Developmental Brain Research, 58 (1991) 257-264 Elsevier
257
BRESD 51224
c-DOPA-induced air-stepping in decerebrate developing rats T. Iwahara ~, C. Van H a r t e s v e l d t 2, E. Garcia-Rill I and R . D . S k i n n e r t 1Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, A R 72205-7199 (U.S.A.) and eDepartment of Psychology, University of Florida, Gainesville, FL 32611 (U.S.A.) (Accepted 30 October 1990) Key words: L-3,4-Dihydroxyphenylalanine; Locomotion; Decerebrate; Rat
Developing rats were anesthetized and a precollicular brainstem transection performed. Administration of L-3,4-dihydroxyphenylalanine (L-DOPA, 100 mg/kg, s.c.) induced locomotion in groups of animals 0-3, 6-8, 14-16 and 20-22 days of age. At early stages in development (0-3 days), continuous, long-lasting air-stepping was induced consisting of 4-limb walking with stronger alternation in the forelimbs compared to the hindlimbs. At 6-8 days of age, continuous air-stepping was characterized by better agreement between forelimb and hindlimb step cycles. By 14-16 days of age, the hindlimbs showed stronger and faster stepping than the forelimbs. The first evidence of consistent galloping was observed in this age group. In some cases, the forelimbs were held extended and only the hindlimbs walked or galloped. At 20-22 days, walking and galloping became episodic and the hindlimbs typically galloped while the forelimbs alternated. Each age group was tested for overground locomotion. Only in the 20-22 day group was weight-bearing overground stepping observed. At earlier ages, limb alternation was evident but of insufficient strength to lift the body off the ground. In general, the duration of the effect of the same dose of L-DOPA decreased with age. Animals in each group which received a mid-thoracic spinal cord transection showed forelimb alternation but no hindlimb stepping. These results indicate that L-DOPA induces stepping by activating brainstem and/or spinal centers via an as yet unknown mechanism. The pattern of the development of gait (forelimb to hindlimb gradient) at various ages was similar to that observed in intact developing rats. INTRODUCTION T h e characteristics of adult locomotion have been described quantitatively for n u m e r o u s v e r t e b r a t e and i n v e r t e b r a t e species, and much of the underlying neural organization mediating stepping has been worked out (see e.g.12). The d e v e l o p m e n t of stepping has been studied less extensively, although information on amphibians ~3"26 and birds 18'19 shows interlimb coordination is present at the last stages of embryonic life. Walking m o v e m e n t s have been described in m a m m a l i a n fetuses (cat 5, human14). In the rat, previous studies have revealed that evidence of interlimb coordination is present at birth 4. In these studies, swimming rather than walking was studied in o r d e r to minimize any effect of weak limb muscles in young rats. Using the limb-attached in vitro b r a i n s t e m - s p i n a l cord p r e p a r a t i o n , we have shown that electrical stimulation of the brainstem of 0 to 4-day-old rats produces an adult-like pattern of locomotor muscular contractions 1. Recent evidence suggests that c o o r d i n a t e d limb alternation in the rat fetus may begin at e m b r y o n i c day 18, although m o r e primitive cocontractions ('kicking' or 'hatching'-type movements) may be present as early as e m b r y o n i c day 1516. On the o t h e r hand, not all aspects of l o c o m o t o r control
may be present at birth. While increasing stimulus amplitude ~'3, a p p r o p r i a t e chemical stimulation x'/s or deafferentation 2 can lead to faster hindlimb alternation in the in vitro p r e p a r a t i o n , evidence of galloping (as defined in the adult) has not been o b s e r v e d so soon after birth. That is, in the adult, the alternating l e f t - r i g h t muscular contractions evident in a walk are shifted to a fast walk and then a gallop, at which point cocontraction of left and right limb muscles is evident. This usually is a c c o m p a n i e d by a decrease in step or stroke cycle duration ~2. Studies of stroke cycle duration in the developing, swimming rat r e p o r t e d a gradual decrease (faster alternation) in duration from birth until two weeks of age without much change t h e r e a f t e r 4, but galloping as such was not described. While swimming is an ideal way to minimize the effects of gravity and c o m p e n s a t e for w e a k limb muscles, it m a y not be a m e n a b l e to studying rapid galloping m o v e m e n t s due to the resistance of limb m o v e m e n t through the water. A i r - s t e p p i n g ( s u s p e n d e d b o d y with limbs free to move) offers an o p p o r t u n i t y to study such movements. M o r e o v e r , the d e c e r e b r a t e p r e p a r a t i o n eliminates the contribution of volational elements and allows the recording of e l e c t r o m y o g r a p h i c activity without the need for chronic i m p l a n t a t i o n techniques. R e c e n t findings suggest that long-lasting locomotion
Correspondence." E. Garcia-Rill, Department of Anatomy, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205-7199, U.S.A. 0165-3806/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
258 can be i n d u c e d in the d e v e l o p i n g rat after a d m i n i s t r a t i o n of L - 3 , 4 - d i h y d r o x p h e n y l a l a n i n e ( L - D O P A ) (subcutan e o u s 21, intraperitoneal6). L - D O P A has b e e n used to
fixative overnight and in 20'~4 sucrose tk)r at least ~ days. Frozen sections (60 **m sagittal) were cut and stained for Cresyl violet to confirm the level of brainstem transection, essentially, precollicularmidnigral in all the cases reported herein.
elicit s t e p p i n g in the adult spinal cat ( i n t r a v e n o u s 11). T h e p r e s e n t studies t o o k a d v a n t a g e of this effect and w e r e d e s i g n e d to test the effects of a single c o n c e n t r a t i o n of
RESULTS
L - D O P A on d e c e r e b r a t e , a i r - s t e p p i n g d e v e l o p i n g rats at v a r i o u s stages in d e v e l o p m e n t . P r e l i m i n a r y results h a v e b e e n r e p o r t e d 8.
MATERIALS AND METHODS The use of timed-pregnant Sprague-Dawley dams allowed the accurate determination of the date of birth of rat pups. The day of birth was designated as 0 day postnatally and litters culled to 8 pups. Four age groups were chosen for study at birth (0-3 days), 1 week of age (6-8 days), 2 weeks of age (14-16 days) and 3 weeks of age (20-22 days). No more than 2 pups from any litter formed part of a group and most litters used contributed at least 1 member to each group. The younger 2 age groups were anesthetized with penthrane while the older 2 groups were anesthetized with halothane. Some subjects in the 14-16 day group (3/7) were anesthetized with penthrane and no differences were found in the effects observed between those subjected to penthrane compared to those anesthetized with haiothane. Once a surgical plane of anesthesia was established (e.g. absence of withdrawal reflex), a brainstem transection such as that used in the adult rat preparation was carried out 23. Typically, anesthesia was maintained for 10-15 min during the surgical procedure. The precollicular decerebration was performed using suction ablation and anesthesia discontinued. Electromyographic (EMG) recordings were carried out using double hookedwire electrodes inserted into one extensor muscle in each limb, usually the m. triceps brachii in the forelimbs and the gastrocnemius-soleus group in the hindlimbs. L-DOPA (100 mg/kg) dissolved in vehicle (50 mg L-DOPA in 0.4 ml 1 N HCI and 0.1 ml distilled water, mixed until clear, then added 4.5 ml 0.1 M Na2HPO 4, pH 6.2) was injected subcutaneously at the nape of the neck, making sure to maintain pressure on the skin after needle withdrawal to prevent leakage. At least 30 rain elapsed following discontinuation of anesthesia before L-DOPA was injected. For the present set of studies, a single dose was chosen pending further determinations of dose-response curves at each age. If an effect (induced stepping) was not present after 20 min, a second injection was administered and, occasionally, a third injection was necessary (see below), again, administered no earlier than 20 min following the second injection. Considering the levels of DOPA decarboxylase present at the ages tested 2°, this period of time was considered sufficient for metabolism of the L-DOPA. Animals were tested for overground stepping on a smooth surface and for air-stepping by suspension using a strip of tape around the midriff which did not interfere with limb movement. In some cases (n = 4, one subject in each age group) following decerebration, a mid-thoracic transection of the spinal cord was performed using suction ablation. This allowed testing of L-DOPA-induced locomotion after spinal transection. The completeness of the transection was confirmed visually as a 2-3 mm gap between the proximal and distal ends of the spinal cord. In another series of experiments, two subjects in each age group were subjected to a spinal cord transection after stepping had been induced by L-DOPA, Because stepping movements can be induced in decerebrate and spinal animals by sensory stimulation, once L-DOPA was administered, no such stimuli were administered in order not to influence the latency to the onset of stepping. Locomotion was recorded on FM tape (EMGs) and on video tape. At the end of the experiment, animals were sacrificed with an overdose of barbiturate and the brainstem removed and stored in
Effects o f decerebration In the a b s e n c e of I _ - D O P A o r f o l l o w i n g an i n j e c t i o n of vehicle
(pH
equally
inactive.
