Brain Research, 582 (1992) 329-334 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
329
BRES 25209
Electric-field-induced reconnection of severed axons Angelio T. Todorov a, David Yogev a, Peinim Qi b, Janos H. Fendler a and Gerard S. Rodziewicz b ~Department of Chemtstry, Syracuse Untversity, Syracuse, NY 13244 (USA) and bDepartment of Neurosurgery, SUNY-HSC College of Medicine, Syracuse, NY 13210 (USA) (Accepted 25 February 1992) Key words" Nerve regeneration; Cell fusion; Electric field; Axon; Lumbrtcus terrestris; Axon reconnection; Nerve injury, Nerve repair
We report the first successful axon reconnectlon in the earthworm (Lumbricus terrestrts) medial giant axon (MGA) by electric fields generated by electrical pulses of 10-100/~s duration and 80-200 V amplitude. Reconnection was documented by light and electron microscopy, and by transport of Lucifer yellow dye across the reconnected MGA segments. Direct repair of a severed nerve axon promises the advantages of preserving axon viability and distal connections. Advances in cell fusion techniques have led to their application to many systemsg; however, only a few reports address their use for the reconnection of axons. Three of these reports detail efforts to reconnect severed segments of the glial-cell ensheathed medial giant axon (MGA) of invertebrates by the application of polyethylene glycol (PEG) 1'4'5. The mechanism of action of PEG has been suggested to involve dehydration and the alteration of membrane structure 6, although PEG does not intrinsically select a direction or location for the fusion process. We have employed two intrinsically directional cell fusion techniques for axon reconnection. We have previously reported initial results of laser pulse induced axon reconnection s, and now report initial results of electric-field-induced reconnection of axons severed by crushing. Electric fields reported to be of 1-5 kV/cm field strength and ns-/~s duration have been used in single-cell systems to produce heterokaryons and to introduce macromolecules into cells9. By keeping the parameters of electric field strength and pulse duration within the above ranges, the cell membranes underwent reversible pore formation that allowed the cell to remain viable. An in° trinsic property of electric field fusion of cells is that it is vectorial, i.e. membranes oriented perpendicular to the field lines are under higher field strength (and are thus more likely to experience reversible breakdown 7) than membranes oriented otherwise. Our group has developed an apparatus in which to perform and evaluate the results of fusion in a single large glial-cell ensheathed axon" the earthworm ( L u m -
bricus terrestris) medial giant axon (MGA) 4. To our knowledge, no group has published results of the application of electric field pulses to reconnect severed axons. The earthworm ventral nerve cord containing the M G A was dissected and prepared according to Krause and Bittner 4. The ventral nerve cord was pinned onto a silicone rubber stage and bathed in earthworm saline 4 at 21°C. The earthworm saline was replaced with mannitol buffer (0.3 M mannitol, 0.5 mM MgC12, 0.5 mM CaCI 2 in distilled water). A double-edged razor blade was used to crush the ventral nerve cord, severing the medial and two lateral giant axons, while leaving the outer nerve sheath intact. This process, carried out under the dissecting microscope, allowed the operator to see the retraction of the medial and two lateral giant axons away from the knife blade, with the outer nerve sheath remaining intact. This ensured apposition of the two severed M G A segments. No enzymatic treatment 1° of the severed axons was performed. Micromanipulators were then used to place two microelectrodes (Microprobe Inc. WE30031.5A), the anode in the rostral M G A segment and the cathode in the caudal M G A segment, at a total distance of 200-300/~m. This electrode design allowed the electric field to be applied perpendicularly through the apposed rostral and caudal cell membranes located between the anode and the cathode. A Hewlett-Packard 8116A pulse generator was attached to an inhouse-built device designed to provide electrical pulses up to 1200 V with rectangular shape and
Correspondence . G.S. Rodziewlcz, Dept. of Neurosurgery, 750 E. Adams St., Syracuse, NY 13210, USA Fax: (1) (315) 464-5520
330
rostral Lucifer yellow electrode
200-300 pm
\
rostral MGA
caudal MGA
\
rostral pulsing electrode
80-2o0 II
I
volts
caudal pulsing electrode
1O0 p.sec Fig. 1. Diagram of the experimental system The rostral and caudal ends of the severed MGA are dmgrammed. Dashed hnes mdtcate that septae may or may not be present m any particular MGA. Although deptcted in the rostral MGA segment, the Lucifer yellow rejection electrode was mserted m either the rostral or the caudal segment of the severed MGA. The pulsing electrodes were placed m both the rostral and the caudal segments of the severed MGA Electrical pulses of 80-200 V amphtude and 10-100 Fts duration were apphed between electrode (A) and electrode (B).
