Brain Research, 570 (1992) 1-10
1
© 1992 Elsevier Science Publishers B.V, All tights reserved. 0006-8993/92/$05.00 BRES 17105
Research Reports
Adaptability of the oxidative capacity of motoneurons Gordon R. Chalmers 1, Roland R. Roy I and V. Reggie Edgerton 1'2 1Brain Research Institute and 2Department of Kinesiology, University of California, Los Angeles, CA 90024 (U.S.A.) (Accepted 11 June 1991)
Key words: Motoneuron; Succinate dehydrogenase; Oxidative capacity; Soma area; Spinal transection; Spinal isolation; Muscle functional overload; Energy metabolism; Cat
Previous studies have demonstrated that a chronic change in neuronal activation can produce a change in soma oxidative capacity, suggesting that: (i) these 2 variables are directly related in neurons and (ii) ion pumping is an important energy requiring activity of a neuron. Most of these studies, however, have focused on reduced activation levels of sensory systems. In the present study the effect of a chronic increase or decrease in motoneuronal activity on motoneuron oxidative capacity and soma size was studied. In addition, the effect of chronic axotomy was studied as an indicator of whether cytoplasmic volume may also be related to the oxidative capacity of motoneurons. A quantitative histochemical assay for succinate dehydrogenase activity was used as a measure of motoneuron oxidative capacity in experimental models in which chronic electromyography has been used to verify neuronal activity levels. Spinal transection reduced, and spinal isolation virtually eliminated lumbar motoneuron electrical activity. Functional overload of the plantaris by removal of its major synergists was used to chronically increase neural activity of the plantaris motor pool. No change in oxidative capacity or soma size resulted from either a chronic increase or decrease in neuronal activity level. These data indicate that the chronic modulation of ionic transport and neurotransmitter turnover associated with action potentials do not induce compensatory metabolic responses in the metabolic capacity of the soma of lumbar motoneurons. Soma oxidative capacity was reduced in the axotomized motoneurons, suggesting that a combination of axoplasmic transport, intraeellular biosynthesis and perhaps neurotransmitter turnover represent the major energy demands on a motoneuron. While soma oxidative capacity may be closely related to neural activity in some neural systems, e.g. visual and auditory, lumbar motoneurons appear to be much less sensitive to modulations in chronic activity levels. INTRODUCTION If the generation of action potentials has a significant impact on m o t o n e u r o n metabolic demands, then one would expect that modulation of n e u r o n a l activity (i.e. n u m b e r and frequency of action potentials) would alter the oxidative potential of a n e u r o n , assuming that the metabolic capacity is able to respond. Other cellular processes, however, including axonal transport, m a i n t e n a n c e of cytoplasmic components and neurotransmitter turnover may also influence the energy demands of a motoneuron. Since each of these cell functions is indirectly or directly affected by cell size, the energy d e m a n d on a m o t o n e u r o n may also be related to one or more measures of size of the m o t o n e u r o n . Presumably those motoneurons with the greatest energy demands per unit of time have the highest oxidative capacity and, if energy demands are chronically modulated, the oxidative potential will adapt accordingly. The effects of increasing activation on m o t o n e u r o n a l oxidative capacity have differed, as have the methods by which the activation is elevated. Swim training in rats for
52 days increased m o t o n e u r o n a l oxidative capacity 19, while 56 days of chronic antidromic electrical stimulation in cats produced no change in m o t o n e u r o n soma succinate dehydrogenase (SDH) activity 12. After 60 days of unilateral functional overload (FO) of rat soleus muscles, a model expected to increase m o t o n e u r o n activation, the oxidative capacity was reduced in the soma of the motoneurons innervating the overloaded muscles 3°. Thus, the only data that suggest that the oxidative capacity of lumbar m o t o n e u r o n s is elevated following increased neuronal activation is in chronically exercised-trained rats. Reduced m o t o n e u r o n a l activity for periods ranging from 6 h to 14 months following spinal transection (ST) resulted in no change in the oxidative capacity of rat m o t o n e u r o n s caudal to the lesionca. In contrast, a decreased neuronal oxidative capacity following reductions in neural activation have been reported in visual49'51, auditory 5° and olfactory 9 systems. However, all of these studies in n o n - m o t o r systems were based on qualitative enzyme assays of chemically fixed tissues, a procedure known to cause marked and variable inhibition of enzymatic activity7.
Correspondence: V.R. Edgerton, Dept. of Kinesiology, 1804 Life Sciences, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-1527, U.S.A. Fax: (1) (213) 206-9184.
T h e p r e s e n t s t u d y w a s u n d e r t a k e n to d e t e r m i n e t h e effect of a c h r o n i c i n c r e a s e o r d e c r e a s e in m o t o n e u r o n a l activity a n d of a x o t o m y o n s o m a o x i d a t i v e c a p a c i t y a n d
Control group for spinal cats (spinal control) The spinal cord lumbar enlargement of 9 control cats (8 females and 1 male, body weights 3.0-5.2 kg) was removed and frozen as described above.
size, a n d t h e r e l a t i o n s h i p b e t w e e n t h e s e m e a s u r e s . M o t o n e u r o n s o m a o x i d a t i v e c a p a c i t y a n d size w e r e u n a f f e c t e d by c h r o n i c a l t e r a t i o n s in a c t i v a t i o n levels, w h i l e s o m a o x i d a t i v e c a p a c i t y , b u t n o t size, was r e d u c e d following axotomy. These results suggest that the oxidative c a p a c i t y of t h e s o m a of cat l u m b a r m o t o n e u r o n s is inf l u e n c e d b y t h e t o t a l v o l u m e of t h e cell, b u t s e e m s r a t h e r i n s e n s i t i v e t o t h e a m o u n t of a c t i v a t i o n .