6.2),
decerebrate No
developing
spontaneous
or
rats
were
rhythmic
limb
m o v e m e n t s w e r e o b s e r v e d at any of the ages testedl M u s c l e t o n e was e v i d e n t as early as 15 min following the discontinuation stimuli
(e.g.
of anesthesia. tail
or
pinna
Responses
pinch)
to
noxious
produced
scratch
r e f l e x - t y p e r e s p o n s e s starting 1 5 - 2 0 min f o l l o w i n g the e n d of anesthesia. observed
Limb alternation
f o l l o w i n g such
stimuli
but
o c c a s i o n a l l y was the
movements
s t o p p e d s o o n after the cessation of stimulation. W h e n tested on a surface, t h e r e was n o e v i d e n c e of standing p o s t u r e . W h e n s u s p e n d e d , all d e c e r e b r a t e animals h u n g limply. For a d e s c r i p t i o n of the b e h a v i o r o f the intact d e v e l o p i n g rat, see the c o m p a n i o n article 21'2s.
£ffects o f L - D O P A T h e m a i n characteristics of the effects o f i n j e c t i o n s of the s a m e d o s e of L - D O P A at v a r i o u s ages are o u t l i n e d in Table I. In g e n e r a l , d e c e r e b r a t e d e v e l o p i n g r a t s e x h i b i t e d L - D O P A - i n d u c e d air-stepping a f t e r o n e or two injections, resulting in s t e p p i n g b e g i n n i n g a p p r o x i m a t e l y 10 min
after
the
injection
and
lasting
for 3 0 - 9 0
min.
H o w e v e r , t h e r e w e r e significant d i f f e r e n c e s b e t w e e n the v a r i o u s age g r o u p s tested and e a c h will be c o n s i d e r e d individually.
0 - 3 Days. Within 5 min a f t e r i n j e c t i o n of L - D O P A , d e c e r e b r a t e subjects b e g a n to s h o w an i n c r e a s e in the background
EMG
and the tail was h e l d h o r i z o n t a l l y
while the h e a d was raised slowly a n d h e l d in a line with
TABLE I
Effects of L-DOPA on neonate rats A KruskaI-Wallis test, which is a non-parametric (distribution-free) analogue of analysis of variance, showed there to be a significant effect (P < 0.05) of age upon duration. Univariate t-tests then showed the 0--3 day group to be different from the 6-8 day group (P < 0.05*) and from the 14-16 day group (P < 0.01 **).
Age (days)
n
Mean no. of injections
Mean latency to effect (rain)
Mean duration of effect (rain)
0-3 6-8 14-16 2{)-22
9 9 7 11
1.4_+0.7 1.9 + 0.8 2.0_+0.8 1.9 -+ tl.7
8.1 +3.9 9.6 + 5.9 8.1-+5.0 9.6 _+ 5.5
80.2_+24.2 42.3 -+ 28.6* 29.3_+22.8** 49.0 _+36.6
259
O-3Days 30min
post
L'DOPA
,,
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R,, ., LHL
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t 4sec Fig. I. L-DOPA induced locomotion in a 0-3 day rat. Recordings of EMGs from muscles of the left forelimb (LFL), right forelimb (RFL), left hindlimb ( L H L ) and righ hindlimb ( R H L ) show good alternation between left and right sides, but without exact agreement between forelimbs and hindlimbs (note out-of-phase relationships). Moreover, there were lapses (arrow) in the regularity of hindlimb stepping. Episode shown was recorded 30 min after an injection of L-DOPA (I00 mg/kg, s.c.). Stepping frequency in this record was 0.7 Hz.
the spine (not completely upright as in a typical standing posture). Gradually, one or two limbs began to alternate until all 4 limbs air-stepped continuously (The mean latency following one injection of L-DOPA was 7.7 _+ 2.6 min while it was 9.0 _+ 6.6 min following a second injection when necessary. When both groups are pooled, 8.1 _+ 3.9 rain was the mean and standard deviation of the latency to 4 limb alternation). In all cases, the forelimbs alternated first, occasionally alternated faster than the hindlimbs at the peak of the effect, and kept alternating after the hindlimbs had stopped when the effect was waning. The mean duration of 4 limb air-stepping was 80.2 _+ 24.2 (S.D.) min in this age group. The mean
frequency of forelimb stepping at the peak of the effect was 0.7 _+ 0.5 (S.D.) Hz. Mean frequency at peak of effect was determined as the highest mean cycle time of 10 consecutive steps. An additional injection of L-DOPA did not induce faster stepping but merely extended the duration of the effect• Fig. 1 is a representative example of the EMGs recorded in each limb in a subject in this age group. The frequency of air-stepping of the forelimbs in this case was 0.7 Hz and quite consistent, contrasting with the hindlimb frequency of 0.6 Hz, which showed inconsistencies (arrow) in terms of agreement between the step cycles of fore- and hindlimbs. In some cases (3/9), it was necessary to administer a second injection in order to induce air-stepping. As evident in Table II, the mean latency was slightly longer (9.0 _+ 6.6 min) in subjects requiring more than one injection than those stepping after a single injection (7.7 +_ 2.6 min). The duration of the effect was longer after more than one injection (84.7 _ 24.4 min) than after a single injection (75.7 +_ 28.4 min). These and all other measures in Table II were not statistically significant but give a good idea of the trends observed. When tested overground, none of the subjects could hold their chins above ground although the tails were held suspended. The limb alternation was insufficient to propel the animal, which rested on its abdomen throughout. 6-8 Days. The postural effects observed at 0-3 days also were seen in the 6-8 day group. The group mean latency to 4 limb air-stepping was 9.6 + 5.9 min (Table I), similar to that of the younger group. However, the mean duration of effect was significantly (t-test, P < 0.05) shorter (42.3 _+ 28.6 min). In this group, the forelimbs also were the first to alternate although there
6-8Days 15min post L-DOPA
L F L , , L ~ k i . kL [ k L. x k L A ~ L [[ -, ~
T A B L E II
TF'rr¢
Effects of single vs multiple injections of L-DOPA Age (days)
n
1 vs > 1
0-3
6 3
6-8
3 6
14-16
2 5
1 inj. >linj. (range 1-2) 1 inj. >1 inj. (range l-3) lin i. >1 inj. (range 1-3) 1 inj. >1 inj. (range 1-3)
20-22
3 8
Mean latency (min)
75.7 + 28.4 84.7_+24.4
11.0 +- 8.2 8.8_+5.3
69.7 +_ 37.3 32.4_+1(1.6
13.5_+4.9 6.0_+3.4
29.0_+ 1.4 29.2_+25.5
13.7 _+ 6.4 8.1 +_4.7
27.5 + 10.6 59.8_+41.6
r '
?rrrrl,I
Mean duration (min)
7.7 + 2.6 9.0_+6.6
''
r
'J
'
I
Ir
v
,
"
RHL
4sec Fig. 2. L-DOPA induced locomotion in a 6-8 day rat. Recordings of EMGs as in Fig. l showing good agreement between left and right sides and in-phase relationship between forelimbs and hindlimbs, 15 rain after an injection of L-DOPA. Stepping frequency in this record was 0.8 Hz.
260 14 - 1 6 D a y s post
A
LHL-
-
~
~
,
C
B
I
L F L .....
L.DOPA
-
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L.I...L. Ll., .l.
f4@ 2sec
Fig. 3. L-DOPA induced locomotion in 14-16 day rats. A: hindlimb alternation with no forelimb alternation evident 12 rain after L-DOPA. Stepping frequency in this record was 1.8 Hz. B: another subject in this group which showed alternation in forelimbs and ~ m b s . The sequence shown occurred 15 min after L-DOPA and includes the transition from walk to gallop in the hindlimbs (note mixed alternation and cocontraction). Stepping frequency 1.5 Hz. C: a third subject which showed forelimb alternation and faster hindlimb cocomractions 10 rain after L-DOPA. Stepping frequency 1.3 Hz.
was not as g r e a t a difference between the rates of alternation o f the two pairs of limbs. Fig. 2 is a representative recording from a subject in this g r o u p showing a stepping frequency of about 0.8 H z for b o t h forelimbs and hindlimbs. A l t h o u g h there was g r e a t e r a g r e e m e n t b e t w e e n the fore- and hindlimb step cycles, the match was not as exact as in the adult d e c e r e b r a t e electrically induced locomotion 23. The mean frequency o f stepping at the p e a k of the effect was 0.7 + 0.5 Hz. W h e n tested overground, these subjects were capable of maintaining both h e a d and tail erect and lifting the t h o r a x from the ground. The limbs varied between alternation and placing, the purchase of the paws on the ground (placing) a p p e a r i n g to interfere with alternation. 14-16 Days. A m a j o r difference between this age group and the younger groups was the shift almost
exclusively to hindlimb air-stepping. In all cases, hindlimb alternation was the first to occur and, in some cases (3/7), no forelimb alternation was evident, as both forelimbs were held in the 'swimming' position 5. T h e latency to hindlimb alternation was similar to that of the o t h e r groups (8.1 + 5.0 min, Table I), although the duration of the effect was s h o r t e r than in the 0 - 3 day group (29.3 + 22.8 min) (t-test, P < 0.01). Most subjects (5/7) in this group required m o r e than one injection (Table II). Fig. 3 A is a representative e x a m p l e o f the recordings o b t a i n e d in this group, showing a p e a k effect of hindlimb alternation at a m e a n frequency o f 1.8 + 0.9 Hz, with n o
20-22Days spinal trans 20rain
post
L.DOPA
20-22Days 15min post L.DOPA
t L,LL,iL
1,1 , i l l llt,tttT,llr!~l
•
RFL
~.~ RHL
LJ,~|ILIlLI=AJI=, l l T t l T t t I It, l i T , w -
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Fig. 4. L-DOPA induced locomotion in a 20-22 day rat, Episodic alternation and coeontraction (mostly in hindlimbs) evident 15 rain after L-DOPA. Stepping for 4-6 s which was interrupted by 4-6 s recurred for prolonged periods. Stepping frequency in this record was 3.5 Hz.
