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Recovery from spinal transection in fish: Regrowth of axons past the transection
Ablumc/olce L~t~s. 38 (1983) 227-231 Elsevier Scientific Publishers h d a n d Ltd.
RICHARD E, ~ E S H A L L
227
and ~ N T H I A S, YOUNGBLOOD
Marine Biomedical Institute and Departments of Anatomy and of Physiology and Biophysics. University of Texas Medical Branch, Galveston, TX 77J30.2772 (U.S.A,) (Rect~ved April 22nd, 1983; Accepted April 27th, 1983)
Key words: horseradish peroxidase - spinal transection - spinal regeneration - goldfish
In fish axons rqx~rtedly cross a spinal transection, form synapses, and then cease growing or grow in abnormal directions. This obviously would not restore pgevious synaptic connections, and since fish recover function, it is often stated that recovery of function in fish seems to be independent of rcztoration of normal synaptic architecture. By contrast, the prc~.nt study mmphasizes that many axons in igoldfish continue srowinlg after they pass a spinal transection. Honmndish ix'roxidase was it~jected into caudal spinal cord in normal or spinal cord-transected fish. Neurons were laheled in the same places in operated and normal fish. Since the site of injection was 4 cm (18 sqpnents) caudal to the transection, it seems clear that the axons of cells labeled in transacted fish ~tm~d~l past the cut for almou the lenjth of the spinal cord. Thus Igrowth did not cea~ for thmc axons. Therefore, even thoullh s y n a ~ undoubtedly form in the nenropil distal to the cut, many axons do not stop srowinlg and restoration of ~ i o u s synaptic connections may well be a requir~qngnt for return of funclion.
Axons grow across a spinal transection in fish but not in mammals ll-5. 8=-11]. it is reasonable to conclude that motor recovery, which also occurs in fish but not in mammals, is the result of this regrowth. An unresolved question, however, i~ whether function returns when the axons return to their previous synap/ic sire, thus partially or completely restoring the original synapfic architecture, or whether runetion returns without the original architecture being restored. Several important studies favor the latter idea for they provide evidence to indicate that regrowing axons form synapses soon after they cross the transection and then cease growing or grow in abnormal directions [2, 3, 8-11], situations t ~ t would not restore pretransection synaptic patterns. By contrast, the present study provides evidence to indicate that a large number of regrowing axons extend the length of the spinal cord and thus do not cease growing after they pass a spinal transection. Goldfish (Carassius auralus), approximately 10 cm from snout to base of tail fin and weighing approxi~,nately 50 g, were used. After anesthesia with Finquel (tricaine methanesulfonate), spinal cords were exposed by laminectomy and t r a ~ e d between segments 6 and S. This is between the caudal end of the operculum and the 0JO4-3940/SJ/S 03.00 © 198J Elsevi,:r--Sck'ntifi¢ Publishers Ireland Ltd.
2211 dmsal fin and is approximately 1.5 cm from the medulla oblongata. It was difficult to transect more craniah'y without damage m the gill cavity. After transection, the caudal pan of the animal was paralyzed. After approximately 1.5 months,
the operated fish were difficult to distinguish from normal. The f'mh were allowed to smvive for 138--160 days (Table D and then reanesthetized. Another lami_~nectomy was done between the anal fin and the base o f the tail and 1 ~ of a ~)'e solution o f horseradish peroxidas¢ OIRP) was injected approximately 4.0 a n caudal to the transection into segments 24-26. Caudal to the injection site the spinal cord became extremely thin. After 7 days the animals were rcanesthetized, sacrificed, and perfused with saline followed by a mixture o f 1.25% glutaraldehyde and 0.75~'e paraformaldehyd¢ in 0.1 M phosphate buffer. The brain and spinal cord were placed in 30% sucrose and horizontal frozen sections of the brain were cut and rcacted with tctramcthylbenzidine. As a control, an acute transection of the spinal cord was done between sqgments 6--8 just before the injection of HRP into sqgments 24-26 in 4 fish. in addition, 4 unoperated fish were injected with HRP into sqgments 24-26 and treated as above. in control animals (animals with an acute transection at sqpnents just prior to
TABLE I N U M B E R S OF I , A B E L E I ) NL:I~'RONS A comparL~n o f n u m t ~ s o f labeled neurons in the reticular formation, v~tibular nuclei, and nuclei o f ~be medial Ionttiludinal fa~'iculu~ follo~'intt HRP injecti.~m inlo the ~inal cord o f Igoldfish ~ i t h a spinal
Iran~,-tion (Ol~'al~J fi.~h) of imo unolx'raled [mldfish (unoperaled fishL
Fish ¢
Days p.~tt ranse~.lion
Reticular formation
V~tibular nuclei
Nuclei o f the medial Ionlgitudinal fasciculu$
+ +
512 447 580 521
99 42 96 93
71 24 79 65
1311 14~ 161) I~ 146
366 341 24J+ 394 172
60 38 61 108 4"1
51 59 33 58 41
161
31
.'St
5a
22 44
Unoperatcdfish 2. ~, 4++
A~eragc
5. 6. 7. 9i !0. ....