MATERIALS AND METHODS Spinal isolation (S1) group To eliminate activity of motoneurons, 9 female adult cats (body weights 2.9-4.0 kg) were sedated initially with a mixture of ketamine (13 mg/kg, i.m.) and acepromazine (0.01 mg/kg, i.m.) and deeply anesthetized using sodium pentobarbital (35 mg/kg, i.v.) sufficient to keep withdrawal and eye blink responses suppressed. Under sterile conditions a skin incision was made from vertebral level Tlx to $3, the fascia incised and the paraspinal muscles retracted. All dorsal spinous processes were removed from T12 to $2 and a narrow midline laminectomy (approximately 2 mm wide) from T12 to S1 was made to expose the spinal cord. All dorsal roots were severed bilaterally within the dura between these vertebral levels. The spinal cord then was transected completely at the junctions of T12_13and again at LT-S 1, with care being taken to preserve the ventral artery. In 2 of the cats the transection points were at L4-L 5 and $2-$3 with deafferentation and laminectomy between these points. The overlying dorsal fascia, muscle and skin were sutured and a topical antiseptic (Furacin) applied. The cats were placed in an incubator for postsurgical recovery and then tranferred to vivarium cages ( 2 x l . 2 x 3 m) in which the floor was covered with shredded newspaper. Postsurgical care followed the procedures previously described for spinally transected (ST) cats 1'3s. The SI cats were maintained for 6 months, with the right leg of each cat receiving passive cyclic oscillations mimicking a step cycle for 30 min/day, 5 days/week during the last 5 months. To quantify the frequency of motoneuronal activation, neuromuscular activity levels in representative muscles in the hindlimb innervated by the isolated segment were determined at 6 months postsurgery by measuring the EMG over a 48-h period in 5 SI and 4 control cats. In the control cats, the soleus, lateral gastrocnemius, extensor digitorum longus, and tibialis anterior muscles were monitored. In the SI cats, because the soleus and tibialis anterior were used for other experiments, activity levels were determined only in the extensor digitorum longus and lateral gastrocnemius muscles. Following a terminal in situ muscle physiological experiment, each SI cat was killed with an overdose of sodium pentobarbital and the lumbar enlargement was rapidly removed, cut into blocks, approximately 1 cm long, quickly frozen in isopentane cooled by liquid nitrogen and stored at -70°C until processed. Spinal transection (ST) group To markedly reduce, but not eliminate activity of motoneurons, 9 adult female cats (body weights 2.7-3.5 kg) were anesthetized as described for the SI cats. Under sterile conditions a laminectomy and complete spinal cord transection were performed at the T12-T13 vertebral level3s. Postoperative care was as described for the SI cats. The cats were maintained for 6 months, 4 cats were untreated and 5 cats were trained to stand while supporting the hind quarters 30 rain/day, 5 days/week for the last 5 months of the 6-month experimental period. Following a terminal in situ muscle physiological experiment, the lumbar enlargement of each ST cat was removed and prepared as described for the SI cats.
Plantaris functional overload (FO) group To increase the activity levels of motoneurons above normal, 5 adult female littermate cats were anesthetized as described for the SI cats. Under aseptic conditions, skin incisions were made to expose the dorsal region of both hindlimbs. The plantaris muscle was bilaterally functionally isolated by removing its major synergists. After ligating the blood supply and cutting the nerve branches to the soleus and medial gastrocnemius, these muscles were completely excised from both legs. The lateral gastrocnemius also was removed except for a small fleshy portion which remained attached near the proximal end of the plantaris. Care was taken to avoid injury to the plantaris muscle, nerve and blood supply. Postsurgical care was as described for the SI cats, except that the cages did not contain shredded newspaper. To elevate the level of motoneuronal activation, the FO cats were started on an exercise program about 4 weeks postsurgery, when it was evident that digitigrade locomotor capabilities had recovered. The exercise program started with the FO cats walking around a room for up to 15 min/day, 6 days/week. Progressively, the excercise duration was increased to 25 min/day and the intensity was increased by eliciting highly active running and jumping activity by throwing table tennis balls around the room in which the cats played. Twelve weeks following the initial surgery each FO cat was anesthetized as described above. The right plantaris muscle was exposed and freed as much as possible from the surrounding tissue, and 750 /~1 of a 2% solution of horseradish peroxidase conjugated with wheat germ agglutinin (HRP, Sigma) was injected into multiple sites throughout the muscle. Care was taken to ensure that other muscles were not exposed to the HRP by isolating the plantaris, injecting the HRP very slowly to prevent reflux from the injection site and using cotton to absorb any HRP observed to leak, although this rarely occurred. The incisions then were closed as described above and the cats were returned to their cages for 96 h. During this period the FO cats were included in the daily exercise session only if fully recovered from surgery. After 96 h each cat was deeply anesthetized and perfused transcardially with 0.5 1 of phosphate-buffered saline. This procedure removed blood from the vasculature of the cord to eliminate any artifacts in the HRP reaction normally produced by red blood cells. A laminectomy was performed to allow the L 5 to S 1 cord segments to be identified by following the appropriate nerve roots. Each cord segment was then removed and prepared for analysis as described above. The plantaris muscles from each of the cats were removed bilaterally, cleaned of fat and connective tissue and weighed (wet weight). Control group for plantaris FO cats (FO control) Four adult female cats (littermates to the cats in the plantaris FO group) were housed in 0.6 x 0.5 × 0.8 m cages and kept in the vivarium for the same duration of time as the plantaris FO group. Injection of HRP, survival times and terminal experimental procedures were the same as described for the FO group, except that only 500/A of HRP was injected because of the smaller muscle mass in the control compared with the FO eats (see below). Soma SDH activity and area measurements in spinal control, S T and SI cats The SDH activity and soma area of a sample of motoneurons from the midportion of the lumbar enlargement of each cat in each group were determined as described in detail previously s. Briefly, a 10 ktm thick spinal cord tissue section was cut in a cryostat and incubated for 8 min in assay media lacking succinate substrate to allow non-specific enzymatic staining to occur and then plateau and cease. The tissue was then transferred to identical media containing
3 succinate substrate and the progression of the SDH reaction was recorded using a digital image processing system for 6 min. Previous tests have demonstrated that the increase in optical density during the incubation with succinate substrate is linear over this period 8. Individual motoneuron soma were identified in the recorded images and the mean optical density of the entire soma cytoplasm, excluding the nuclear region if present in the cell section, was determined in each picture. The rate of increase in optical density over the reaction period, i.e. their SDH activity, was then determined for each cell. SDH activity was determined in soma sections both with and without a nucleus in the section. Previous testss have demonstrated that the SDH activity of a cell soma is equally represented in a section of the soma either with or without the nucleus included. Soma area was determined only for those neurons which had a visible nucleus in the tissue section. Since the mean diameter of cat lumbar a-motoneurons is approximately 60/,tm 1°'43, 60 pm of tissue was cut and discarded between each tissue section examined to reduce the probability of sampling any individual motoneuron twice.
