THE JOURNAL OF COMPARATIVE NEUROLOGY 306:409-416 (1991)
Axonal Regeneration in the Adult Lamprey Spinal Cord DIANA I. LURIE
AND
MICHAEL E. SELZER
Department of Neurology (M.E.S.)and the David Mahoney Institute of Neurological Sciences (D.I.L., M.E.S.), University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283
ABSTRACT Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Miiller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodesand injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%)neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92%were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration. Key words: Miiller cells, Mauthner cells, directional specificity,transection, behavioral recovery, axon guidance
The larval sea lamprey (Petromyzonmarinus) is capable MATERIALS AND METHODS of functional recovery following spinal cord transection Staging of animals (Rovainen, 1976; Selzer, 1978; Cohen et al., 1986). Spinal Animals were considered fully transformed adults if they axons regenerate across the transection zone (Hibbard, 1963; Rovainen, 1976; Selzer, 1978; Wood and Cohen, met the following criteria (Hardisty and Potter, 1971). The 1979; 1981; Borgens et al., 1981) and this regeneration is characteristic brown skin color of the larva had changed to specific in that axons grow selectively in their normal a dark gray on the dorsal surface and silver on the ventral surface. The eye had fully differentiated with a dark pupil directions (Yin and Selzer, 1983;Yin et al., 1984;Mackler et and silver iris. The oral disk had enlarged and developed al., 1986)and form appropriate synaptic connections (Mack- tooth-like structures. The fimbria had become enlarged and ler and Selzer, 1987). In other vertebrates, the capacity for fully differentiated. The fins had enlarged. The animal no axonal outgrowth diminishes greatly with age (Piatt and longer burrowed in the sand at the bottom of the tank but Piatt, 1958; Bernstein, 1964; Kiernan, 1979; Pestronk et attached itself to the side of the tank with its sucker mouth. al., 1980; Fagg et al., 1981). However, recent studies Surgery indicate that adult sea lampreys can also recover behaviorally from spinal transection (Ayers et al., 1982; Margolin Fourteen adult lampreys received complete spinal transecand Ayers, 1987; Cohen and Baker, 1988). Preliminary tions at the level of the 5th gill. Briefly, animals were findings based on retrograde transport of HRP suggest that anesthetized in tricaine methansulfonate (MS 222, Finguel, reticulospinal neurites cross the lesion site (Lurie and Ayerst) 1:4,000and pinned to a Sylgard plate (184 silicone Selzer, 1988; Ayers, 1989). elastomer, Dow Corning). The skin, muscle, and fibrous In the present study, giant reticulospinal axons (GRAs) canal were cut by a dorsal incision with a fine scalpel in previously transected adult lampreys were injected with exposing the spinal cord. The exposed spinal cords were horseradish peroxidase (HRP) in order to visualize their transected under direct stereomicroscopicvision with iridecregenerated neurites, measure their distances of regeneration, and determine whether they were oriented in their Accepted January 10,1991 correct directions.
o 1991 WILEY-LISS, TNC.
Figure 1
AXONAL REGENERATION IN ADULT LAMPREYS tomy scissors and the cut ends allowed to re-appose. The completeness of the transections were verified by direct observation as well as by the fact that post-operative animals were paralyzed below the transection. The wounds were healed by air drying with the animals resting on ice for 1-2 hours post-operatively. Animals were then returned to fresh water aerated aquaria and allowed to recover for 10 weeks at 16°C.
Retrograde tracer studies After 10 weeks of recovery, two animals were retransected 5 mm caudal to the original transection and a gelfoam pleget soaked in 50%HRP was placed in the second transection. The animals were allowed to recover for an additional 2 weeks, after which the brain and spinal cord were removed and processed for HRP histochemistry (see below).
Intracellular HRP injection In the 12 remaining recovered animals, the rostral brain and 5 cm of cord was removed together with the underlying notocord. This preparation included the lesion site. The tissue was then placed in 0.01% collagenase (Sigma Chemical Go., St. Louis, MO) for 10 minutes, rinsed three times in cold lamprey Ringer (Selzer, 19781, and pinned dorsal side up to the bottom of a Sylgard-lined experimental chamber. The tissue was transilluminated from below (Selzer, 1978) and the meninx primitiva was stripped away with jewelers forceps. Microelectrodes were backfilled with 6% HRP in 0.2 M potassium acetate and 0.1 M tris buffer, pH 7.4. Those with resistances of at least 40 Mfl were selected. Muller and Mauthner cells in the brain and their giant axons in the cord were impaled under stereomicroscopic vision. No attempt was made to limit the study to one cell type and the data presented here are pooled from all cells. HRP was iontophoretically injected with 15 nA pulses of 200 msec duration at 3.3 Hz for 20 minutes. At the end of the experiment, the spinal cord was placed in fresh Ringer and kept overnight at 4°C.
Histological methods
411
Behavioral observations Each transected animal was examined 24 hours after surgery to ascertain that there was no movement caudal to the lesion site. A transection was tentatively considered complete if the animal could move only its head and body rostral to the lesion. Each week thereafter, animals were observed and classified into stages of recovery as described below.
RESULTS Behavioral recovery After 1 week, paralysis caudal to the lesion continued. However, the animals tended to undulate slowly when attached to the bottom of the tank by their oral sucker. This movement has been analyzed by Ayers and determined to originate caudal to the lesion (Ayers, 1989). If the animals were forcibly detached, they were completely immobile. At 2 weeks post-transection, violent head oscillations propelled the animals forward, although they were unable to move vertically and exhibited no righting reflex. Body segments caudal to the lesion moved only passively (Selzer, 1978; Ayers, 1989).At weeks 3 and 4, the animals displayed weak, poorly coordinated swimming movements, which could not be sustained for more than 5 seconds. By week 6, two thirds of all the animals showed coordinated swimming in all planes and by week 7, all animals could swim well.
Probability of regeneration of GRAs GRAs are normally unbranched and extend almost the entire length of the lamprey spinal cord. Therefore, high spinal transection axotomizes all GRAs. Thirty Muller and Mauthner axons were labeled with HRP in 12 transected adults. These axons gave rise to 121 neurites of which 61 (50%) grew beyond the level of the scar (Fig. 1, Table 1). Fifteen of the thirty s o n s sent at least one branch across the scar and it was common for an axon to give rise to some neurites that regenerated and others that did not. These results are similar to those reported previously for larval lampreys (Yin and Selzer, 1983).
