. I o u r i ~ dof Ncirr.ochotii~ti.!~. 1915. Vol. 25, pp. 921-923. Pergamon Press. Printed in Great Britain.
SHORT COMMUNICATION The effect of electrical stimulation on the distribution of labelled proteins in isolated segments of rabbit vagus nerve (Received 21 March 1975. 4ccepted 28 May 1975)
After 1 h,3 h,4 hor6 hofelectrical stimulation, nerves were T H ~EFFECTS . of neuronal activity on the fast axonal transport of proteins have been studied in several ways and removed from the chamber along with non-stimulated conwith various results. Decreased activity has little effect on trols, cut into 5 mm segments and placed in I ml 10% TCA et ul., 1972; KARLSSON& SJOSTRAND, at 4°C. One day later the segments were rinsed again in transport (GRAFSTEIN 1971; GEFFEN & RUSH: 1968) and some workers have TCA and dissolved in 500pl Soluene (Packard) at room reported no effect of direct stimulation (DAHLSTROM, 1971; tcmperature. Radioactivity was measured in a Packard TriJANKOWSKA et al., 1969; GARCIA et al., 1974). In other carb liquid scintillation counter. D.p.m. in each 5mm cases, however, increased activity has increased the amount segmcnt were expressed as a percentage of the total d.p.m. of transported material arriving at a terminal or ligature in the 4 cm of nerve between the 2 ligatures. (NORSTROM & SJ~STRAND, 1972iqb; KEEN & MCLEAN, 1974; KISSet al., 1974). We have here examined the effects RESULTS of an electrical field on the distribution of labelled proteins The distribution of labelled proteins in the nerve segin isolated segments of vagus nerve. In this way changes mcnts at the time of tying double ligatures (0 h) and after in axonal transport could be detected at short time intera further 3 h of incubation are shown in Fig. 1. During vals and free of alterations in protein synthcsis. incubation the proteins between the 2 ligatures have moved in an anterograde direction and accumulated at MATERIALS AND METHODS the distal ligature. Similar profiles were obtained for nerves Male albino rabbits (weight 1.8-2.5 kg) were killed by incubated for 1 h, 3 h, 4 h and 6 h with or without overdose of pentobarbitonc-sodium and their circulation electrical stimulation. Figure 2 shows the change with time perfused with Ringer solution. Nodose ganglia and about of the accumulations at proximal and distal ligatures in control and stimulated nerves. Accumulation at the proxi7 cm pcripheral vagus nerve were removed and transferred to a two-cornpartmcnt pcrspex chamber containing mal ligature showed only a slight increasc with time and medium 199 (Flow LabordtorieS, Irvine, Scotland). To 1 was unaffected by electrical stimulation. Accumulation at compartment, containing the nodose ganglion, was added the distal ligature in the non-stimulated nerves increased 15 pCi [3HJleucine (L-4.5 ['Hlleucine; Radiochemical sharply with time with a maximum value at 4 h. Thereafter Centre, Amersham, England) in 300 pl medium. The remainder of the nerve lay in the second, larger, compartment, which containcd only medium. The chamber was r maintained in a moist atmosphere of 95% 0,/5% C 0 2 30 at 38°C for 3.5 h. Nerves were thcn removed, and 2 liga20 tures tied with 4/0 silk thread, one 2 cm from the nodose ganglion and thc other 4 cm distal to the first. The gang10 lion and adjoining 1.5 cm of each nerve werc discarded and the remaining segmcnt incubated for a further period 0 of time with or without electrical stimulation. ae 30 Oh The apparatus used for electrical stimulation was a closed perspex chambcr, volume adjustable from 0 7 to 4.5 ml with 1 cm diameter platinum-grid electrodes at each end. In these studies the electrodes were fixed 3 cm apart. A perspex jacket containing water at 37°C surrounded the chamber. A superfusion pump allowed continuous renewal (1.2 ml/min) of oxygenated medium 199 within the chamber; the used medium could he collected. lmmedi- FIG.1. The profile of [3H]leucine-labelled proteins in isoately after tying of the double ligatures, 3-6 nerves were lated segments of rabbit vagus nerves. Vagus nerves/ placed in the chamber already filled with medium. At the nodose ganglia preparations were labelled by incubation same time a group of similarly-treated nerves was placed of the ganglia with 15 pCi [3H]leucine; 3.5 h later, nodose in a gauze-covered dish filled with medium from the same ganglia and 