- Email: [email protected]
Sparing and recovery of function in spinal larval frogs (Rana catesbeiana): Effect of level of transection
BEHAVIORAL AND NEURAL BIOLOGY
56, 292-306 (1991)
Sparing and Recovery of Function in Spinal Larval Frogs (Rana Catesbeiana):Effect of Level of Transection PAUL R . BRENNER AND DONALD J. STEHOUWER 1
Department of Psychology and The Centerfor Neurobiological Sciences, University of Florida, Gainesville, Florida 32611 Bullfrog tadpoles with cervical or midthoracic transection of the spinal cord were allowed to recover for 5 weeks, at which time axonal growth across the transection site was assessed by transport of horseradish peroxidase. Weekly behavioral tests included those for posture, spontaneous locomotion, cutaneously elicited swimming, and intersegmental coordination. Behavioral and electrophysiological assessments suggest that behavioral recovery depends, at least in part, on the growth of fibers across the transection site. Anatomical and behavioral recovery does not appear to differ with the level of spinal transection, but there was greater sparing of posture, spontaneous locomotion, and stimulus-induced locomotion in tadpoles with thoracic transection of the spinal cords. © 1991Academic Press, Inc.
The behavioral capacity of an animal following damage to the central nervous system (CNS) depends on those abilities that persist despite the damage (sparing) and on those that are initially lost but subsequently return (recovery). The degree and nature of sparing depends on the amount and locus of damage, the particular species under consideration, and, frequently, on the stage of development at which the damage occurs (e.g., Bregman & Goldberger, 1983a,b; Goldman & Rosvold, 1972; Johnson & Almli, 1978; Puchala & Windle, 1977; Windle, 1956). Mechanisms of behavioral sparing include behavioral compensation or substitution (Stein, Finger & Hart, 1983) and continued function of undamaged tissue (Eidelberg, Straehley, Erspamer, & Watkins, 1977). The amount of sparing is usually directly proportional to the amount of remaining neural tissue, as first suggested by Lashley (1938). Specific mechanisms of recovery are often undetermined (Goldberger, Bregman, Vierck, & Brown, 1990), but may include enhanced function of remaining tissue via deneri D.J.S. was supported by PHS-24442 from the National Institute of Neurobiological Diseases and Stroke. P.R.B. was supported by training grant PHS-2271. Address correspondence and reprint requests to Donald J. Stehouwer, Dept. of Psychology, Univ. of Florida, Gainesville, FL. 32611. 292
0163-1047/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
RECOVERY FROM SPINAL TRANSECTION
293
vation supersensitivity (Stelzner, Weber, & Prendergast, 1979), collateral sprouting of axons (Goldberger, 1973; Ichikawa, 1987, 1989; Murray, 1973), assumption of new functions by remaining tissue, and actual repair of damaged tissue (see Freed, Medinaceli, & Wyatt, 1985; Stein et al., 1983). It is often difficult to distinguish between the recovery due to anatomical reorganization within the CNS and that due to assumption of compensatory strategies (Sperry, 1947). Behavioral competence following spinal transection in tadpoles appears to depend on the level of spinal transection, with greater competence being associated with cervical than with thoracic spinal transections (cf. Forehand & Farel, 1982a; Sims, 1962; Piatt & Piatt, 1958; Stehouwer, 1986). However, no attempt was made to distinguish between sparing and recovery in those studies, and both sparing and recovery may have varied with the level of transection. For example, there may be greater sparing of function following cervical than following thoracic transection of the spinal cord because of the greater amount of CNS tissue caudal to the transection site in the former. Consistent with this idea, the capacity for normal function of the central pattern generators for swimming has been shown to be roughly proportional to the number of intact spinal cord segments in the lamprey (Cohen & Wallen, 1980) and bullfrog tadpole (Stehouwer & Farel, 1980). Behavioral recovery in tadpoles may vary with the level of spinal transection for a variety of reasons. First, there are more ascending and descending axons at cervical than at thoracic levels, so that more axons would have the opportunity to regenerate following the more rostral transection. Second, greater denervation of the spinal cord following cervical transections may stimulate greater denervation supersensitivity and collateral sprouting. Third, axons from different populations may grow across the two transection sites because, within limits, regeneration of severed axons is greater following lesions proximal to the cell body than following more distal lesions (Grafstein & McQuarrie, 1978; Richardson, Issa, & Aguayo, 1984, Yin & Selzer, 1983). Thus, brain stem neurons associated with locomotor command systems (Currie & Ayers, 1983; Grillner, 1981; McClellan, 1988) may cross cervical but not thoracic transections of the spinal cord. This study was undertaken to evaluate sparing and recovery of function following spinal transection in bullfrog tadpoles. Specifically, we measured (1) posture (2) the amount of spontaneous locomotion (3) locomotor response threshold to tactile stimulation rostral and caudal to the transection, and (4) intersegmental coupling of locomotor activity across the transection site. METHODS
Subjects. Subjects were 448 larval bullfrogs (Rana catesbeiana) obtained from Carolina Biological Supply. Larvae (tadpoles) ranged from developmental Stages IV to XIV according to the criteria of Taylor and Kollros
294
BRENNER AND STEHOUWER
(1946) and were maintained in oxygenated 38-liter aquaria at ambient room temperature on a diet of alfalfa for at least 1 week prior to surgery. Following surgery tadpoles were housed individually in oxygenated 3-liter aquaria and water was changed daily. Because spinally transected tadpoles were unable to feed during the postoperative period, food was withheld from 14 intact tadpoles for 4 weeks to control for the effects of food deprivation on performance on the behavioral tests used in this study. Surgery. All surgical procedures were performed following anesthesia induced by submerging the subject in a 0.2% tricaine methanesulfonate solution for 2 min, or by placing the subject in an ice and water slurry for 30 min, prior to surgery. Using a dissection microscope, the spinal cord was exposed by laminectomy and the dorsal vertebral artery was carefully deflected to one side to allow access to the spinal cord, which was then transected immediately below the obex (cervical transection) or between spinal roots 5 and 7 (midthoracic transection). Complete transection was verified visually by retracting the cut ends of the spinal cord. Function. Following spinal transection, behavioral competence of tadpoles was assessed once a week for 4 postoperative weeks on a battery of tests that included tests of posture, spontaneous locomotion, and cutaneously elicited swimming. Posture was evaluated by recording whether or not each tadpole was in an upright position. After recording posture, tadpoles were placed individually in 22.8-cm diameter containers marked into quadrants and filled with 1000 ml of water. After 30 min of adaptation to the apparatus, 4 h of spontaneous locomotion was recorded on time-lapse videotape at 100 frames per minute. The amount of locomotion was measured by recording the number of times a tadpole crossed quadrant markings. Finally, the ability of cutaneous stimuli to elicit swimming, defined as undulations of the tail caudal to the transection site, was measured. Tadpoles were stimulated on the snout and on the base of the tail with a set of 10 calibrated von Frey hairs (range of pressures, 0.03 to 3.9 g). Thresholds for eliciting swimming tadpoles with cervically and thoracically transected spinal cords were calculated by averaging the results of an ascending and a descending stimulus series. To assess intersegmental coordination across the transection site, two 0.18-mm Teflon-insulated, silver wire-hook electrodes were implanted in the axial musculature, one approximately 2 mm above the transection site and the other at the base of the tail. Electromyographic activity was recorded from freely swimming intact tadpoles and from those whose spinal cords had been thoracically transected either 4 or 28 days earlier. To determine whether intersegmental coordination could be achieved exclusively by growth of axons across the transection site, fictive locomotion was recorded in vitro from homolateral ventral roots above and below
RECOVERY FROM SPINAL TRANSECTION
295
the transection site of one isolated CNS preparation (see Stehouwer & Farel, 1980) obtained 28 days postoperatively. Anatomy. Immediately following the behavioral tests on Postoperative Week 4, H R P (Sigma, Type VI) was recrystallized onto the tip of a 00 insect pin. With the aid of a micromanipulator, the pin was inserted into the spinal cord approximately 2 mm caudal to the transection site and held in place until the H R P dissolved. In addition, H R P was inserted into the cervical and thoracic spinal cord in intact tadpoles to ascertain the source of descending spinal projections in normal animals. One week after the H R P application, the animals were sacrificed and the brains were perfused according to a modification of a procedure described by Rosen and Mesulam (1977). The CNS was dissected and placed in a 2.5% glutaraldehyde solution for 24 h and embedded in either albumin or gelatin. Sections were cut at 45/xm in the horizontal place mounted, and counterstained with cresyl violet. The total number of HRP-labeled axons and cell bodies rostral to the transection site were counted 1 week after H R P injection. Two tadpoles with thoracically transected spinal cords were discarded because of extracellular diffusion of H R P across the transection site. The nuclear localization of HRP-labeled cell bodies was based on the atlas of Kemali and Braitenberg (1969), and on H R P (Forehand & Farel, 1982b), fluorescence (Parent, 1973; Soller, 1977), and immunohistochemical (Ueda, Nojyo, & Sano, 1984) studies of the bullfrog brain stem. Statistical analysis. At the time of sacrifice, continuity of the spinal cord across the transection site was assessed visually and the data were analyzed by dividing tadpoles into groups that had or had not established gross continuity of the spinal cord. Analyses of variance were used to determine whether locomotor behavior changed as a function of postoperative recovery interval, lesion site, or spinal cord continuity. Differences in posture between cervical and thoracic tadpoles were analyzed by a X2 test. Student's t tests were used to evaluate differences in the number of labeled processes and brain stem cell bodies rostral to the cervical and thoracic spinal cord transection sites. All statistical tests were evaluated at the .01 level of significance. RESULTS
Anatomy. Of 370 tadpoles that received spinal transections followed by weekly behavioral testing, 107 survived the 5-week postoperative period. Of those, 36 of 62 tadpoles with thoracic transections and 33 of 45 tadpoles with cervical transections had reestablished gross continuity of the spinal cord. Most were constricted at the lesion site, and rostral and caudal stumps of cords that had not reestablished continuity were separated by gaps 1-3 segments in length.