Distance of regeneration of GRAs
The tissue was processed for HRP histochemistry with a In larval sea lampreys, cut GRAs die back an average of modified Hanker-Yates mixture (Selzer, 1978) for 20 minutes at room temperature and rinsed in lamprey Ringer. 1-2 mm before regenerating (Roederer et al., 1983). HowThey were dehydrated for 5 minutes in 35% ethanol, fixed ever, because of the large variability in the distance of in 70% ethanol at 4°C for 1 hour, and the notocord dieback (as much as 2 cm in our laboratory) and because the separated from the spinal cord and discarded. The spinal extent of dieback has not been determined in adults, cords were dehydrated in serial ethanols, cleared in cedar- distance of regeneration was measured relative to the wood oil, and mounted on slides with a Permount (Fischer)/ center of the lesion. This had the added advantage that it allowed comparison with previously reported data from this xylene mixture. laboratory on larval lampreys (Yin and Selzer, 1983). The Light microscopic analysis neurites of GRAs in adult lampreys regenerated to a mean The regenerating HRP-injected branches of GRAs were examined for the following: 1)the distance of the neurite tip TABLE 1. Neurites of GRAs in Transected Cords proximal or distal to the center of the lesion and 2 ) the Neurite paths orientation of the neurite tip (ipsilateral vs. contralateral to the parent axon; rostral vs. caudal). Neurite
Fig. 1. Wholemounts of adult transected spinal cords. A, B, C: Most labeled neurites regenerate through the scar (arrows) although some neurites loop and grow rostralward (arrowhead in B). Left, rostral. cc, central canal.
terminations
Looped
Proximal to the scar Within t h e scar Distal to the scar Total
0 (0%) 9 (30%) 4 (6%) 13 (11%)
Decussating
Ipsilateral
Total
0 (0%) 4 114%) 1(2%) 5 (44%)
30 (100%) 17 (56%) 56 (92%) 103 (85%)
30 30 61 121
Summary of neurite projection paths in 10 week post-transection spinal cords. Note that 5 0 4 of all labeled neurites regenerated beyond t h e level of t h e scar and, of these, 92% remained on the ipsilaterai side of t h e spinal cord.
D.I. LURIE AND M.E. SELZER
412 distance of +0.15 mm beyond the center of the transection scar. The range was between -5.46 mm (i.e., 5.46 mm proximal to the scar) and +3.20 mm (i,e., 3.20 mm distal to the scar). This is comparable to the distances reported for larval GRAs (Rovainen, 1976; Selzer, 1978; Yin and Selzer, 1983; Wood and Cohen, 1979).
TABLE 2. Effect of Neurite Orientation on Distance of Regeneration Straight (ipsilateral) Distance (mmY
1.05 t 0.08 n = 56
Corrected distance (mmIb
1.03 ? 0.08 n = 56
Directional specificity of GRA regeneration Of those neurites which terminated distal to the lesion, 92% grew in their appropriate direction, ipsilateral to their parent axon and pointing caudalward (Fig. 1, Table 1).Of those neurites which failed to regenerate beyond the level of the scar, 77% remained correctly oriented (Table 1).These results are similar to those reported previously in larval animals transected at the level of the 7th gill or at the level of the third gill (Yin and Selzer, 1983; Yin et al., 1984; Mackler et al., 1986).
Looped
Decussating
0.53 ? 0.14 n = 16 P < 0.01 0.52 t 0.14
0.25
?
0.14
n=5 P < 0.0001
n = 16 P < 0.01
eDistance (mm) f SEM of terminations of straight and decussating GRA neurites relative to the center of the scar. Negative values were assigned to terminations rostral to the scar. Distance of loowed neurites was measured from the point of looping . -to the end of the looped neurite. 'Corrected distance (mm). GRAs die back after transection and the point at which they start their forward mowth is unknown. In order to compare the growth of straight neurites with those chat looped, the average location at which looping neurites turned rostralward (0.026 mm distal to t h e scar) was defined as zero for those axons which remained straight and those that looped. Pvalues are for comparison with control.
L1 Non-looping neurites
Directional specificity and distance of regeneration
Looping neurites c
Although most regenerating giant reticulospinal neurites which terminated distal to the lesion grew on the correct side of the cord and in the correct rostrocaudal direction, several neurites crossed the midline in the region of the scar, while others looped rostralward towards the brain (Fig. lB, Table 1). If directional specificity of neurite regeneration resulted merely from the tendency of axons to grow in the direction they are already facing, rather than from specific guidance mechanisms, then neurites that had become misoriented should continue to grow as readily as those which remain oriented caudalward. However, as in larval lampreys (Mackler et al., 19861,this was not the case. GRAs which regenerated in their correct direction grew longer than those which had either decussated or looped backwards. In order to make this comparison, the mean location at which looping s o n s turned rostralward was determined and defined as zero both for those axons which remained straight and those that looped. This location was +.026 mm (i.e., 26 p,m caudal to the center of the scar); range -2.72 to + 1.02 mm. The average distance of regeneration for neurites which grew beyond this point and remained on the correct side of the cord was + 1.03 mm ? 0.08 SEM (n = 56). Neurites which looped rostralward (incorrectly) grew approximately half as far (0.53 mm ? 0.14 SEM; n = 16) and this difference was significant (Student's t test P < 0.05; Table 2). Four neurites decussated and terminated almost immediately within the scar. Only one neurite crossed the midline and extended in the distal stump for 0.86 mm. These results are similar to those obtained for GRAs and other spinal neurites in larval animals (Mackler et al., 1986) Approximately half of the labeled neurites failed to grow into the distal stump. An examination of their tip locations suggested that they were concentrated in the region of the scar (Fig. 2A). A more detailed examination of the tip locations revealed that they were distributed evenly within the scar and in the 0.5 mm of spinal cord proximal and distal to it and were not backed up just proximal to the scar (Fig. 2B,C,D). Thus it is unlikely that the scar represents a hostile environment for axon outgrowth.
Regeneration of other axons Although their large size make GRAs convenient to study, their relationship to locomotor behavior is not clear (Rovainen, 1967; 1982; Buchanan and Cohen, 1982) and
5 10 5 =
$
5
z -6
-5
-4
-3 -2 -1
,
0,
1
Z
3 4
Distance from scar (mm)
a c ._
Looping
z
I)
E
3
z
10 D
-1
Non-Looping
-5 0
.5
1
i scar i
II(mm)1I Fig. 2. Distribution of neurite tips in transected spinal cords of adult lampreys. A: Neurite terminations tended to be concentrated in the region of the scar rather than being backed up just proximal to it. For looping neurites (dark bars) the distance plotted represents the location of the termination of the looped neurite. B: Expanded scale to highlight the distribution of neurite tips in the region of the scar. Neurites were distributed fairly uniformly within the scar. C: Distribution of the terminations of looping neurites. Most terminations were located just rostral to the scar in the proximal stump. D: Distribution of nonlooping neurite tips. The tendency for neurites to terminate fairly evenly throughout the vicinity of the scar is not altered by excluding the looping neurites.
other neurons are probably more important to recovery of locomotion following spinal transection (McClellan, 1988; Ayers, 1989). Therefore, it is important to demonstrate whether regeneration characterizes other brainstem neurons with spinal projections. Two adults were retransected
AXONAL REGENERATION IN ADULT LAMPREYS approximately 5 mm distal to the original transection and HRP injected into the second lesion. Work in our laboratory has demonstrated that HRP can diffuse extracellularly and label neurons whose axons terminate within 2.5 mm of the injection site but not beyond (Snedeker and Selzer, 1986). Thus, any retrogradely labeled brainstem neurons had axons that regenerated at least 2.5 mm beyond the center of the scar (Snedeker and Selzer, 1986; Croop et al., 1988). Figure 3 shows a wholemounted adult brain from such a preparation. Many neurons of all sizes were labeled with HRP, indicating that the axons of these neurons had regenerated across the first transection. Retransection eliminated all recovered swimming in these animals, confirming that recovery of function was dependent at least in part upon spinal regeneration.