1.5 cin of axon wcre rcmoved. and the remainsource and similarly kept at 37°C. A period of 5 min was ing 5 cm of axons were (0 h) cut up into 5 mm segments allowed for renewal of the medium in the chamber. Bipha- and placed in ice-cold TCA or incubated in uitro in sic pulses, frequency 80 Hz and pulse-duration 5.8 ms, were medium 199 for a further 3 h with ligatures at points A then delivered to the electrodes for 60 s every 3 min. Cur- (proximal) and B (distal), before being cut into segments. rent was monitored on an oscilloscope and kept constant Protein-bound radioactivity in each 5 mm segment was expressed as a percentage of that between ligatures A and B. at 20 mA. 921
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P
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922
Short communication
time hours
FIG.2. The change with time of incubation of the percentage accumulation of labelled proteins at distal (anterograde) and proximal (retrograde) ligatures on rabbit vagus nerve. Nerves were treated as in Fig. 1. Double-ligated segments of nerve containing labelled proteins were then incubated for up to 12 h in medium 199, with or without electrical stimulation. The percentage accumulation of labelled proteins in the 5 mm segments distal to the proximal ligature (retrograde) and proximal to the distal ligature (anterograde) were calculated for each time point. Differences between stimulated (a)and control (0)anterograde values at 3 h and 4 h were statistically significant ( P < 0.05 and < 0Ol5, respectively) by Student t-test. The number of nerves at each point is indicated. the percentage accumulation decreased with a lesser increase again at later intervals. The maximum increase in accumulation in stimulated nerves was seen after 3 h of incubation, and the accumulation had begun to decrease at 4 h. Thus electrical stimulation produced an acceleration of the early changes in distribution. DISCUSSION In uitro studies on isolated segments of vagus nerve containing labelled proteins in the motor fibres have demonstrated a retrograde redistribution of fast-rate labelled proteins from a ligature (MCLEANet al., 1975). In the sensory fibres studied in this work no marked retrograde accumulations were seen. However, the accumulations at the distal ligature showed a temporary decrease after 6 h of incubation following the initial sharp increase due to anterograde flow. The fate of the proteins which left the distal ligature at that time is unknown. Some may be destined for accumulation at the proximal ligature and give rise to the gradual small increase at the proximal ligature after 9 h (Fig. 2). Others may be incorporated into membrane or become stationary axoplasmic proteins forming the background level between the two ligatures. The electrical parameters for field stimulation were chosen on the basis of previous results on release of putative transmitters from brain and single nerve cells in oitro unpublished results). In the work (BADR& SELLSTROM, reported here a continuous release of labelled material from the nerve segments was observed throughout the experiment and the release was unchanged during the periods of stimulation. Figure 2 indicates that as a result of electrical stimulation the initial distribution of proteins both towards and
then away from the distal ligature was accelerated. The total amount of labelled proteins able to accumulate was unchanged. The accelerated transport rate was not due to changes in temperature in the stimulation chamber which were kept minimal by intermittent stimulation and the fast diffusion rate. An indicator in the superfusion medium showed no alterations in pH. While some of the released radioactivity was probably in the form of proteins, no significant change in the total protein-bound radioactivity of the nerves was caused by stimulation for 3 h or 4 h. This and the fact that the activity of a 5 mm segment of nerve immediately distal to the ligature (results not shown) remaincd unchanged suggests that thc results at 4 h and 3 h cannot be explained as a selective release or prevention of release respectively of proteins at the ligature zone. Electrical stimulation induces a depolarization of brain 1957). There slices (HILLMAN et a/., 1963; LI & MC~LWAIN, is a discriminate increase in membrane permeability (SKINIVASAN et a!., 1969) and as a consequence exchange diffusion is facilitated (KATZ rt al., 1969; JONES & BANKS.1970). 