296
BRENNER AND STEHOUWER
The white and gray matter lacked heavy glial scarring normally associated with mammalian spinal transections (Windle, 1956), but we found wide variability in the amount of growth across the transections, as have others (e.g., Forehand & Farel, 1982a; Hooker, 1925). Despite gross continuity of the spinal cord, axonal growth was abortive in many tadpoles, but many processes crossed the lesion sites in others (Fig. 1). Cell bodies were labeled in 14 tadpoles, exclusively supraspinally and primarily in the brain stem, and processes were labeled in 26 tadpoles. There were no differences between tadpoles with cervical and thoracic transection of the spinal cord with respect to the number of cell bodies (means = 3.0 and 1.75, respectively) or processes (means = 16.5 and 12.67, respectively), t's(30) < 1.33, p's > .05 that were labeled. Cells were labeled in the reticular formation, the raphe nuclei and vestibular nuclei in tadpoles transected at either level of the spinal cord (Figs. 2 and 3). Intact tadpoles injected with H R P cervically or thoracically had heavy labeling cells in the spinal cord, reticular formation, raphe nuclei, and vestibular nuclei, with sparse labeling of cells in the optic tectum. Function. Cervical and thoracic transection of the spinal cord severely disrupted posture, and many tadpoles lay on their backs or sides. There were no differences in posture as a function either of the degree of continuity of the spinal cord or of postoperative recovery interval in either group. However, more tadpoles with thoracic transections (70.0%) than cervical transections (42.7%) to the spinal cord maintained an upright posture X2(1, n = 340) = 25.67, p < .001. Because posture of bullfrog tadpoles is maintained by regulating inflation of the lungs, which in turn is controlled via cervical sympathetic and vagal nerves (Carlson & Luckhardt, 1920), one would expect greater sparing of posture in thoracic tadpoles because the cervical sympathetic nerves exit the cord between the cervical and thoracic transection sites. Spinal transection at either thoracic or cervical levels severely depressed spontaneous locomotion. Recovery of spontaneous locomotion was greater at longer postoperative recovery intervals F(3,345) = 8.64, p < .001 and in tadpoles that had reestablished anatomical continuity of the spinal cord F(2, 115) = 139.13, p < .001. Furthermore, the effect of postoperative recovery interval interacted with the level of transection F(3, 345) = 5.33, p < .01 and continuity F(3, 345) = 14.36, p < .001. Analyses of simple main effects revealed that recovery was greater in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those without continuity F(3,345) = 13.69, p < .001, whereas recovery of spontaneous locomotion was no greater in tadpoles with thoracically transected spinal cords that had reestablished gross continuity of the cord than in those that had not F(3, 345) = 1.95, p > .01. Sparing of locomotion was greater in tadpoles with thoracically transeeted spinal cords than in tadpoles with cervical transections to the
FIG. 1. This Stage IX tadpole with a thoracic transection of the spinal cord had a large number of processes crossing the transection site (arrowhead). In this and all subsequent photomicrographs, sections were cut in the horizontal plane and rostral is to the right. Calibration bar, 125 /~m; magnification, 100×.
--.a
Z
Z
©
¢b 0 <
Fl6. 2. HRP-labeled cells (arrows) were found in the vestibular nucleus of thoracic tadpoles. The lateral margin of the spinal cord is at th e top and the edge of the fourth ventricle is at the bottom of this photomicrograph. The eighth cranial nerve (asterisk) can be seen entering the brain stem immediately rostral to the labeled cell bodies of this Stage V tadpole. Calibration bar, 50/zm; magnification, 260 x .
b~
bl
U: 0 C
r~
>
7~
:Z
O0
F~. 3. Dark-field photomicrograph (left) reveals axonal processes labeled with HRP in the brain stem of a Stage V tadpole whose spinal cord had been thoracically transected. Calibration bar, 175 /xm; magnification, 102 x . Several cell bodies of the raphe nuclei (inset) are visible under greater magnification in the bright-field photomicrograph (right). Calibration bar, 25/zm; magnification, 408 x .
t~
Z
> Z r~
K
©
<
0
300
BRENNER AND STEHOUWER
&-~ e~e
Gross continuity
O~O
No continuity
°it
A~-.~..•Thoroclc
Cervical T r a n s e c t i o n s
20
•"
9
I
1
2
,
3
Transections
--0
,
4
I 1
I 2
I 3
I 4
POSTOPERATIVE WEEKS Fic. 4. The amount of spontaneous locomotion in each weekly test is plotted as the mean number of quadrants entered in 4 h for intact tadpoles and those with cervical and thoracic transections of the spinal cord. In this and subsequent figures, error bars represent standard errors of the means.
spinal cord, as indicated by a higher m e a n level of locomotion at Postoperative week 1. By Postoperative weeks 3 and 4 the level of spontaneous locomotion in tadpoles that had reestablished continuity of the spinal cord was similar regardless of level of transection (Fig. 4). These observations are supported by a significant main effect of level of transection F ( 1 , 1 1 5 ) = 20.09, p < .001 and a level X recovery interval interaction F(3, 345) = 11.33, p < .001. Figure 5 shows that the threshold for swimming elicited by cutaneous stimulation of the snout declined in all groups of spinally transected tadpoles as a function of recovery interval F ( 3 , 3 1 2 ) = 15.37, p < .001. The m e a n threshold of each group of tadpoles with cervical transections was higher than those with thoracic transections F ' s ( 1 , 1 0 4 ) > 9.72, p ' s < .01. Thresholds were lower in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those that had not F(1, 104) = 9.15, p < .01 whereas no such difference was found in tadpoles with thoracically transected spinal cords F(1, 104) = .21, p >
4.0 D "I" ttl w
3.0
O
~
0
T~-----T~
Cervical Transectlons e--e
^
O~O
&~&
T h o r a c i c Tronsections Gross continuity No continuity Intoct
!