DISCUSSION Behavioral recovery The present study demonstrates that spinal axons in adult sea lampreys regenerate almost as readily as those of the larval form (Yin and Selzer, 1983; Lurie and Selzer, 1988).In addition, the animals recovered normal-appearing swimming and this was reversed upon retransection of the cord. These observations are consistent with the more detailed behavioral measurements of Margolin and Ayers (1987, Ayers, 1989) and with the observations on fictive swimming by Cohen and Baker (1988). The latter study suggested that recoupling of central pattern generators in the adult was less secure than in the larva (Cohen et al., 1986) because synchronization of ventral root discharges across a healed transection could be demonstrated only after overlappingstaged partial lesions. It should be pointed out that the present experiments do not identify the axons whose regeneration mediates the recovered behavior. Muller and Mauthner cells do not appear to function as locomotor command neurons (Rovainen, 1967; 1982; Buchanan and Cohen, 1982), although the Mauthner cells may be involved in triggering startle responses (Currie and Carlsen, 1987). Microstimulation and lesion experiments suggest the existance of clusters of neurons that constitute brainstem locomotor command centers (McClellanand Grillner, 1984; Ayers, 1989)and neurons of adult lampreys that are located in the region of lowest threshold for triggering locomotor responses were labeled retrogradely in these experiments (top arrows, Fig. 3). Swain (1989) has also demonstrated retrograde labeling of these neurons across a transection scar in both recovered larvae and adult lampreys. In larval lampreys, McClellan (1988) has been able to activate the central pattern generators caudal t o a healed transection by microstimulation of brainstem locomotor command centers, even when the spinal cord rostral to and including the scar was bathed in low calcium to block synaptic transmission. He concluded that the locomotor activity must have been triggered by regenerated axons of locomotor command neurons. However, the moment by moment coordination of central pattern generators at different levels of the cord involves local coordinating axons and it is entirely possible that the behavioral recovery seen in the present study and most studies in larvae (e.g., Currie and Ayers, 1983; Cohen et al., 1986) is due mainly to regeneration of axons of intersegmental neurons that coordinate the central pattern generators at different levels (Cohen et al., 1988). In the larva, behavioral recovery is accompanied by regeneration of synaptic connections selectively between
413 appropriate types of neurons on opposite sides of the transection scar (Mackler and Selzer, 1985; 1987). Here again it is not clear that the synapses studied were involved in the behavioral recovery. Whether regenerated neurites of adult spinal axons also form appropriate synaptic contacts remains to be determined. If they do, they clearly will not reproduce the original pattern of innervation, since as in the larval studies, many of the original synaptic targets are located beyond the range of regeneration of these neurites.
Probability of GRA regeneration Approximately 50% of HRP-injected GRAs in adult lampreys regenerated beyond the center of the transection scar. In a previous study on larvae, the probability of regeneration was only slightly greater (57%;Yin and Selzer, 1983). More recent work from this laboratory on regeneration of GRAs in both hemisected and transected larval spinal cords suggests a similar value (59%;Lurie and Selzer, submitted manuscript). There are many possible reasons why not every axon regenerated beyond the scar. These may relate to limitations in the intrinsic regenerative capacities of neurons, to inhibitory influences of the spinal cord extracellular environment, or to a barrier effect on the part of the scar toward regeneration. If the scar were acting as a barrier, one would expect a disproportionate number of the tips of regenerating neurites to be backed up just proximal to the scar. However, within the general region of the lesion, neurite tips were uniformly distributed proximal to, within, and distal to the scar, suggesting that the scar did not act as a barrier. On the other hand, the few neurites which looped or crossed the midline tended to do so within the scar, indicating that the cues which guide directional specificity may be less accessible to growing axons within the scar.
Distance of GRA regeneration In the larval sea lamprey, cut GRAs die back during the first 2 postoperative weeks. The distance of dieback has been estimated to average 1-2 mm (Roederer et al., 1983) but these distances are variable and evidence for dieback as much as .02 m has been observed occasionally in our laboratory. Thus, the process of regeneration begins before axons have crossed the lesion site. Because of the variability of dieback and because much of the current interest in the lamprey derives from the ability of its axons to grow through the lesion, distances of regeneration have always been measured relative to the center of the scar. Thus they underestimate the actual distance of neuritic extension. This convention was followed in the present study as well, especially because dieback in the adult has not been quantified. The average distance of regeneration for neurites of adult GRAs was to a point 0.15 mm distal to the center of the scar within a range -5.4 to +3.2 mm relative to the center of the scar. Similar maximal distances of regeneration were reported for larval GRAs by Rovainen (1976; more than 1 mm but less than 9 mm), Selzer (1978; 3.6 mm), Yin and Selzer (1983; 5.3 mm), and Wood and Cohen (1979; 3.1 mm). Even these maxima are likely to be underestimates because they were based either on reconstructions of serial light microscopic sections or on observations of intracellularly injected axons in spinal cord wholemounts. A s neurites become narrower and longer, they become increasingly difficult to follow with these techniques. In this laboratory, distances of regeneration of up to 21 mm have been observed by antidromic activation of
Figure 3
AXONAL REGENERATION IN ADULT LAMPREYS
GRAs (unpublished)and 30 mm by retrograde transport of HRP (Croop et al., 1988). Nevertheless, it is clear that GRAs do not regrow to their original length since in untransected animals these large unbranched axons extend almost the entire length of the cord, about 150 mm in large larvae and recently transformed adults.
Directional specificity of regeneration The axonal regeneration observed in the present study was directionally specific. This result was similar to that obtained in larval animals where the majority of regenerating spinal axons grew through the transection site and remained correctly oriented (Yin and Selzer, 1983; Yin et al., 1984; Mackler et al., 1986; Lurie and Selzer, 1987). In tissue culture, axons tend to grow forward rather than meander randomly (Katz, 1985). Such a tendency may have contributed to the preferential growth of reticulospinal axons caudalward and ipsilateral to their parent axons. However, the long distances that regenerating axons must travel relative to the width of the ipsilateral hemicord suggests that other guidance mechanisms must be involved. This is supported by the fact that neurites which looped rostralward grew approximately half as far as those that did not and that only one neurite crossed the midline distal to the scar. Previous reports on giant reticulospinal neurons and other cell types in the larval CNS also concluded that the tendency toward forward motion cannot fully explain the specificity of regeneration of lamprey spinal axons Win et al., 1984; Mackler et al., 1986).
Regeneration vs. development The growth of spinal axons across the transection in the adult lamprey is not likely to be due to late developing axons or new neurons. Although the diameters of GRAs and of their perykaria can be more than twice as large in the adult as in the larva, larval GRAs already extend almost the full length of the spinal cord and few, if any neurons are added to this population of cells during transformation (Rovainen, 1982). In addition, retrograde labeling of all types of reticulospinal neurons with HRP yielded similar total counts in the adult and in the larva (Ayers, 1989).
Comparison with other vertebrates It is generally accepted that regenerative capacity decreases progressively with maturation (Windle, 1955). For example, spinal transection in tadpoles is followed by axonal regeneration but in postmetamorphic frogs, it is not. Even in teleost fish, where regeneration of spinal axons occurs in the adult, an age-dependent reduction in the robustness of regeneration has been described (Bernstein, 1964). The very slight degree of reduction in axonal regeneration that accompanies transformation from larval lamprey to the adult form was therefore somewhat surprising. It may be that further reductions in regenerative capacity occur with aging in adult lampreys but we were not in a position to assess this because of the difficulty in obtaining and maintaining older adults.
Fig. 3. Wholemounted hindbrain of a n adult 10 weeks posttransection. Many reticulospinal neurons have been back-filled with HRP. The arrows point to groups of labeled neurons demonstrating t h a t t h e axons of these neurons must have regenerated across t h e transection (see text).
