemmal membrane or the formation of a membranous partition at the cut end. Rather, our data are consistent with the hypothesis that a tightly packed vesicular plug is responsible for sealing of earthworm giant axons.

We are able to morphologically fuse the severed halves of an invertebrate-myelinated axon by application of polyethylene glycol (PEG) Neurons are unique among metazoan cell types in having long cytoplasmic processes (axons). Nerve axons typically degenerate within 12-48 hr when severed from their cell bodies in mammals, many other vertebrates, and some invertebrates (1-4). In the mammalian central nervous system such myelinated axons usually do not regenerate. Outside the central nervous system, myelinated axons can regenerate but may take months to years to reach denervated tissues, and the regenerated connections are often not correct. Repair (if any) occurs by the slow (1-2 mm/day) outgrowth of axonal processes regenerating from proximal stumps to contact denervated target cells. This repair of some invertebrate axons and many vertebrate axons is complicated (5, 6) by a myelin sheath, defined as tight wrappings of glial cell membranes around individual axons. We have employed polyethylene glycol (PEG), a substrate used by molecular biologists to fuse plasma membranes of cells suspended in aqueous medium (7-9), to repair the cut ends of an invertebratemyelinated central nervous system axon in the earthworm. Our procedure takes about a minute to morphologically reconnect the cytoplasm and plasma membranes of two severed axonal segments. MATERIALS AND METHODSEarthworms, Lumbricus terrestris, were obtained from a local wholesale bait distributor. The animals were maintained in plastic trays containing moist soil and peat moss at 130C to promote a more favorable fatty acid ratio for membrane fusion (10,11).Operations. After anesthesia [4% (wt/vol) Chlorotone (Kodak)], adult earthworms 100-150 segments in length were pinned on a paraffin dish containing 50-100 ,uM carbachol in an earthworm saline (40.1 mM NaCi/10 mM Na2SO4/25 mM sodium acetate/0.5 mM K2SO4/3.0 mM CaC12/0.5 mM MgCl2/1.25 mM Tris/1.5 mM Hepes, pH 7.4). sheath, the cut ends of all severed axons remained approximately in their original orientations and were tightly apposed as the outer sheath retracted back to its original length upon removal of the blade.In cases of axonal severance with complete VNC transection, the two cut ends ofthe VNC with protruded MGAs were physically separated and reoriented with glass micropipettes or minutenadelen to appose the severed MGAs as tightly as possible without distorting them. Pins were placed in nerve roots arising from the severed VNC segments several millimeters from the potential fusion site to hold the MGAs in place. In cases of axonal severance with an intact VNC sheath, the cord was left undisturbed after lesioning. After both types of lesion, the normal saline (175 milliequivalents/ liter) was usually replaced by a hypotonic saline (120-160 milliequivalents/liter) containing NaCl reduced to 13.9-28.1 mM, CaCl2 reduced to 0-0.5 mM, and MgCl2 raised to 3.5-5 mM.PEG Application. A glass micropipette (25-to 100-,m tip diameter) filled with 50% (wt/wt) PEG (1-10 kDa; Baker) in distilled water was posi...

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Ionic conduction in the axolemmal and septal membranes of the medial giant fiber (MGF) of the earthworm (EW) Lumbricus terrestris was assessed by impedance spectroscopy in the frequency range 2.5-1000 Hz. Impedance loci in the complex plane were described by two semi-circular arcs, one at a lower characteristic frequency (100 Hz) and the other at a higher frequency (500 Hz). The lower frequency arc had a chord resistance of 53 k omega and was not affected by membrane potential changes or ion channel blockers [tetrodotoxin (TTX), 3,4-diaminopyridine (3,4-DAP), 4-aminopyridine (4-AP), and tetraethylammonium (TEA)]. The higher frequency arc had a chord resistance of 274 k omega at resting potential, was voltage-dependent, and was affected by the addition of TTX, 3,4-DAP, 4-AP, and TEA to the physiological EW salines. When all four blockers were added to the bathing solution, the impedance locus was described by two voltage-independent arcs. Considering the effects of these and other (i.e., Cd and Ni) ion channel blockers, we conclude that: 1) the higher frequency locus reflects conduction by voltage-sensitive ion channels in the axolemmal membrane, which contains at least four ion channels selective for sodium, calcium, and potassium (delayed rectifier and calcium-dependent), and 2) the lower frequency locus reflects voltage-insensitive channels in the septal membrane, which separates adjacent MGFs.