2.0 0
I 1
I
2
I 3
I 4
I
I
POSTOPERATIVE WEEKS FxG. 5. Mean thresholds for locomotion elicited by cutaneous stimulation rostral to the transection site are plotted as a function of postoperative interval.
301
RECOVERY FROM SPINAL TRANSECTION
, ~ 4.0
Thoracic Tronsections
Cervical T r a n s a c t i o n s
0 "1o') laJ
e--e Gross continuity 0--0 NO continuity A - - A , Intoct
3.0 2.0
I-. Z
"5
er ~ ,
1 i.o
~
.
A~
T
~@~---0
T
T 0
3
4
o
v
I
P 2
~ 0.0 1
2
I 3
I 4
POSTOPERATIVE WEEKS FI6. 6. M e a n thresholds for locomotion elicited by cutaneous stimulation caudal to the transection site are plotted as a function of postoperative interval.
.01. The threshold for swimming elicited by cutaneous stimulation at the base of the tail (Fig. 6) was elevated only slightly by either transection and declined as a function of postoperative recovery interval F(3,312) = 10.79, p < .001. Myotomal activity recorded from homolateral axial muscles of the cervical and tail regions was coupled during locomotion of all 4 intact tadpoles (Fig. 7a), in 4 of 10 tadpoles whose cords were thoracically transected 4 days previously (Fig. 7b), and in 11 of 17 tadpoles with cords transected 28 days previously (Fig. 7C). Four days of recovery is not enough time for axons to grow across the transection site (Forehand & Farel, 1982a; Hooker, 1925), so any coordination across the transection within 4 postoperative days m u s t be mediated by the peripheral transmission of locomotor activity around the transection site (see Discussion). Some of the transected animals could not be induced to swim, but in no case did we observe uncoupled activity. However, initiation and coordination of locomotion can be mediated centrally in recovered tadpoles, because tictive locomotion recorded from homolateral ventral roots of isolated CNS preparations in vitro may also be coupled across the transection site (Fig. 7d). However, anatomical restitution of the spinal cord occurs infrequently and involves only modest numbers of processes crossing the transection site (vide supra). Many attempts to obtain a viable preparation for study in vitro failed either because there was no anatomical repair or because the few axons that may have grown across the transection site were damaged during dissection. DISCUSSION
These experiments demonstrate sparing and recovery of function and growth of axons across spinal transections in tadpoles. We found greater sparing of posture, spontaneous locomotion, and stimulus-induced locomotion in tadpoles with thoracic transections than in tadpoles with cervical
302
BRENNER AND STEHOUWER
.O.T.AL .Jdk TAIL
If. I dih~.
. . ~/l h . . .
d ..~ill~,l.
TAIL
. . . . .
100 ms
i00 ms a
C
INTACT
ROSTRAL
~
Jill,
If
'T . . . . . . .
TAIL
28 DAYS POST-OP
r
/ ....
100 ms
b 4 D A Y S POST-OP
'~, i~,, d 5orris 28 DAYS POST-OP
FIG. 7. Electromyograms show that during swimming, activity of ipsilateral myotomes of cervical and tail segments was coupled in (a) intact tadpoles and in tadpoles which had been thoracically transected (b) 4 days and (c) 28 days previously. Ventral root recordings obtained 28 days postoperatively during an episode of flctive locomotion in vitro (d) show that activity of homolateral ventral roots rostral (VR4) and caudal (VR8) to a thoracic transection was coupled in the isolated CNS.
transections of the spinal cord. However, behavior of tadpoles with cervical spinal transections improved more than those with thoracic transection, so that performance after 4 weeks of recovery was similar. Two lines of evidence suggest that anatomical repair contributes behavioral recovery in the present study. First, recovery of spontaneous locomotion and locomotion elicited by stimulation of the snout was greater in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those that had not. Second, synchronous activity of homolateral ventral roots was recorded across the transection site during fictive swimming of the isolated CNS, providing direct evidence that anatomical repair was functionally effective. The ability of so few axons to initiate and coordinate locomotor activity across the transection site is not surprising, because relatively few spinal axons are necessary to mediate considerable function in other systems (Eidelberg et al., 1977; Windle, Smart & Beers, 1958), and gross electrical (Stehouwer & Farel, 1980) or pharmacological stimulation (McClellan & Farel, 1985) of the isolated tadpole spinal cord can elicit fictive locomotion. To the best of our knowledge, the present study offers the only demonstration
RECOVERY FROM SPINAL TRANSECTION
303
of coordination of fictive locomotion across the transection site of an isolated preparation in an animal other than the lamprey (McClellan, 1988). Although reestablishment of specific connections is not necessary to excite spinal locomotor circuits, a few nonspecific connections cannot orchestrate rapid and precise adjustments during locomotion, and our tadpoles showed no evidence of vestibular control during swimming. The amount of anatomical repair found in this study was similar to that reported following mammalian spinal cord injuries (cf. Bernstein, Bechard, & Stelzner, 1981; Brown & McCouch, 1947; Clemente & Windle, 1954). We may have found relatively modest central repair because tadpoles at the developmental stages we studied are relatively static with respect to growth of descending projections (Forehand & Farel, 1982c; Gona, 1972; 1975), and the amount of repair appears to be directly related to the number of proliferating and differentiating neurons available both in amphibians (Davis, Duffy, & Simpson, 1989; Forehand & Farel, 1982a; Holtzer, 1955) and in mammals (Bregman & Goldberger, 1983a,b; Holder & Clarke, 1988). Although we hypothesized that the greater amount of tissue caudal to cervical than to thoracic transection of the spinal cord may result in greater sparing of locomotion in the former, we found exactly the opposite. Greater sparing of locomotion may occur in tadpoles with thoracic transection of the cord because each ventral and dorsal root projects to more than one myotome (Fetcho, 1987; Letinsky; 1974), so that roots immediately rostral to the transection innervate myotomes caudal to the transection and vice versa. This overlapping innervation allows for reflex transmission of locomotion around the transection site. Because no spinal roots exit rostral to our cervical transections, this mechanism of functional sparing was unavailable to those tadpoles and the initial loss of behavior was greater. As central axons grew across the transection site, locomotor performance of tadpoles with cervical transection of the cord improved until it was equal to that of those with thoracic transections 3 and 4 weeks after surgery. Published reports indicate that there is less recovery following thoracic transection (Forehand & Farel, 1982a; Sims, 1962) than following cervical transection (Piatt & Piatt, 1958; Stehouwer, 1986) in tadpoles. The present study suggests that those apparent differences result from differences in sparing and not recovery; following thoracic transections, there is considerable sparing of function, which masks recovery resulting from central repair. The qualitative behavioral assessments used to assess function in other studies were probably not sensitive enough to detect that recovery. Following cervical transection there is so little sparing that recovery is readily apparent. The results of the present study show that sparing and recovery both contribute to behavioral competence following spinal cord transection and
304
BRENNER AND STEHOUWER
u n d e r s c o r e t h e i m p o r t a n c e o f u s i n g t e s t p r o c e d u r e s t h a t a l l o w o n e to distinguish between those two phenomena, which are often confounded in studies of CNS injury.