415 In both neonatal and adult mammals, cut axons are not able to regenerate across the injury scar (Gearhart et al., 1979; Kalil and Reh, 1979; 1982; Guth et al., 1981; Reh and Kalil, 1982; Bregman and Goldberger, 1982; 1983; Bernstein and Stelzner, 1983; Reier and Houle, 1988; Goldberg and Frank, 1980). The reason for this is not known, but properties of reactive glia are usually invoked. However, several features of the adult CNS in addition to the scar are thought to limit regeneration. For example, axons can grow along a scaffold formed by implants of embryonic glial cells but not adult glia (Silver and Ogawa, 1983). The work of Schwab and colleagues also suggests that molecules on the surface of mature CNS myelin block axon outgrowth and that this can be reversed by application of antibodies to those molecules (Schwab and Caroni, 1988). The fact that the lamprey CNS contains no myelin or oligodendrocytes may help to explain the relatively robust axonal regeneration. However, the scar itself clearly exerts a major inhibitory influence on regeneration of mammalian CNS axons. While damage to adult CNS often results in collateral sprouting (Gall and Lynch, 1978, 1981; Crutcher and Marfurt, 1988; Pestronk and Drachman, 1988), growing neurites are unable to cross a transection scar. Even in early postnatal mammals, late developing axons of the corticospinal and rubrospinal tracts cannot cross a scar produced by spinal hemisection but can grow around it (Bernstein and Stelzner, 1983; Bregman and Goldberger, 1983; Kalil and Reh, 1979, 1982; Reh and Kalil, 1982). Similarly, when the inhibitory effect of myelin was reduced by application of blocking antibodies, corticospinal axons grew around a hemisection scar rather than through it (Schnell and Schwab, 1990). In contrast, regenerating neurites in the adult lamprey grew through the scar and maintained directional specificity. In experiments on lamprey larvae, cut axons grew around a spinal hemisection rather than around it (Lurie and Selzer, submitted). Whether GRAs of adult lampreys would behave similarly is not known but the present results demonstrate that the ability of lamprey neurites to cross a spinal lesion is not dependent upon the developmental stage of the animal. Nor are the cues which guide directional specificity lost or significantly diminished following transformation.
ACKNOWLEDGMENTS We thank Joseph Snedeker for his expert technical assistance. This work was supported by NIH grants R 0 1 NS14837 and R 0 1 NS25581.
LITERATURE CITED Ayers, J. (1989) Recovery of oscillator function following spinal regeneration in the sea lamprey. In J.W. Jacklet (ed): Cellular and Neurond Oscillators. New York: Marcel Decker, pp. 371405. Ayers, J., S. Currie, and J. Kinch (1982) Adult lamprey can recover from complete spinal cord transection. Soc. Neurosci. Abstr. 8:868. Bernstein, J.J. (1964) Relation of spinal cord regeneration to age in adult goldfish. Exp. Neurol. 9:161-174. Bernstein, D.R., and D.J. Stelzner (1983) Plasticity of the corticospinal tract following midthoracic spinal injury in the postnatal rat. J. Comp. Neurol. 221:382400. Borgens, R.B., E. Roederer, and M.J. Cohen (1981) Enhanced spinal cord regeneration in lamprey by applied electrical fields. Science 213:611617. Bregman, B.S., and M.E. Goldberger (1982) Anatomical plasticity and sparing of function after spinal cord damage in neonatal cats. Science 217:553-555.
416 Bregman, B.S., and M.E. Goldberger (1983) Infant lesion effect: 111. anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats. Dev. Brain Res. 9rl37-154. Buchanan, J.T., and A.H. Cohen (1982) Activities of identified interneurons, motoneurons and muscle fibers during fictive swimming in the lamprey and effects of reticulospinal and dorsal cell stimulation. J. Neurophysiol. 47~948-960. Cohen, A.H., and M.T. Baker (1988) Functional and non-functional regeneration in the spinal cord of adult lampreys. In P.J. Reier, R.P. Bunge, and F.J. Seil (eds): Current Issues in Neural Regeneration Research. New York: Alan R. Liss, pp. 387-396. Cohen, A.H., S.A. Mackler, and M.E. Selzer (1986) Functional regeneration following spinal transection demonstrated in the isolated spinal cord of the larval sea lamprey. Proc. Natl. Acad. Sci. 83:2763-2766. Croop, R.S., J.A. Snedeker, and M.E. Selzer (1988) Unequal probabilities of regeneration among lamprey reticulospinal neurons. Soc. Neurosci. Abs. 14.656. Crutcher, K.A., and C.F. Marfurt (1988) Nonregenerative axonal growth within the mature mammalian brain: Ultrastructural identification of sympathohippocampal sprouts. J. Neurosci. 8:2289-2302. Currie, S.N., and J. Ayers (1983) Regeneration of locomotor command systems in the sea lamprey. Brain Res. 279t238-240. Currie, S.N., and R.C. Carlsen (1987) Functional significance and neural basis of larval lamprey startle behavior. J. Exp. Biol. 133~121-135. Fagg, G.E., S.W. Scheff, and C.W. Cotman (1981) Axonal sprouting a t the neuromuscular junction of adult and aged rats. Exp. Neurol. 74:847854. Gall, C., and G. Lynch (1978) Rapid axon sprouting in the neonatal rat hippocampus. Brain Res. 153:357-362. Gall, C., and G. Lynch (1981) Fiber architecture of the dentate gyrus following ablation of the entorhinal cortex in rats of different ages: Evidence for two forms of axon sprouting in immature brain. Neurosci. 6~903-910. Gearhart, J., M.L. Oster-Granite, and L. Guth (1979) Histological changes after transection of the spinal cord of fetal and neonatal mice. Exp. Neurol. 66:l-15. Goldberg, S., and B. Frank (1980) Will central nervous system in the adult mammal regenerate after bypassing a lesion? A study in the mouse and chick visual systems. Exp. Neurol. 70:675-689. Guth, L., C. Barret, E.J. Donati, S.S. Deshpande, and E.X. Albuquerque (1981) Histopathological reactions and axonal regeneration in the transected spinal cord of hibernating ground squirrels. J. Comp. Neurol. 203:297-308. Hardisty, M.W., and I.C. Potter (1971) The general biology of adult lampreys. In M.W. Hardisty and I.C. Potter (eds): The Biology of Lampreys, Val. 1. New York: Academic Press, pp. 127-206. Hibbard, E. (1963) Regeneration of the severed spinal cord of chordate larvae ofPetromyzorz marinus. Exp. Neurol. 7t175-185. Kalil, K., and T. Reh (1979) Regrowth of severed axons in the neonatal central nervous system: Establishment of normal connections. Science 2O5:1158-1161. Kalil, K., T. Reh (1982) A light and electron microscope study of regrowing pyramidal tract fibers. J. Comp. Neurol. 21lt265-275. Katz, M.J. (1985) HOWstraight do axons grow? J. Neurosci. 5:589-595. Kiernan, J.A. (1979) Hypothesis concerned with axonal regeneration in the mammalian newous system. Biol. Rev. 54~153-197. Lurie, D.I., and M.E. Selzer (1987) Preferential regeneration of spinal axons through the scar in hemisected lamprey spinal cord. Soc. Neurosci. Abs. 13:467. Lurie, D.I., and M.E. Selzer (1988) Directionally specific axonal regeneration in the adult lamprey spinal cord. Soc. Neurosci. Abs. 14:452. Mackler, S.A., and M.E. Selzer (1985) Regeneration of functional synapses between individual recognizable neurons in the lamprey spinal cord. Science 229: 774--776. Mackler, S.A., and M.E. Selzer (1987) Specificity of synaptic regeneration in the spinal cord of the larval sea lamprey. J. Physiol. (London) 388~183198. Mackler, S.A., H.S. Yin, and M.E. Selzer (1986) Determinants of directional