Average
143
229
6 - 8 to the injection of HRP), no cells were labeled in the brain, indicating that the HRP was not falsely labeling by being t r a n s p o ~ in cerebrospinal fluid or blood. Following HRP injection in transected and non-transected animals, labeled neurons were seen in 3 locations: (1) the reticular formation; (2) the vestibular
neurons following spinal injection of HRP in zebrafish [6]. The numbers of labeled cells in operated and unoperated fish are given in Table 1. Note that in unoperated fish there are an average of 521 labeled cells in the reticular formation, almost 100 in the vestibular nuclei and 63 cells in the nuclei of the medial longitudinal fasiculus, In operated fish, there are an average of 281 labeled cells in the reticular formation, 58 in the vestibular nuclei and 44 in the nuclei of the medial longitudinal fasiculus. The percentages of labeled cells in transected as compared to normal fish are 54~e for the reticular formation, 62070 for the vestibular nuclei, and 68e/e for the nuclei of the medial longitudinal fasiculus. The overall percentage of labeled cells in transected as compared to normal fish is 55%. The possibility that function may be restored without restoration of 'normal' synaptic connections has been discussed for many years. For regeneration of fish spinal cord following transection, a possible example of such a restoration, a key issue is whether regenerating axons stop growing after they pass the transection and form synapses. The first important papers on this point indicated that many regenerating axons in fish did seem to form synapses at the first available sites caudal to a transection and then largely ceased growing [2, 3]. Furthermore when a teflon sheet was interposed between the two ends of the transection, the axons that gathered next to the teflon formed synapses with one another and growth did not resume when the teflon was removed 131. The inhibitory effect of the teflon could be negated if the spinal cord was retransected, however, the retransection presumably removing those parts of the axons in synaptic contact with one another [31. The phrase 'contact inhibition' was used to describe these phenomena and it was hypothesized that this renders the neuron incapable of resuming axonal growth [2,
31. More recent work on lamprey was confirmatory. These animals have large, individually identifiable spinal axons, and these axons regenerated past a transection and then seemed to end suddenly or turned and went for a short distance in an abnormal direction 18-11]. Thus the general belief that fish behavioral recovery is associated with axonal regrowth, but the axonal regrowth does not seem to result in the restoration ~)f a pretransection synaptic architecture. By contrast, however, there are indications that axonal growth following synapse formation does not cease. For example, in studies on goldfish [4] and tadpoles 151, it was shown that many cells in the brain were labeled by the retrograde transport of HRP injected several segments caudal to a spinal transection. The conclusion in both these studies was that the cells thaf projected to particular areas of spinal cord
in the normal sent their axem back to them areas after mmegtion. It would seem, therefore, that axons continued growing after Imssing the ttamection, in another study it was shown that function retmtmd when tranmcted Mauthner cell axons in ~ grew ~ to t l ~ - ~ ~ ~ m. ~ mautlmer axon growth did not seem to be inhibited by ~ fomation near tbe tmmetaion. The present mm'y extends the above work by ddiberatdy separating the site of injec;ion of HIgP from the transection by as great a distance as conveniently possible. When this is dome, more than half of the number of brain neurons that are labeled following HlgP injection into the normal caudal spinal cord are labeled with the same type of H R P injection following recovery from transection of the cranial spinal cord. Furthermore, the labeled