Brain Research, 143 (1978) 397-391 C~ Elsevier/North-Holland Biomedical Press
387
The effect of D-amphetamine on gamma efferent activity in the acute decerebrate rat
CHARLES A. SORENSON, STEPHEN A. GEORGE and ANDREW J. FRIEDMAN Neuroscience Program, Amherst College, Amherst, Mass. 01002 (U.S.A.)
(Accepted October 6th, 1977)
Attempts to evaluate the function of reticulospinal (descending) norepinephrine (NE)-containing neurons in rats have focused on the effects of various pharmacological agents on hindlimb reflexes. These studies have shown that drug treatments which increase noradrenergic receptor activity (e.g. amphetamine is, DOPA 1° or clonidine 1) potentiate the flexion reflex in spinal or decerebrate preparations, while treatments that decrease noradrenergic transmission (e.g. reserpine 1, alpha-methyl-para-tyrosine ~, haloperidol ~,4 or phenoxybenzamine 2,4) greatly reduce the amplitude of the flexion reflex in these preparations. A series of studies by And6n et al. was carried out using cats as subjects to analyze electrophysiologically the neural mechanisms involved. Their studies showed that the administration of DOPA depressed the short latency 3,7 but enhanced the long latencyS, 6 transmission from flexor reflex afferents to alpha and gamma motor neurons, to ascending pathways and to primary afferents. More recently, Grillner and his colleagues 14,16 have suggested that spinal NEcontaining neurons contribute to the control of locomotor movements, based upon their work with cats. They have shown that stepping movements are produced in the acute spinal cat when its hindlimbs are placed on a treadmill after the administration of D O P A or clonidine la. The pattern of muscle activation during these movements is virtually identical to that of the intact animal walking on a treadmilP 3. Extensor contraction occurs 30-60 msec before the foot touches the treadmill, which shows that the timing of the action is intrinsic to the spinal cord and not controlled by stimulation of the foot. The gait of the stepping movements depends upon the speed of the treadmill - - it changes from a walk to a gallop as the speed of the treadmill increases. Stepping movements were also produced in decerebrate cats by low intensity electrical stimulation of a region in the midbrainX4,16 (which they called the 'mesencephalic locomotor region (MLR)') near the nucleus locus coeruleus iv. This effect is blocked by low doses of the noradrenergic receptor blocker phenoxybenzamine 13. Both of these treatments, electrical stimulation of the M LR and the administration of drugs such as DOPA, are associated with characteristic electrophysiological changes in the resting bias of gamma efferent motor neurons - - an increase in static gamma efferent activity but a decrease in dynamic activity s,9,14.
388 TABLE 1
The effect of intravenously applied D-amphetamine on baseline hindlimb flexor la activity Rat number
1 2 3 4
Average frequency (spikes~see) Vehicle
D-Amphetamine
24.2 100.6 27.4 82.9
42.2 122.9 197.7 257.0
Per cent change compared to vehicle L 74.4 22.2 ~ 621.5 1210.0
The suggestion of Griliner et al. that descending NE-containing neurons might play a role in feline locomotion is particularly intriguing, since it is generally believed that the locomotor stimulating effect of amphetamine in rats is produced by activation of ascending catecholamine-containing pathways, especially the mesolimbic dopaminergic pathway through its connections with the nucleus accumbens. It is possible that the rat possesses no similar descending locomotor system. However, if it does possess such a system, then administration of amphetamine to the decerebrate rat should produce a pattern ofelectrophysiological changes identical to those seen when DOPA is administered to the spinal cat or when the M L R is stimulated in the decerebrate cat. This study presents the results of the test of this prediction. The output of the gamma efferent system was measured by recording the responses of muscle spindle afferents, which are in series with intrafusal muscle elements innervated by gamma efferent neurons, to applied stretch of flexor muscles in the rat's hindlimb. Rats were anesthetized with chloral hydrate (385 mg/kg) given intraperitoneally. Cutaneous afferents from the left hindtimb were eliminated by removing the skin covering the limb. A laminectomy was performed to expose dorsal roots in the lumbar region of the spinal cord. The rat was decerebrated by aspiration of a 1 mm strip of brain tissue just rostral to the colliculi in order to remove any possible influences from ascending catecholaminergic pathways that could ultimately return to the spinal cord. The animal was placed in a holder constructed to fix the lower torso while allowing the left hindlimb to be stretched to a known position. The dorsal root serving the left hindlimb was cut, the distal end divided, and one branch picked up on a wick electrode. The rootlet was alternately tested and divided further until it contained no more than about 5 active units, all from flexor muscle spindles. Signals from the electrode were amplified and displayed conventionally, and stored on F M tape for later analysis by a peak detector circuit whose output pulses were timed and counted by a PDP-11 computer. The computer also generated histograms showing the spike frequency measured in consecutive time bins. In each experiment control records were first made of the response to 3 cycles of stretching (10 sec) and relaxation (5 sec) after injection of 150 #1 of saline in the tail vein. Then 150 #1 of a 1 mg/ml o-amphetamine solution was administered, and 5 min later the same sequence of 3 stretches was repeated.