D.I. LURIE AND M.E. SELZER specificity in the regeneration of lamprey spinal axons. J. Neurosci. 6:1814-1821. Margolin, L., and J. Ayers (1987) The effect of temperature on the quality of recovered swimming following spinal transection in metamorphosing sea lamprey. Soc. Neurosci. Abstr. 11~1287. McClellan, A.D. (1987) In vitro CNS preparations: unique approaches to the study of command and pattern generation systems in motor control. J. Neurosci. Meth. 21.251-264. McClellan, A.D. (1988) Functional regeneration of descending command pathways for locomotion demonstrated in the in vitro CNS. Brain Res. 448:339-345. McClellan, A.D., and S. Grillner (1984)Activation of “fictive swimming” by electrical microstimulation of brainstem locomotor regions in an in uitro preparation of the lamprey central nervous system. Brain Res. 300r357361. Pestronk, A., and D.B. Drachman (1988)Motor nerve outgrowth: Reduced capacity for sprouting in the terminals of longer axons. Brain Res. 463:218-222. Pestronk, A., D.B. Drachman, and J.W. Griffin (1980) Effects of aging on nerve sprouting and regeneration. Exp. Neurol. 70~65-82. Piatt, J., and M. Piatt (1958) Transection of the spinal cord in the adult frog. Anat. Rec. 131:81-91. Reh, T., and K. Kalil (1982) Functional role of regrowing pyramidal tract fibers. J. Comp. Neurol. 211t276-283. Reier, P.J., and J.D. Houle (1988) The glial scar: Its bearing on axonal elongation and transplantation approaches to CNS repair. In S.G. Waxman (ed): Advances in Neurology, Vol. 47. New York: Raven Press, pp. 87-138. Roederer, E., N.H. Goldberg, and M.J. Cohen (1983) Modification of retrograde degeneration in transected spinal axons of the lamprey by applied DC current. J. Neurosci. 3.153-160. Rovainen, C.M. (1967) Physiological and anatomical studies on large neurons of the central nervous system of the sea lamprey. 1. Muller and Mauthner cells. J. Neurophysiol. 30:lOOO-1023. Rovainen, C.M. (1976) Regeneration of Miiller and Mauthner axons after spinal transection in larval lampreys. J. Comp. Neurol. 168:545-554. Rovainen, C.M. (1982) Neurophysiology. In M.W. Hardisty and I.C. Potter (eds): The Biology of Lampreys, Vol. 3. New York: Academic Press, pp. 1-136. Schwab, M.E., and P. Caroni (1988) Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in uitro.J. Neurosci. 8,2381-2393. Schnell, L., and M.E. Schwab (1990) Axonal regeneration in the rat spinal cord produced by a n antibody against myelin-associated neurite growth inhibitors. Nature 343t269-272. Selzer, M.E. (1978) Mechanisms of functional recovery and regeneration after spinal cord transection in larval sea lamprey. J. Physiol. (London) 277:395408. Silver, J., and M.Y. Ogawa (1983) Postnatally induced formation of the corpus callosum in acallosal mice on glia-coated cellulose bridges. Science 220:1067-1069. Snedeker, J.A., and M.E. Selzer (1986) Probability of regeneration as a function of distance of axotomy from the cell body in lamprey brain neurons. Soc. Neurosci. Abstr. 121575. Swain, G. (1989) Development of Descending Reticulospinal Systems in the Sea Lamprey. Doctoral dissertation, Northeastern University. Windle, W.F. (ed) (1955) Regeneration in the Central Nervous System. Springfield, Ill.: Charles C. Thomas. Wood, M.R., and M.J. Cohen (1979) Synaptic regeneration in identified neurons of the lamprey spinal cord. Science 206t344-347. Wood, M.R., and M.J. Cohen (1981) Synaptic regeneration and glial reactions in the transected spinal cord of the lamprey. J. Neurocytol. 10:57-79. Yin, H.S., S.A. Mackler, and M.E. Selzer (1984) Directional specificity in the regeneration of lamprey spinal axons. Science 224:894-896. Yin, H.S., and M.E. Selzer (1983) Axonal regeneration in the lamprey spinal cord. J. Neurosci. 3:1135-1144.
Axonal Regeneration in the Adult Lamprey Spinal Cord DIANA I. LURIE
AND
MICHAEL E. SELZER
Department of Neurology (M.E.S.)and the David Mahoney Institute of Neurological Sciences (D.I.L., M.E.S.), University of Pennsylvania School of Medicine, Philadelphia, PA 19104-4283
ABSTRACT Larval sea lampreys recover from complete spinal transection by a process involving directionally specific axonal regeneration. In order to determine whether this is also true of adults, 14 adult lampreys were transected at the level of the 5th gill and allowed to recover for 10 weeks. Miiller and Mauthner cells and their giant reticulospinal axons (GRAs) were impaled with microelectrodesand injected with horseradish peroxidase (HRP). The tissue was processed for HRP histochemistry and wholemounts of brain and spinal cord were prepared. All animals recovered coordinated swimming; 61 of 121 (50%)neurites emanating from 30 axons regenerated caudal to the scar into the distal stump. Of the neurites which had grown beyond the scar, 92%were correctly oriented, i.e., caudalward and ipsilateral to the parent axon. Retransection in two additional animals eliminated the recovered swimming. Thus, behavioral recovery in adult sea lampreys is accompanied by directionally specific axonal regeneration. Key words: Miiller cells, Mauthner cells, directional specificity,transection, behavioral recovery, axon guidance
The larval sea lamprey (Petromyzonmarinus) is capable MATERIALS AND METHODS of functional recovery following spinal cord transection Staging of animals (Rovainen, 1976; Selzer, 1978; Cohen et al., 1986). Spinal Animals were considered fully transformed adults if they axons regenerate across the transection zone (Hibbard, 1963; Rovainen, 1976; Selzer, 1978; Wood and Cohen, met the following criteria (Hardisty and Potter, 1971). The 1979; 1981; Borgens et al., 1981) and this regeneration is characteristic brown skin color of the larva had changed to specific in that axons grow selectively in their normal a dark gray on the dorsal surface and silver on the ventral surface. The eye had fully differentiated with a dark pupil directions (Yin and Selzer, 1983;Yin et al., 1984;Mackler et and silver iris. The oral disk had enlarged and developed al., 1986)and form appropriate synaptic connections (Mack- tooth-like structures. The fimbria had become enlarged and ler and Selzer, 1987). In other vertebrates, the capacity for fully differentiated. The fins had enlarged. The animal no axonal outgrowth diminishes greatly with age (Piatt and longer burrowed in the sand at the bottom of the tank but Piatt, 1958; Bernstein, 1964; Kiernan, 1979; Pestronk et attached itself to the side of the tank with its sucker mouth. al., 1980; Fagg et al., 1981). However, recent studies Surgery indicate that adult sea lampreys can also recover behaviorally from spinal transection (Ayers et al., 1982; Margolin Fourteen adult lampreys received complete spinal transecand Ayers, 1987; Cohen and Baker, 1988). Preliminary tions at the level of the 5th gill. Briefly, animals were findings based on retrograde transport of HRP suggest that anesthetized in tricaine methansulfonate (MS 222, Finguel, reticulospinal neurites cross the lesion site (Lurie and Ayerst) 1:4,000and pinned to a Sylgard plate (184 silicone Selzer, 1988; Ayers, 1989). elastomer, Dow Corning). The skin, muscle, and fibrous In the present study, giant reticulospinal axons (GRAs) canal were cut by a dorsal incision with a fine scalpel in previously transected adult lampreys were injected with exposing the spinal cord. The exposed spinal cords were horseradish peroxidase (HRP) in order to visualize their transected under direct stereomicroscopicvision with iridecregenerated neurites, measure their distances of regeneration, and determine whether they were oriented in their Accepted January 10,1991 correct directions.
o 1991 WILEY-LISS, TNC.