approximately 10-100/~s time duration. The input voltage was varied such that electrical pulses with an amplitude of 80-200 V were applied through both electrodes and the tissue between the electrode tips (Fig. 1). Five minutes after severing, 3 - 8 electric field pulses were applied. Five minutes subsequent to electric field application, the mannitol buffer was washed off with
earthworm saline, and the ventral nerve cord containing the M G A was allowed to survive 0.5-2 h at 21°C. Illumination and monitoring of dye injection was provided by a 100 watt mercury lamp attached to a Zeiss microscope. The images obtained were viewed and stored by a video camera (MTI CCD-72) attached to a viewing monitor (Hitachi VM1721U) and video recorder (Mag-
t 100 I1
A
100 t~
B
Fig 2. Lucifer yellow A axon reconnecuon. 30 mm after 3-5 electncal pulses of 80-200 V and 10-100/~s, the Lucifer yellow dye passed the crushed area (white arrowheads) freely, filling the caudal MGA segment B: control The Lucifer yellow dye stopped at the crushed area (white arrowheads)
331 dye was injected into the axon through a drawn-glass
navox VR3235AT01). B e t w e e n 30-60 min after axon reconnection, Lucifer
micropipette, and its flow was controlled by a Hewlett-
yellow C H (Sigma), 3% w/v in water, was injected ion-
Packard 8116A pulse g en er at o r and a high-voltage elec-
tophoretically into either the rostral or caudal M G A seg-
t r o m e t e r (W-P Instruments Inc. M707). The diffusion of
ment, 1-2 m m away from the crushed area. T h e injec-
Lucifer yellow through the crushed M G A segment was
tion duration varied from 5 - 2 0 min. T h e Lucifer yellow
followed with real-time videotaping.
i
100 p
A1
100 p
A2
100 t~
B
100
C
Fig. 3. Light microscopy. AI: axon reconnectlon. After 3-5 electrical pulses of 80-200 V and 10-100/zs, serial longitudinal sections 1 ~m thick demonstrated that the axoplasm of the rostrai and caudal MGA segments appear to be in continuity (open black arrowhead) across the crushed area (sohd white arrowheads). No cell membrane/gllal-cell sheath appears to be partitioning rostral from caudal MGA. The lateral giant axons on either side of the MGA are still not connected. A2: axon reconnectlon The reconnected segment (white arrowheads) has a diameter less than 25% of the diameter of the unsevered MGA This narrow reconnected segment still passed Lucifer yellow dye B: control. The ghal-cell sheaths covenng the rostral and caudal axons (open black arrowhead) demarcate the severed segment of the MGA (solid white arrowheads) No axoplasmic continuity exists between rostral and caudal axons C: axon reconnectlon. The reconnected segment (white arrowheads) in this figure represents the area examined by electron microscopy of an adjacent shce in Fig. 4.
t,~
333 Fig. 4. Electron microscopy Upper panel: axon reconnection After 3-5 electrical pulses of 80-200 V and 10-100/zs, the axoplasm of the rostral and caudal MGA appear to be m continmty across the line of reconnectlon. The axoplasm of rostral and caudal axons is continuous across the hne of fusion (large box, also shown in Fig. 4B), without a visible cell membrane or septum23 partition between rostral and caudal axons. Lower panel' axon reconnection, fusion site. The axoplasm of rostral and caudal axons is continuous across the line of fusion, with no evidence of a cell membrane partition.