1966), The metabolism of the cells is increased (MCILWAIN, the respiration and the rate of glycolysis stimulated (BRADFORD,1970; HILLMANet a/., 1963). Fast axoplasmic transport is known to be dependent on energy metabolism (O~HS, 1974). It is suggested that, possibly as a result of such metabolic changes, increased electrical activity and depolarization of the nerve membrane can give rise to accelerated axonal transport. Acknowledgements-The authors thank Prof. H. H Y D ~ N for their interest and advice, and and Dr. J. SJOSTRAND Mrs. L. ESKILSONfor technical help. The work was supported by grants from Swedish Research Council (874I3X-2226-08C). Science Research Council, Great Britain (W.G.McL.) and the Medical Faculty, University of Goteborg (G.G.B.). Institute of Neurobiology, University of Gotehorg, Goteborg, Sweden
G. G. BADR W. G. MCLEAN
REFERENCES H. F. (1970) Brain Res. 19. 239-247. BRADFORD A. (1971) Phil. Trans. R . SOC.Ser. B. 261. 32% DAHLSTROM 358. GARCIAA. G., KIRPEKARS. M., PRATJ. C. & WAKADE A. R. (1974) J . Physiol., h n d . 241. 809-821. GEFFEN L. B. & RUSHR. (1968) J . Nrurochem. 15. 925-930. M. & INCAGLIA N. A. (1972) Brain GRAFSTEIN B., MURRAY Res. 44. 3748. HILLMAN H. H., CAMPBELL W. J. & MCILWAIN H. (1963) J . Neurochrm. 10. 325-339. JANKOWSKA E., LUBINSKA L. & NIEMERKO S. (1969) Comp. Biochem. Physiol. 28. 907-913. JONES C. T. & BANKSP. (1970) Biochem. J . 118. 801-812. KARLSSON J . - 0 . & SJOYlX4ND J. (1971) Brain R ~ s 29. . 31% 321. KATZR. I., CHASET. N. & KOPINI. J. (1969) J . Neurochem. 16. 167-168. KEENP. & MCLEANW. G. (1974) Br. J. Pharmac. 52. 527-53 I . KISSJ.. LANGE. & HLMORIJ. (1974) J . Neurol. Transmiss., SUPPI. XI. 125-133. LI C. 1. & MCILWAINH. (1957) J . PhysioL, Lond. 139. 178-190.
Short communication
M C~ LWAH. I NBiochemistry mid the central nervous system (1966) 3rd ed. pp. 49-77. J. & A. Churchill, London. MCLEANW. G., FRIZELL M. & SJ~STRAND J. (1975) J. Neurochem. NORSTROM A. & SJ~STRAND J. (1972~)d. Endocr. 52. 87105.
923
NOI~STROM A. & SJOSTRAND J. (19726) J. Endocr. 52. 107117. OCHs S . (1974) Fedn Proc. Fedn Am. SOCS exp. Biol. 33. 105&1058. SRINIVASAN V., NEALM. J. & MITCHELL J. F. (1969) J. Neurochem. 16. 1235-1244.
SHORT COMMUNICATION The effect of electrical stimulation on the distribution of labelled proteins in isolated segments of rabbit vagus nerve (Received 21 March 1975. 4ccepted 28 May 1975)
After 1 h,3 h,4 hor6 hofelectrical stimulation, nerves were T H ~EFFECTS . of neuronal activity on the fast axonal transport of proteins have been studied in several ways and removed from the chamber along with non-stimulated conwith various results. Decreased activity has little effect on trols, cut into 5 mm segments and placed in I ml 10% TCA et ul., 1972; KARLSSON& SJOSTRAND, at 4°C. One day later the segments were rinsed again in transport (GRAFSTEIN 1971; GEFFEN & RUSH: 1968) and some workers have TCA and dissolved in 500pl Soluene (Packard) at room reported no effect of direct stimulation (DAHLSTROM, 1971; tcmperature. Radioactivity was measured in a Packard TriJANKOWSKA et al., 1969; GARCIA et al., 1974). In other carb liquid scintillation counter. D.p.m. in each 5mm cases, however, increased activity has increased the amount segmcnt were expressed as a percentage of the total d.p.m. of transported material arriving at a terminal or ligature in the 4 cm of nerve between the 2 ligatures. (NORSTROM & SJ~STRAND, 1972iqb; KEEN & MCLEAN, 1974; KISSet al., 1974). We have here examined the effects RESULTS of an electrical field on the distribution of labelled proteins The distribution of labelled proteins in the nerve segin isolated segments of vagus nerve. In this way changes mcnts at the time of tying double ligatures (0 h) and after in axonal transport could be detected at short time intera further 3 h of incubation are shown in Fig. 1. During vals and free of alterations in protein synthcsis. incubation the proteins between the 2 ligatures have moved in an anterograde direction and accumulated at MATERIALS AND METHODS the distal ligature. Similar profiles were obtained for nerves Male albino rabbits (weight 1.8-2.5 kg) were killed by incubated for 1 h, 3 h, 4 h and 6 h with or without overdose of pentobarbitonc-sodium and their circulation electrical