neurons are in the same location following transection as they are in the normal. These results are essentially the same as in our previous experiments where the transection and injection sites were much closer together. The interpretation of the data from the normal animals is that there are 3 populations of cells in the brainstem (the reticular formation, the vestibular nuclei. and the nuclei of the medial longitudinal fasiculus) whose axons project the length of the spinal cord. The interpretation of the data m,. transected fish is that a majority of cells from the areas that project to caudal spinal cord in the normal project there after transection. Thus it would seem safe to assume that a significant number of neurons send axons across the transection back to their original locations and probably to their original synaptic sites. Since the original locations are at least 4 cm from the transection, this would strongly favor the idea that axonal growht does not cease n e a r the tran~gtion. An understanding of sncce~sful regeneration in frsh and lower vertebJates should be of great value in suggesting ways to deal with unsuccessful regeneration in higher organisms. It seems clear from earlier work that axons grow across a spinal transection in lower vertebrates and then form synapses when they enter the denervated neuropi[ on the other side of the transection [2, 3, I0, I 1]. Furthermore, some of tl~e axons probably cease growth at this point because quamitative studies indicate that fe~'er axons cross Ill and fewer cells label [4] (also this study) in transected than in normal animals. This study emphasizes that not all axons cease to grow, however. Sim:e a significant number of axons cross the transection and grow for a considerable distance, it is a reasonable hypothesis that function returns when the original synapti¢ connections are restored, as suggested by the physiological data on Mauthner axon regrowth [7]. If this is the case, then the synapses that are formed early, near the transection site, might deter normal function because they could be a source of synaptic noise. Another possibility is that after the 'right' synaptic connections are made, the 'wrong' synaptic connections are eliminated. Further experiments designed to test these hypotheses are feasible. This ~ r c h is ~ ~ l y supported by Grants NS 10161, 17039, 11255 and 07377 from the United States Public Health Service, and a support grant from the Moody Foundation.
231 I Bernstein, J.J., Relatimt of spinal cord regeneration to age in adult goldfish, Exp. Neurol., 9 (1964) 161-174. 2 Bernstein, J.J. and ~ , M.D., Effect of fglial-epend3mtal scar and teflon arrest on the regataafive calmcity of goldfish spinal cord, Exp. Neurol., 19 (198'7) 25-32. 3 Bernstein, J.J. and Bernstein, M.E., Uitmstrucmre of normal regeucration and loss of regenerative
Neurosci. Lett., 32(1982) 259-7,64. $ Forehand, C.J. and Farel, P.B., Anatomical and bebavioral recovery from the effects of spinal cord transection: depglldettg~ on inetamo~hosis in Anuran larvae, J. Neurosci., 2 (1982) 654-662. 6 Kimntel, C.B., Powell, $.L. and Metculfe, W.K., Brain neurons which project to the spinal cord in youn$ larvae in zebrafish, J. comp. Neurol., 205 (1982) 112-137. 7 Lee, M.T., Rejeneration and functional reconnection of an ickntified vertebrate central neuron, J. Neurmci., 2 (1982) 1793-1811. 8 Rovainin, C.M., ~ a t i o n of Muller and Mauthner axons after spinal transection in law-al lainpreys, J. comp. Neurol., 168 (1976) 545-554. 9 Seizer, M.E., Mechanism of functiO,~d+ recovery and rclgeneration after spinal cord tran~ction in larvae sea lamprey, J. Physiol. (Lond.~, 227 (1978) 395-408. 10 Wood, M.R. and Coheu, M.J., Synaptic refgeneration in identified neurons in the lamprey spinal cord, Scieuc¢, 206 (1979) 344-346. I I Wood, M.R. and Cohen, M.J., Synaptic relgCnerationand fglial reactions in the transects! spinal cord of the lamprey, J. Neurocytol., I0 (1981) .~7-79.