REFERENCES Bernstein, D. R., Bechard, D. E., & Stelzner, D. J. (1981). Neuritic growth maintained near the lesion site long after spinal transection. Neuroscience Letters, 26, 55-60. Bregman, B. S., & Goldberger, M. E. (1983a). Infant lesion effect: II. Sparing and recovery of function after spinal cord damage in newborn and adult cats. Developmental Brain Research, 9, 119-135. Bregman, B. S., & Goldberger, M. E. (1983b). Infant lesion effect: III. Anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats. Developmental Brain Research, 9, 137-154. Brown, J. O., & McCouch, G. P. (1947). Abortive regeneration of the transected spinal cord. Journal of Comparative Neurology, 87, 131-137. Carlson, A. J., & Luckhardt, A. B. (1920). Studies on the visceral sensory nervous system. I. Lung automatism and lung reflexes in the frog (R. pipiens and R. catesbiana). American Journal of Physiology, 54, 55-95. Clemente, C. D., & Windle, W. F. (1954). Regeneration of severed nerve fibers in the spinal cord of the adult cat. Journal of Comparative Physiology A, 160, 181-193. Cohen, A. H., & Wallen, P. (1980). The neural correlate of locomotion in fish. Experimental Brain Research, 41, 11-18. Currie, S. N., & Ayers, J. (1983). Regeneration of locomotor command systems in the sea lamprey. Brain Research, 279, 238-240. Davis, B. M., Duffy, M. T., & Simpson, S. B. (1989). Bulbospinal and intraspinal connections in normal and regenerated salamander spinal cord. Experimental Neurology, 103, 41-51. Eidelberg, E., Straehley, D., Erspamer, R., & Watkins, C. J. (1977). Relationship between residual hind limb-assisted locomotion and surviving axons after incomplete spinal cord injuries. Experimental Neurology, 56, 312-322. Fetcho, J. R. (1987). A review of the organization and evolution of motoneurons innervating the axial musculature of vertebrates, Brain Research Reviews, 12, 243-280. Forehand, C. J., & Farel, P. B. (1982a). Anatomical and behavioral recovery from the effects of spinal cord transection: Dependence on metamorphosis in anuran larvae. Journal of Neuroscienee, 2, 654-662. Forehand, C. J., & Farel, P. B. (1982b). Spinal cord development in anuran larvae: I. Primary and secondary neurons. Journal of Comparative Neurology, 209, 386-394. Forehand, C. J., & Farel, P. B. (1982c). Spinal cord development in anuran larvae: II. Ascending and descending pathways. Journal of Comparative Neurology, 209, 386-394. Freed, W. J., Medinaceli, L., & Wyatt, R. J. (1985). Promoting functional plasticity in the damaged nervous system. Science, 227, 1544-1552. Goldberger, M. E. (1973). Restitution of function and collateral sprouting in the cat spinal cord: The deafferented animal. Anatomical Record, 175, 329. Goldberger, M. E., Bregman, B. S., Vierck, C. J., & Brown, M. (1990). Criteria for assessing recovery of function after spinal cord injury: Behavioral methods. Experimental Neurology, 107, 113-117. Goldman, P. S., & Rosvold, H. E. (1972). The effect of selective caudate lesions in infant and juvenile rhesus monkeys. Brain Research, 43, 53-66. Gona, A. G. (1972). Morphogenesis of the cerebellum of the frog tadpole during spontaneous metamorphosis. Journal of Comparative Neurology, 146, 133-142.
RECOVERY FROM SPINAL TRANSECTION
305
Gona, A. G. (1975). Golgi studies of cerebellar maturation in frog tadpoles. Brain Research, 95, 132-136. Grafstein, B., & McQuarrie, I. G. (1978). Role of the nerve cell body in axonal regeneration. In C. W. Cotman (Ed.), Axonal regeneration (pp. 155-195). New York: Raven Press. Grillner, S. (1981). Control of locomotion in bipeds, tetrapods, and fish. In V. Brooks (Ed.), Handbook of physiology: Motor control (pp. 1179-1236). American Physiological Society. Holder, N., & Clarke, J. D. W. (1988). Is there a correlation between continuous neurogenesis and directed axon regeneration in the vertebrate nervous system. Trends in Neuroscience, 11, 94-99. Holtzer, H. (1955). Comments on regeneration as opposed to neogenesis of amphibian neurons. In W. F. Windle (Ed.), Regeneration in the central nervous system (pp. 8183). Springfield, IL: Thomas. Hooker, D. (1925). Studies on regeneration in the spinal cord: III. Journal of Comparative Neurology, 38, 315-345. Ichikawa, M. (1987). Synaptic reorganization in the medial amygdaloid nucleus after lesion of the accessory olfactory bulb of adult rat. I. Quantitative and electron microscopic study of the recovery of synaptic of density. Brain Research, 420, 243-252. Ichikawa, M. (1989). Recovery of olfactory behavior following removal of accessory olfactory bulb in adult rat. Brain Research, 498, 45-52. Johnson, D. A., & Almli, C. R. (1978). Age, brain damage and performance. In S. Finger (Ed.), Recovery from brain damage: Research and theory (pp. 115-134). New York: Plenum Press. Kemali, M., & Braitenberg, B. (1969). Atlas of the frog's brain. New York: Springer-Verlag. Lashley, K. S. (1938). Factors limiting recovery after central nervous system lesions. Journal of Nervous and Mental Disorders, 88, 733-755. Letinsky, M. S. (1974). The development of nerve-muscle junctions in Rana catesbiana tadpoles. Development Biology, 40, 129-153. McClellan, A. D. (1988). Functional regeneration of descending brainstem command pathways for locomotion demonstrated in the in vitro lamprey CNS. Brain Research, 448, 339-345. McClellan, A. D., and Farel, P. B. (1985). Pharmacological activation of locomotor patterns in larval and adult frog spinal cords. Brain Research, 332, 119-130. Murray, M. (1973). Restitution of function and collateral sprouting in the cat spinal cord: The hemisected animal. Anatomical Record, 175, 395. Parent, A. (1973). Distribution of monamine containing neurons in the brainstem of the frog, Rana temporaria. Journal of Morphology, 139, 67-78. Piatt, J., & Piatt, M. (1958). Transection of the spinal cord in the adult frog. Anatomical Record, 131, 81-95. Puchala, E., & Windle, W. F. (1977). The possibility of structural and functional restitution after spinal cord injury. A review. Experimental Neurology, 55, 1-42. Richardson, P. M., Issa, V. M. K., & Aguayo, A. J. (1984). Regeneration of long spinal axons in the rat. Journal of Neurocytology, 13, 165-182. Rosen, C. D., & Mesulam, M. M. (1977). Fixation variables in horseradish peroxidase neurohistochemistry: The effects of fixation time and perfusion procedure upon enzyme activities. Journal of Histochemistry, 26, 28-39. Sims, R. T. (1962). Transection of the spinal cord in developing Xenopus laevis. Journal of Embryology and Experimental Morphology, 10, part 2, 115-126. Soller, W. R. (1977). Monoaminergic inputs to frog motoneurons: An anatomical study using fluorescence histochemical and silver degeneration techniques. Brain Research, 122, 445-458.
306
BRENNER AND STEHOUWER
Sperry, R. W. (1947). Effects of crossing nerves to antagonistic limb muscles in the monkey. Archives of Neurological Psychiatry, 58, 452-473. Stehouwer, D. J. (1986). Behavior of larval and juvenile bullfrogs (Rana catesbeiana) following chronic spinal transection. Behavioral and Neural Biology, 45, 120-134. Stehouwer, D. J., & Farel, P. B. (1980). Central and peripheral controls of swimming in anuran larvae. Brain Research, 195, 323-335. Stein, D. G., Finger, S., & Hart, T. (1983). Brain damage and recovery: Problems and perspectives. Behavioral and Neural Biology, 37, 185-222. Stelzner, D. J., Weber, E. D., & Prendergast, J. (1979). A comparison of the effect of thoracic spinal hemisection in the neonatal or weanling rat on the distribution and density of dorsal root axons in the lumbosacral spinal cord of the rat. Brain Research, 172, 407-426. Taylor, A. C., & Kollros, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anatomical Record, 947 7-24. Ueda, S. Nojyo, Y., & Sano, Y. (1984). Immunohistochemical demonstration of the serotonin neuron system in the central nervous system of the bullfrog, Rana catesbeiana. Anatomical Embryology, 169, 219-229. Windle, W. F. (1956). Regeneration of axons in the vertebrate central nervous system. Physiological Reviews, 36, 427-440. Windle, W. F., Smart, J. O., & Beers, J. J. (1958). Residual function after subtotal spinal cord transection in adult cats. Neurology, 8, 518-521. Yin, H. S., & Seizer, M, E. (1983). Axonal regeneration in lamprey spinal cord. Journal of Neuroscience, 3, 1135-1144.