Soma SDH activity and area measurements in FO control and FO cats A 10-ttm tissue cross-section was cut from a spinal cord tissue block and placed in a tray in the cryostat for HRP processing. A serial 10-/~m tissue section was cut and reacted for the measurement of SDH activity as described above, with the results being stored on computer tape for later comparison with HRP-stained sections. Then 60 pm of tissue was cut and discarded. This process was repeated until an average of 73 plantaris motoneurons were sampled in each cat. The tissue sections stored in the cryostat then were reacted for HRP histochemistry using the technique of Sickles and Oblak 41 with the following modifications. The tetramethyl benzidine incubation consisted of 4 changes of 12,5 min each of the following media (mixed immediately before use): 195 ml solution A; 7.5 ml solution B (solution A and B mixed as described by Sickles and Oblak 41) and 3.5 ml of 0.3% H20 2. Following 5 rinses in 0.01 M cold acetate buffer (pH 3.3) the slides were air-dried and mounted in a glycerol/phosphate buffer (0.1 M, pH 7.4) mixture (9:1) and the coverslips then were sealed with nail polish. Digitized images recording the SDH reaction were analyzed as previously described 8 and compared with serial HRP-stained sections to identify plantaris motoneurons. Motoneurons that innervated the soleus and medial and lateral gastrocnemius muscles (triceps surae) in FO control cats and equivalent triceps surae neurons that were axotomized in FO cats, were analyzed separately from other non-labeled motoneurons. These cells could be identified by their position directly ventral to the labeled plantaris pool, and as the most medial cells in the middle portion of the ventral horn at the L 7 level in the spinal c o r d 36. Statistics Mean motoneuron SDH activity and soma area of the spinal control, SI and ST groups were compared using an ANOVA. SDH activity and soma area of the FO control and FO groups were compared using a t-test. In both cases the tests were for unpaired samples and the number of cats determined the degrees of freedom for the test. For cells in which there was a visible nucleus in the tissue section, the relationship between soma area and SDH activity was examined. To test for differences in the variability of SDH activity as a function of cell size, the data were divided into bins along the soma area axis, with an approximately equal number of observations in each bin for each experimental group. The variance of the SDH activities observed in each bin was determined and a confidence interval for the variance estimate for each bin was calculated using a chi square distribution. All procedures followed the Guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use Committee at UCLA.
TABLE I
Motoneuron mean succinate dehydrogenase (SDH) activity and soma area for spinal control, spinal isolated (SI) and spinal transected (ST) cats Group
Nucleus
N(n) a
SDH activity b ( O. D./min ×
Spinal control Spinal control SI SI ST ST
yes c no yes no yes no
9(437) 9(620) 9(356) 9(751) 9(447) 9(697)
Soma area b (l~m2)
lo-')
14.47d+ 14.91 + 13.87 + 13.58 + 13.62 + 13.42 +
3.54 e 3.58 2.70 g 2.67 2.95 i 2.26
3108d+ 412~ 3070 + 398 h 3296 ___ 40P
a Number of cats and (number of neurons), b Values are mean ___ standard deviation (S.D.). c Data in the first row for each group are from neurons with a visible nucleus, the second row from neurons without a visible nucleus in the tissue section. d The mean of the means for each cat. e Mean S.D. for SDH activities within control cats for motoneurons with and without a nucleus were 3.63 and 4.03 x 10 -3 O.D./min, respectively. Mean S.D. of areas within control cats was 945 #m 2. g Mean S.D. for SDH activities within SI cats for motoneurons with and without a nucleus were 4.30 and 3.72 x 10 -3 O.D./min, respectively, h Mean S.D. of areas within SI cats was 975/tin 2. i Mean S.D. for SDH activities within ST cats for motoneurons with and without a nucleus were 4.21 and 3.81 × 10 - 3 0 . D . / m i n , respectively. J Mean S.D. of areas within ST cats was 1118/~m 2.