389 800
VEHICLE
AMPHETAMINE
?ZO
G40 5G0 SPIKES
488 PER
4N
SECOND ~40 IGO
~.0
25.0
50.0
0~0
25.0
50.0
TIHE SINCE START OF ROll (SECONDS)
Fig. 1. Spike frequency profiles of hindlimb flexor la activity of rat 3 after decerebration and subsequent sequential intravenous injections of vehicle and D-amphetamine solutions. Profiles show responses in the relaxed condition and to three successive stretches applied to the hindlimb. Frequency was measured over 500 msec intervals.
The effect of amphetamine treatment on static gamma motoneuron activity can be determined by comparing the baseline level of activity after saline treatment to that following amphetamine treatment (Table I). In each of the four animals tested the amphetamine treatment produced an increase in muscle spindle spike frequency. This shows that amphetamine facilitates the activity of static gamma-motoneurons in the decerebrate rat. Dynamic gamma-motoneuron activity can be measured by comparing the spike frequency from muscle spindles while stretch is being applied (i.e. when the muscle is subject to velocity as well as length changes) to the frequency at a given time (3.0 sec in this case) after the stretching is complete and while the muscle is being held at its new length (i.e., when it is undergoing no dynamic changes). The difference between these two measurements of spike frequency, called the 'dynamic index', must result entirely from dynamic gamma-motoneuron activity, since the static gamma-motoneurons contribute equally to both measurements. To determine the effect of amphetamine on dynamic gamma-motoneuron activity, we compared the dynamic index before and after administration of amphetamine. Fig. 1 shows a representative case in which the peak level of activity declines from an average of 365 to 227 spikes/sec after saline (dynamic index = 138) and from 627 to 588 (dynamic index -- 39) after amphetamine. Of the three other rats tested, two showed comparable decreases in dynamic index. One animal, which had a very low dynamic index prior to drug administration, showed no change in dynamic index after amphetamine treatment. These results therefore indicate that amphetamine inhibits the activity of dynamic gamma-efferents in the decerebrate rat.
390 These results show t h a t the a d m i n i s t r a t i o n of a m p h e t a m i n e to the decerebrate rat p r o d u c e d the same p a t t e r n o f changes in static a n d d y n a m i c gamma-efferents as seen in the spinal cat t r e a t e d with D O P A or the decerebrate cat stimulated in the ' m e s e n c e p h a l i c l o c o m o t o r region'. These results therefore s u p p o r t the hypothesis t h a t a m p h e t a m i n e - i n d u c e d b e h a v i o r a l s t i m u l a t i o n in the rat as well as in the cat is due at least in p a r t to the a c t i v a t i o n o f a 'mesencephalic l o c o m o t o r region' which exerts its influence t h r o u g h the release o f N E at the segmental level. These results also lead to the p r e d i c t i o n t h a t the a d m i n i s t r a t i o n o f a m p h e t a m i n e would stimulate the l o c o m o t o r activity o f the d e c e r e b r a t e rat as it does t h a t o f the t h a l a m i c rat 1~, which lacks all structures a b o v e the diencephalon. I n light o f these findings, c a u t i o n s h o u l d be exercised in a s s u m i n g t h a t it is the ascending p a t h w a y s which m e d i a t e the behavioral effects o f p h a r m a c o l o g i c a l agents acting on c a t e c h o l a m i n e r g i c systems in i n t a c t animals. This investigation was s u p p o r t e d in p a r t by N I M H Small G r a n t MH27467 a n d by a g r a n t f r o m the A l f r e d P. Sloan F o u n d a t i o n to A m h e r s t College. W e t h a n k D a v i d N. M a s t r o n a r d e for assistance with the c o m p u t e r analysis o f the e l e c t r o p h y s i o l o g i c a l data.