RICHARD E, ~ E S H A L L
227
and ~ N T H I A S, YOUNGBLOOD
Marine Biomedical Institute and Departments of Anatomy and of Physiology and Biophysics. University of Texas Medical Branch, Galveston, TX 77J30.2772 (U.S.A,) (Rect~ved April 22nd, 1983; Accepted April 27th, 1983)
Key words: horseradish peroxidase - spinal transection - spinal regeneration - goldfish
In fish axons rqx~rtedly cross a spinal transection, form synapses, and then cease growing or grow in abnormal directions. This obviously would not restore pgevious synaptic connections, and since fish recover function, it is often stated that recovery of function in fish seems to be independent of rcztoration of normal synaptic architecture. By contrast, the prc~.nt study mmphasizes that many axons in igoldfish continue srowinlg after they pass a spinal transection. Honmndish ix'roxidase was it~jected into caudal spinal cord in normal or spinal cord-transected fish. Neurons were laheled in the same places in operated and normal fish. Since the site of injection was 4 cm (18 sqpnents) caudal to the transection, it seems clear that the axons of cells labeled in transacted fish ~tm~d~l past the cut for almou the lenjth of the spinal cord. Thus Igrowth did not cea~ for thmc axons. Therefore, even thoullh s y n a ~ undoubtedly form in the nenropil distal to the cut, many axons do not stop srowinlg and restoration of ~ i o u s synaptic connections may well be a requir~qngnt for return of funclion.
Axons grow across a spinal transection in fish but not in mammals ll-5. 8=-11]. it is reasonable to conclude that motor recovery, which also occurs in fish but not in mammals, is the result of this regrowth. An unresolved question, however, i~ whether function returns when the axons return to their previous synap/ic sire, thus partially or completely restoring the original synapfic architecture, or whether runetion returns without the original architecture being restored. Several important studies favor the latter idea for they provide evidence to indicate that regrowing axons form synapses soon after they cross the transection and then cease growing or grow in abnormal directions [2, 3, 8-11], situations t ~ t would not restore pretransection synaptic patterns. By contrast, the present study provides evidence to indicate that a large number of regrowing axons extend the length of the spinal cord and thus do not cease growing after they pass a spinal transection. Goldfish (Carassius auralus), approximately 10 cm from snout to base of tail fin and weighing approxi~,nately 50 g, were used. After anesthesia with Finquel (tricaine methanesulfonate), spinal cords were exposed by laminectomy and t r a ~ e d between segments 6 and S. This is between the caudal end of the operculum and the 0JO4-3940/SJ/S 03.00 © 198J Elsevi,:r--Sck'ntifi¢ Publishers Ireland Ltd.
2211 dmsal fin and is approximately 1.5 cm from the medulla oblongata. It was difficult to transect more craniah'y without damage m the gill cavity. After transection, the caudal pan of the animal was paralyzed. After approximately 1.5 months,
the operated fish were difficult to distinguish from normal. The f'mh were allowed to smvive for 138--160 days (Table D and then reanesthetized. Another lami_~nectomy was done between the anal fin and the base o f the tail and 1 ~ of a ~)'e solution o f horseradish peroxidas¢ OIRP) was injected approximately 4.0 a n caudal to the transection into segments 24-26. Caudal to the injection site the spinal cord became extremely thin. After 7 days the animals were rcanesthetized, sacrificed, and perfused with saline followed by a mixture o f 1.25% glutaraldehyde and 0.75~'e paraformaldehyd¢ in 0.1 M phosphate buffer. The brain and spinal cord were placed in 30% sucrose and horizontal frozen sections of the brain were cut and rcacted with tctramcthylbenzidine. As a control, an acute transection of the spinal cord was done between sqgments 6--8 just before the injection of HRP into sqgments 24-26 in 4 fish. in addition, 4 unoperated fish were injected with HRP into sqgments 24-26 and treated as above. in control animals (animals with an acute transection at sqpnents just prior to
TABLE I N U M B E R S OF I , A B E L E I ) NL:I~'RONS A comparL~n o f n u m t ~ s o f labeled neurons in the reticular formation, v~tibular nuclei, and nuclei o f ~be medial Ionttiludinal fa~'iculu~ follo~'intt HRP injecti.~m inlo the ~inal cord o f Igoldfish ~ i t h a spinal
Iran~,-tion (Ol~'al~J fi.~h) of imo unolx'raled [mldfish (unoperaled fishL
Fish ¢
Days p.~tt ranse~.lion
Reticular formation
V~tibular nuclei
Nuclei o f the medial Ionlgitudinal fasciculu$
+ +
512 447 580 521
99 42 96 93
71 24 79 65
1311 14~ 161) I~ 146
366 341 24J+ 394 172
60 38 61 108 4"1
51 59 33 58 41
161
31
.'St
5a
22 44
Unoperatcdfish 2. ~, 4++
A~eragc
5. 6. 7. 9i !0. ....