RESULTS
Effects o f chronically decreased activity: S I and S T EMG
r e c o r d i n g s o v e r a 4 8 - h p e r i o d in e a c h SI cat
demonstrated
that the extensor digitorum longus and
l a t e r a l g a s t r o c n e m i u s m u s c l e s in t h e SI c a t s , r e p r e s e n t a tive o f t h e m o t o n e u r o n a l
p o o l s w i t h i n t h e i s o l a t e d seg-
m e n t o f t h e s p i n a l c o r d , e x h i b i t e d a daily l e v e l o f electrical a c t i v i t y ( m i l l i v o l t s e c o n d s ) w h i c h w a s 0 . 0 0 3 % o f t h e c o n t r o l l e v e l 33'37. S i m i l a r l y , it h a s p r e v i o u s l y b e e n d e m o n s t r a t e d t h a t S T r e s u l t s in a r e d u c t i o n in d a i l y i n t e grated EMG
activity o f a p p r o x i m a t e l y 50% a n d 7 5 % in
l a t e r a l g a s t r o c n e m i u s a n d s o l e u s m o t o n e u r o n s c a u d a l to t h e l e s i o n , r e s p e c t i v e l y 1. T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in m e a n
mo-
t o n e u r o n S D H activity o r s o m a a r e a b e t w e e n : (i) t h e 2 sides o f t h e s p i n a l c o r d in a n y g r o u p ; (ii) t h e 2 S T g r o u p s ; (iii) t h e SI cats w i t h s p i n a l t r a n s e c t i o n s at d i f f e r e n t levels, o r (iv) t h e m a l e a n d f e m a l e s p i n a l c o n t r o l cats. T h u s e a c h o f t h e s e d a t a sets w e r e p o o l e d ( T a b l e I). T h e r e w e r e n o significant d i f f e r e n c e s a m o n g t h e SI, S T a n d s p i n a l c o n t r o l cats in m o t o n e u r o n m e a n S D H a c t i v i t y o r m e a n s o m a c r o s s - s e c t i o n a l a r e a ( T a b l e I). I n all g r o u p s , t h e s m a l l e r n e u r o n s h a d a w i d e r a n g e o f S D H a c t i v i t i e s (i.e. low to h i g h v a l u e s ) w h i l e t h e l a r g e r n e u r o n s w e r e g e n e r a l l y l i m i t e d t o o n l y l o w S D H activities (Figs. 1, 2). T h i s p a t t e r n is e m p h a s i z e d in t h e c o n t r o l g r o u p as a d e n s i t y p l o t in Fig. l B . N o t e in Fig. 1 A t h a t
very few large cells are above the b o t t o m third of the observed range of S D H activities. F o r SI and ST cats (Fig. 2) the density plots (not shown) of the scatter plots were similar to the control cats. The data were divided into 3 bins along the soma area axis with an approximately equal n u m b e r of observations in each bin for each group (136, 110, 140 cells/bin for control, SI and ST groups, respectively). In all 3 groups the variance in S D H activities in the bin containing the smallest cells was significantly greater ( P < 0.01) than the variance of the cells in the bin containing the largest cells. In the SI and ST groups the variance in S D H activities in the bin containing the smallest cells was also significantly greater (P
< 0.01) than the variance in the middle bin containing the medium-sized cells. If a soma d i a m e t e r of 38 ~ m (1134 /~m2 area) is used as a cutoff between a- and 7-motoneurons 5'~°, it is a p p a r e n t from Figs. 1 and 2 that almost all of the neurons examined which had a nucleus visible in the tissue section were a - m o t o n e u r o n s .
Effects of chronically increased activity: FO There were no significant differences in m e a n S D H activity or soma area of plantaris m o t o n e u r o n s in F O control and F O cats (Table II). The relationships between S D H activity and soma area for F O control (Fig. 3A) and F O (Fig. 3B) plantaris m o t o n e u r o n s were similar. As observed in the SI, ST and spinal control cats
SO"
A
50'
CONTROL
.
A
SPINAL
ISOLATED
40
40. + ÷
4+ 4`
4,
+
4` 4-
+ ++
÷
4`:4÷4`~+
~'%4,~,.¢
" ~ % ":'.i~'~.
+
4`;
4, . ~
+4, ÷ 4" 4`
4`
+n
+4, + 4,+ + ÷.K
4,
i'*~÷Ll**
41- +4.41"
10"
+ +
• 4`
20.
~
÷
4,
~ " 20-
*
÷ 4, 4, 4 , . ~ ' ' ~ . F ' +
+ ~ + 4 . + 4`
4, 4,, 4,
4,
• ~+
4`.4,
4, ÷ ÷
"~ 10
÷
+
o
÷
X
4,4,++ 4,
4, ~ ; :
0
~
+
>. _
>.50
o
~_.,~,
I.-,
B
B '~ 4 0
SPINAL
TRANSECTED
ffl 40-
÷.* ÷
3oi 30-
++++÷+ ++++
i:
+
20 20.
,÷ +
+
+
÷!
÷
*~ , ÷:~"
~ ÷ . ~ . ++'n. 4 + +
!-,
÷
*
+.
10 100
0
i
1540
3080
I 4620
I
j
6160
7700
SOMA A R E A ( g m 2)
Fig. 1. Relationship between succinate dehydrogenase (SDH) activity and soma area in lumbar motoneurons of spinal control cats. A: each symbol represents a single cell, Dotted lines separate bins used for variance analysis on SDH activities (see text for further explanation)• B: density plot of the observations in (A). Note the triangular pattern of the plot demonstrating the wider range in SDH activities for the smaller compared to the larger motoneurons.
+
0
0
1540
3080
4620
6160
7700
SOMA A R E A (btm2)
Fig. 2. Relationship between succinate dehydrogenase (SDH) activity and soma area in lumbar motoneurons of spinal isolated (A) and spinal transected (B) cats. Each symbol represents a single cell. Dotted lines separate bins used for variance analysis on SDH activities (see text for further explanation).
50,
TABLE II
CONTROL
A
Plantaris motoneuron mean succinate dehydrogenase (SDH) activity and soma area in functional overload control (FO control) and functional overload (FO) cats
40.
Group
Nucleus
N(n) ~
30. FO Control FO Control FO FO
4.
2O .;.+', 4. "~
.
4-
.:4- • "~;~. "P:'÷~
.
.
.
.
E e~ O
4.
~.~
.~.,~,+~
:
4. 4,*. ":".~'.~-:,.,,,:.,~
.
,..
4-'
0
i
>" 50 _P >
FUNCTIONAL OVERLOAD
B
IO
4(81) 4(211) 5(106) 5(258)
Soma areab
11.49d+I.09 e 11.42 +0.13 10.13 +1.13 g 10.68 +1.24
3613d+358 f
(Itm2)
3910 +309 h
a Number of cats and (number of neurons)• b Values are mean _+ standard deviation (S.D.). c Data in the first row for each group are from neurons with a visible nucleus, the second row from neurons without a visible nucleus in the tissue section• d The mean of the means for each cat. e Mean S.D. for SDH activities within control cats for motoneurons with and without a nucleus were 2.94 and 2.76 x 10 - 3 0 . D . / m i n , respectively• f Mean S.D. of areas within control cats was 1154/~m 2. g Mean S.D. for SDH activities within FO cats for motoneurons with and without a nucleus were 2.64 and 3.31 x 10-30.D./min, respectively, h Mean S.D. of areas within FO cats was 1128/Jm 2.