For light and electron microscopy, the ventral nerve cord containing the M G A was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), osmicated, dehydrated, then embedded in low viscosity Spurr's medium (Polysciences Co., Warrington, PA). Longitudinal horizontal serial sections were cut. For light microscopy, sections 1 /~m thick were stained with 0.1% Toluidine blue dye, mounted, and examined with an Olympus Vanox-T microscope. For electron microscopy, 90 nm sections were cut, stained with uranyl acetate and lead citrate, then examined with a JEOL JEM-100CX II transmission electron microscope. Successful axon reconnection was achieved in 20 of 100 experiments (20%) using the experimental system described above, with criteria for successful axon reconnection being either serial longitudinal sections (18/20) or passage of Lucifer yellow dye (2/20). Serial longitudinal sections 1-2/~m thick were examined in 18/20 successfully reconnected cells, and 51/80 non-reconnected cells. Three reconnected cells studied with serial longitudinal sections were also processed for electron microscopy. Lucifer yellow dye injection was performed in 9/20 successfully reconnected cells, and 36/80 non-reconnected cells. Lucifer yellow dye, iontophoretically injected into one M G A segment, was seen to pass through the reconnected M G A segment into the other M G A segment (Fig. 2A). In contrast, the Lucifer yellow dye did not diffuse into the other M G A segments which were not exposed to the electric field pulses (21/21 control preparations) or which did not fuse after exposure to electric field pulses (Fig. 2B). Some leakage of Lucifer yellow dye into the surrounding ventral nerve cord, but not into the caudal MGA, occurred in the controls when the nerves were injected with Lucifer yellow dye within 15 min of severing but not if injected greater than 30 min after severing, possibly because the severed M G A cell membranes did not completely seal after the 15 min in this experimental system. We observed this leakage of dye both in mannitol and earthworm saline solutions. Longitudinal serial sections using light microscopy demonstrated the absence of a cell membrane/glial-cell sheath boundary between the rostral and caudal seg-
ments in the successfully reconnected axons (Fig. 3,A1), whereas a cell membrane/glial-cell sheath boundary was found to exist between the unconnected axons (Fig. 3B). The diameter of the reconnected segment itself can vary from over half the unsevered MGA diameter to approximately 10% of the unsevered M G A diameter (Fig. 3,A2). Local conditions influencing the re-annealing process of the cell membranes probably account for this variation in the diameter of the reconnected segments and the exact point of axon reconnection (position of electrical breakdown). We noted that in 4/100 experiments, the M G A appeared to be in continuity by serial longitudinal sections examined by light microscopy, but no Lucifer yellow dye was seen to pass during injection. These reconnection experiments were considered to be unsuccessful. Transmission electron microscopy of three axons confirmed the light microscopic finding in 18/20 reconnected axons: there was continuity between the axoplasm of rostral and caudal axons (Fig. 4A and B). We have documented the persistence of connection for 24 h after axon reconnection, and are currently studying the physiology of the reconnected axons. In single cell systems 9, cell membrane apposition and the magnitude of the field delivered to the membranes are critical parameters affecting cell fusion yields: we suggest that this is also true in our experimental system. The functional recovery of neurological systems such as severed spinal cord and peripheral nerve depends on the integrity of the axons transmitting information across the injured segment. Axon reconnection offers the promise of rapid return of axon integrity, a necessary condition for the functional recovery of these systems.
Support of this research by a grant from the National Science Foundation (to D.Y., A,T.T. and J.H.F) and by the SUNY Research Foundation (to G S.R. and P.Q.), is gratefully acknowledged. D Y. is the holder of a Chalm Weizmann Postdoctoral Fellowship for Scientific Research. A.T.T. thanks the Bulgarian Ministry of Science and Education (Project No. 587) for partial support We thank Dr. John Robson, Ph.D. for his invaluable assistance w~th neuroanatom~cal preparations We thank Dr. Joel Greenspan, Ph D. for his advice and assistance with physiological preparations.