56, 292-306 (1991)
Sparing and Recovery of Function in Spinal Larval Frogs (Rana Catesbeiana):Effect of Level of Transection PAUL R . BRENNER AND DONALD J. STEHOUWER 1
Department of Psychology and The Centerfor Neurobiological Sciences, University of Florida, Gainesville, Florida 32611 Bullfrog tadpoles with cervical or midthoracic transection of the spinal cord were allowed to recover for 5 weeks, at which time axonal growth across the transection site was assessed by transport of horseradish peroxidase. Weekly behavioral tests included those for posture, spontaneous locomotion, cutaneously elicited swimming, and intersegmental coordination. Behavioral and electrophysiological assessments suggest that behavioral recovery depends, at least in part, on the growth of fibers across the transection site. Anatomical and behavioral recovery does not appear to differ with the level of spinal transection, but there was greater sparing of posture, spontaneous locomotion, and stimulus-induced locomotion in tadpoles with thoracic transection of the spinal cords. © 1991Academic Press, Inc.
The behavioral capacity of an animal following damage to the central nervous system (CNS) depends on those abilities that persist despite the damage (sparing) and on those that are initially lost but subsequently return (recovery). The degree and nature of sparing depends on the amount and locus of damage, the particular species under consideration, and, frequently, on the stage of development at which the damage occurs (e.g., Bregman & Goldberger, 1983a,b; Goldman & Rosvold, 1972; Johnson & Almli, 1978; Puchala & Windle, 1977; Windle, 1956). Mechanisms of behavioral sparing include behavioral compensation or substitution (Stein, Finger & Hart, 1983) and continued function of undamaged tissue (Eidelberg, Straehley, Erspamer, & Watkins, 1977). The amount of sparing is usually directly proportional to the amount of remaining neural tissue, as first suggested by Lashley (1938). Specific mechanisms of recovery are often undetermined (Goldberger, Bregman, Vierck, & Brown, 1990), but may include enhanced function of remaining tissue via deneri D.J.S. was supported by PHS-24442 from the National Institute of Neurobiological Diseases and Stroke. P.R.B. was supported by training grant PHS-2271. Address correspondence and reprint requests to Donald J. Stehouwer, Dept. of Psychology, Univ. of Florida, Gainesville, FL. 32611. 292
0163-1047/91 $3.00 Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
RECOVERY FROM SPINAL TRANSECTION
293
vation supersensitivity (Stelzner, Weber, & Prendergast, 1979), collateral sprouting of axons (Goldberger, 1973; Ichikawa, 1987, 1989; Murray, 1973), assumption of new functions by remaining tissue, and actual repair of damaged tissue (see Freed, Medinaceli, & Wyatt, 1985; Stein et al., 1983). It is often difficult to distinguish between the recovery due to anatomical reorganization within the CNS and that due to assumption of compensatory strategies (Sperry, 1947). Behavioral competence following spinal transection in tadpoles appears to depend on the level of spinal transection, with greater competence being associated with cervical than with thoracic spinal transections (cf. Forehand & Farel, 1982a; Sims, 1962; Piatt & Piatt, 1958; Stehouwer, 1986). However, no attempt was made to distinguish between sparing and recovery in those studies, and both sparing and recovery may have varied with the level of transection. For example, there may be greater sparing of function following cervical than following thoracic transection of the spinal cord because of the greater amount of CNS tissue caudal to the transection site in the former. Consistent with this idea, the capacity for normal function of the central pattern generators for swimming has been shown to be roughly proportional to the number of intact spinal cord segments in the lamprey (Cohen & Wallen, 1980) and bullfrog tadpole (Stehouwer & Farel, 1980). Behavioral recovery in tadpoles may vary with the level of spinal transection for a variety of reasons. First, there are more ascending and descending axons at cervical than at thoracic levels, so that more axons would have the opportunity to regenerate following the more rostral transection. Second, greater denervation of the spinal cord following cervical transections may stimulate greater denervation supersensitivity and collateral sprouting. Third, axons from different populations may grow across the two transection sites because, within limits, regeneration of severed axons is greater following lesions proximal to the cell body than following more distal lesions (Grafstein & McQuarrie, 1978; Richardson, Issa, & Aguayo, 1984, Yin & Selzer, 1983). Thus, brain stem neurons associated with locomotor command systems (Currie & Ayers, 1983; Grillner, 1981; McClellan, 1988) may cross cervical but not thoracic transections of the spinal cord. This study was undertaken to evaluate sparing and recovery of function following spinal transection in bullfrog tadpoles. Specifically, we measured (1) posture (2) the amount of spontaneous locomotion (3) locomotor response threshold to tactile stimulation rostral and caudal to the transection, and (4) intersegmental coupling of locomotor activity across the transection site. METHODS
Subjects. Subjects were 448 larval bullfrogs (Rana catesbeiana) obtained from Carolina Biological Supply. Larvae (tadpoles) ranged from developmental Stages IV to XIV according to the criteria of Taylor and Kollros
294
BRENNER AND STEHOUWER
(1946) and were maintained in oxygenated 38-liter aquaria at ambient room temperature on a diet of alfalfa for at least 1 week prior to surgery. Following surgery tadpoles were housed individually in oxygenated 3-liter aquaria and water was changed daily. Because spinally transected tadpoles were unable to feed during the postoperative period, food was withheld from 14 intact tadpoles for 4 weeks to control for the effects of food deprivation on performance on the behavioral tests used in this study. Surgery. All surgical procedures were performed following anesthesia induced by submerging the subject in a 0.2% tricaine methanesulfonate solution for 2 min, or by placing the subject in an ice and water slurry for 30 min, prior to surgery. Using a dissection microscope, the spinal cord was exposed by laminectomy and the dorsal vertebral artery was carefully deflected to one side to allow access to the spinal cord, which was then transected immediately below the obex (cervical transection) or between spinal roots 5 and 7 (midthoracic transection). Complete transection was verified visually by retracting the cut ends of the spinal cord. Function. Following spinal transection, behavioral competence of tadpoles was assessed once a week for 4 postoperative weeks on a battery of tests that included tests of posture, spontaneous locomotion, and cutaneously elicited swimming. Posture was evaluated by recording whether or not each tadpole was in an upright position. After recording posture, tadpoles were placed individually in 22.8-cm diameter containers marked into quadrants and filled with 1000 ml of water. After 30 min of adaptation to the apparatus, 4 h of spontaneous locomotion was recorded on time-lapse videotape at 100 frames per minute. The amount of locomotion was measured by recording the number of times a tadpole crossed quadrant markings. Finally, the ability of cutaneous stimuli to elicit swimming, defined as undulations of the tail caudal to the transection site, was measured. Tadpoles were stimulated on the snout and on the base of the tail with a set of 10 calibrated von Frey hairs (range of pressures, 0.03 to 3.9 g). Thresholds for eliciting swimming tadpoles with cervically and thoracically transected spinal cords were calculated by averaging the results of an ascending and a descending stimulus series. To assess intersegmental coordination across the transection site, two 0.18-mm Teflon-insulated, silver wire-hook electrodes were implanted in the axial musculature, one approximately 2 mm above the transection site and the other at the base of the tail. Electromyographic activity was recorded from freely swimming intact tadpoles and from those whose spinal cords had been thoracically transected either 4 or 28 days earlier. To determine whether intersegmental coordination could be achieved exclusively by growth of axons across the transection site, fictive locomotion was recorded in vitro from homolateral ventral roots above and below
RECOVERY FROM SPINAL TRANSECTION
295
the transection site of one isolated CNS preparation (see Stehouwer & Farel, 1980) obtained 28 days postoperatively. Anatomy. Immediately following the behavioral tests on Postoperative Week 4, H R P (Sigma, Type VI) was recrystallized onto the tip of a 00 insect pin. With the aid of a micromanipulator, the pin was inserted into the spinal cord approximately 2 mm caudal to the transection site and held in place until the H R P dissolved. In addition, H R P was inserted into the cervical and thoracic spinal cord in intact tadpoles to ascertain the source of descending spinal projections in normal animals. One week after the H R P application, the animals were sacrificed and the brains were perfused according to a modification of a procedure described by Rosen and Mesulam (1977). The CNS was dissected and placed in a 2.5% glutaraldehyde solution for 24 h and embedded in either albumin or gelatin. Sections were cut at 45/xm in the horizontal place mounted, and counterstained with cresyl violet. The total number of HRP-labeled axons and cell bodies rostral to the transection site were counted 1 week after H R P injection. Two tadpoles with thoracically transected spinal cords were discarded because of extracellular diffusion of H R P across the transection site. The nuclear localization of HRP-labeled cell bodies was based on the atlas of Kemali and Braitenberg (1969), and on H R P (Forehand & Farel, 1982b), fluorescence (Parent, 1973; Soller, 1977), and immunohistochemical (Ueda, Nojyo, & Sano, 1984) studies of the bullfrog brain stem. Statistical analysis. At the time of sacrifice, continuity of the spinal cord across the transection site was assessed visually and the data were analyzed by dividing tadpoles into groups that had or had not established gross continuity of the spinal cord. Analyses of variance were used to determine whether locomotor behavior changed as a function of postoperative recovery interval, lesion site, or spinal cord continuity. Differences in posture between cervical and thoracic tadpoles were analyzed by a X2 test. Student's t tests were used to evaluate differences in the number of labeled processes and brain stem cell bodies rostral to the cervical and thoracic spinal cord transection sites. All statistical tests were evaluated at the .01 level of significance. RESULTS
Anatomy. Of 370 tadpoles that received spinal transections followed by weekly behavioral testing, 107 survived the 5-week postoperative period. Of those, 36 of 62 tadpoles with thoracic transections and 33 of 45 tadpoles with cervical transections had reestablished gross continuity of the spinal cord. Most were constricted at the lesion site, and rostral and caudal stumps of cords that had not reestablished continuity were separated by gaps 1-3 segments in length.