'l,',"
xt 10
yes" no yes no
SDH activityb (0. D. ~rain X 10-3)
< 40 .i-
g,
30'
P
1
© 1992 Elsevier Science Publishers B.V, All tights reserved. 0006-8993/92/$05.00 BRES 17105
Research Reports
Adaptability of the oxidative capacity of motoneurons Gordon R. Chalmers 1, Roland R. Roy I and V. Reggie Edgerton 1'2 1Brain Research Institute and 2Department of Kinesiology, University of California, Los Angeles, CA 90024 (U.S.A.) (Accepted 11 June 1991)
Key words: Motoneuron; Succinate dehydrogenase; Oxidative capacity; Soma area; Spinal transection; Spinal isolation; Muscle functional overload; Energy metabolism; Cat
Previous studies have demonstrated that a chronic change in neuronal activation can produce a change in soma oxidative capacity, suggesting that: (i) these 2 variables are directly related in neurons and (ii) ion pumping is an important energy requiring activity of a neuron. Most of these studies, however, have focused on reduced activation levels of sensory systems. In the present study the effect of a chronic increase or decrease in motoneuronal activity on motoneuron oxidative capacity and soma size was studied. In addition, the effect of chronic axotomy was studied as an indicator of whether cytoplasmic volume may also be related to the oxidative capacity of motoneurons. A quantitative histochemical assay for succinate dehydrogenase activity was used as a measure of motoneuron oxidative capacity in experimental models in which chronic electromyography has been used to verify neuronal activity levels. Spinal transection reduced, and spinal isolation virtually eliminated lumbar motoneuron electrical activity. Functional overload of the plantaris by removal of its major synergists was used to chronically increase neural activity of the plantaris motor pool. No change in oxidative capacity or soma size resulted from either a chronic increase or decrease in neuronal activity level. These data indicate that the chronic modulation of ionic transport and neurotransmitter turnover associated with action potentials do not induce compensatory metabolic responses in the metabolic capacity of the soma of lumbar motoneurons. Soma oxidative capacity was reduced in the axotomized motoneurons, suggesting that a combination of axoplasmic transport, intraeellular biosynthesis and perhaps neurotransmitter turnover represent the major energy demands on a motoneuron. While soma oxidative capacity may be closely related to neural activity in some neural systems, e.g. visual and auditory, lumbar motoneurons appear to be much less sensitive to modulations in chronic activity levels. INTRODUCTION If the generation of action potentials has a significant impact on m o t o n e u r o n metabolic demands, then one would expect that modulation of n e u r o n a l activity (i.e. n u m b e r and frequency of action potentials) would alter the oxidative potential of a n e u r o n , assuming that the metabolic capacity is able to respond. Other cellular processes, however, including axonal transport, m a i n t e n a n c e of cytoplasmic components and neurotransmitter turnover may also influence the energy demands of a motoneuron. Since each of these cell functions is indirectly or directly affected by cell size, the energy d e m a n d on a m o t o n e u r o n may also be related to one or more measures of size of the m o t o n e u r o n . Presumably those motoneurons with the greatest energy demands per unit of time have the highest oxidative capacity and, if energy demands are chronically modulated, the oxidative potential will adapt accordingly. The effects of increasing activation on m o t o n e u r o n a l oxidative capacity have differed, as have the methods by which the activation is elevated. Swim training in rats for
52 days increased m o t o n e u r o n a l oxidative capacity 19, while 56 days of chronic antidromic electrical stimulation in cats produced no change in m o t o n e u r o n soma succinate dehydrogenase (SDH) activity 12. After 60 days of unilateral functional overload (FO) of rat soleus muscles, a model expected to increase m o t o n e u r o n activation, the oxidative capacity was reduced in the soma of the motoneurons innervating the overloaded muscles 3°. Thus, the only data that suggest that the oxidative capacity of lumbar m o t o n e u r o n s is elevated following increased neuronal activation is in chronically exercised-trained rats. Reduced m o t o n e u r o n a l activity for periods ranging from 6 h to 14 months following spinal transection (ST) resulted in no change in the oxidative capacity of rat m o t o n e u r o n s caudal to the lesionca. In contrast, a decreased neuronal oxidative capacity following reductions in neural activation have been reported in visual49'51, auditory 5° and olfactory 9 systems. However, all of these studies in n o n - m o t o r systems were based on qualitative enzyme assays of chemically fixed tissues, a procedure known to cause marked and variable inhibition of enzymatic activity7.
Correspondence: V.R. Edgerton, Dept. of Kinesiology, 1804 Life Sciences, UCLA, 405 Hilgard Ave., Los Angeles, CA 90024-1527, U.S.A. Fax: (1) (213) 206-9184.
T h e p r e s e n t s t u d y w a s u n d e r t a k e n to d e t e r m i n e t h e effect of a c h r o n i c i n c r e a s e o r d e c r e a s e in m o t o n e u r o n a l activity a n d of a x o t o m y o n s o m a o x i d a t i v e c a p a c i t y a n d
Control group for spinal cats (spinal control) The spinal cord lumbar enlargement of 9 control cats (8 females and 1 male, body weights 3.0-5.2 kg) was removed and frozen as described above.
size, a n d t h e r e l a t i o n s h i p b e t w e e n t h e s e m e a s u r e s . M o t o n e u r o n s o m a o x i d a t i v e c a p a c i t y a n d size w e r e u n a f f e c t e d by c h r o n i c a l t e r a t i o n s in a c t i v a t i o n levels, w h i l e s o m a o x i d a t i v e c a p a c i t y , b u t n o t size, was r e d u c e d following axotomy. These results suggest that the oxidative c a p a c i t y of t h e s o m a of cat l u m b a r m o t o n e u r o n s is inf l u e n c e d b y t h e t o t a l v o l u m e of t h e cell, b u t s e e m s r a t h e r i n s e n s i t i v e t o t h e a m o u n t of a c t i v a t i o n .