4sec
Fig. 5. L-DOPA induced locomotion in a mid-thoracic spinal rat 20-22 days. Consistent forelimb alternation was evident, with the segment shown occurring 20 min after L-DOPA. Note absence of hindlimb movement in a mid-thoracic spinal transccted subject, Stepping frequency in this record was 1.3 Hz.
261 Days
6-8
post
L-DOPA
B
C
post trans l
Llkl,lltl,., ,rrTrr • _J,. ''|'
t =,, ,e,rr,r,,
•
,
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15rain post
trans
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4sec Fig. 6. L-DOPA induced locomotion before and after spinal transection at 6-8 days. A: 4-limb alternation elicited 20 min after L-DOPA administration. B: perseverance of forelimb alternation immediately after mid-thoracic transection. C: forelimb alternation effect was waning 15 rain after transection, accounting for the slower frequency of stepping. Stepping frequency was 1.3 Hz in A and 1.2 Hz in B. Although slower, forelimb alternation after transection appeared more vigorous.
forelimb alternation in this case. A second major difference in this group was the presence of galloping (4/7). The mean frequency during these brief episodes of galloping was 1.5 + 0.9 Hz. Fig. 3B and 3C are different examples of the transitions in stepping frequency obtained in this group, showing cocontraction of the hindlimbs mixed with alternation, as well as cocontractions of the hindlimbs simultaneously with alternation of the forelimbs.
14-16
Days
A 8min
13
post
~Tr-V r'l~+r ~ ~
L.DOPA
r ~,-1,-r-,
15min
post
tr;lul'.;
r I,
~ttiJJl~ALiiaLi. LJi~t p[~rTr,1'TltrT~lr~v,1 •
i
....
,.LLIL,
. . . .
,*l~,-l--lr~qr,l-r|"
,iJl,,Ja~l,,Jli 2sec Fig. 7. L-DOPA induced locomotion before and after spinal transection at 14-16 days. A: 4-limb alternation elicited 8 min after L-DOPA administration. Stepping frequency was 2 Hz. B: forelimb alternation 15 min after transection (and 25 min after L-DOPA) in the same rat. Stepping frequency after transection increased to 3 Hz, Potentiation of the cervical cord after mid-thoracic transection appeared similar to a Schiff-Sherrington effect.
Most of the subjects in this group (5/7) also required more than one injection (Table II) but, regardless of the number of injections required, the effect lasted about 30 min. Overground testing in these subjects revealed similar manifestations to those of the 6-8 day group, including forelimb flexion and rapid hindlimb alternation or galloping. No weight-bearing stepping was evident although the head and tail were held erect. 20-22 Days. This age group also showed hindlimb bias with earlier and faster alternation of the hindlimbs. While the latency to the effect was similar to that of other groups (9.6 + 5.5 min), the duration of effect was intermediate between the 0-3 day and the other two groups (49.0 + 36.6 min). The duration of the effect was highly variable, making it lack statistical significance. For the first time, however, the effect of L-DOPA became episodic compared to the continuous alternation observed at earlier ages. Episodes of air-stepping in this group occasionally were preceded by a slight jerk or startle followed by very fast hindlimb galloping and forelimb alternation. As the effect took hold (first 5-10 min) or waned (last 5-10 min), the episodes lasted 5-15 s with stops of about 10 s. At the peak of the effect (middle 10-15 min), the episodes were shorter (2-5 s) as were the off periods (2-5 s). Almost all subjects (9/11) galloped, while the remainder showed very rapid hindlimb alternation (>2.5 Hz). Overground testing revealed the presence of weight-bearing locomotion. Characteristically, the subject would undergo a slight jerk or startle (intrinsically generated since loud sounds, tail pinch, etc. did not induce this effect) and walk or gallop rapidly
262 forward in a straight line until an obstacle was encountered, at which point weight support ceased. Fig. 4 is an example of the EMG activity observed in this group showing hindlimb alternation slowly building to fast cocontractions (galloping) of the hindlimbs, and coupled with alternation in the forelimbs. Of note is the episodic nature of this effect and the regularity of the episodes observed. The mean frequency of the galloping in the present studies was narrowly defined as cocontractions occurring at a faster rate than the alternation occurring during a walk. In this age group, several animals (not included in the above data) died within minutes of the second or third injection of L-DOPA. This was attributed to particularly high rates of respiration and heart rate, including arrhythmia, induced, perhaps, by the peripheral effects following decarboxylation of L-DOPA. Although increases in respiration and heart rate appeared qualitatively higher in all L-DOPA groups compared to uninjected decerebrates, this age group appeared particularly sensitive to high doses of L-DOPA. Such an effect may be due to the loss of central regulation induced by decerebration and is evident across strains of rats. Similar effects are observed in spinal cats, in which L-DOPA (especially intravenously) can induce marked autonomic changes and can lead to cardiovascular compromise.
Spinal transections One subject in each group underwent a mid-thoracic spinal cord transection and was tested 1 h after surgery. Following administration of L-DOPA, forelimb alternation was present in every case within 10 min of the injection. No hindlimb stepping was evident after spinal transection or following additional administration of L-DOPA. Conversely, forelimb alternation could be prolonged by additional injections of L-DOPA. Fig. 5 is an example of recordings from a 20-22 day group subject in which a spinal transection was performed. Only forelimb alternation was evident following L-DOPA administration. In the case shown, forelimb alternation appeared qualitatively stronger than before the transection. Two additional subjects in each age group were tested by first inducing stepping with L-DOPA and then transecting the spinal cord. Fig. 6 is an example from a 6-8 day group subject in which four-limb stepping was induced by L-DOPA (6A). Immediately following the spinal transection (6B) only forelimb alternation was evident, and this was still present 15 min later (6C) although at a slower frequency. Interestingly, one of the 6-8 day subjects showed only hindlimb alternation before transection and it was abolished by the transection. Fig. 7 is an example from a subject in the 14-16 day group in
which 4-limb locomotion was induced by ~-DOPA. Following a mid-thoracic spinal transection, forelimb alternation increased in frequency while hindlimb alternation ceased altogether. DISCUSSION Previous studies on the development of interlimb coordination in the rat have shown that this coordination was already present on postnatal day 14. Because of the inability of limb muscles to support and propel the body at this age, a swimming task was used to analyze limb movements and phase relationships between limb movements. Findings reported in the companion article to the present one show that air-stepping can be induced by L-DOPA at early stages (from postnatal day 0) in development zs. Suspension of the body allowed free limb movement and L-DOPA administration allowed longterm induction of stepping during which limb movements could be analyzed. In the present investigation, it was found that L-DOPA induced air-stepping in the decerebrate developing rat. This stepping showed characteristics similar to those observed in swimming and in L-DOPA-treated intact animals. All of these studies showed that coordinated stepping, (a) is present as early as postnatal day 1, (b) phase relationships between pairs of limbs were fairly stable, (c) there was a better phase relationship between all 4 limbs with age, (d) there was faster alternation with age, and (e) early emphasis on forelimb use changed to hindlimb bias by 2 weeks of age. Regarding each of these findings, it is evident that neural pattern generators for each limb are present and active at birth. This has been confirmed in the in vitro brainstem-spinal cord preparation, in which ventral root discharges 25 and, especially hindlimb EMGs ~ have demonstrated the presence of an adult-like step cycle. That is, there was a proper phase relationship between agonists in opposite limbs, between antagonists in the same limb and a proximo-distal delay in the contractions of agonists at different joints of the same limb 1-3. These investigations, however, have intrinsic limitations in being able to study only hindlimb EMGs (forelimbs are very difficult to leave attached in vitro) and only early stages in development can be investigated, i.e. embryonic 16 or 0-4 days of age 1-3'2~. A comparison of phase relationships between forelimbs and hindlimbs shows that, in the intact and decerebrate developing rat, L-DOPA-induced locomotion is characterized by good agreement between left and right sides but, at early stages, forelimbs and hindlimbs are not well synchronized. By 1 week of age, however, there is better agreement between forelimbs and hindlimbs. However, there is a superimposed shift from early