296
BRENNER AND STEHOUWER
The white and gray matter lacked heavy glial scarring normally associated with mammalian spinal transections (Windle, 1956), but we found wide variability in the amount of growth across the transections, as have others (e.g., Forehand & Farel, 1982a; Hooker, 1925). Despite gross continuity of the spinal cord, axonal growth was abortive in many tadpoles, but many processes crossed the lesion sites in others (Fig. 1). Cell bodies were labeled in 14 tadpoles, exclusively supraspinally and primarily in the brain stem, and processes were labeled in 26 tadpoles. There were no differences between tadpoles with cervical and thoracic transection of the spinal cord with respect to the number of cell bodies (means = 3.0 and 1.75, respectively) or processes (means = 16.5 and 12.67, respectively), t's(30) < 1.33, p's > .05 that were labeled. Cells were labeled in the reticular formation, the raphe nuclei and vestibular nuclei in tadpoles transected at either level of the spinal cord (Figs. 2 and 3). Intact tadpoles injected with H R P cervically or thoracically had heavy labeling cells in the spinal cord, reticular formation, raphe nuclei, and vestibular nuclei, with sparse labeling of cells in the optic tectum. Function. Cervical and thoracic transection of the spinal cord severely disrupted posture, and many tadpoles lay on their backs or sides. There were no differences in posture as a function either of the degree of continuity of the spinal cord or of postoperative recovery interval in either group. However, more tadpoles with thoracic transections (70.0%) than cervical transections (42.7%) to the spinal cord maintained an upright posture X2(1, n = 340) = 25.67, p < .001. Because posture of bullfrog tadpoles is maintained by regulating inflation of the lungs, which in turn is controlled via cervical sympathetic and vagal nerves (Carlson & Luckhardt, 1920), one would expect greater sparing of posture in thoracic tadpoles because the cervical sympathetic nerves exit the cord between the cervical and thoracic transection sites. Spinal transection at either thoracic or cervical levels severely depressed spontaneous locomotion. Recovery of spontaneous locomotion was greater at longer postoperative recovery intervals F(3,345) = 8.64, p < .001 and in tadpoles that had reestablished anatomical continuity of the spinal cord F(2, 115) = 139.13, p < .001. Furthermore, the effect of postoperative recovery interval interacted with the level of transection F(3, 345) = 5.33, p < .01 and continuity F(3, 345) = 14.36, p < .001. Analyses of simple main effects revealed that recovery was greater in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those without continuity F(3,345) = 13.69, p < .001, whereas recovery of spontaneous locomotion was no greater in tadpoles with thoracically transected spinal cords that had reestablished gross continuity of the cord than in those that had not F(3, 345) = 1.95, p > .01. Sparing of locomotion was greater in tadpoles with thoracically transeeted spinal cords than in tadpoles with cervical transections to the
FIG. 1. This Stage IX tadpole with a thoracic transection of the spinal cord had a large number of processes crossing the transection site (arrowhead). In this and all subsequent photomicrographs, sections were cut in the horizontal plane and rostral is to the right. Calibration bar, 125 /~m; magnification, 100×.
--.a
Z
Z
©
¢b 0 <
Fl6. 2. HRP-labeled cells (arrows) were found in the vestibular nucleus of thoracic tadpoles. The lateral margin of the spinal cord is at th e top and the edge of the fourth ventricle is at the bottom of this photomicrograph. The eighth cranial nerve (asterisk) can be seen entering the brain stem immediately rostral to the labeled cell bodies of this Stage V tadpole. Calibration bar, 50/zm; magnification, 260 x .
b~
bl
U: 0 C
r~
>
7~
:Z
O0
F~. 3. Dark-field photomicrograph (left) reveals axonal processes labeled with HRP in the brain stem of a Stage V tadpole whose spinal cord had been thoracically transected. Calibration bar, 175 /xm; magnification, 102 x . Several cell bodies of the raphe nuclei (inset) are visible under greater magnification in the bright-field photomicrograph (right). Calibration bar, 25/zm; magnification, 408 x .
t~
Z
> Z r~
K
©
<
0
300
BRENNER AND STEHOUWER
&-~ e~e
Gross continuity
O~O
No continuity
°it
A~-.~..•Thoroclc
Cervical T r a n s e c t i o n s
20
•"
9
I
1
2
,
3
Transections
--0
,
4
I 1
I 2
I 3
I 4
POSTOPERATIVE WEEKS Fic. 4. The amount of spontaneous locomotion in each weekly test is plotted as the mean number of quadrants entered in 4 h for intact tadpoles and those with cervical and thoracic transections of the spinal cord. In this and subsequent figures, error bars represent standard errors of the means.
spinal cord, as indicated by a higher m e a n level of locomotion at Postoperative week 1. By Postoperative weeks 3 and 4 the level of spontaneous locomotion in tadpoles that had reestablished continuity of the spinal cord was similar regardless of level of transection (Fig. 4). These observations are supported by a significant main effect of level of transection F ( 1 , 1 1 5 ) = 20.09, p < .001 and a level X recovery interval interaction F(3, 345) = 11.33, p < .001. Figure 5 shows that the threshold for swimming elicited by cutaneous stimulation of the snout declined in all groups of spinally transected tadpoles as a function of recovery interval F ( 3 , 3 1 2 ) = 15.37, p < .001. The m e a n threshold of each group of tadpoles with cervical transections was higher than those with thoracic transections F ' s ( 1 , 1 0 4 ) > 9.72, p ' s < .01. Thresholds were lower in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those that had not F(1, 104) = 9.15, p < .01 whereas no such difference was found in tadpoles with thoracically transected spinal cords F(1, 104) = .21, p >
4.0 D "I" ttl w
3.0
O
~
0
T~-----T~
Cervical Transectlons e--e
^
O~O
&~&
T h o r a c i c Tronsections Gross continuity No continuity Intoct
!
2.0 0
I 1
I
2
I 3
I 4
I
I
POSTOPERATIVE WEEKS FxG. 5. Mean thresholds for locomotion elicited by cutaneous stimulation rostral to the transection site are plotted as a function of postoperative interval.