I And6n, N.-E., Effects of amphetamine and some other drugs on central catecholamine mechanisms. In E. Costa and S. Garattini (Eds.), Amphetamines and Related Compounds, Raven Press, New York, 1970, pp. 447-462. 2 And6n, N.-E., Corrodi, H., Fuxe, K. and H6kfelt, T., Increased impulse flow in bulbospinal noradrenaline neurons produced by catecholamine receptor blocking agents, Europ. J. PharmacoL, 2 (1967) 59-64. 3 And6n, N.-E., Jukes, M. G. M. and Lundberg, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 4 And6n, N.-E., Jukes, M. G. M. and Lundberg, A., The effect of DOPA on the spinal cord. 2. A pharmacological analysis, Aeta physiol, scand., 67 (1966) 387-397. 5 And6n, N.-E., Jukes, M. G. M., Lundberg, A. and Vyklick3~,L., A new spinal flexor reflex, Nature (Lond.), 202 (1964) 1344-1345. 6 And6n, N.-E., Jukes, M. G, M., Lundberg, A. and Vyklick3~, L., The effect of DOPA on the spinal cord. 1. Influence on transmission from primary afferents, Actaphysiol. scand., 67 (t966) 373-386. 7 And6n, N.-E., Lundberg, A., Rosengren, E. and Vyklick~, L., The effect of DOPA on spinal reflexes from the FRA (flexor reflex afferents), Experientia (Basel), 19 (1963) 654-655. 8 Bergmans, J. and Grillner, S., Changes in dynamic sensitivity of primary endings of muscle spindle afferents induced by DOPA, Acta physiol, scand., 74 (1968) 629-636. 9 Bergmans, J. and Grillner, S., Reciprocal control of spontaneous activity and reflex effects in static and dynamic gamma-motoneurons revealed by an injection of DOPA, Acta physiol, scand., 77 (t969) 106-124. 10 Carlsson, A., Magnusson, T. and Rosengren, E., 5-Hydroxytryptamine of the spinal cord norreally and after transection, Experientia (Basel), 19 (t963) 359. 11 Forssberg, H. and Grillner, S., The locomotion of the acute spinal cat injected with clonidine, Brain Research, 50 (1973) 184-186. 12 Grillner, S., Supraspinal and segmental control of static and dynamic gamma-motoneurones in the cat, ,4cta physiol, scand., Suppl. 327 (1969) 1-34. 13 Grillner, S., Locomotion in the spinal cat. In Stein, R. B., Pearson, K. G., Smith, R. S. and Redford, J. B. (Eds.), Control of Posture and Locomotion, Plenum Press, New York, 1974, pp. 515-536. 14 Grillner, S. and Shik, M. L., On the descending control of the lumbosacral spinal cord from the 'mesencephalic locomotor region', ,4cta physiol, scand., 87 (1973) 320-333. 15 Huston, J. P. and Borb~ly, A. A., The thalamic rat: general behavior, operant learning with rewarding hypothalamic stimulation, and effects of amphetamine, Physiol. Behav., 12 (1974) 433-448.
391 16 Shik, M. L., Severin, F. V. and Orlovsky, G. N., Control of walking and running by means of electrical stimulation of the midbrain, Biofizika, 11 (1966) 756-765. 17 Steeves, J. D., Jordan, L. M. and Lake, N., The close proximity of catecholamine-containing cells to the 'mesencephalic locomotor region' (MLR), Brain Research, 100 (1975) 663 670. 18 Vyklick~, L. and Tabin, V., Effect of amphetamine on spinal reflexes, Nature (Lond.), 204 (1964) 384 385.