334 1 Blttner, G.D., Balllnger, M L. and Raymond, M . A , Reconnectlon of severed nerve axons with polyethylene glycol, Brain Res, 367 (1986) 351-355. 2 Gunther, J , Neuronal syncyua in the giant fibres of earthworms, J Neurocytol, 4 (1975) 55-62 3 Kensler, R , Bnnk, P and Dewey, M , The septum of the lateral axon of the earthworm: a thm section and freeze-fracture study, J Neurocytol, 8 (1979) 565-590. 4 Krause, T.L and Blttner, G . D , Rapid morphological fusion of severed myehnated axons by polyethylene glycol, Proc Natl Acad Scl. USA, 87 (1990) 1471-1475 5 Krause, T L., Marquis, R E , Lyckman, A W , Balhnger, M.L and Blttner, G D., Rapid artificial restorauon of electncal contmmty across a crush lesion of a grant axon, Bram Res., 561 (1991) 350-353 6 Parente, R A. and Lentz, B R , Rate and extent of poly(eth-
7
8
9
10
ylene glycol)-mduced large vesicle fusion momtored by bdayer and internal contents mixing, Blochemtstry, 25 (1986) 66786688 Telssle, J and Blangero, C , D~rect experimental evidence of the vectorial character of the interaction between electric pulses and cells m cell electrofusmn, Btochtrn Bgophys Acta, 775 (1984) 446-448 Yogev, D., Todorov, A T , Q1, P, Fendler, Y H and RodzleWlCZ, G , Laser-reduced reconnectlon of severed axons, Bgochem Btophys Res Commun , 180 (1991)874-880 Zlmmerman, U , Electrical breakdown, electropermeabfllzatlon, and electrofuslon, Rev. Btochem Pharmacol , 105 (1986) 175-256 Zlmmermann, U and Pdwat, G , Electric-field-stimulated fusion' increased field stabdlty of cells reduced by pronase, Naturwtssenschaften, 68 (1981) 577-579
329
BRES 25209
Electric-field-induced reconnection of severed axons Angelio T. Todorov a, David Yogev a, Peinim Qi b, Janos H. Fendler a and Gerard S. Rodziewicz b ~Department of Chemtstry, Syracuse Untversity, Syracuse, NY 13244 (USA) and bDepartment of Neurosurgery, SUNY-HSC College of Medicine, Syracuse, NY 13210 (USA) (Accepted 25 February 1992) Key words" Nerve regeneration; Cell fusion; Electric field; Axon; Lumbrtcus terrestris; Axon reconnection; Nerve injury, Nerve repair
We report the first successful axon reconnectlon in the earthworm (Lumbricus terrestrts) medial giant axon (MGA) by electric fields generated by electrical pulses of 10-100/~s duration and 80-200 V amplitude. Reconnection was documented by light and electron microscopy, and by transport of Lucifer yellow dye across the reconnected MGA segments. Direct repair of a severed nerve axon promises the advantages of preserving axon viability and distal connections. Advances in cell fusion techniques have led to their application to many systemsg; however, only a few reports address their use for the reconnection of axons. Three of these reports detail efforts to reconnect severed segments of the glial-cell ensheathed medial giant axon (MGA) of invertebrates by the application of polyethylene glycol (PEG) 1'4'5. The mechanism of action of PEG has been suggested to involve dehydration and the alteration of membrane structure 6, although PEG does not intrinsically select a direction or location for the fusion process. We have employed two intrinsically directional cell fusion techniques for axon reconnection. We have previously reported initial results of laser pulse induced axon reconnection s, and now report initial results of electric-field-induced reconnection of axons severed by crushing. Electric fields reported to be of 1-5 kV/cm field strength and ns-/~s duration have been used in single-cell systems to produce heterokaryons and to introduce macromolecules into cells9. By keeping the parameters of electric field strength and pulse duration within the above ranges, the cell membranes underwent reversible pore formation that allowed the cell to remain viable. An in° trinsic property of electric field fusion of cells is that it is vectorial, i.e. membranes oriented perpendicular to the field lines are under higher field strength (and are thus more likely to experience reversible breakdown 7) than membranes oriented otherwise. Our group has developed an apparatus in which to perform and evaluate the results of fusion in a single large glial-cell ensheathed axon" the earthworm ( L u m -