263 forelimb to later hindlimb bias. Therefore, while the ability to coordinate 4-limb stepping is present at 6-8 days, this shift makes it difficult to follow the degree of agreement between all 4 limbs developmentally, especially due to the galloping induced at 2 weeks of age by L-DOPA. The shift from forelimb to hindlimb bias is present at the same age range in the intact swimming rat4; therefore, this does not appear to be an effect induced by L-DOPA. It is possible that this forelimb to hindlimb shift is due to the maturation of descending control pathways. For example, corticospinal tract axons reach cervical levels of the spinal cord by 1 day of age, whereas the lumbar cord is not reached until 5 days of age 7. However, other, yet undetermined, mechanisms also may be involved. In terms of the rate of alternation, studies in the intact rat induced to step by punctate or noxious stimuli or the presence of a littermate or mother, showed that forward locomotion appeared at 6-8 days 27. Running and rapid changes in the rate of forward locomotion were not seen until after eye opening at 14-16 days 27. Studies in the swimming, developing rat revealed a gradual decrease in stroke cycle duration (increase in frequency) during the first two postnatal weeks and was attributed to maturing sensory input 4. L-DOPA appears to induce alternation at fairly slow rates at 0-3 days. Additional administration of L-DOPA did not induce faster cycling, merely a longerlasting effect. Not until 14-16 days was galloping, as defined herein, observed, suggesting that, indeed, some time between 1 and 2 weeks of age, the capability for galloping arises. This is supported by the observations that supramaximal electrical stimulation 1"3, chemical stimulation ~5 or electrical stimulation after deafferentation 2 all lead to faster cycling in the in vitro preparation (0-4 days) but never to galloping. This suggestion is tempered by the difficulty in interpreting negative results, i.e. the absence of galloping at 0-3 days and 6-8 days. It may be that the appropriate stimuli or conditions have not been incorporated into the experimental designs to allow galloping to be manifested. Despite the fact that the companion study 2~'2s was performed on a different strain of rat (Long-Evans) the results obtained in intact compared to decerebrate developing rats are very similar. The latency to effect, duration of effect across ages, the type of air-stepping produced, etc. all were virtually identical. The only difference appeared to be the frequency of stepping produced, with step cycle frequency being higher in intact t,-DOPA treated 21 and swimming 4, developing rats. The lower frequency of stepping, therefore, could be attributed to the effects of decerebration. It should be noted that at 2 weeks of age there is a peak in anatomical development within brainstem and
cerebellar cell groups involved in stepping. The cells of the locus coeruleus 22 and of the pedunculopontine nucleus 24 (part of the mesencephalic locomotor region) undergo a sharp increase in soma size at this time, indicative of increased metabolic activity attendant to proliferation of processes ~7. It is not known if the sudden increase, especially of catecholaminergic projections downstream, is directly responsible for some of the effects observed in terms of increased stepping frequency and galloping. An additional observation which warrants discussion is the episodic nature of the effect of L-DOPA at 20-22 days. These episodes were extremely regular and could, in fact, be predicted in timing. This kind of result has been observed in the adult decerebrate rat 9'~5. That is, localized injections of neuroactive agents into locomotion-related brainstem regions resulted in episodic locomotion in the rat. This is in contrast to the continuous stepping produced by most neuroactive agents injected into the brainstem of the cat 1°. Only in the case of the excitatory amino acid agonist, N-methyl-D-aspartic acid (NMDA), is episodic stepping observed in the cat 9'~5. It has not been determined if, (a) the L-DOPA effect at 20-22 days (or, for that matter, any of the ages tested) is mediated by NMDA, or (b) why there is a change from continuous to episodic activity at this age, or (c) whether this early episodic activity is equivalent or related to that observed in the adult. While a mechanism for the generation of episodic stepping by N M D A has been proposed 9, much additional evidence also is needed in that area to arrive at a firm conclusion. A final item to be considered is the effect of L-DOPA on mid-thoracic transected animals. Only forelimb locomotion was evident in each age group, whether or not the transection was performed before L-DOPA administration or afterwards. This suggests a site of action between the midbrain and the mid-thoracic cord rather than directly on the lumbar cord. L-DOPA injected intravenously in the decerebrate or high spinal cat induces stepping under certain circumstances but is more effective in inducing stepping in the low spinal cat Ij. At the moment, there is no satisfactory explanation for the difference in the response of spinal animals injected neonataily (forelimb alternation) and in adulthood (hindlimb alternation). Various possibilities exist, including a different site of action, a different response of the spinal cord to transection and subsequent altered response to L-DOPA, etc. Overall, the effects of L-DOPA, whether through catecholaminergic systems or other as yet undetermined mechanisms, appear reliable and reproducible. The air-stepping elicited at various ages reflects the normal developmental patterns of stepping seen in the intact
264 animal. The decerebrate preparation allows the study of a limited behavioral measure which can be recalled for
form of d o s e - r e s p o n s e curves are required for the continued characterization of this m o d e l
prolonged periods or L-DOPA and thus promises to be a useful model for the study of the details of the development of the control of stepping. Additional studies in the REFERENCES 1 Atsuta, Y., Garcia-Rill, E. and Skinner, R.D., Electrically induced locomotion in the in vitro brainstem-spinal cord preparation, Dev. Brain Res., 42 (1988) 309-312. 2 Atsuta, Y., Garcia-Rili, E. and Skinner, R.D., Electrically induced locomotion in the deafferented in vitro brainstem-spinal cord preparation Soc. Neurosci. Abstr., 14 (1988) 1145. 3 Atsuta, Y., Garcia-Riil, E. and Skinner, R.D., Characteristics of electrically induced locomotion in the rat in vitro brainstemspinal cord preparation, 3. Neurophysiol., 64 (1990) 727-735. 4 Bekoff, A., The development of interlimb co-ordination during swimming in postnatal rats, J. Exp. Biol., 83 (1979) 1-11. 5 Brown, T.G., On the activities of the central nervous system of the unborn foetus of the cat; with a discussion of the question whether progression (walking, etc.) is a 'learnt' complex, J. Physiol. (Lond.), 49 (1915) 208-215. 6 Bursian, A.V., Role of catecholaminergic mechanisms in regulating the autogenic periodic motor activity of rat pups, Zhurnal evol. biok. fiziol., 23 (1988) 755-760. 7 Donatelle, J.M., Growth of the corticospinal tract and the development of placing reactions in the postnatal rat, J. Comp. Neurol., 175 (1976) 207-231. 8 Garcia-Rill, E., Atsuta, Y., Iwahara, T. and Skinner, R.D., Development of brainstem modulation of locomotion. In S. Mori (Ed.), Brainstem Control of Posture and Movement, First Asahikawa Symposium, Japan, 1989. 9 Garcia-Rill, E., Kinjo, N., Atsuta, Y., Ishikawa, Y., Webber, M. and Skinner, R.D., Posterior midbrain-induced locomotion, Brain Res. Bull., 24 (1990) 499-508. 10 Garcia-Rill, E., Skinner, R.D. and Fitzgerald, J.A., Chemical activation of the mesencephalic locomotor region, Brain Res., 330 (1985) 43-54. 11 Grillner, S., Supraspinal and segmental control of static and dynamic y-motoneurones in the cat, Acta Physiol. Scand., 327 (1969) 1-34. 12 Grillner, S., Control of locomotion in bipeds, tetrapods, and fish. In V.E. Brooks (Eds.), Handbook of Physiology -- the Nervous System II, Waverly Press, Baltimore, 1981, pp. 11991236. 13 Hughes, A. and Prestige, M.C., Development of behavior in the hindlimb of ,':enopus laevis, J. Zool. (Lond.), 152 (1967) 347-359. 14 Humphrey, T., The development of human fetal activity and its
Acknowledgement. This work was supported by USPHS Grant NS 20246.