301
RECOVERY FROM SPINAL TRANSECTION
, ~ 4.0
Thoracic Tronsections
Cervical T r a n s a c t i o n s
0 "1o') laJ
e--e Gross continuity 0--0 NO continuity A - - A , Intoct
3.0 2.0
I-. Z
"5
er ~ ,
1 i.o
~
.
A~
T
~@~---0
T
T 0
3
4
o
v
I
P 2
~ 0.0 1
2
I 3
I 4
POSTOPERATIVE WEEKS FI6. 6. M e a n thresholds for locomotion elicited by cutaneous stimulation caudal to the transection site are plotted as a function of postoperative interval.
.01. The threshold for swimming elicited by cutaneous stimulation at the base of the tail (Fig. 6) was elevated only slightly by either transection and declined as a function of postoperative recovery interval F(3,312) = 10.79, p < .001. Myotomal activity recorded from homolateral axial muscles of the cervical and tail regions was coupled during locomotion of all 4 intact tadpoles (Fig. 7a), in 4 of 10 tadpoles whose cords were thoracically transected 4 days previously (Fig. 7b), and in 11 of 17 tadpoles with cords transected 28 days previously (Fig. 7C). Four days of recovery is not enough time for axons to grow across the transection site (Forehand & Farel, 1982a; Hooker, 1925), so any coordination across the transection within 4 postoperative days m u s t be mediated by the peripheral transmission of locomotor activity around the transection site (see Discussion). Some of the transected animals could not be induced to swim, but in no case did we observe uncoupled activity. However, initiation and coordination of locomotion can be mediated centrally in recovered tadpoles, because tictive locomotion recorded from homolateral ventral roots of isolated CNS preparations in vitro may also be coupled across the transection site (Fig. 7d). However, anatomical restitution of the spinal cord occurs infrequently and involves only modest numbers of processes crossing the transection site (vide supra). Many attempts to obtain a viable preparation for study in vitro failed either because there was no anatomical repair or because the few axons that may have grown across the transection site were damaged during dissection. DISCUSSION
These experiments demonstrate sparing and recovery of function and growth of axons across spinal transections in tadpoles. We found greater sparing of posture, spontaneous locomotion, and stimulus-induced locomotion in tadpoles with thoracic transections than in tadpoles with cervical
302
BRENNER AND STEHOUWER
.O.T.AL .Jdk TAIL
If. I dih~.
. . ~/l h . . .
d ..~ill~,l.
TAIL
. . . . .
100 ms
i00 ms a
C
INTACT
ROSTRAL
~
Jill,
If
'T . . . . . . .
TAIL
28 DAYS POST-OP
r
/ ....
100 ms
b 4 D A Y S POST-OP
'~, i~,, d 5orris 28 DAYS POST-OP
FIG. 7. Electromyograms show that during swimming, activity of ipsilateral myotomes of cervical and tail segments was coupled in (a) intact tadpoles and in tadpoles which had been thoracically transected (b) 4 days and (c) 28 days previously. Ventral root recordings obtained 28 days postoperatively during an episode of flctive locomotion in vitro (d) show that activity of homolateral ventral roots rostral (VR4) and caudal (VR8) to a thoracic transection was coupled in the isolated CNS.
transections of the spinal cord. However, behavior of tadpoles with cervical spinal transections improved more than those with thoracic transection, so that performance after 4 weeks of recovery was similar. Two lines of evidence suggest that anatomical repair contributes behavioral recovery in the present study. First, recovery of spontaneous locomotion and locomotion elicited by stimulation of the snout was greater in tadpoles with cervically transected spinal cords that had reestablished gross continuity of the cord than in those that had not. Second, synchronous activity of homolateral ventral roots was recorded across the transection site during fictive swimming of the isolated CNS, providing direct evidence that anatomical repair was functionally effective. The ability of so few axons to initiate and coordinate locomotor activity across the transection site is not surprising, because relatively few spinal axons are necessary to mediate considerable function in other systems (Eidelberg et al., 1977; Windle, Smart & Beers, 1958), and gross electrical (Stehouwer & Farel, 1980) or pharmacological stimulation (McClellan & Farel, 1985) of the isolated tadpole spinal cord can elicit fictive locomotion. To the best of our knowledge, the present study offers the only demonstration
RECOVERY FROM SPINAL TRANSECTION
303
of coordination of fictive locomotion across the transection site of an isolated preparation in an animal other than the lamprey (McClellan, 1988). Although reestablishment of specific connections is not necessary to excite spinal locomotor circuits, a few nonspecific connections cannot orchestrate rapid and precise adjustments during locomotion, and our tadpoles showed no evidence of vestibular control during swimming. The amount of anatomical repair found in this study was similar to that reported following mammalian spinal cord injuries (cf. Bernstein, Bechard, & Stelzner, 1981; Brown & McCouch, 1947; Clemente & Windle, 1954). We may have found relatively modest central repair because tadpoles at the developmental stages we studied are relatively static with respect to growth of descending projections (Forehand & Farel, 1982c; Gona, 1972; 1975), and the amount of repair appears to be directly related to the number of proliferating and differentiating neurons available both in amphibians (Davis, Duffy, & Simpson, 1989; Forehand & Farel, 1982a; Holtzer, 1955) and in mammals (Bregman & Goldberger, 1983a,b; Holder & Clarke, 1988). Although we hypothesized that the greater amount of tissue caudal to cervical than to thoracic transection of the spinal cord may result in greater sparing of locomotion in the former, we found exactly the opposite. Greater sparing of locomotion may occur in tadpoles with thoracic transection of the cord because each ventral and dorsal root projects to more than one myotome (Fetcho, 1987; Letinsky; 1974), so that roots immediately rostral to the transection innervate myotomes caudal to the transection and vice versa. This overlapping innervation allows for reflex transmission of locomotion around the transection site. Because no spinal roots exit rostral to our cervical transections, this mechanism of functional sparing was unavailable to those tadpoles and the initial loss of behavior was greater. As central axons grew across the transection site, locomotor performance of tadpoles with cervical transection of the cord improved until it was equal to that of those with thoracic transections 3 and 4 weeks after surgery. Published reports indicate that there is less recovery following thoracic transection (Forehand & Farel, 1982a; Sims, 1962) than following cervical transection (Piatt & Piatt, 1958; Stehouwer, 1986) in tadpoles. The present study suggests that those apparent differences result from differences in sparing and not recovery; following thoracic transections, there is considerable sparing of function, which masks recovery resulting from central repair. The qualitative behavioral assessments used to assess function in other studies were probably not sensitive enough to detect that recovery. Following cervical transection there is so little sparing that recovery is readily apparent. The results of the present study show that sparing and recovery both contribute to behavioral competence following spinal cord transection and
304
BRENNER AND STEHOUWER
u n d e r s c o r e t h e i m p o r t a n c e o f u s i n g t e s t p r o c e d u r e s t h a t a l l o w o n e to distinguish between those two phenomena, which are often confounded in studies of CNS injury.