bricus terrestris) medial giant axon (MGA) 4. To our knowledge, no group has published results of the application of electric field pulses to reconnect severed axons. The earthworm ventral nerve cord containing the M G A was dissected and prepared according to Krause and Bittner 4. The ventral nerve cord was pinned onto a silicone rubber stage and bathed in earthworm saline 4 at 21°C. The earthworm saline was replaced with mannitol buffer (0.3 M mannitol, 0.5 mM MgC12, 0.5 mM CaCI 2 in distilled water). A double-edged razor blade was used to crush the ventral nerve cord, severing the medial and two lateral giant axons, while leaving the outer nerve sheath intact. This process, carried out under the dissecting microscope, allowed the operator to see the retraction of the medial and two lateral giant axons away from the knife blade, with the outer nerve sheath remaining intact. This ensured apposition of the two severed M G A segments. No enzymatic treatment 1° of the severed axons was performed. Micromanipulators were then used to place two microelectrodes (Microprobe Inc. WE30031.5A), the anode in the rostral M G A segment and the cathode in the caudal M G A segment, at a total distance of 200-300/~m. This electrode design allowed the electric field to be applied perpendicularly through the apposed rostral and caudal cell membranes located between the anode and the cathode. A Hewlett-Packard 8116A pulse generator was attached to an inhouse-built device designed to provide electrical pulses up to 1200 V with rectangular shape and
Correspondence . G.S. Rodziewlcz, Dept. of Neurosurgery, 750 E. Adams St., Syracuse, NY 13210, USA Fax: (1) (315) 464-5520
330
rostral Lucifer yellow electrode
200-300 pm
\
rostral MGA
caudal MGA
\
rostral pulsing electrode
80-2o0 II
I
volts
caudal pulsing electrode
1O0 p.sec Fig. 1. Diagram of the experimental system The rostral and caudal ends of the severed MGA are dmgrammed. Dashed hnes mdtcate that septae may or may not be present m any particular MGA. Although deptcted in the rostral MGA segment, the Lucifer yellow rejection electrode was mserted m either the rostral or the caudal segment of the severed MGA. The pulsing electrodes were placed m both the rostral and the caudal segments of the severed MGA Electrical pulses of 80-200 V amphtude and 10-100 Fts duration were apphed between electrode (A) and electrode (B).
approximately 10-100/~s time duration. The input voltage was varied such that electrical pulses with an amplitude of 80-200 V were applied through both electrodes and the tissue between the electrode tips (Fig. 1). Five minutes after severing, 3 - 8 electric field pulses were applied. Five minutes subsequent to electric field application, the mannitol buffer was washed off with
earthworm saline, and the ventral nerve cord containing the M G A was allowed to survive 0.5-2 h at 21°C. Illumination and monitoring of dye injection was provided by a 100 watt mercury lamp attached to a Zeiss microscope. The images obtained were viewed and stored by a video camera (MTI CCD-72) attached to a viewing monitor (Hitachi VM1721U) and video recorder (Mag-
t 100 I1
A
100 t~
B
Fig 2. Lucifer yellow A axon reconnecuon. 30 mm after 3-5 electncal pulses of 80-200 V and 10-100/~s, the Lucifer yellow dye passed the crushed area (white arrowheads) freely, filling the caudal MGA segment B: control The Lucifer yellow dye stopped at the crushed area (white arrowheads)
331 dye was injected into the axon through a drawn-glass
navox VR3235AT01). B e t w e e n 30-60 min after axon reconnection, Lucifer
micropipette, and its flow was controlled by a Hewlett-
yellow C H (Sigma), 3% w/v in water, was injected ion-
Packard 8116A pulse g en er at o r and a high-voltage elec-
tophoretically into either the rostral or caudal M G A seg-
t r o m e t e r (W-P Instruments Inc. M707). The diffusion of
ment, 1-2 m m away from the crushed area. T h e injec-
Lucifer yellow through the crushed M G A segment was
tion duration varied from 5 - 2 0 min. T h e Lucifer yellow
followed with real-time videotaping.