MATERIALS AND METHODS Spinal isolation (S1) group To eliminate activity of motoneurons, 9 female adult cats (body weights 2.9-4.0 kg) were sedated initially with a mixture of ketamine (13 mg/kg, i.m.) and acepromazine (0.01 mg/kg, i.m.) and deeply anesthetized using sodium pentobarbital (35 mg/kg, i.v.) sufficient to keep withdrawal and eye blink responses suppressed. Under sterile conditions a skin incision was made from vertebral level Tlx to $3, the fascia incised and the paraspinal muscles retracted. All dorsal spinous processes were removed from T12 to $2 and a narrow midline laminectomy (approximately 2 mm wide) from T12 to S1 was made to expose the spinal cord. All dorsal roots were severed bilaterally within the dura between these vertebral levels. The spinal cord then was transected completely at the junctions of T12_13and again at LT-S 1, with care being taken to preserve the ventral artery. In 2 of the cats the transection points were at L4-L 5 and $2-$3 with deafferentation and laminectomy between these points. The overlying dorsal fascia, muscle and skin were sutured and a topical antiseptic (Furacin) applied. The cats were placed in an incubator for postsurgical recovery and then tranferred to vivarium cages ( 2 x l . 2 x 3 m) in which the floor was covered with shredded newspaper. Postsurgical care followed the procedures previously described for spinally transected (ST) cats 1'3s. The SI cats were maintained for 6 months, with the right leg of each cat receiving passive cyclic oscillations mimicking a step cycle for 30 min/day, 5 days/week during the last 5 months. To quantify the frequency of motoneuronal activation, neuromuscular activity levels in representative muscles in the hindlimb innervated by the isolated segment were determined at 6 months postsurgery by measuring the EMG over a 48-h period in 5 SI and 4 control cats. In the control cats, the soleus, lateral gastrocnemius, extensor digitorum longus, and tibialis anterior muscles were monitored. In the SI cats, because the soleus and tibialis anterior were used for other experiments, activity levels were determined only in the extensor digitorum longus and lateral gastrocnemius muscles. Following a terminal in situ muscle physiological experiment, each SI cat was killed with an overdose of sodium pentobarbital and the lumbar enlargement was rapidly removed, cut into blocks, approximately 1 cm long, quickly frozen in isopentane cooled by liquid nitrogen and stored at -70°C until processed. Spinal transection (ST) group To markedly reduce, but not eliminate activity of motoneurons, 9 adult female cats (body weights 2.7-3.5 kg) were anesthetized as described for the SI cats. Under sterile conditions a laminectomy and complete spinal cord transection were performed at the T12-T13 vertebral level3s. Postoperative care was as described for the SI cats. The cats were maintained for 6 months, 4 cats were untreated and 5 cats were trained to stand while supporting the hind quarters 30 rain/day, 5 days/week for the last 5 months of the 6-month experimental period. Following a terminal in situ muscle physiological experiment, the lumbar enlargement of each ST cat was removed and prepared as described for the SI cats.
Plantaris functional overload (FO) group To increase the activity levels of motoneurons above normal, 5 adult female littermate cats were anesthetized as described for the SI cats. Under aseptic conditions, skin incisions were made to expose the dorsal region of both hindlimbs. The plantaris muscle was bilaterally functionally isolated by removing its major synergists. After ligating the blood supply and cutting the nerve branches to the soleus and medial gastrocnemius, these muscles were completely excised from both legs. The lateral gastrocnemius also was removed except for a small fleshy portion which remained attached near the proximal end of the plantaris. Care was taken to avoid injury to the plantaris muscle, nerve and blood supply. Postsurgical care was as described for the SI cats, except that the cages did not contain shredded newspaper. To elevate the level of motoneuronal activation, the FO cats were started on an exercise program about 4 weeks postsurgery, when it was evident that digitigrade locomotor capabilities had recovered. The exercise program started with the FO cats walking around a room for up to 15 min/day, 6 days/week. Progressively, the excercise duration was increased to 25 min/day and the intensity was increased by eliciting highly active running and jumping activity by throwing table tennis balls around the room in which the cats played. Twelve weeks following the initial surgery each FO cat was anesthetized as described above. The right plantaris muscle was exposed and freed as much as possible from the surrounding tissue, and 750 /~1 of a 2% solution of horseradish peroxidase conjugated with wheat germ agglutinin (HRP, Sigma) was injected into multiple sites throughout the muscle. Care was taken to ensure that other muscles were not exposed to the HRP by isolating the plantaris, injecting the HRP very slowly to prevent reflux from the injection site and using cotton to absorb any HRP observed to leak, although this rarely occurred. The incisions then were closed as described above and the cats were returned to their cages for 96 h. During this period the FO cats were included in the daily exercise session only if fully recovered from surgery. After 96 h each cat was deeply anesthetized and perfused transcardially with 0.5 1 of phosphate-buffered saline. This procedure removed blood from the vasculature of the cord to eliminate any artifacts in the HRP reaction normally produced by red blood cells. A laminectomy was performed to allow the L 5 to S 1 cord segments to be identified by following the appropriate nerve roots. Each cord segment was then removed and prepared for analysis as described above. The plantaris muscles from each of the cats were removed bilaterally, cleaned of fat and connective tissue and weighed (wet weight). Control group for plantaris FO cats (FO control) Four adult female cats (littermates to the cats in the plantaris FO group) were housed in 0.6 x 0.5 × 0.8 m cages and kept in the vivarium for the same duration of time as the plantaris FO group. Injection of HRP, survival times and terminal experimental procedures were the same as described for the FO group, except that only 500/A of HRP was injected because of the smaller muscle mass in the control compared with the FO eats (see below). Soma SDH activity and area measurements in spinal control, S T and SI cats The SDH activity and soma area of a sample of motoneurons from the midportion of the lumbar enlargement of each cat in each group were determined as described in detail previously s. Briefly, a 10 ktm thick spinal cord tissue section was cut in a cryostat and incubated for 8 min in assay media lacking succinate substrate to allow non-specific enzymatic staining to occur and then plateau and cease. The tissue was then transferred to identical media containing
3 succinate substrate and the progression of the SDH reaction was recorded using a digital image processing system for 6 min. Previous tests have demonstrated that the increase in optical density during the incubation with succinate substrate is linear over this period 8. Individual motoneuron soma were identified in the recorded images and the mean optical density of the entire soma cytoplasm, excluding the nuclear region if present in the cell section, was determined in each picture. The rate of increase in optical density over the reaction period, i.e. their SDH activity, was then determined for each cell. SDH activity was determined in soma sections both with and without a nucleus in the section. Previous testss have demonstrated that the SDH activity of a cell soma is equally represented in a section of the soma either with or without the nucleus included. Soma area was determined only for those neurons which had a visible nucleus in the tissue section. Since the mean diameter of cat lumbar a-motoneurons is approximately 60/,tm 1°'43, 60 pm of tissue was cut and discarded between each tissue section examined to reduce the probability of sampling any individual motoneuron twice.