relation to postnatal behavior, Adv. Child Devel. Behav., 5 (1970) 1-57. 15 Kinjo, N., Atsuta, Y., Webber, M., Kyle, R., Skinner, R.D. and Garcia-Rill, E., Medioventral medulla-induced locomotion, Brain Res. Bull., 24 (1990) 509-518. 16 Kudo, N., Ontogeny of rhythmic activities in the spinal cord, In M. Shimamura (Ed.), Neurobiological Basis of Human Locomotion, Wiley, New York, in press. 17 Murray, M. and Grafstein, B., Changes in the morphology and amino acid incorporation of regenerating goldfish optic neurons, Exp. Neurol., 23 (1969) 544-560. 18 O'Donovan, M.J., Experimental analysis of motor development in the chick embryo. In S. Grillner, P.S.G. Stein, D. Stmirt and R. Herman (Eds.), Neurobiology of Vertebrate Locomotion, MacMillan Press, London, 1986, pp. 415-431. 19 Oppenheim, R., Prehatching and hatching behavior: a comparative and physiological consideration. In G . Gottlieb (Ed.), Behavioral Embryology, Academic Press, New York, 1973, pp. 163-244. 20 Porcher, W. and Heller, A., Regional development of catecholamine biosynthesis in rat brain, J. Neurochem., 19 (1972) 1917-1930. 21 Sickles, A.E., Porter, J., Stehouwer, D.J. and Van Hartesveldt, C., Ontogeny of L-DOPA-induced air-stepping in pre-weanling rats, Int. Soc. Devel. Psychobiol., 1989. 22 Sievers, J., Lolova, I., Jenner, S., Klemm, H.P. and Sievers, H., Morphological and biochemical studies on the ontogenesis of the nucleus locus coeruleus, Bibl. Anat., 19 (1981) 52-130. 23 Skinner, R.D. and Garcia-Rill, E., The mesencephalic locomotor region (MLR) in the rat, Brain Res., 323 (1984) 385-389. 24 Skinner, R.D,, Conrad, C., Henderson, V., Gilmore, S:A. and Garcia-RiU, E., Development of NADPH diaphorase-positive peduneulopontine neurons, Exp. Neurol., 104 (1989) I5-21. 25 Smith, J.C., Garcia-Rill, E. and Feldman, J.L., Chemical activation of mammalian locomotion in vitro, Soc Neurosci. Abstr., 12 (1986) 386. 26 Stehouwer, O. and Farel, P., Development of locomotor mechanisms in the frog, J. Neurophysiol., 53 (1985) 1453-1466. 27 Stelzner, D.J., The normal postnatal development of synaptic end-feet in the lumbosacral spinal cord and of responses in the hindlimbs of the albino rat, Exp. Neurol., 31 (1971) 337-357. 28 Van Hartesveldt, C., Sickles, A., Porter, J. and Stehouwer, D., ,_-DOPA-induced air-stepping in developing rats, Dev. Brain Res., 58 (1991) 251-255.
257
BRESD 51224
c-DOPA-induced air-stepping in decerebrate developing rats T. Iwahara ~, C. Van H a r t e s v e l d t 2, E. Garcia-Rill I and R . D . S k i n n e r t 1Department of Anatomy, University of Arkansas for Medical Sciences, Little Rock, A R 72205-7199 (U.S.A.) and eDepartment of Psychology, University of Florida, Gainesville, FL 32611 (U.S.A.) (Accepted 30 October 1990) Key words: L-3,4-Dihydroxyphenylalanine; Locomotion; Decerebrate; Rat
Developing rats were anesthetized and a precollicular brainstem transection performed. Administration of L-3,4-dihydroxyphenylalanine (L-DOPA, 100 mg/kg, s.c.) induced locomotion in groups of animals 0-3, 6-8, 14-16 and 20-22 days of age. At early stages in development (0-3 days), continuous, long-lasting air-stepping was induced consisting of 4-limb walking with stronger alternation in the forelimbs compared to the hindlimbs. At 6-8 days of age, continuous air-stepping was characterized by better agreement between forelimb and hindlimb step cycles. By 14-16 days of age, the hindlimbs showed stronger and faster stepping than the forelimbs. The first evidence of consistent galloping was observed in this age group. In some cases, the forelimbs were held extended and only the hindlimbs walked or galloped. At 20-22 days, walking and galloping became episodic and the hindlimbs typically galloped while the forelimbs alternated. Each age group was tested for overground locomotion. Only in the 20-22 day group was weight-bearing overground stepping observed. At earlier ages, limb alternation was evident but of insufficient strength to lift the body off the ground. In general, the duration of the effect of the same dose of L-DOPA decreased with age. Animals in each group which received a mid-thoracic spinal cord transection showed forelimb alternation but no hindlimb stepping. These results indicate that L-DOPA induces stepping by activating brainstem and/or spinal centers via an as yet unknown mechanism. The pattern of the development of gait (forelimb to hindlimb gradient) at various ages was similar to that observed in intact developing rats. INTRODUCTION T h e characteristics of adult locomotion have been described quantitatively for n u m e r o u s v e r t e b r a t e and i n v e r t e b r a t e species, and much of the underlying neural organization mediating stepping has been worked out (see e.g.12). The d e v e l o p m e n t of stepping has been studied less extensively, although information on amphibians ~3"26 and birds 18'19 shows interlimb coordination is present at the last stages of embryonic life. Walking m o v e m e n t s have been described in m a m m a l i a n fetuses (cat 5, human14). In the rat, previous studies have revealed that evidence of interlimb coordination is present at birth 4. In these studies, swimming rather than walking was studied in o r d e r to minimize any effect of weak limb muscles in young rats. Using the limb-attached in vitro b r a i n s t e m - s p i n a l cord p r e p a r a t i o n , we have shown that electrical stimulation of the brainstem of 0 to 4-day-old rats produces an adult-like pattern of locomotor muscular contractions 1. Recent evidence suggests that c o o r d i n a t e d limb alternation in the rat fetus may begin at e m b r y o n i c day 18, although m o r e primitive cocontractions ('kicking' or 'hatching'-type movements) may be present as early as e m b r y o n i c day 1516. On the o t h e r hand, not all aspects of l o c o m o t o r control
may be present at birth. While increasing stimulus amplitude ~'3, a p p r o p r i a t e chemical stimulation x'/s or deafferentation 2 can lead to faster hindlimb alternation in the in vitro p r e p a r a t i o n , evidence of galloping (as defined in the adult) has not been o b s e r v e d so soon after birth. That is, in the adult, the alternating l e f t - r i g h t muscular contractions evident in a walk are shifted to a fast walk and then a gallop, at which point cocontraction of left and right limb muscles is evident. This usually is a c c o m p a n i e d by a decrease in step or stroke cycle duration ~2. Studies of stroke cycle duration in the developing, swimming rat r e p o r t e d a gradual decrease (faster alternation) in duration from birth until two weeks of age without much change t h e r e a f t e r 4, but galloping as such was not described. While swimming is an ideal way to minimize the effects of gravity and c o m p e n s a t e for w e a k limb muscles, it m a y not be a m e n a b l e to studying rapid galloping m o v e m e n t s due to the resistance of limb m o v e m e n t through the water. A i r - s t e p p i n g ( s u s p e n d e d b o d y with limbs free to move) offers an o p p o r t u n i t y to study such movements. M o r e o v e r , the d e c e r e b r a t e p r e p a r a t i o n eliminates the contribution of volational elements and allows the recording of e l e c t r o m y o g r a p h i c activity without the need for chronic i m p l a n t a t i o n techniques. R e c e n t findings suggest that long-lasting locomotion
Correspondence." E. Garcia-Rill, Department of Anatomy, University of Arkansas for Medical Sciences, 4301 West Markham, Little Rock, AR 72205-7199, U.S.A. 0165-3806/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
258 can be i n d u c e d in the d e v e l o p i n g rat after a d m i n i s t r a t i o n of L - 3 , 4 - d i h y d r o x p h e n y l a l a n i n e ( L - D O P A ) (subcutan e o u s 21, intraperitoneal6). L - D O P A has b e e n used to
fixative overnight and in 20'~4 sucrose tk)r at least ~ days. Frozen sections (60 **m sagittal) were cut and stained for Cresyl violet to confirm the level of brainstem transection, essentially, precollicularmidnigral in all the cases reported herein.
elicit s t e p p i n g in the adult spinal cat ( i n t r a v e n o u s 11). T h e p r e s e n t studies t o o k a d v a n t a g e of this effect and w e r e d e s i g n e d to test the effects of a single c o n c e n t r a t i o n of
RESULTS
L - D O P A on d e c e r e b r a t e , a i r - s t e p p i n g d e v e l o p i n g rats at v a r i o u s stages in d e v e l o p m e n t . P r e l i m i n a r y results h a v e b e e n r e p o r t e d 8.