i
100 p
A1
100 p
A2
100 t~
B
100
C
Fig. 3. Light microscopy. AI: axon reconnectlon. After 3-5 electrical pulses of 80-200 V and 10-100/zs, serial longitudinal sections 1 ~m thick demonstrated that the axoplasm of the rostrai and caudal MGA segments appear to be in continuity (open black arrowhead) across the crushed area (sohd white arrowheads). No cell membrane/gllal-cell sheath appears to be partitioning rostral from caudal MGA. The lateral giant axons on either side of the MGA are still not connected. A2: axon reconnectlon The reconnected segment (white arrowheads) has a diameter less than 25% of the diameter of the unsevered MGA This narrow reconnected segment still passed Lucifer yellow dye B: control. The ghal-cell sheaths covenng the rostral and caudal axons (open black arrowhead) demarcate the severed segment of the MGA (solid white arrowheads) No axoplasmic continuity exists between rostral and caudal axons C: axon reconnectlon. The reconnected segment (white arrowheads) in this figure represents the area examined by electron microscopy of an adjacent shce in Fig. 4.
t,~
333 Fig. 4. Electron microscopy Upper panel: axon reconnection After 3-5 electrical pulses of 80-200 V and 10-100/zs, the axoplasm of the rostral and caudal MGA appear to be m continmty across the line of reconnectlon. The axoplasm of rostral and caudal axons is continuous across the hne of fusion (large box, also shown in Fig. 4B), without a visible cell membrane or septum23 partition between rostral and caudal axons. Lower panel' axon reconnection, fusion site. The axoplasm of rostral and caudal axons is continuous across the line of fusion, with no evidence of a cell membrane partition.
For light and electron microscopy, the ventral nerve cord containing the M G A was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), osmicated, dehydrated, then embedded in low viscosity Spurr's medium (Polysciences Co., Warrington, PA). Longitudinal horizontal serial sections were cut. For light microscopy, sections 1 /~m thick were stained with 0.1% Toluidine blue dye, mounted, and examined with an Olympus Vanox-T microscope. For electron microscopy, 90 nm sections were cut, stained with uranyl acetate and lead citrate, then examined with a JEOL JEM-100CX II transmission electron microscope. Successful axon reconnection was achieved in 20 of 100 experiments (20%) using the experimental system described above, with criteria for successful axon reconnection being either serial longitudinal sections (18/20) or passage of Lucifer yellow dye (2/20). Serial longitudinal sections 1-2/~m thick were examined in 18/20 successfully reconnected cells, and 51/80 non-reconnected cells. Three reconnected cells studied with serial longitudinal sections were also processed for electron microscopy. Lucifer yellow dye injection was performed in 9/20 successfully reconnected cells, and 36/80 non-reconnected cells. Lucifer yellow dye, iontophoretically injected into one M G A segment, was seen to pass through the reconnected M G A segment into the other M G A segment (Fig. 2A). In contrast, the Lucifer yellow dye did not diffuse into the other M G A segments which were not exposed to the electric field pulses (21/21 control preparations) or which did not fuse after exposure to electric field pulses (Fig. 2B). Some leakage of Lucifer yellow dye into the surrounding ventral nerve cord, but not into the caudal MGA, occurred in the controls when the nerves were injected with Lucifer yellow dye within 15 min of severing but not if injected greater than 30 min after severing, possibly because the severed M G A cell membranes did not completely seal after the 15 min in this experimental system. We observed this leakage of dye both in mannitol and earthworm saline solutions. Longitudinal serial sections using light microscopy demonstrated the absence of a cell membrane/glial-cell sheath boundary between the rostral and caudal seg-