Soma SDH activity and area measurements in FO control and FO cats A 10-ttm tissue cross-section was cut from a spinal cord tissue block and placed in a tray in the cryostat for HRP processing. A serial 10-/~m tissue section was cut and reacted for the measurement of SDH activity as described above, with the results being stored on computer tape for later comparison with HRP-stained sections. Then 60 pm of tissue was cut and discarded. This process was repeated until an average of 73 plantaris motoneurons were sampled in each cat. The tissue sections stored in the cryostat then were reacted for HRP histochemistry using the technique of Sickles and Oblak 41 with the following modifications. The tetramethyl benzidine incubation consisted of 4 changes of 12,5 min each of the following media (mixed immediately before use): 195 ml solution A; 7.5 ml solution B (solution A and B mixed as described by Sickles and Oblak 41) and 3.5 ml of 0.3% H20 2. Following 5 rinses in 0.01 M cold acetate buffer (pH 3.3) the slides were air-dried and mounted in a glycerol/phosphate buffer (0.1 M, pH 7.4) mixture (9:1) and the coverslips then were sealed with nail polish. Digitized images recording the SDH reaction were analyzed as previously described 8 and compared with serial HRP-stained sections to identify plantaris motoneurons. Motoneurons that innervated the soleus and medial and lateral gastrocnemius muscles (triceps surae) in FO control cats and equivalent triceps surae neurons that were axotomized in FO cats, were analyzed separately from other non-labeled motoneurons. These cells could be identified by their position directly ventral to the labeled plantaris pool, and as the most medial cells in the middle portion of the ventral horn at the L 7 level in the spinal c o r d 36. Statistics Mean motoneuron SDH activity and soma area of the spinal control, SI and ST groups were compared using an ANOVA. SDH activity and soma area of the FO control and FO groups were compared using a t-test. In both cases the tests were for unpaired samples and the number of cats determined the degrees of freedom for the test. For cells in which there was a visible nucleus in the tissue section, the relationship between soma area and SDH activity was examined. To test for differences in the variability of SDH activity as a function of cell size, the data were divided into bins along the soma area axis, with an approximately equal number of observations in each bin for each experimental group. The variance of the SDH activities observed in each bin was determined and a confidence interval for the variance estimate for each bin was calculated using a chi square distribution. All procedures followed the Guidelines published in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Animal Use Committee at UCLA.
TABLE I
Motoneuron mean succinate dehydrogenase (SDH) activity and soma area for spinal control, spinal isolated (SI) and spinal transected (ST) cats Group
Nucleus
N(n) a
SDH activity b ( O. D./min ×
Spinal control Spinal control SI SI ST ST
yes c no yes no yes no
9(437) 9(620) 9(356) 9(751) 9(447) 9(697)
Soma area b (l~m2)
lo-')
14.47d+ 14.91 + 13.87 + 13.58 + 13.62 + 13.42 +
3.54 e 3.58 2.70 g 2.67 2.95 i 2.26
3108d+ 412~ 3070 + 398 h 3296 ___ 40P
a Number of cats and (number of neurons), b Values are mean ___ standard deviation (S.D.). c Data in the first row for each group are from neurons with a visible nucleus, the second row from neurons without a visible nucleus in the tissue section. d The mean of the means for each cat. e Mean S.D. for SDH activities within control cats for motoneurons with and without a nucleus were 3.63 and 4.03 x 10 -3 O.D./min, respectively. Mean S.D. of areas within control cats was 945 #m 2. g Mean S.D. for SDH activities within SI cats for motoneurons with and without a nucleus were 4.30 and 3.72 x 10 -3 O.D./min, respectively, h Mean S.D. of areas within SI cats was 975/tin 2. i Mean S.D. for SDH activities within ST cats for motoneurons with and without a nucleus were 4.21 and 3.81 × 10 - 3 0 . D . / m i n , respectively. J Mean S.D. of areas within ST cats was 1118/~m 2.