Figure 1
AXONAL REGENERATION IN ADULT LAMPREYS tomy scissors and the cut ends allowed to re-appose. The completeness of the transections were verified by direct observation as well as by the fact that post-operative animals were paralyzed below the transection. The wounds were healed by air drying with the animals resting on ice for 1-2 hours post-operatively. Animals were then returned to fresh water aerated aquaria and allowed to recover for 10 weeks at 16°C.
Retrograde tracer studies After 10 weeks of recovery, two animals were retransected 5 mm caudal to the original transection and a gelfoam pleget soaked in 50%HRP was placed in the second transection. The animals were allowed to recover for an additional 2 weeks, after which the brain and spinal cord were removed and processed for HRP histochemistry (see below).
Intracellular HRP injection In the 12 remaining recovered animals, the rostral brain and 5 cm of cord was removed together with the underlying notocord. This preparation included the lesion site. The tissue was then placed in 0.01% collagenase (Sigma Chemical Go., St. Louis, MO) for 10 minutes, rinsed three times in cold lamprey Ringer (Selzer, 19781, and pinned dorsal side up to the bottom of a Sylgard-lined experimental chamber. The tissue was transilluminated from below (Selzer, 1978) and the meninx primitiva was stripped away with jewelers forceps. Microelectrodes were backfilled with 6% HRP in 0.2 M potassium acetate and 0.1 M tris buffer, pH 7.4. Those with resistances of at least 40 Mfl were selected. Muller and Mauthner cells in the brain and their giant axons in the cord were impaled under stereomicroscopic vision. No attempt was made to limit the study to one cell type and the data presented here are pooled from all cells. HRP was iontophoretically injected with 15 nA pulses of 200 msec duration at 3.3 Hz for 20 minutes. At the end of the experiment, the spinal cord was placed in fresh Ringer and kept overnight at 4°C.
Histological methods
411
Behavioral observations Each transected animal was examined 24 hours after surgery to ascertain that there was no movement caudal to the lesion site. A transection was tentatively considered complete if the animal could move only its head and body rostral to the lesion. Each week thereafter, animals were observed and classified into stages of recovery as described below.
RESULTS Behavioral recovery After 1 week, paralysis caudal to the lesion continued. However, the animals tended to undulate slowly when attached to the bottom of the tank by their oral sucker. This movement has been analyzed by Ayers and determined to originate caudal to the lesion (Ayers, 1989). If the animals were forcibly detached, they were completely immobile. At 2 weeks post-transection, violent head oscillations propelled the animals forward, although they were unable to move vertically and exhibited no righting reflex. Body segments caudal to the lesion moved only passively (Selzer, 1978; Ayers, 1989).At weeks 3 and 4, the animals displayed weak, poorly coordinated swimming movements, which could not be sustained for more than 5 seconds. By week 6, two thirds of all the animals showed coordinated swimming in all planes and by week 7, all animals could swim well.
Probability of regeneration of GRAs GRAs are normally unbranched and extend almost the entire length of the lamprey spinal cord. Therefore, high spinal transection axotomizes all GRAs. Thirty Muller and Mauthner axons were labeled with HRP in 12 transected adults. These axons gave rise to 121 neurites of which 61 (50%) grew beyond the level of the scar (Fig. 1, Table 1). Fifteen of the thirty s o n s sent at least one branch across the scar and it was common for an axon to give rise to some neurites that regenerated and others that did not. These results are similar to those reported previously for larval lampreys (Yin and Selzer, 1983).
Distance of regeneration of GRAs
The tissue was processed for HRP histochemistry with a In larval sea lampreys, cut GRAs die back an average of modified Hanker-Yates mixture (Selzer, 1978) for 20 minutes at room temperature and rinsed in lamprey Ringer. 1-2 mm before regenerating (Roederer et al., 1983). HowThey were dehydrated for 5 minutes in 35% ethanol, fixed ever, because of the large variability in the distance of in 70% ethanol at 4°C for 1 hour, and the notocord dieback (as much as 2 cm in our laboratory) and because the separated from the spinal cord and discarded. The spinal extent of dieback has not been determined in adults, cords were dehydrated in serial ethanols, cleared in cedar- distance of regeneration was measured relative to the wood oil, and mounted on slides with a Permount (Fischer)/ center of the lesion. This had the added advantage that it allowed comparison with previously reported data from this xylene mixture. laboratory on larval lampreys (Yin and Selzer, 1983). The Light microscopic analysis neurites of GRAs in adult lampreys regenerated to a mean The regenerating HRP-injected branches of GRAs were examined for the following: 1)the distance of the neurite tip TABLE 1. Neurites of GRAs in Transected Cords proximal or distal to the center of the lesion and 2 ) the Neurite paths orientation of the neurite tip (ipsilateral vs. contralateral to the parent axon; rostral vs. caudal). Neurite
Fig. 1. Wholemounts of adult transected spinal cords. A, B, C: Most labeled neurites regenerate through the scar (arrows) although some neurites loop and grow rostralward (arrowhead in B). Left, rostral. cc, central canal.
terminations
Looped
Proximal to the scar Within t h e scar Distal to the scar Total
0 (0%) 9 (30%) 4 (6%) 13 (11%)
Decussating
Ipsilateral
Total
0 (0%) 4 114%) 1(2%) 5 (44%)
30 (100%) 17 (56%) 56 (92%) 103 (85%)
30 30 61 121
Summary of neurite projection paths in 10 week post-transection spinal cords. Note that 5 0 4 of all labeled neurites regenerated beyond t h e level of t h e scar and, of these, 92% remained on the ipsilaterai side of t h e spinal cord.
D.I. LURIE AND M.E. SELZER
412 distance of +0.15 mm beyond the center of the transection scar. The range was between -5.46 mm (i.e., 5.46 mm proximal to the scar) and +3.20 mm (i,e., 3.20 mm distal to the scar). This is comparable to the distances reported for larval GRAs (Rovainen, 1976; Selzer, 1978; Yin and Selzer, 1983; Wood and Cohen, 1979).
TABLE 2. Effect of Neurite Orientation on Distance of Regeneration Straight (ipsilateral) Distance (mmY
1.05 t 0.08 n = 56
Corrected distance (mmIb
1.03 ? 0.08 n = 56
Directional specificity of GRA regeneration Of those neurites which terminated distal to the lesion, 92% grew in their appropriate direction, ipsilateral to their parent axon and pointing caudalward (Fig. 1, Table 1).Of those neurites which failed to regenerate beyond the level of the scar, 77% remained correctly oriented (Table 1).These results are similar to those reported previously in larval animals transected at the level of the 7th gill or at the level of the third gill (Yin and Selzer, 1983; Yin et al., 1984; Mackler et al., 1986).