MATERIALS AND METHODS The use of timed-pregnant Sprague-Dawley dams allowed the accurate determination of the date of birth of rat pups. The day of birth was designated as 0 day postnatally and litters culled to 8 pups. Four age groups were chosen for study at birth (0-3 days), 1 week of age (6-8 days), 2 weeks of age (14-16 days) and 3 weeks of age (20-22 days). No more than 2 pups from any litter formed part of a group and most litters used contributed at least 1 member to each group. The younger 2 age groups were anesthetized with penthrane while the older 2 groups were anesthetized with halothane. Some subjects in the 14-16 day group (3/7) were anesthetized with penthrane and no differences were found in the effects observed between those subjected to penthrane compared to those anesthetized with haiothane. Once a surgical plane of anesthesia was established (e.g. absence of withdrawal reflex), a brainstem transection such as that used in the adult rat preparation was carried out 23. Typically, anesthesia was maintained for 10-15 min during the surgical procedure. The precollicular decerebration was performed using suction ablation and anesthesia discontinued. Electromyographic (EMG) recordings were carried out using double hookedwire electrodes inserted into one extensor muscle in each limb, usually the m. triceps brachii in the forelimbs and the gastrocnemius-soleus group in the hindlimbs. L-DOPA (100 mg/kg) dissolved in vehicle (50 mg L-DOPA in 0.4 ml 1 N HCI and 0.1 ml distilled water, mixed until clear, then added 4.5 ml 0.1 M Na2HPO 4, pH 6.2) was injected subcutaneously at the nape of the neck, making sure to maintain pressure on the skin after needle withdrawal to prevent leakage. At least 30 rain elapsed following discontinuation of anesthesia before L-DOPA was injected. For the present set of studies, a single dose was chosen pending further determinations of dose-response curves at each age. If an effect (induced stepping) was not present after 20 min, a second injection was administered and, occasionally, a third injection was necessary (see below), again, administered no earlier than 20 min following the second injection. Considering the levels of DOPA decarboxylase present at the ages tested 2°, this period of time was considered sufficient for metabolism of the L-DOPA. Animals were tested for overground stepping on a smooth surface and for air-stepping by suspension using a strip of tape around the midriff which did not interfere with limb movement. In some cases (n = 4, one subject in each age group) following decerebration, a mid-thoracic transection of the spinal cord was performed using suction ablation. This allowed testing of L-DOPA-induced locomotion after spinal transection. The completeness of the transection was confirmed visually as a 2-3 mm gap between the proximal and distal ends of the spinal cord. In another series of experiments, two subjects in each age group were subjected to a spinal cord transection after stepping had been induced by L-DOPA, Because stepping movements can be induced in decerebrate and spinal animals by sensory stimulation, once L-DOPA was administered, no such stimuli were administered in order not to influence the latency to the onset of stepping. Locomotion was recorded on FM tape (EMGs) and on video tape. At the end of the experiment, animals were sacrificed with an overdose of barbiturate and the brainstem removed and stored in
Effects o f decerebration In the a b s e n c e of I _ - D O P A o r f o l l o w i n g an i n j e c t i o n of vehicle
(pH
equally
inactive.
6.2),
decerebrate No
developing
spontaneous
or
rats
were
rhythmic
limb
m o v e m e n t s w e r e o b s e r v e d at any of the ages testedl M u s c l e t o n e was e v i d e n t as early as 15 min following the discontinuation stimuli
(e.g.
of anesthesia. tail
or
pinna
Responses
pinch)
to
noxious
produced
scratch
r e f l e x - t y p e r e s p o n s e s starting 1 5 - 2 0 min f o l l o w i n g the e n d of anesthesia. observed
Limb alternation
f o l l o w i n g such
stimuli
but
o c c a s i o n a l l y was the
movements
s t o p p e d s o o n after the cessation of stimulation. W h e n tested on a surface, t h e r e was n o e v i d e n c e of standing p o s t u r e . W h e n s u s p e n d e d , all d e c e r e b r a t e animals h u n g limply. For a d e s c r i p t i o n of the b e h a v i o r o f the intact d e v e l o p i n g rat, see the c o m p a n i o n article 21'2s.
£ffects o f L - D O P A T h e m a i n characteristics of the effects o f i n j e c t i o n s of the s a m e d o s e of L - D O P A at v a r i o u s ages are o u t l i n e d in Table I. In g e n e r a l , d e c e r e b r a t e d e v e l o p i n g r a t s e x h i b i t e d L - D O P A - i n d u c e d air-stepping a f t e r o n e or two injections, resulting in s t e p p i n g b e g i n n i n g a p p r o x i m a t e l y 10 min
after
the
injection
and
lasting
for 3 0 - 9 0
min.
H o w e v e r , t h e r e w e r e significant d i f f e r e n c e s b e t w e e n the v a r i o u s age g r o u p s tested and e a c h will be c o n s i d e r e d individually.
0 - 3 Days. Within 5 min a f t e r i n j e c t i o n of L - D O P A , d e c e r e b r a t e subjects b e g a n to s h o w an i n c r e a s e in the background
EMG
and the tail was h e l d h o r i z o n t a l l y
while the h e a d was raised slowly a n d h e l d in a line with
TABLE I
Effects of L-DOPA on neonate rats A KruskaI-Wallis test, which is a non-parametric (distribution-free) analogue of analysis of variance, showed there to be a significant effect (P < 0.05) of age upon duration. Univariate t-tests then showed the 0--3 day group to be different from the 6-8 day group (P < 0.05*) and from the 14-16 day group (P < 0.01 **).
Age (days)
n
Mean no. of injections
Mean latency to effect (rain)
Mean duration of effect (rain)
0-3 6-8 14-16 2{)-22
9 9 7 11
1.4_+0.7 1.9 + 0.8 2.0_+0.8 1.9 -+ tl.7
8.1 +3.9 9.6 + 5.9 8.1-+5.0 9.6 _+ 5.5
80.2_+24.2 42.3 -+ 28.6* 29.3_+22.8** 49.0 _+36.6
259
O-3Days 30min
post
L'DOPA
,,
-,.
R,, ., LHL
~I "~
r
'
'r
?
I'F ~ ' " I ~ T ff
~
~
"I
~" '~
Pr -
"
~
~ . 7! ~
m
"
t 4sec Fig. I. L-DOPA induced locomotion in a 0-3 day rat. Recordings of EMGs from muscles of the left forelimb (LFL), right forelimb (RFL), left hindlimb ( L H L ) and righ hindlimb ( R H L ) show good alternation between left and right sides, but without exact agreement between forelimbs and hindlimbs (note out-of-phase relationships). Moreover, there were lapses (arrow) in the regularity of hindlimb stepping. Episode shown was recorded 30 min after an injection of L-DOPA (I00 mg/kg, s.c.). Stepping frequency in this record was 0.7 Hz.
the spine (not completely upright as in a typical standing posture). Gradually, one or two limbs began to alternate until all 4 limbs air-stepped continuously (The mean latency following one injection of L-DOPA was 7.7 _+ 2.6 min while it was 9.0 _+ 6.6 min following a second injection when necessary. When both groups are pooled, 8.1 _+ 3.9 rain was the mean and standard deviation of the latency to 4 limb alternation). In all cases, the forelimbs alternated first, occasionally alternated faster than the hindlimbs at the peak of the effect, and kept alternating after the hindlimbs had stopped when the effect was waning. The mean duration of 4 limb air-stepping was 80.2 _+ 24.2 (S.D.) min in this age group. The mean
frequency of forelimb stepping at the peak of the effect was 0.7 _+ 0.5 (S.D.) Hz. Mean frequency at peak of effect was determined as the highest mean cycle time of 10 consecutive steps. An additional injection of L-DOPA did not induce faster stepping but merely extended the duration of the effect• Fig. 1 is a representative example of the EMGs recorded in each limb in a subject in this age group. The frequency of air-stepping of the forelimbs in this case was 0.7 Hz and quite consistent, contrasting with the hindlimb frequency of 0.6 Hz, which showed inconsistencies (arrow) in terms of agreement between the step cycles of fore- and hindlimbs. In some cases (3/9), it was necessary to administer a second injection in order to induce air-stepping. As evident in Table II, the mean latency was slightly longer (9.0 _+ 6.6 min) in subjects requiring more than one injection than those stepping after a single injection (7.7 +_ 2.6 min). The duration of the effect was longer after more than one injection (84.7 _ 24.4 min) than after a single injection (75.7 +_ 28.4 min). These and all other measures in Table II were not statistically significant but give a good idea of the trends observed. When tested overground, none of the subjects could hold their chins above ground although the tails were held suspended. The limb alternation was insufficient to propel the animal, which rested on its abdomen throughout. 6-8 Days. The postural effects observed at 0-3 days also were seen in the 6-8 day group. The group mean latency to 4 limb air-stepping was 9.6 + 5.9 min (Table I), similar to that of the younger group. However, the mean duration of effect was significantly (t-test, P < 0.05) shorter (42.3 _+ 28.6 min). In this group, the forelimbs also were the first to alternate although there
6-8Days 15min post L-DOPA
L F L , , L ~ k i . kL [ k L. x k L A ~ L [[ -, ~
T A B L E II
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20-22
3 8
Mean latency (min)
75.7 + 28.4 84.7_+24.4
11.0 +- 8.2 8.8_+5.3
69.7 +_ 37.3 32.4_+1(1.6
13.5_+4.9 6.0_+3.4
29.0_+ 1.4 29.2_+25.5
13.7 _+ 6.4 8.1 +_4.7
27.5 + 10.6 59.8_+41.6
r '
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Mean duration (min)
7.7 + 2.6 9.0_+6.6
''
r
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I
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v
,
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4sec Fig. 2. L-DOPA induced locomotion in a 6-8 day rat. Recordings of EMGs as in Fig. l showing good agreement between left and right sides and in-phase relationship between forelimbs and hindlimbs, 15 rain after an injection of L-DOPA. Stepping frequency in this record was 0.8 Hz.
260 14 - 1 6 D a y s post
A
LHL-
-
~
~
,
C
B
I
L F L .....