387
The effect of D-amphetamine on gamma efferent activity in the acute decerebrate rat
CHARLES A. SORENSON, STEPHEN A. GEORGE and ANDREW J. FRIEDMAN Neuroscience Program, Amherst College, Amherst, Mass. 01002 (U.S.A.)
(Accepted October 6th, 1977)
Attempts to evaluate the function of reticulospinal (descending) norepinephrine (NE)-containing neurons in rats have focused on the effects of various pharmacological agents on hindlimb reflexes. These studies have shown that drug treatments which increase noradrenergic receptor activity (e.g. amphetamine is, DOPA 1° or clonidine 1) potentiate the flexion reflex in spinal or decerebrate preparations, while treatments that decrease noradrenergic transmission (e.g. reserpine 1, alpha-methyl-para-tyrosine ~, haloperidol ~,4 or phenoxybenzamine 2,4) greatly reduce the amplitude of the flexion reflex in these preparations. A series of studies by And6n et al. was carried out using cats as subjects to analyze electrophysiologically the neural mechanisms involved. Their studies showed that the administration of DOPA depressed the short latency 3,7 but enhanced the long latencyS, 6 transmission from flexor reflex afferents to alpha and gamma motor neurons, to ascending pathways and to primary afferents. More recently, Grillner and his colleagues 14,16 have suggested that spinal NEcontaining neurons contribute to the control of locomotor movements, based upon their work with cats. They have shown that stepping movements are produced in the acute spinal cat when its hindlimbs are placed on a treadmill after the administration of D O P A or clonidine la. The pattern of muscle activation during these movements is virtually identical to that of the intact animal walking on a treadmilP 3. Extensor contraction occurs 30-60 msec before the foot touches the treadmill, which shows that the timing of the action is intrinsic to the spinal cord and not controlled by stimulation of the foot. The gait of the stepping movements depends upon the speed of the treadmill - - it changes from a walk to a gallop as the speed of the treadmill increases. Stepping movements were also produced in decerebrate cats by low intensity electrical stimulation of a region in the midbrainX4,16 (which they called the 'mesencephalic locomotor region (MLR)') near the nucleus locus coeruleus iv. This effect is blocked by low doses of the noradrenergic receptor blocker phenoxybenzamine 13. Both of these treatments, electrical stimulation of the M LR and the administration of drugs such as DOPA, are associated with characteristic electrophysiological changes in the resting bias of gamma efferent motor neurons - - an increase in static gamma efferent activity but a decrease in dynamic activity s,9,14.
388 TABLE 1
The effect of intravenously applied D-amphetamine on baseline hindlimb flexor la activity Rat number
1 2 3 4
Average frequency (spikes~see) Vehicle
D-Amphetamine
24.2 100.6 27.4 82.9
42.2 122.9 197.7 257.0
Per cent change compared to vehicle L 74.4 22.2 ~ 621.5 1210.0
The suggestion of Griliner et al. that descending NE-containing neurons might play a role in feline locomotion is particularly intriguing, since it is generally believed that the locomotor stimulating effect of amphetamine in rats is produced by activation of ascending catecholamine-containing pathways, especially the mesolimbic dopaminergic pathway through its connections with the nucleus accumbens. It is possible that the rat possesses no similar descending locomotor system. However, if it does possess such a system, then administration of amphetamine to the decerebrate rat should produce a pattern ofelectrophysiological changes identical to those seen when DOPA is administered to the spinal cat or when the M L R is stimulated in the decerebrate cat. This study presents the results of the test of this prediction. The output of the gamma efferent system was measured by recording the responses of muscle spindle afferents, which are in series with intrafusal muscle elements innervated by gamma efferent neurons, to applied stretch of flexor muscles in the rat's hindlimb. Rats were anesthetized with chloral hydrate (385 mg/kg) given intraperitoneally. Cutaneous afferents from the left hindtimb were eliminated by removing the skin covering the limb. A laminectomy was performed to expose dorsal roots in the lumbar region of the spinal cord. The rat was decerebrated by aspiration of a 1 mm strip of brain tissue just rostral to the colliculi in order to remove any possible influences from ascending catecholaminergic pathways that could ultimately return to the spinal cord. The animal was placed in a holder constructed to fix the lower torso while allowing the left hindlimb to be stretched to a known position. The dorsal root serving the left hindlimb was cut, the distal end divided, and one branch picked up on a wick electrode. The rootlet was alternately tested and divided further until it contained no more than about 5 active units, all from flexor muscle spindles. Signals from the electrode were amplified and displayed conventionally, and stored on F M tape for later analysis by a peak detector circuit whose output pulses were timed and counted by a PDP-11 computer. The computer also generated histograms showing the spike frequency measured in consecutive time bins. In each experiment control records were first made of the response to 3 cycles of stretching (10 sec) and relaxation (5 sec) after injection of 150 #1 of saline in the tail vein. Then 150 #1 of a 1 mg/ml o-amphetamine solution was administered, and 5 min later the same sequence of 3 stretches was repeated.