REFERENCES Bernstein, D. R., Bechard, D. E., & Stelzner, D. J. (1981). Neuritic growth maintained near the lesion site long after spinal transection. Neuroscience Letters, 26, 55-60. Bregman, B. S., & Goldberger, M. E. (1983a). Infant lesion effect: II. Sparing and recovery of function after spinal cord damage in newborn and adult cats. Developmental Brain Research, 9, 119-135. Bregman, B. S., & Goldberger, M. E. (1983b). Infant lesion effect: III. Anatomical correlates of sparing and recovery of function after spinal cord damage in newborn and adult cats. Developmental Brain Research, 9, 137-154. Brown, J. O., & McCouch, G. P. (1947). Abortive regeneration of the transected spinal cord. Journal of Comparative Neurology, 87, 131-137. Carlson, A. J., & Luckhardt, A. B. (1920). Studies on the visceral sensory nervous system. I. Lung automatism and lung reflexes in the frog (R. pipiens and R. catesbiana). American Journal of Physiology, 54, 55-95. Clemente, C. D., & Windle, W. F. (1954). Regeneration of severed nerve fibers in the spinal cord of the adult cat. Journal of Comparative Physiology A, 160, 181-193. Cohen, A. H., & Wallen, P. (1980). The neural correlate of locomotion in fish. Experimental Brain Research, 41, 11-18. Currie, S. N., & Ayers, J. (1983). Regeneration of locomotor command systems in the sea lamprey. Brain Research, 279, 238-240. Davis, B. M., Duffy, M. T., & Simpson, S. B. (1989). Bulbospinal and intraspinal connections in normal and regenerated salamander spinal cord. Experimental Neurology, 103, 41-51. Eidelberg, E., Straehley, D., Erspamer, R., & Watkins, C. J. (1977). Relationship between residual hind limb-assisted locomotion and surviving axons after incomplete spinal cord injuries. Experimental Neurology, 56, 312-322. Fetcho, J. R. (1987). A review of the organization and evolution of motoneurons innervating the axial musculature of vertebrates, Brain Research Reviews, 12, 243-280. Forehand, C. J., & Farel, P. B. (1982a). Anatomical and behavioral recovery from the effects of spinal cord transection: Dependence on metamorphosis in anuran larvae. Journal of Neuroscienee, 2, 654-662. Forehand, C. J., & Farel, P. B. (1982b). Spinal cord development in anuran larvae: I. Primary and secondary neurons. Journal of Comparative Neurology, 209, 386-394. Forehand, C. J., & Farel, P. B. (1982c). Spinal cord development in anuran larvae: II. Ascending and descending pathways. Journal of Comparative Neurology, 209, 386-394. Freed, W. J., Medinaceli, L., & Wyatt, R. J. (1985). Promoting functional plasticity in the damaged nervous system. Science, 227, 1544-1552. Goldberger, M. E. (1973). Restitution of function and collateral sprouting in the cat spinal cord: The deafferented animal. Anatomical Record, 175, 329. Goldberger, M. E., Bregman, B. S., Vierck, C. J., & Brown, M. (1990). Criteria for assessing recovery of function after spinal cord injury: Behavioral methods. Experimental Neurology, 107, 113-117. Goldman, P. S., & Rosvold, H. E. (1972). The effect of selective caudate lesions in infant and juvenile rhesus monkeys. Brain Research, 43, 53-66. Gona, A. G. (1972). Morphogenesis of the cerebellum of the frog tadpole during spontaneous metamorphosis. Journal of Comparative Neurology, 146, 133-142.
RECOVERY FROM SPINAL TRANSECTION
305
Gona, A. G. (1975). Golgi studies of cerebellar maturation in frog tadpoles. Brain Research, 95, 132-136. Grafstein, B., & McQuarrie, I. G. (1978). Role of the nerve cell body in axonal regeneration. In C. W. Cotman (Ed.), Axonal regeneration (pp. 155-195). New York: Raven Press. Grillner, S. (1981). Control of locomotion in bipeds, tetrapods, and fish. In V. Brooks (Ed.), Handbook of physiology: Motor control (pp. 1179-1236). American Physiological Society. Holder, N., & Clarke, J. D. W. (1988). Is there a correlation between continuous neurogenesis and directed axon regeneration in the vertebrate nervous system. Trends in Neuroscience, 11, 94-99. Holtzer, H. (1955). Comments on regeneration as opposed to neogenesis of amphibian neurons. In W. F. Windle (Ed.), Regeneration in the central nervous system (pp. 8183). Springfield, IL: Thomas. Hooker, D. (1925). Studies on regeneration in the spinal cord: III. Journal of Comparative Neurology, 38, 315-345. Ichikawa, M. (1987). Synaptic reorganization in the medial amygdaloid nucleus after lesion of the accessory olfactory bulb of adult rat. I. Quantitative and electron microscopic study of the recovery of synaptic of density. Brain Research, 420, 243-252. Ichikawa, M. (1989). Recovery of olfactory behavior following removal of accessory olfactory bulb in adult rat. Brain Research, 498, 45-52. Johnson, D. A., & Almli, C. R. (1978). Age, brain damage and performance. In S. Finger (Ed.), Recovery from brain damage: Research and theory (pp. 115-134). New York: Plenum Press. Kemali, M., & Braitenberg, B. (1969). Atlas of the frog's brain. New York: Springer-Verlag. Lashley, K. S. (1938). Factors limiting recovery after central nervous system lesions. Journal of Nervous and Mental Disorders, 88, 733-755. Letinsky, M. S. (1974). The development of nerve-muscle junctions in Rana catesbiana tadpoles. Development Biology, 40, 129-153. McClellan, A. D. (1988). Functional regeneration of descending brainstem command pathways for locomotion demonstrated in the in vitro lamprey CNS. Brain Research, 448, 339-345. McClellan, A. D., and Farel, P. B. (1985). Pharmacological activation of locomotor patterns in larval and adult frog spinal cords. Brain Research, 332, 119-130. Murray, M. (1973). Restitution of function and collateral sprouting in the cat spinal cord: The hemisected animal. Anatomical Record, 175, 395. Parent, A. (1973). Distribution of monamine containing neurons in the brainstem of the frog, Rana temporaria. Journal of Morphology, 139, 67-78. Piatt, J., & Piatt, M. (1958). Transection of the spinal cord in the adult frog. Anatomical Record, 131, 81-95. Puchala, E., & Windle, W. F. (1977). The possibility of structural and functional restitution after spinal cord injury. A review. Experimental Neurology, 55, 1-42. Richardson, P. M., Issa, V. M. K., & Aguayo, A. J. (1984). Regeneration of long spinal axons in the rat. Journal of Neurocytology, 13, 165-182. Rosen, C. D., & Mesulam, M. M. (1977). Fixation variables in horseradish peroxidase neurohistochemistry: The effects of fixation time and perfusion procedure upon enzyme activities. Journal of Histochemistry, 26, 28-39. Sims, R. T. (1962). Transection of the spinal cord in developing Xenopus laevis. Journal of Embryology and Experimental Morphology, 10, part 2, 115-126. Soller, W. R. (1977). Monoaminergic inputs to frog motoneurons: An anatomical study using fluorescence histochemical and silver degeneration techniques. Brain Research, 122, 445-458.
306
BRENNER AND STEHOUWER
Sperry, R. W. (1947). Effects of crossing nerves to antagonistic limb muscles in the monkey. Archives of Neurological Psychiatry, 58, 452-473. Stehouwer, D. J. (1986). Behavior of larval and juvenile bullfrogs (Rana catesbeiana) following chronic spinal transection. Behavioral and Neural Biology, 45, 120-134. Stehouwer, D. J., & Farel, P. B. (1980). Central and peripheral controls of swimming in anuran larvae. Brain Research, 195, 323-335. Stein, D. G., Finger, S., & Hart, T. (1983). Brain damage and recovery: Problems and perspectives. Behavioral and Neural Biology, 37, 185-222. Stelzner, D. J., Weber, E. D., & Prendergast, J. (1979). A comparison of the effect of thoracic spinal hemisection in the neonatal or weanling rat on the distribution and density of dorsal root axons in the lumbosacral spinal cord of the rat. Brain Research, 172, 407-426. Taylor, A. C., & Kollros, J. J. (1946). Stages in the normal development of Rana pipiens larvae. Anatomical Record, 947 7-24. Ueda, S. Nojyo, Y., & Sano, Y. (1984). Immunohistochemical demonstration of the serotonin neuron system in the central nervous system of the bullfrog, Rana catesbeiana. Anatomical Embryology, 169, 219-229. Windle, W. F. (1956). Regeneration of axons in the vertebrate central nervous system. Physiological Reviews, 36, 427-440. Windle, W. F., Smart, J. O., & Beers, J. J. (1958). Residual function after subtotal spinal cord transection in adult cats. Neurology, 8, 518-521. Yin, H. S., & Seizer, M, E. (1983). Axonal regeneration in lamprey spinal cord. Journal of Neuroscience, 3, 1135-1144.