Looped
Decussating
0.53 ? 0.14 n = 16 P < 0.01 0.52 t 0.14
0.25
?
0.14
n=5 P < 0.0001
n = 16 P < 0.01
eDistance (mm) f SEM of terminations of straight and decussating GRA neurites relative to the center of the scar. Negative values were assigned to terminations rostral to the scar. Distance of loowed neurites was measured from the point of looping . -to the end of the looped neurite. 'Corrected distance (mm). GRAs die back after transection and the point at which they start their forward mowth is unknown. In order to compare the growth of straight neurites with those chat looped, the average location at which looping neurites turned rostralward (0.026 mm distal to t h e scar) was defined as zero for those axons which remained straight and those that looped. Pvalues are for comparison with control.
L1 Non-looping neurites
Directional specificity and distance of regeneration
Looping neurites c
Although most regenerating giant reticulospinal neurites which terminated distal to the lesion grew on the correct side of the cord and in the correct rostrocaudal direction, several neurites crossed the midline in the region of the scar, while others looped rostralward towards the brain (Fig. lB, Table 1). If directional specificity of neurite regeneration resulted merely from the tendency of axons to grow in the direction they are already facing, rather than from specific guidance mechanisms, then neurites that had become misoriented should continue to grow as readily as those which remain oriented caudalward. However, as in larval lampreys (Mackler et al., 19861,this was not the case. GRAs which regenerated in their correct direction grew longer than those which had either decussated or looped backwards. In order to make this comparison, the mean location at which looping s o n s turned rostralward was determined and defined as zero both for those axons which remained straight and those that looped. This location was +.026 mm (i.e., 26 p,m caudal to the center of the scar); range -2.72 to + 1.02 mm. The average distance of regeneration for neurites which grew beyond this point and remained on the correct side of the cord was + 1.03 mm ? 0.08 SEM (n = 56). Neurites which looped rostralward (incorrectly) grew approximately half as far (0.53 mm ? 0.14 SEM; n = 16) and this difference was significant (Student's t test P < 0.05; Table 2). Four neurites decussated and terminated almost immediately within the scar. Only one neurite crossed the midline and extended in the distal stump for 0.86 mm. These results are similar to those obtained for GRAs and other spinal neurites in larval animals (Mackler et al., 1986) Approximately half of the labeled neurites failed to grow into the distal stump. An examination of their tip locations suggested that they were concentrated in the region of the scar (Fig. 2A). A more detailed examination of the tip locations revealed that they were distributed evenly within the scar and in the 0.5 mm of spinal cord proximal and distal to it and were not backed up just proximal to the scar (Fig. 2B,C,D). Thus it is unlikely that the scar represents a hostile environment for axon outgrowth.
Regeneration of other axons Although their large size make GRAs convenient to study, their relationship to locomotor behavior is not clear (Rovainen, 1967; 1982; Buchanan and Cohen, 1982) and
5 10 5 =
$
5
z -6
-5
-4
-3 -2 -1
,
0,
1
Z
3 4
Distance from scar (mm)
a c ._
Looping
z
I)
E
3
z
10 D
-1
Non-Looping
-5 0
.5
1
i scar i
II(mm)1I Fig. 2. Distribution of neurite tips in transected spinal cords of adult lampreys. A: Neurite terminations tended to be concentrated in the region of the scar rather than being backed up just proximal to it. For looping neurites (dark bars) the distance plotted represents the location of the termination of the looped neurite. B: Expanded scale to highlight the distribution of neurite tips in the region of the scar. Neurites were distributed fairly uniformly within the scar. C: Distribution of the terminations of looping neurites. Most terminations were located just rostral to the scar in the proximal stump. D: Distribution of nonlooping neurite tips. The tendency for neurites to terminate fairly evenly throughout the vicinity of the scar is not altered by excluding the looping neurites.
other neurons are probably more important to recovery of locomotion following spinal transection (McClellan, 1988; Ayers, 1989). Therefore, it is important to demonstrate whether regeneration characterizes other brainstem neurons with spinal projections. Two adults were retransected
AXONAL REGENERATION IN ADULT LAMPREYS approximately 5 mm distal to the original transection and HRP injected into the second lesion. Work in our laboratory has demonstrated that HRP can diffuse extracellularly and label neurons whose axons terminate within 2.5 mm of the injection site but not beyond (Snedeker and Selzer, 1986). Thus, any retrogradely labeled brainstem neurons had axons that regenerated at least 2.5 mm beyond the center of the scar (Snedeker and Selzer, 1986; Croop et al., 1988). Figure 3 shows a wholemounted adult brain from such a preparation. Many neurons of all sizes were labeled with HRP, indicating that the axons of these neurons had regenerated across the first transection. Retransection eliminated all recovered swimming in these animals, confirming that recovery of function was dependent at least in part upon spinal regeneration.
DISCUSSION Behavioral recovery The present study demonstrates that spinal axons in adult sea lampreys regenerate almost as readily as those of the larval form (Yin and Selzer, 1983; Lurie and Selzer, 1988).In addition, the animals recovered normal-appearing swimming and this was reversed upon retransection of the cord. These observations are consistent with the more detailed behavioral measurements of Margolin and Ayers (1987, Ayers, 1989) and with the observations on fictive swimming by Cohen and Baker (1988). The latter study suggested that recoupling of central pattern generators in the adult was less secure than in the larva (Cohen et al., 1986) because synchronization of ventral root discharges across a healed transection could be demonstrated only after overlappingstaged partial lesions. It should be pointed out that the present experiments do not identify the axons whose regeneration mediates the recovered behavior. Muller and Mauthner cells do not appear to function as locomotor command neurons (Rovainen, 1967; 1982; Buchanan and Cohen, 1982), although the Mauthner cells may be involved in triggering startle responses (Currie and Carlsen, 1987). Microstimulation and lesion experiments suggest the existance of clusters of neurons that constitute brainstem locomotor command centers (McClellanand Grillner, 1984; Ayers, 1989)and neurons of adult lampreys that are located in the region of lowest threshold for triggering locomotor responses were labeled retrogradely in these experiments (top arrows, Fig. 3). Swain (1989) has also demonstrated retrograde labeling of these neurons across a transection scar in both recovered larvae and adult lampreys. In larval lampreys, McClellan (1988) has been able to activate the central pattern generators caudal t o a healed transection by microstimulation of brainstem locomotor command centers, even when the spinal cord rostral to and including the scar was bathed in low calcium to block synaptic transmission. He concluded that the locomotor activity must have been triggered by regenerated axons of locomotor command neurons. However, the moment by moment coordination of central pattern generators at different levels of the cord involves local coordinating axons and it is entirely possible that the behavioral recovery seen in the present study and most studies in larvae (e.g., Currie and Ayers, 1983; Cohen et al., 1986) is due mainly to regeneration of axons of intersegmental neurons that coordinate the central pattern generators at different levels (Cohen et al., 1988). In the larva, behavioral recovery is accompanied by regeneration of synaptic connections selectively between
413 appropriate types of neurons on opposite sides of the transection scar (Mackler and Selzer, 1985; 1987). Here again it is not clear that the synapses studied were involved in the behavioral recovery. Whether regenerated neurites of adult spinal axons also form appropriate synaptic contacts remains to be determined. If they do, they clearly will not reproduce the original pattern of innervation, since as in the larval studies, many of the original synaptic targets are located beyond the range of regeneration of these neurites.