389 800
VEHICLE
AMPHETAMINE
?ZO
G40 5G0 SPIKES
488 PER
4N
SECOND ~40 IGO
~.0
25.0
50.0
0~0
25.0
50.0
TIHE SINCE START OF ROll (SECONDS)
Fig. 1. Spike frequency profiles of hindlimb flexor la activity of rat 3 after decerebration and subsequent sequential intravenous injections of vehicle and D-amphetamine solutions. Profiles show responses in the relaxed condition and to three successive stretches applied to the hindlimb. Frequency was measured over 500 msec intervals.
The effect of amphetamine treatment on static gamma motoneuron activity can be determined by comparing the baseline level of activity after saline treatment to that following amphetamine treatment (Table I). In each of the four animals tested the amphetamine treatment produced an increase in muscle spindle spike frequency. This shows that amphetamine facilitates the activity of static gamma-motoneurons in the decerebrate rat. Dynamic gamma-motoneuron activity can be measured by comparing the spike frequency from muscle spindles while stretch is being applied (i.e. when the muscle is subject to velocity as well as length changes) to the frequency at a given time (3.0 sec in this case) after the stretching is complete and while the muscle is being held at its new length (i.e., when it is undergoing no dynamic changes). The difference between these two measurements of spike frequency, called the 'dynamic index', must result entirely from dynamic gamma-motoneuron activity, since the static gamma-motoneurons contribute equally to both measurements. To determine the effect of amphetamine on dynamic gamma-motoneuron activity, we compared the dynamic index before and after administration of amphetamine. Fig. 1 shows a representative case in which the peak level of activity declines from an average of 365 to 227 spikes/sec after saline (dynamic index = 138) and from 627 to 588 (dynamic index -- 39) after amphetamine. Of the three other rats tested, two showed comparable decreases in dynamic index. One animal, which had a very low dynamic index prior to drug administration, showed no change in dynamic index after amphetamine treatment. These results therefore indicate that amphetamine inhibits the activity of dynamic gamma-efferents in the decerebrate rat.
390 These results show t h a t the a d m i n i s t r a t i o n of a m p h e t a m i n e to the decerebrate rat p r o d u c e d the same p a t t e r n o f changes in static a n d d y n a m i c gamma-efferents as seen in the spinal cat t r e a t e d with D O P A or the decerebrate cat stimulated in the ' m e s e n c e p h a l i c l o c o m o t o r region'. These results therefore s u p p o r t the hypothesis t h a t a m p h e t a m i n e - i n d u c e d b e h a v i o r a l s t i m u l a t i o n in the rat as well as in the cat is due at least in p a r t to the a c t i v a t i o n o f a 'mesencephalic l o c o m o t o r region' which exerts its influence t h r o u g h the release o f N E at the segmental level. These results also lead to the p r e d i c t i o n t h a t the a d m i n i s t r a t i o n o f a m p h e t a m i n e would stimulate the l o c o m o t o r activity o f the d e c e r e b r a t e rat as it does t h a t o f the t h a l a m i c rat 1~, which lacks all structures a b o v e the diencephalon. I n light o f these findings, c a u t i o n s h o u l d be exercised in a s s u m i n g t h a t it is the ascending p a t h w a y s which m e d i a t e the behavioral effects o f p h a r m a c o l o g i c a l agents acting on c a t e c h o l a m i n e r g i c systems in i n t a c t animals. This investigation was s u p p o r t e d in p a r t by N I M H Small G r a n t MH27467 a n d by a g r a n t f r o m the A l f r e d P. Sloan F o u n d a t i o n to A m h e r s t College. W e t h a n k D a v i d N. M a s t r o n a r d e for assistance with the c o m p u t e r analysis o f the e l e c t r o p h y s i o l o g i c a l data.