Average
143
229
6 - 8 to the injection of HRP), no cells were labeled in the brain, indicating that the HRP was not falsely labeling by being t r a n s p o ~ in cerebrospinal fluid or blood. Following HRP injection in transected and non-transected animals, labeled neurons were seen in 3 locations: (1) the reticular formation; (2) the vestibular
neurons following spinal injection of HRP in zebrafish [6]. The numbers of labeled cells in operated and unoperated fish are given in Table 1. Note that in unoperated fish there are an average of 521 labeled cells in the reticular formation, almost 100 in the vestibular nuclei and 63 cells in the nuclei of the medial longitudinal fasiculus, In operated fish, there are an average of 281 labeled cells in the reticular formation, 58 in the vestibular nuclei and 44 in the nuclei of the medial longitudinal fasiculus. The percentages of labeled cells in transected as compared to normal fish are 54~e for the reticular formation, 62070 for the vestibular nuclei, and 68e/e for the nuclei of the medial longitudinal fasiculus. The overall percentage of labeled cells in transected as compared to normal fish is 55%. The possibility that function may be restored without restoration of 'normal' synaptic connections has been discussed for many years. For regeneration of fish spinal cord following transection, a possible example of such a restoration, a key issue is whether regenerating axons stop growing after they pass the transection and form synapses. The first important papers on this point indicated that many regenerating axons in fish did seem to form synapses at the first available sites caudal to a transection and then largely ceased growing [2, 3]. Furthermore when a teflon sheet was interposed between the two ends of the transection, the axons that gathered next to the teflon formed synapses with one another and growth did not resume when the teflon was removed 131. The inhibitory effect of the teflon could be negated if the spinal cord was retransected, however, the retransection presumably removing those parts of the axons in synaptic contact with one another [31. The phrase 'contact inhibition' was used to describe these phenomena and it was hypothesized that this renders the neuron incapable of resuming axonal growth [2,
31. More recent work on lamprey was confirmatory. These animals have large, individually identifiable spinal axons, and these axons regenerated past a transection and then seemed to end suddenly or turned and went for a short distance in an abnormal direction 18-11]. Thus the general belief that fish behavioral recovery is associated with axonal regrowth, but the axonal regrowth does not seem to result in the restoration ~)f a pretransection synaptic architecture. By contrast, however, there are indications that axonal growth following synapse formation does not cease. For example, in studies on goldfish [4] and tadpoles 151, it was shown that many cells in the brain were labeled by the retrograde transport of HRP injected several segments caudal to a spinal transection. The conclusion in both these studies was that the cells thaf projected to particular areas of spinal cord
in the normal sent their axem back to them areas after mmegtion. It would seem, therefore, that axons continued growing after Imssing the ttamection, in another study it was shown that function retmtmd when tranmcted Mauthner cell axons in ~ grew ~ to t l ~ - ~ ~ ~ m. ~ mautlmer axon growth did not seem to be inhibited by ~ fomation near tbe tmmetaion. The present mm'y extends the above work by ddiberatdy separating the site of injec;ion of HIgP from the transection by as great a distance as conveniently possible. When this is dome, more than half of the number of brain neurons that are labeled following HlgP injection into the normal caudal spinal cord are labeled with the same type of H R P injection following recovery from transection of the cranial spinal cord. Furthermore, the labeled