ments in the successfully reconnected axons (Fig. 3,A1), whereas a cell membrane/glial-cell sheath boundary was found to exist between the unconnected axons (Fig. 3B). The diameter of the reconnected segment itself can vary from over half the unsevered MGA diameter to approximately 10% of the unsevered M G A diameter (Fig. 3,A2). Local conditions influencing the re-annealing process of the cell membranes probably account for this variation in the diameter of the reconnected segments and the exact point of axon reconnection (position of electrical breakdown). We noted that in 4/100 experiments, the M G A appeared to be in continuity by serial longitudinal sections examined by light microscopy, but no Lucifer yellow dye was seen to pass during injection. These reconnection experiments were considered to be unsuccessful. Transmission electron microscopy of three axons confirmed the light microscopic finding in 18/20 reconnected axons: there was continuity between the axoplasm of rostral and caudal axons (Fig. 4A and B). We have documented the persistence of connection for 24 h after axon reconnection, and are currently studying the physiology of the reconnected axons. In single cell systems 9, cell membrane apposition and the magnitude of the field delivered to the membranes are critical parameters affecting cell fusion yields: we suggest that this is also true in our experimental system. The functional recovery of neurological systems such as severed spinal cord and peripheral nerve depends on the integrity of the axons transmitting information across the injured segment. Axon reconnection offers the promise of rapid return of axon integrity, a necessary condition for the functional recovery of these systems.
Support of this research by a grant from the National Science Foundation (to D.Y., A,T.T. and J.H.F) and by the SUNY Research Foundation (to G S.R. and P.Q.), is gratefully acknowledged. D Y. is the holder of a Chalm Weizmann Postdoctoral Fellowship for Scientific Research. A.T.T. thanks the Bulgarian Ministry of Science and Education (Project No. 587) for partial support We thank Dr. John Robson, Ph.D. for his invaluable assistance w~th neuroanatom~cal preparations We thank Dr. Joel Greenspan, Ph D. for his advice and assistance with physiological preparations.
334 1 Blttner, G.D., Balllnger, M L. and Raymond, M . A , Reconnectlon of severed nerve axons with polyethylene glycol, Brain Res, 367 (1986) 351-355. 2 Gunther, J , Neuronal syncyua in the giant fibres of earthworms, J Neurocytol, 4 (1975) 55-62 3 Kensler, R , Bnnk, P and Dewey, M , The septum of the lateral axon of the earthworm: a thm section and freeze-fracture study, J Neurocytol, 8 (1979) 565-590. 4 Krause, T.L and Blttner, G . D , Rapid morphological fusion of severed myehnated axons by polyethylene glycol, Proc Natl Acad Scl. USA, 87 (1990) 1471-1475 5 Krause, T L., Marquis, R E , Lyckman, A W , Balhnger, M.L and Blttner, G D., Rapid artificial restorauon of electncal contmmty across a crush lesion of a grant axon, Bram Res., 561 (1991) 350-353 6 Parente, R A. and Lentz, B R , Rate and extent of poly(eth-
7
8
9
10
ylene glycol)-mduced large vesicle fusion momtored by bdayer and internal contents mixing, Blochemtstry, 25 (1986) 66786688 Telssle, J and Blangero, C , D~rect experimental evidence of the vectorial character of the interaction between electric pulses and cells m cell electrofusmn, Btochtrn Bgophys Acta, 775 (1984) 446-448 Yogev, D., Todorov, A T , Q1, P, Fendler, Y H and RodzleWlCZ, G , Laser-reduced reconnectlon of severed axons, Bgochem Btophys Res Commun , 180 (1991)874-880 Zlmmerman, U , Electrical breakdown, electropermeabfllzatlon, and electrofuslon, Rev. Btochem Pharmacol , 105 (1986) 175-256 Zlmmermann, U and Pdwat, G , Electric-field-stimulated fusion' increased field stabdlty of cells reduced by pronase, Naturwtssenschaften, 68 (1981) 577-579