RESULTS
Effects o f chronically decreased activity: S I and S T EMG
r e c o r d i n g s o v e r a 4 8 - h p e r i o d in e a c h SI cat
demonstrated
that the extensor digitorum longus and
l a t e r a l g a s t r o c n e m i u s m u s c l e s in t h e SI c a t s , r e p r e s e n t a tive o f t h e m o t o n e u r o n a l
p o o l s w i t h i n t h e i s o l a t e d seg-
m e n t o f t h e s p i n a l c o r d , e x h i b i t e d a daily l e v e l o f electrical a c t i v i t y ( m i l l i v o l t s e c o n d s ) w h i c h w a s 0 . 0 0 3 % o f t h e c o n t r o l l e v e l 33'37. S i m i l a r l y , it h a s p r e v i o u s l y b e e n d e m o n s t r a t e d t h a t S T r e s u l t s in a r e d u c t i o n in d a i l y i n t e grated EMG
activity o f a p p r o x i m a t e l y 50% a n d 7 5 % in
l a t e r a l g a s t r o c n e m i u s a n d s o l e u s m o t o n e u r o n s c a u d a l to t h e l e s i o n , r e s p e c t i v e l y 1. T h e r e w e r e n o s i g n i f i c a n t d i f f e r e n c e s in m e a n
mo-
t o n e u r o n S D H activity o r s o m a a r e a b e t w e e n : (i) t h e 2 sides o f t h e s p i n a l c o r d in a n y g r o u p ; (ii) t h e 2 S T g r o u p s ; (iii) t h e SI cats w i t h s p i n a l t r a n s e c t i o n s at d i f f e r e n t levels, o r (iv) t h e m a l e a n d f e m a l e s p i n a l c o n t r o l cats. T h u s e a c h o f t h e s e d a t a sets w e r e p o o l e d ( T a b l e I). T h e r e w e r e n o significant d i f f e r e n c e s a m o n g t h e SI, S T a n d s p i n a l c o n t r o l cats in m o t o n e u r o n m e a n S D H a c t i v i t y o r m e a n s o m a c r o s s - s e c t i o n a l a r e a ( T a b l e I). I n all g r o u p s , t h e s m a l l e r n e u r o n s h a d a w i d e r a n g e o f S D H a c t i v i t i e s (i.e. low to h i g h v a l u e s ) w h i l e t h e l a r g e r n e u r o n s w e r e g e n e r a l l y l i m i t e d t o o n l y l o w S D H activities (Figs. 1, 2). T h i s p a t t e r n is e m p h a s i z e d in t h e c o n t r o l g r o u p as a d e n s i t y p l o t in Fig. l B . N o t e in Fig. 1 A t h a t
very few large cells are above the b o t t o m third of the observed range of S D H activities. F o r SI and ST cats (Fig. 2) the density plots (not shown) of the scatter plots were similar to the control cats. The data were divided into 3 bins along the soma area axis with an approximately equal n u m b e r of observations in each bin for each group (136, 110, 140 cells/bin for control, SI and ST groups, respectively). In all 3 groups the variance in S D H activities in the bin containing the smallest cells was significantly greater ( P < 0.01) than the variance of the cells in the bin containing the largest cells. In the SI and ST groups the variance in S D H activities in the bin containing the smallest cells was also significantly greater (P
< 0.01) than the variance in the middle bin containing the medium-sized cells. If a soma d i a m e t e r of 38 ~ m (1134 /~m2 area) is used as a cutoff between a- and 7-motoneurons 5'~°, it is a p p a r e n t from Figs. 1 and 2 that almost all of the neurons examined which had a nucleus visible in the tissue section were a - m o t o n e u r o n s .
Effects of chronically increased activity: FO There were no significant differences in m e a n S D H activity or soma area of plantaris m o t o n e u r o n s in F O control and F O cats (Table II). The relationships between S D H activity and soma area for F O control (Fig. 3A) and F O (Fig. 3B) plantaris m o t o n e u r o n s were similar. As observed in the SI, ST and spinal control cats
SO"
A
50'
CONTROL
.
A
SPINAL
ISOLATED
40
40. + ÷
4+ 4`
4,
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+ ++
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4,
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0
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>.50
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I.-,
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SPINAL
TRANSECTED
ffl 40-
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3oi 30-
++++÷+ ++++
i:
+
20 20.
,÷ +
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÷
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+.
10 100
0
i
1540
3080
I 4620
I
j
6160
7700
SOMA A R E A ( g m 2)
Fig. 1. Relationship between succinate dehydrogenase (SDH) activity and soma area in lumbar motoneurons of spinal control cats. A: each symbol represents a single cell, Dotted lines separate bins used for variance analysis on SDH activities (see text for further explanation)• B: density plot of the observations in (A). Note the triangular pattern of the plot demonstrating the wider range in SDH activities for the smaller compared to the larger motoneurons.
+
0
0
1540
3080
4620
6160
7700
SOMA A R E A (btm2)
Fig. 2. Relationship between succinate dehydrogenase (SDH) activity and soma area in lumbar motoneurons of spinal isolated (A) and spinal transected (B) cats. Each symbol represents a single cell. Dotted lines separate bins used for variance analysis on SDH activities (see text for further explanation).
50,
TABLE II
CONTROL
A
Plantaris motoneuron mean succinate dehydrogenase (SDH) activity and soma area in functional overload control (FO control) and functional overload (FO) cats
40.
Group
Nucleus
N(n) ~
30. FO Control FO Control FO FO
4.
2O .;.+', 4. "~
.
4-
.:4- • "~;~. "P:'÷~
.
.
.
.
E e~ O
4.
~.~
.~.,~,+~
:
4. 4,*. ":".~'.~-:,.,,,:.,~
.
,..
4-'
0
i
>" 50 _P >
FUNCTIONAL OVERLOAD
B
IO
4(81) 4(211) 5(106) 5(258)
Soma areab
11.49d+I.09 e 11.42 +0.13 10.13 +1.13 g 10.68 +1.24
3613d+358 f
(Itm2)
3910 +309 h
a Number of cats and (number of neurons)• b Values are mean _+ standard deviation (S.D.). c Data in the first row for each group are from neurons with a visible nucleus, the second row from neurons without a visible nucleus in the tissue section• d The mean of the means for each cat. e Mean S.D. for SDH activities within control cats for motoneurons with and without a nucleus were 2.94 and 2.76 x 10 - 3 0 . D . / m i n , respectively• f Mean S.D. of areas within control cats was 1154/~m 2. g Mean S.D. for SDH activities within FO cats for motoneurons with and without a nucleus were 2.64 and 3.31 x 10-30.D./min, respectively, h Mean S.D. of areas within FO cats was 1128/Jm 2.
'l,',"
xt 10
yes" no yes no
SDH activityb (0. D. ~rain X 10-3)
< 40 .i-
g,
30'
P