neurons are in the same location following transection as they are in the normal. These results are essentially the same as in our previous experiments where the transection and injection sites were much closer together. The interpretation of the data from the normal animals is that there are 3 populations of cells in the brainstem (the reticular formation, the vestibular nuclei. and the nuclei of the medial longitudinal fasiculus) whose axons project the length of the spinal cord. The interpretation of the data m,. transected fish is that a majority of cells from the areas that project to caudal spinal cord in the normal project there after transection. Thus it would seem safe to assume that a significant number of neurons send axons across the transection back to their original locations and probably to their original synaptic sites. Since the original locations are at least 4 cm from the transection, this would strongly favor the idea that axonal growht does not cease n e a r the tran~gtion. An understanding of sncce~sful regeneration in frsh and lower vertebJates should be of great value in suggesting ways to deal with unsuccessful regeneration in higher organisms. It seems clear from earlier work that axons grow across a spinal transection in lower vertebrates and then form synapses when they enter the denervated neuropi[ on the other side of the transection [2, 3, I0, I 1]. Furthermore, some of tl~e axons probably cease growth at this point because quamitative studies indicate that fe~'er axons cross Ill and fewer cells label [4] (also this study) in transected than in normal animals. This study emphasizes that not all axons cease to grow, however. Sim:e a significant number of axons cross the transection and grow for a considerable distance, it is a reasonable hypothesis that function returns when the original synapti¢ connections are restored, as suggested by the physiological data on Mauthner axon regrowth [7]. If this is the case, then the synapses that are formed early, near the transection site, might deter normal function because they could be a source of synaptic noise. Another possibility is that after the 'right' synaptic connections are made, the 'wrong' synaptic connections are eliminated. Further experiments designed to test these hypotheses are feasible. This ~ r c h is ~ ~ l y supported by Grants NS 10161, 17039, 11255 and 07377 from the United States Public Health Service, and a support grant from the Moody Foundation.
231 I Bernstein, J.J., Relatimt of spinal cord regeneration to age in adult goldfish, Exp. Neurol., 9 (1964) 161-174. 2 Bernstein, J.J. and ~ , M.D., Effect of fglial-epend3mtal scar and teflon arrest on the regataafive calmcity of goldfish spinal cord, Exp. Neurol., 19 (198'7) 25-32. 3 Bernstein, J.J. and Bernstein, M.E., Uitmstrucmre of normal regeucration and loss of regenerative
Neurosci. Lett., 32(1982) 259-7,64. $ Forehand, C.J. and Farel, P.B., Anatomical and bebavioral recovery from the effects of spinal cord transection: depglldettg~ on inetamo~hosis in Anuran larvae, J. Neurosci., 2 (1982) 654-662. 6 Kimntel, C.B., Powell, $.L. and Metculfe, W.K., Brain neurons which project to the spinal cord in youn$ larvae in zebrafish, J. comp. Neurol., 205 (1982) 112-137. 7 Lee, M.T., Rejeneration and functional reconnection of an ickntified vertebrate central neuron, J. Neurmci., 2 (1982) 1793-1811. 8 Rovainin, C.M., ~ a t i o n of Muller and Mauthner axons after spinal transection in law-al lainpreys, J. comp. Neurol., 168 (1976) 545-554. 9 Seizer, M.E., Mechanism of functiO,~d+ recovery and rclgeneration after spinal cord tran~ction in larvae sea lamprey, J. Physiol. (Lond.~, 227 (1978) 395-408. 10 Wood, M.R. and Coheu, M.J., Synaptic refgeneration in identified neurons in the lamprey spinal cord, Scieuc¢, 206 (1979) 344-346. I I Wood, M.R. and Cohen, M.J., Synaptic relgCnerationand fglial reactions in the transects! spinal cord of the lamprey, J. Neurocytol., I0 (1981) .~7-79.