I And6n, N.-E., Effects of amphetamine and some other drugs on central catecholamine mechanisms. In E. Costa and S. Garattini (Eds.), Amphetamines and Related Compounds, Raven Press, New York, 1970, pp. 447-462. 2 And6n, N.-E., Corrodi, H., Fuxe, K. and H6kfelt, T., Increased impulse flow in bulbospinal noradrenaline neurons produced by catecholamine receptor blocking agents, Europ. J. PharmacoL, 2 (1967) 59-64. 3 And6n, N.-E., Jukes, M. G. M. and Lundberg, A., Spinal reflexes and monoamine liberation, Nature (Lond.), 202 (1964) 1222-1223. 4 And6n, N.-E., Jukes, M. G. M. and Lundberg, A., The effect of DOPA on the spinal cord. 2. A pharmacological analysis, Aeta physiol, scand., 67 (1966) 387-397. 5 And6n, N.-E., Jukes, M. G. M., Lundberg, A. and Vyklick3~,L., A new spinal flexor reflex, Nature (Lond.), 202 (1964) 1344-1345. 6 And6n, N.-E., Jukes, M. G, M., Lundberg, A. and Vyklick3~, L., The effect of DOPA on the spinal cord. 1. Influence on transmission from primary afferents, Actaphysiol. scand., 67 (t966) 373-386. 7 And6n, N.-E., Lundberg, A., Rosengren, E. and Vyklick~, L., The effect of DOPA on spinal reflexes from the FRA (flexor reflex afferents), Experientia (Basel), 19 (1963) 654-655. 8 Bergmans, J. and Grillner, S., Changes in dynamic sensitivity of primary endings of muscle spindle afferents induced by DOPA, Acta physiol, scand., 74 (1968) 629-636. 9 Bergmans, J. and Grillner, S., Reciprocal control of spontaneous activity and reflex effects in static and dynamic gamma-motoneurons revealed by an injection of DOPA, Acta physiol, scand., 77 (t969) 106-124. 10 Carlsson, A., Magnusson, T. and Rosengren, E., 5-Hydroxytryptamine of the spinal cord norreally and after transection, Experientia (Basel), 19 (t963) 359. 11 Forssberg, H. and Grillner, S., The locomotion of the acute spinal cat injected with clonidine, Brain Research, 50 (1973) 184-186. 12 Grillner, S., Supraspinal and segmental control of static and dynamic gamma-motoneurones in the cat, ,4cta physiol, scand., Suppl. 327 (1969) 1-34. 13 Grillner, S., Locomotion in the spinal cat. In Stein, R. B., Pearson, K. G., Smith, R. S. and Redford, J. B. (Eds.), Control of Posture and Locomotion, Plenum Press, New York, 1974, pp. 515-536. 14 Grillner, S. and Shik, M. L., On the descending control of the lumbosacral spinal cord from the 'mesencephalic locomotor region', ,4cta physiol, scand., 87 (1973) 320-333. 15 Huston, J. P. and Borb~ly, A. A., The thalamic rat: general behavior, operant learning with rewarding hypothalamic stimulation, and effects of amphetamine, Physiol. Behav., 12 (1974) 433-448.
391 16 Shik, M. L., Severin, F. V. and Orlovsky, G. N., Control of walking and running by means of electrical stimulation of the midbrain, Biofizika, 11 (1966) 756-765. 17 Steeves, J. D., Jordan, L. M. and Lake, N., The close proximity of catecholamine-containing cells to the 'mesencephalic locomotor region' (MLR), Brain Research, 100 (1975) 663 670. 18 Vyklick~, L. and Tabin, V., Effect of amphetamine on spinal reflexes, Nature (Lond.), 204 (1964) 384 385.