L.DOPA
-
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L.I...L. Ll., .l.
f4@ 2sec
Fig. 3. L-DOPA induced locomotion in 14-16 day rats. A: hindlimb alternation with no forelimb alternation evident 12 rain after L-DOPA. Stepping frequency in this record was 1.8 Hz. B: another subject in this group which showed alternation in forelimbs and ~ m b s . The sequence shown occurred 15 min after L-DOPA and includes the transition from walk to gallop in the hindlimbs (note mixed alternation and cocontraction). Stepping frequency 1.5 Hz. C: a third subject which showed forelimb alternation and faster hindlimb cocomractions 10 rain after L-DOPA. Stepping frequency 1.3 Hz.
was not as g r e a t a difference between the rates of alternation o f the two pairs of limbs. Fig. 2 is a representative recording from a subject in this g r o u p showing a stepping frequency of about 0.8 H z for b o t h forelimbs and hindlimbs. A l t h o u g h there was g r e a t e r a g r e e m e n t b e t w e e n the fore- and hindlimb step cycles, the match was not as exact as in the adult d e c e r e b r a t e electrically induced locomotion 23. The mean frequency o f stepping at the p e a k of the effect was 0.7 + 0.5 Hz. W h e n tested overground, these subjects were capable of maintaining both h e a d and tail erect and lifting the t h o r a x from the ground. The limbs varied between alternation and placing, the purchase of the paws on the ground (placing) a p p e a r i n g to interfere with alternation. 14-16 Days. A m a j o r difference between this age group and the younger groups was the shift almost
exclusively to hindlimb air-stepping. In all cases, hindlimb alternation was the first to occur and, in some cases (3/7), no forelimb alternation was evident, as both forelimbs were held in the 'swimming' position 5. T h e latency to hindlimb alternation was similar to that of the o t h e r groups (8.1 + 5.0 min, Table I), although the duration of the effect was s h o r t e r than in the 0 - 3 day group (29.3 + 22.8 min) (t-test, P < 0.01). Most subjects (5/7) in this group required m o r e than one injection (Table II). Fig. 3 A is a representative e x a m p l e o f the recordings o b t a i n e d in this group, showing a p e a k effect of hindlimb alternation at a m e a n frequency o f 1.8 + 0.9 Hz, with n o
20-22Days spinal trans 20rain
post
L.DOPA
20-22Days 15min post L.DOPA
t L,LL,iL
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Fig. 4. L-DOPA induced locomotion in a 20-22 day rat, Episodic alternation and coeontraction (mostly in hindlimbs) evident 15 rain after L-DOPA. Stepping for 4-6 s which was interrupted by 4-6 s recurred for prolonged periods. Stepping frequency in this record was 3.5 Hz.
4sec
Fig. 5. L-DOPA induced locomotion in a mid-thoracic spinal rat 20-22 days. Consistent forelimb alternation was evident, with the segment shown occurring 20 min after L-DOPA. Note absence of hindlimb movement in a mid-thoracic spinal transccted subject, Stepping frequency in this record was 1.3 Hz.
261 Days
6-8
post
L-DOPA
B
C
post trans l
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4sec Fig. 6. L-DOPA induced locomotion before and after spinal transection at 6-8 days. A: 4-limb alternation elicited 20 min after L-DOPA administration. B: perseverance of forelimb alternation immediately after mid-thoracic transection. C: forelimb alternation effect was waning 15 rain after transection, accounting for the slower frequency of stepping. Stepping frequency was 1.3 Hz in A and 1.2 Hz in B. Although slower, forelimb alternation after transection appeared more vigorous.
forelimb alternation in this case. A second major difference in this group was the presence of galloping (4/7). The mean frequency during these brief episodes of galloping was 1.5 + 0.9 Hz. Fig. 3B and 3C are different examples of the transitions in stepping frequency obtained in this group, showing cocontraction of the hindlimbs mixed with alternation, as well as cocontractions of the hindlimbs simultaneously with alternation of the forelimbs.
14-16
Days
A 8min
13
post
~Tr-V r'l~+r ~ ~
L.DOPA
r ~,-1,-r-,
15min
post
tr;lul'.;
r I,
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i
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,iJl,,Ja~l,,Jli 2sec Fig. 7. L-DOPA induced locomotion before and after spinal transection at 14-16 days. A: 4-limb alternation elicited 8 min after L-DOPA administration. Stepping frequency was 2 Hz. B: forelimb alternation 15 min after transection (and 25 min after L-DOPA) in the same rat. Stepping frequency after transection increased to 3 Hz, Potentiation of the cervical cord after mid-thoracic transection appeared similar to a Schiff-Sherrington effect.
Most of the subjects in this group (5/7) also required more than one injection (Table II) but, regardless of the number of injections required, the effect lasted about 30 min. Overground testing in these subjects revealed similar manifestations to those of the 6-8 day group, including forelimb flexion and rapid hindlimb alternation or galloping. No weight-bearing stepping was evident although the head and tail were held erect. 20-22 Days. This age group also showed hindlimb bias with earlier and faster alternation of the hindlimbs. While the latency to the effect was similar to that of other groups (9.6 + 5.5 min), the duration of effect was intermediate between the 0-3 day and the other two groups (49.0 + 36.6 min). The duration of the effect was highly variable, making it lack statistical significance. For the first time, however, the effect of L-DOPA became episodic compared to the continuous alternation observed at earlier ages. Episodes of air-stepping in this group occasionally were preceded by a slight jerk or startle followed by very fast hindlimb galloping and forelimb alternation. As the effect took hold (first 5-10 min) or waned (last 5-10 min), the episodes lasted 5-15 s with stops of about 10 s. At the peak of the effect (middle 10-15 min), the episodes were shorter (2-5 s) as were the off periods (2-5 s). Almost all subjects (9/11) galloped, while the remainder showed very rapid hindlimb alternation (>2.5 Hz). Overground testing revealed the presence of weight-bearing locomotion. Characteristically, the subject would undergo a slight jerk or startle (intrinsically generated since loud sounds, tail pinch, etc. did not induce this effect) and walk or gallop rapidly
262 forward in a straight line until an obstacle was encountered, at which point weight support ceased. Fig. 4 is an example of the EMG activity observed in this group showing hindlimb alternation slowly building to fast cocontractions (galloping) of the hindlimbs, and coupled with alternation in the forelimbs. Of note is the episodic nature of this effect and the regularity of the episodes observed. The mean frequency of the galloping in the present studies was narrowly defined as cocontractions occurring at a faster rate than the alternation occurring during a walk. In this age group, several animals (not included in the above data) died within minutes of the second or third injection of L-DOPA. This was attributed to particularly high rates of respiration and heart rate, including arrhythmia, induced, perhaps, by the peripheral effects following decarboxylation of L-DOPA. Although increases in respiration and heart rate appeared qualitatively higher in all L-DOPA groups compared to uninjected decerebrates, this age group appeared particularly sensitive to high doses of L-DOPA. Such an effect may be due to the loss of central regulation induced by decerebration and is evident across strains of rats. Similar effects are observed in spinal cats, in which L-DOPA (especially intravenously) can induce marked autonomic changes and can lead to cardiovascular compromise.