stimulation. Figure 2 shows the change with time perfused with Ringer solution. Nodose ganglia and about of the accumulations at proximal and distal ligatures in control and stimulated nerves. Accumulation at the proxi7 cm pcripheral vagus nerve were removed and transferred to a two-cornpartmcnt pcrspex chamber containing mal ligature showed only a slight increasc with time and medium 199 (Flow LabordtorieS, Irvine, Scotland). To 1 was unaffected by electrical stimulation. Accumulation at compartment, containing the nodose ganglion, was added the distal ligature in the non-stimulated nerves increased 15 pCi [3HJleucine (L-4.5 ['Hlleucine; Radiochemical sharply with time with a maximum value at 4 h. Thereafter Centre, Amersham, England) in 300 pl medium. The remainder of the nerve lay in the second, larger, compartment, which containcd only medium. The chamber was r maintained in a moist atmosphere of 95% 0,/5% C 0 2 30 at 38°C for 3.5 h. Nerves were thcn removed, and 2 liga20 tures tied with 4/0 silk thread, one 2 cm from the nodose ganglion and thc other 4 cm distal to the first. The gang10 lion and adjoining 1.5 cm of each nerve werc discarded and the remaining segmcnt incubated for a further period 0 of time with or without electrical stimulation. ae 30 Oh The apparatus used for electrical stimulation was a closed perspex chambcr, volume adjustable from 0 7 to 4.5 ml with 1 cm diameter platinum-grid electrodes at each end. In these studies the electrodes were fixed 3 cm apart. A perspex jacket containing water at 37°C surrounded the chamber. A superfusion pump allowed continuous renewal (1.2 ml/min) of oxygenated medium 199 within the chamber; the used medium could he collected. lmmedi- FIG.1. The profile of [3H]leucine-labelled proteins in isoately after tying of the double ligatures, 3-6 nerves were lated segments of rabbit vagus nerves. Vagus nerves/ placed in the chamber already filled with medium. At the nodose ganglia preparations were labelled by incubation same time a group of similarly-treated nerves was placed of the ganglia with 15 pCi [3H]leucine; 3.5 h later, nodose in a gauze-covered dish filled with medium from the same ganglia and 1.5 cin of axon wcre rcmoved. and the remainsource and similarly kept at 37°C. A period of 5 min was ing 5 cm of axons were (0 h) cut up into 5 mm segments allowed for renewal of the medium in the chamber. Bipha- and placed in ice-cold TCA or incubated in uitro in sic pulses, frequency 80 Hz and pulse-duration 5.8 ms, were medium 199 for a further 3 h with ligatures at points A then delivered to the electrodes for 60 s every 3 min. Cur- (proximal) and B (distal), before being cut into segments. rent was monitored on an oscilloscope and kept constant Protein-bound radioactivity in each 5 mm segment was expressed as a percentage of that between ligatures A and B. at 20 mA. 921
-
P
-
922
Short communication
time hours
FIG.2. The change with time of incubation of the percentage accumulation of labelled proteins at distal (anterograde) and proximal (retrograde) ligatures on rabbit vagus nerve. Nerves were treated as in Fig. 1. Double-ligated segments of nerve containing labelled proteins were then incubated for up to 12 h in medium 199, with or without electrical stimulation. The percentage accumulation of labelled proteins in the 5 mm segments distal to the proximal ligature (retrograde) and proximal to the distal ligature (anterograde) were calculated for each time point. Differences between stimulated (a)and control (0)anterograde values at 3 h and 4 h were statistically significant ( P < 0.05 and < 0Ol5, respectively) by Student t-test. The number of nerves at each point is indicated. the percentage accumulation decreased with a lesser increase again at later intervals. The maximum increase in accumulation in stimulated nerves was seen after 3 h of incubation, and the accumulation had begun to decrease at 4 h. Thus electrical stimulation produced an acceleration of the early changes in distribution. DISCUSSION In uitro studies on isolated segments of vagus nerve containing labelled proteins in the motor fibres have demonstrated a retrograde redistribution of fast-rate labelled proteins from a ligature (MCLEANet al., 1975). In the sensory fibres studied in this work no marked retrograde accumulations were seen. However, the accumulations at the distal