Probability of GRA regeneration Approximately 50% of HRP-injected GRAs in adult lampreys regenerated beyond the center of the transection scar. In a previous study on larvae, the probability of regeneration was only slightly greater (57%;Yin and Selzer, 1983). More recent work from this laboratory on regeneration of GRAs in both hemisected and transected larval spinal cords suggests a similar value (59%;Lurie and Selzer, submitted manuscript). There are many possible reasons why not every axon regenerated beyond the scar. These may relate to limitations in the intrinsic regenerative capacities of neurons, to inhibitory influences of the spinal cord extracellular environment, or to a barrier effect on the part of the scar toward regeneration. If the scar were acting as a barrier, one would expect a disproportionate number of the tips of regenerating neurites to be backed up just proximal to the scar. However, within the general region of the lesion, neurite tips were uniformly distributed proximal to, within, and distal to the scar, suggesting that the scar did not act as a barrier. On the other hand, the few neurites which looped or crossed the midline tended to do so within the scar, indicating that the cues which guide directional specificity may be less accessible to growing axons within the scar.
Distance of GRA regeneration In the larval sea lamprey, cut GRAs die back during the first 2 postoperative weeks. The distance of dieback has been estimated to average 1-2 mm (Roederer et al., 1983) but these distances are variable and evidence for dieback as much as .02 m has been observed occasionally in our laboratory. Thus, the process of regeneration begins before axons have crossed the lesion site. Because of the variability of dieback and because much of the current interest in the lamprey derives from the ability of its axons to grow through the lesion, distances of regeneration have always been measured relative to the center of the scar. Thus they underestimate the actual distance of neuritic extension. This convention was followed in the present study as well, especially because dieback in the adult has not been quantified. The average distance of regeneration for neurites of adult GRAs was to a point 0.15 mm distal to the center of the scar within a range -5.4 to +3.2 mm relative to the center of the scar. Similar maximal distances of regeneration were reported for larval GRAs by Rovainen (1976; more than 1 mm but less than 9 mm), Selzer (1978; 3.6 mm), Yin and Selzer (1983; 5.3 mm), and Wood and Cohen (1979; 3.1 mm). Even these maxima are likely to be underestimates because they were based either on reconstructions of serial light microscopic sections or on observations of intracellularly injected axons in spinal cord wholemounts. A s neurites become narrower and longer, they become increasingly difficult to follow with these techniques. In this laboratory, distances of regeneration of up to 21 mm have been observed by antidromic activation of
Figure 3
AXONAL REGENERATION IN ADULT LAMPREYS
GRAs (unpublished)and 30 mm by retrograde transport of HRP (Croop et al., 1988). Nevertheless, it is clear that GRAs do not regrow to their original length since in untransected animals these large unbranched axons extend almost the entire length of the cord, about 150 mm in large larvae and recently transformed adults.
Directional specificity of regeneration The axonal regeneration observed in the present study was directionally specific. This result was similar to that obtained in larval animals where the majority of regenerating spinal axons grew through the transection site and remained correctly oriented (Yin and Selzer, 1983; Yin et al., 1984; Mackler et al., 1986; Lurie and Selzer, 1987). In tissue culture, axons tend to grow forward rather than meander randomly (Katz, 1985). Such a tendency may have contributed to the preferential growth of reticulospinal axons caudalward and ipsilateral to their parent axons. However, the long distances that regenerating axons must travel relative to the width of the ipsilateral hemicord suggests that other guidance mechanisms must be involved. This is supported by the fact that neurites which looped rostralward grew approximately half as far as those that did not and that only one neurite crossed the midline distal to the scar. Previous reports on giant reticulospinal neurons and other cell types in the larval CNS also concluded that the tendency toward forward motion cannot fully explain the specificity of regeneration of lamprey spinal axons Win et al., 1984; Mackler et al., 1986).
Regeneration vs. development The growth of spinal axons across the transection in the adult lamprey is not likely to be due to late developing axons or new neurons. Although the diameters of GRAs and of their perykaria can be more than twice as large in the adult as in the larva, larval GRAs already extend almost the full length of the spinal cord and few, if any neurons are added to this population of cells during transformation (Rovainen, 1982). In addition, retrograde labeling of all types of reticulospinal neurons with HRP yielded similar total counts in the adult and in the larva (Ayers, 1989).
Comparison with other vertebrates It is generally accepted that regenerative capacity decreases progressively with maturation (Windle, 1955). For example, spinal transection in tadpoles is followed by axonal regeneration but in postmetamorphic frogs, it is not. Even in teleost fish, where regeneration of spinal axons occurs in the adult, an age-dependent reduction in the robustness of regeneration has been described (Bernstein, 1964). The very slight degree of reduction in axonal regeneration that accompanies transformation from larval lamprey to the adult form was therefore somewhat surprising. It may be that further reductions in regenerative capacity occur with aging in adult lampreys but we were not in a position to assess this because of the difficulty in obtaining and maintaining older adults.
Fig. 3. Wholemounted hindbrain of a n adult 10 weeks posttransection. Many reticulospinal neurons have been back-filled with HRP. The arrows point to groups of labeled neurons demonstrating t h a t t h e axons of these neurons must have regenerated across t h e transection (see text).
415 In both neonatal and adult mammals, cut axons are not able to regenerate across the injury scar (Gearhart et al., 1979; Kalil and Reh, 1979; 1982; Guth et al., 1981; Reh and Kalil, 1982; Bregman and Goldberger, 1982; 1983; Bernstein and Stelzner, 1983; Reier and Houle, 1988; Goldberg and Frank, 1980). The reason for this is not known, but properties of reactive glia are usually invoked. However, several features of the adult CNS in addition to the scar are thought to limit regeneration. For example, axons can grow along a scaffold formed by implants of embryonic glial cells but not adult glia (Silver and Ogawa, 1983). The work of Schwab and colleagues also suggests that molecules on the surface of mature CNS myelin block axon outgrowth and that this can be reversed by application of antibodies to those molecules (Schwab and Caroni, 1988). The fact that the lamprey CNS contains no myelin or oligodendrocytes may help to explain the relatively robust axonal regeneration. However, the scar itself clearly exerts a major inhibitory influence on regeneration of mammalian CNS axons. While damage to adult CNS often results in collateral sprouting (Gall and Lynch, 1978, 1981; Crutcher and Marfurt, 1988; Pestronk and Drachman, 1988), growing neurites are unable to cross a transection scar. Even in early postnatal mammals, late developing axons of the corticospinal and rubrospinal tracts cannot cross a scar produced by spinal hemisection but can grow around it (Bernstein and Stelzner, 1983; Bregman and Goldberger, 1983; Kalil and Reh, 1979, 1982; Reh and Kalil, 1982). Similarly, when the inhibitory effect of myelin was reduced by application of blocking antibodies, corticospinal axons grew around a hemisection scar rather than through it (Schnell and Schwab, 1990). In contrast, regenerating neurites in the adult lamprey grew through the scar and maintained directional specificity. In experiments on lamprey larvae, cut axons grew around a spinal hemisection rather than around it (Lurie and Selzer, submitted). Whether GRAs of adult lampreys would behave similarly is not known but the present results demonstrate that the ability of lamprey neurites to cross a spinal lesion is not dependent upon the developmental stage of the animal. Nor are the cues which guide directional specificity lost or significantly diminished following transformation.
ACKNOWLEDGMENTS We thank Joseph Snedeker for his expert technical assistance. This work was supported by NIH grants R 0 1 NS14837 and R 0 1 NS25581.
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