Spinal transections One subject in each group underwent a mid-thoracic spinal cord transection and was tested 1 h after surgery. Following administration of L-DOPA, forelimb alternation was present in every case within 10 min of the injection. No hindlimb stepping was evident after spinal transection or following additional administration of L-DOPA. Conversely, forelimb alternation could be prolonged by additional injections of L-DOPA. Fig. 5 is an example of recordings from a 20-22 day group subject in which a spinal transection was performed. Only forelimb alternation was evident following L-DOPA administration. In the case shown, forelimb alternation appeared qualitatively stronger than before the transection. Two additional subjects in each age group were tested by first inducing stepping with L-DOPA and then transecting the spinal cord. Fig. 6 is an example from a 6-8 day group subject in which four-limb stepping was induced by L-DOPA (6A). Immediately following the spinal transection (6B) only forelimb alternation was evident, and this was still present 15 min later (6C) although at a slower frequency. Interestingly, one of the 6-8 day subjects showed only hindlimb alternation before transection and it was abolished by the transection. Fig. 7 is an example from a subject in the 14-16 day group in
which 4-limb locomotion was induced by ~-DOPA. Following a mid-thoracic spinal transection, forelimb alternation increased in frequency while hindlimb alternation ceased altogether. DISCUSSION Previous studies on the development of interlimb coordination in the rat have shown that this coordination was already present on postnatal day 14. Because of the inability of limb muscles to support and propel the body at this age, a swimming task was used to analyze limb movements and phase relationships between limb movements. Findings reported in the companion article to the present one show that air-stepping can be induced by L-DOPA at early stages (from postnatal day 0) in development zs. Suspension of the body allowed free limb movement and L-DOPA administration allowed longterm induction of stepping during which limb movements could be analyzed. In the present investigation, it was found that L-DOPA induced air-stepping in the decerebrate developing rat. This stepping showed characteristics similar to those observed in swimming and in L-DOPA-treated intact animals. All of these studies showed that coordinated stepping, (a) is present as early as postnatal day 1, (b) phase relationships between pairs of limbs were fairly stable, (c) there was a better phase relationship between all 4 limbs with age, (d) there was faster alternation with age, and (e) early emphasis on forelimb use changed to hindlimb bias by 2 weeks of age. Regarding each of these findings, it is evident that neural pattern generators for each limb are present and active at birth. This has been confirmed in the in vitro brainstem-spinal cord preparation, in which ventral root discharges 25 and, especially hindlimb EMGs ~ have demonstrated the presence of an adult-like step cycle. That is, there was a proper phase relationship between agonists in opposite limbs, between antagonists in the same limb and a proximo-distal delay in the contractions of agonists at different joints of the same limb 1-3. These investigations, however, have intrinsic limitations in being able to study only hindlimb EMGs (forelimbs are very difficult to leave attached in vitro) and only early stages in development can be investigated, i.e. embryonic 16 or 0-4 days of age 1-3'2~. A comparison of phase relationships between forelimbs and hindlimbs shows that, in the intact and decerebrate developing rat, L-DOPA-induced locomotion is characterized by good agreement between left and right sides but, at early stages, forelimbs and hindlimbs are not well synchronized. By 1 week of age, however, there is better agreement between forelimbs and hindlimbs. However, there is a superimposed shift from early
263 forelimb to later hindlimb bias. Therefore, while the ability to coordinate 4-limb stepping is present at 6-8 days, this shift makes it difficult to follow the degree of agreement between all 4 limbs developmentally, especially due to the galloping induced at 2 weeks of age by L-DOPA. The shift from forelimb to hindlimb bias is present at the same age range in the intact swimming rat4; therefore, this does not appear to be an effect induced by L-DOPA. It is possible that this forelimb to hindlimb shift is due to the maturation of descending control pathways. For example, corticospinal tract axons reach cervical levels of the spinal cord by 1 day of age, whereas the lumbar cord is not reached until 5 days of age 7. However, other, yet undetermined, mechanisms also may be involved. In terms of the rate of alternation, studies in the intact rat induced to step by punctate or noxious stimuli or the presence of a littermate or mother, showed that forward locomotion appeared at 6-8 days 27. Running and rapid changes in the rate of forward locomotion were not seen until after eye opening at 14-16 days 27. Studies in the swimming, developing rat revealed a gradual decrease in stroke cycle duration (increase in frequency) during the first two postnatal weeks and was attributed to maturing sensory input 4. L-DOPA appears to induce alternation at fairly slow rates at 0-3 days. Additional administration of L-DOPA did not induce faster cycling, merely a longerlasting effect. Not until 14-16 days was galloping, as defined herein, observed, suggesting that, indeed, some time between 1 and 2 weeks of age, the capability for galloping arises. This is supported by the observations that supramaximal electrical stimulation 1"3, chemical stimulation ~5 or electrical stimulation after deafferentation 2 all lead to faster cycling in the in vitro preparation (0-4 days) but never to galloping. This suggestion is tempered by the difficulty in interpreting negative results, i.e. the absence of galloping at 0-3 days and 6-8 days. It may be that the appropriate stimuli or conditions have not been incorporated into the experimental designs to allow galloping to be manifested. Despite the fact that the companion study 2~'2s was performed on a different strain of rat (Long-Evans) the results obtained in intact compared to decerebrate developing rats are very similar. The latency to effect, duration of effect across ages, the type of air-stepping produced, etc. all were virtually identical. The only difference appeared to be the frequency of stepping produced, with step cycle frequency being higher in intact t,-DOPA treated 21 and swimming 4, developing rats. The lower frequency of stepping, therefore, could be attributed to the effects of decerebration. It should be noted that at 2 weeks of age there is a peak in anatomical development within brainstem and
cerebellar cell groups involved in stepping. The cells of the locus coeruleus 22 and of the pedunculopontine nucleus 24 (part of the mesencephalic locomotor region) undergo a sharp increase in soma size at this time, indicative of increased metabolic activity attendant to proliferation of processes ~7. It is not known if the sudden increase, especially of catecholaminergic projections downstream, is directly responsible for some of the effects observed in terms of increased stepping frequency and galloping. An additional observation which warrants discussion is the episodic nature of the effect of L-DOPA at 20-22 days. These episodes were extremely regular and could, in fact, be predicted in timing. This kind of result has been observed in the adult decerebrate rat 9'~5. That is, localized injections of neuroactive agents into locomotion-related brainstem regions resulted in episodic locomotion in the rat. This is in contrast to the continuous stepping produced by most neuroactive agents injected into the brainstem of the cat 1°. Only in the case of the excitatory amino acid agonist, N-methyl-D-aspartic acid (NMDA), is episodic stepping observed in the cat 9'~5. It has not been determined if, (a) the L-DOPA effect at 20-22 days (or, for that matter, any of the ages tested) is mediated by NMDA, or (b) why there is a change from continuous to episodic activity at this age, or (c) whether this early episodic activity is equivalent or related to that observed in the adult. While a mechanism for the generation of episodic stepping by N M D A has been proposed 9, much additional evidence also is needed in that area to arrive at a firm conclusion. A final item to be considered is the effect of L-DOPA on mid-thoracic transected animals. Only forelimb locomotion was evident in each age group, whether or not the transection was performed before L-DOPA administration or afterwards. This suggests a site of action between the midbrain and the mid-thoracic cord rather than directly on the lumbar cord. L-DOPA injected intravenously in the decerebrate or high spinal cat induces stepping under certain circumstances but is more effective in inducing stepping in the low spinal cat Ij. At the moment, there is no satisfactory explanation for the difference in the response of spinal animals injected neonataily (forelimb alternation) and in adulthood (hindlimb alternation). Various possibilities exist, including a different site of action, a different response of the spinal cord to transection and subsequent altered response to L-DOPA, etc. Overall, the effects of L-DOPA, whether through catecholaminergic systems or other as yet undetermined mechanisms, appear reliable and reproducible. The air-stepping elicited at various ages reflects the normal developmental patterns of stepping seen in the intact
264 animal. The decerebrate preparation allows the study of a limited behavioral measure which can be recalled for
form of d o s e - r e s p o n s e curves are required for the continued characterization of this m o d e l
prolonged periods or L-DOPA and thus promises to be a useful model for the study of the details of the development of the control of stepping. Additional studies in the REFERENCES 1 Atsuta, Y., Garcia-Rill, E. and Skinner, R.D., Electrically induced locomotion in the in vitro brainstem-spinal cord preparation, Dev. Brain Res., 42 (1988) 309-312. 2 Atsuta, Y., Garcia-Rili, E. and Skinner, R.D., Electrically induced locomotion in the deafferented in vitro brainstem-spinal cord preparation Soc. Neurosci. Abstr., 14 (1988) 1145. 3 Atsuta, Y., Garcia-Riil, E. and Skinner, R.D., Characteristics of electrically induced locomotion in the rat in vitro brainstemspinal cord preparation, 3. Neurophysiol., 64 (1990) 727-735. 4 Bekoff, A., The development of interlimb co-ordination during swimming in postnatal rats, J. Exp. Biol., 83 (1979) 1-11. 5 Brown, T.G., On the activities of the central nervous system of the unborn foetus of the cat; with a discussion of the question whether progression (walking, etc.) is a 'learnt' complex, J. Physiol. (Lond.), 49 (1915) 208-215. 6 Bursian, A.V., Role of catecholaminergic mechanisms in regulating the autogenic periodic motor activity of rat pups, Zhurnal evol. biok. fiziol., 23 (1988) 755-760. 7 Donatelle, J.M., Growth of the corticospinal tract and the development of placing reactions in the postnatal rat, J. Comp. Neurol., 175 (1976) 207-231. 8 Garcia-Rill, E., Atsuta, Y., Iwahara, T. and Skinner, R.D., Development of brainstem modulation of locomotion. In S. Mori (Ed.), Brainstem Control of Posture and Movement, First Asahikawa Symposium, Japan, 1989. 9 Garcia-Rill, E., Kinjo, N., Atsuta, Y., Ishikawa, Y., Webber, M. and Skinner, R.D., Posterior midbrain-induced locomotion, Brain Res. Bull., 24 (1990) 499-508. 10 Garcia-Rill, E., Skinner, R.D. and Fitzgerald, J.A., Chemical activation of the mesencephalic locomotor region, Brain Res., 330 (1985) 43-54. 11 Grillner, S., Supraspinal and segmental control of static and dynamic y-motoneurones in the cat, Acta Physiol. Scand., 327 (1969) 1-34. 12 Grillner, S., Control of locomotion in bipeds, tetrapods, and fish. In V.E. Brooks (Eds.), Handbook of Physiology -- the Nervous System II, Waverly Press, Baltimore, 1981, pp. 11991236. 13 Hughes, A. and Prestige, M.C., Development of behavior in the hindlimb of ,':enopus laevis, J. Zool. (Lond.), 152 (1967) 347-359. 14 Humphrey, T., The development of human fetal activity and its
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