ligature showed a temporary decrease after 6 h of incubation following the initial sharp increase due to anterograde flow. The fate of the proteins which left the distal ligature at that time is unknown. Some may be destined for accumulation at the proximal ligature and give rise to the gradual small increase at the proximal ligature after 9 h (Fig. 2). Others may be incorporated into membrane or become stationary axoplasmic proteins forming the background level between the two ligatures. The electrical parameters for field stimulation were chosen on the basis of previous results on release of putative transmitters from brain and single nerve cells in oitro unpublished results). In the work (BADR& SELLSTROM, reported here a continuous release of labelled material from the nerve segments was observed throughout the experiment and the release was unchanged during the periods of stimulation. Figure 2 indicates that as a result of electrical stimulation the initial distribution of proteins both towards and
then away from the distal ligature was accelerated. The total amount of labelled proteins able to accumulate was unchanged. The accelerated transport rate was not due to changes in temperature in the stimulation chamber which were kept minimal by intermittent stimulation and the fast diffusion rate. An indicator in the superfusion medium showed no alterations in pH. While some of the released radioactivity was probably in the form of proteins, no significant change in the total protein-bound radioactivity of the nerves was caused by stimulation for 3 h or 4 h. This and the fact that the activity of a 5 mm segment of nerve immediately distal to the ligature (results not shown) remaincd unchanged suggests that thc results at 4 h and 3 h cannot be explained as a selective release or prevention of release respectively of proteins at the ligature zone. Electrical stimulation induces a depolarization of brain 1957). There slices (HILLMAN et a/., 1963; LI & MC~LWAIN, is a discriminate increase in membrane permeability (SKINIVASAN et a!., 1969) and as a consequence exchange diffusion is facilitated (KATZ rt al., 1969; JONES & BANKS.1970). 1966), The metabolism of the cells is increased (MCILWAIN, the respiration and the rate of glycolysis stimulated (BRADFORD,1970; HILLMANet a/., 1963). Fast axoplasmic transport is known to be dependent on energy metabolism (O~HS, 1974). It is suggested that, possibly as a result of such metabolic changes, increased electrical activity and depolarization of the nerve membrane can give rise to accelerated axonal transport. Acknowledgements-The authors thank Prof. H. H Y D ~ N for their interest and advice, and and Dr. J. SJOSTRAND Mrs. L. ESKILSONfor technical help. The work was supported by grants from Swedish Research Council (874I3X-2226-08C). Science Research Council, Great Britain (W.G.McL.) and the Medical Faculty, University of Goteborg (G.G.B.). Institute of Neurobiology, University of Gotehorg, Goteborg, Sweden
G. G. BADR W. G. MCLEAN
REFERENCES H. F. (1970) Brain Res. 19. 239-247. BRADFORD A. (1971) Phil. Trans. R . SOC.Ser. B. 261. 32% DAHLSTROM 358. GARCIAA. G., KIRPEKARS. M., PRATJ. C. & WAKADE A. R. (1974) J . Physiol., h n d . 241. 809-821. GEFFEN L. B. & RUSHR. (1968) J . Nrurochem. 15. 925-930. M. & INCAGLIA N. A. (1972) Brain GRAFSTEIN B., MURRAY Res. 44. 3748. HILLMAN H. H., CAMPBELL W. J. & MCILWAIN H. (1963) J . Neurochrm. 10. 325-339. JANKOWSKA E., LUBINSKA L. & NIEMERKO S. (1969) Comp. Biochem. Physiol. 28. 907-913. JONES C. T. & BANKSP. (1970) Biochem. J . 118. 801-812. KARLSSON J . - 0 . & SJOYlX4ND J. (1971) Brain R ~ s 29. . 31% 321. KATZR. I., CHASET. N. & KOPINI. J. (1969) J . Neurochem. 16. 167-168. KEENP. & MCLEANW. G. (1974) Br. J. Pharmac. 52. 527-53 I . KISSJ.. LANGE. & HLMORIJ. (1974) J . Neurol. Transmiss., SUPPI. XI. 125-133. LI C. 1. & MCILWAINH. (1957) J . PhysioL, Lond. 139. 178-190.
Short communication
M C~ LWAH. I NBiochemistry mid the central nervous system (1966) 3rd ed. pp. 49-77. J. & A. Churchill, London. MCLEANW. G., FRIZELL M. & SJ~STRAND J. (1975) J. Neurochem. NORSTROM A. & SJ~STRAND J. (1972~)d. Endocr. 52. 87105.
923
NOI~STROM A. & SJOSTRAND J. (19726) J. Endocr. 52. 107117. OCHs S . (1974) Fedn Proc. Fedn Am. SOCS exp. Biol. 33. 105&1058. SRINIVASAN V., NEALM. J. & MITCHELL J. F. (1969) J. Neurochem. 16. 1235-1244.
