European Journal of Pharmacology, 57 (1979) 83--92
83
© Elsevier/North-Holland Biomedical Press
MODULATION OF THE TONIC STRETCH REFLEX BY MONOAMINES JOHN W. COMMISSIONG * and E. MICHAEL SEDGWICK **
School of Biochemical and Physiological Sciences, Medical and Biological. Sciences Building, Basset Crescent East, Southampton S09 3TU, U.K. Received 18 December 1978, revised MS received 4 April 1979, accepted 17 April 1979
J.W. COMMISSIONG and E.M. SEDGWICK, Modulation of the tonic stretch reflex by monoamines, European J. Pharmacol. 57 (1979) 83--92. The effects of L-DOPA and 5-HTP on the tonic stretch reflex (TSR) in the decerebrate rat were studied. L-DOPA facilitated the TSR in a dose-dependent manner. The facilitation of the TSR was blocked by pimozide. A sensitive electromygraphic (EMG) technique capable of recording single motor unit discharges was used. The EMG results suggest that gamma m o t o n e u r o n sensitivity was increased to a greater degree than alpha motoneuron sensitivity during the facilitation by L-DOPA. The L-DOPA-induced facilitation persisted in animals partly depleted of 5-hydroxytryptamine (5-HT) by reserpine, p-chlorophenylalanine or 5,6-dihydroxytryptamine. 5-HTP inhibited the TSR in a dose-dependent manner. It is concluded that DA not 5-HT is the amine which normally mediates facilitation of the TSR after L-DOPA and that gamma m o t o n e u r o n activation is more likely to be involved to a greater degree than alpha motoneuron activation in the neural mechanisms of the facilitation. L-DOPA
5-HTP
Pimozide
Tonic stretch reflex
1. Introduction We have reported previously that L-DOPA initially facilitates and then depresses the tonic stretch reflex (TSR) in the decerebrate rat (Commissiong and Sedgwick, 1974a). The present investigation had two objectives. The first was to determine more precisely which amine was responsible for the initial facilitation of the TSR. The second was to get a better understanding of the neural mechanisms associated with the facilitation of the reflex. Our previous results suggested that dopamine (DA) synthesized from L-DOPA was the cause of the facilitation (Commissiong and Sedgwick, 1974a). However, it was unclear whether DA was acting directly to facilitate * Present address: Department of Physiology, McIntyre Medical Sciences Building, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada. ** Present address: Wessex Neurological Center, Southampton General Hospital, Shirley, Southampton S09 4XY, U.K.
Electromyography
the reflex. An alternative possibility is that DA mediated the release of 5-hydroxytryptamine (5-HT) from serotonergic terminals in the cord, and that 5-HT was the active agent responsible for the facilitation. It is known that L-DOPA after metabolism to DA mediates the release of 5-HT elsewhere in the central nervous system (CNS) (Bartholini et al., 1968; Maj et al., 1971; Okada et al., 1972). Also, other workers have shown that 5-hydroxytryptophan (5-HTP), the precursor of 5-HT facilitates the stretch reflex in the decerebrate, spinal cat (Ahlman et al., 1971; Ellaway and Trott, 1975). The idea of an indirect effect of DA in the cord has been attractive, because it had previously been thought that dopaminergic neurons were not present in the cord (Osterholm, 1974). However, recent evidence has suggested that there are dopaminergic neurons in the cord (Commissiong et al., 1978a,b). Moreover, Geger and Dupelj (1977) have shown that in the spinal cat the potentiation of the monosynaptic reflex by
84 L-DOPA is blocked by pimozide, a dopamine receptor blocker. Also in the spinal cat apomorphine, a dopaminergic agonist mimics the effect of L-DOPA in potentiating the flexor reflex (Nygren and Olsen, 1976). It is therefore possible t h a t DA in the cord could have a direct effect in facilitating the TSR. Concerning the neural basis of the facilitation of the TSR by L-DOPA, an increased gamma m o t o n e u r o n activation, leading to increased sensitization of the peripheral muscle spindle receptor, and an increased afferent input during stretch would increase the size of the reflex response. Alternatively, if the alpha m o t o n e u r o n s , but n o t the gamma m o t o n e u r o n s became sensitized, the peripheral input from the receptor would remain constant, but be amplified centrally, leading again to an increased o u t p u t . We have tried to distinguish between these two alternatives, by the use of a very sensitive, integrated, electromyographic m e t h o d , capable of detecting single m o t o r unit discharges. L-DOPA is k n o w n to cause a selective activation of gamma m o t o n e u r o n s in the decerebrate, spinal cat (Grillner, 1969a; Ahlman et al., 1971). Moreover our present electromyographic evidence clearly indicates that in the rat, ~-motoneurons are n o t greatly sensitized when the L-DOPA-induced facilitation of the reflex is most intense. Also in the present experiments, we have demonstrated t h a t 5-HTP depresses the TSR. We, therefore, propose as a working hypothesis that in the decerebrate rat, the facilitation of the TSR by L-DOPA is mediated by DA, and results primarily from an activation of ~f-motoneurons.
2. Materials and methods
2.1. Animals Six adult cats of either sex, weighing 2.5-3.0 kg and 59 male Wistar rats, 250--300 g were used for the stretch reflex experiments.
J.W. COMMISSIONG, E.M. SEDGWICK The animals were decerebrated intercollicularly under halothane or ether anesthesia. Following decerebration the anesthesia was discontinued and the animals were cannulated and prepared for recording the stretch reflex as previously described (Commissiong and Sedgwick, 1974a).
2.2. Electromyographic recording Fine wire silver electrodes were used. One electrode was inserted into the muscle near to the m o t o r point and the other near to the Achilles tendon. The signal was externally amplified using a Tektronix 122 Differential Amplifier and then fed into an integrator. True integration followed half wave rectification. The integrator was designed to reset itself either when the integrated voltage reached 80% of the power supply voltage of the integrator (+15 V) or when the input signal ceased for 200 msec. Both the raw and integrated EMG signals were monitored on a Tektronix 502A oscilloscope and by an audio amplifier. Permanent records of the EMG were made on a Grass Model 7 Polygraph. A more intense EMG is indicated by (1) greater slope of the integrated voltage curve and (2) a greater total number of displacements of the curve.
2.3. Assay of 5-HT 5-HT was assayed by the m e t h o d of Snyder et al. (1965), with an exception that a cation exchange column was used to separate any 5-HTP present from 5-HT. Following preparation of the tissues as described by Snyder et al. (1965) the supernatant was introduced on to an Amberlite CG (50) Type 2, Mesh 200400 (BDH) column. The resin was previously cycled through the H ÷ and Na ÷ forms as described by Hirs et al. (1953), and finally used in a N d form. 15 × 0.6 cm Glass columns were used. The height of the resin packing was 1.5 cm. Each column was first eluted with 30 ml 0.02 M phosphate buffer, pH 6.1 to facilitate settling. Operation was at atmos-
MONOAMINES AND THE TONIC STRETCH REFLEX
pheric pressure. After adding the solution (0.8 ml) containing 5-HT and 5-HTP to the column, 1.0 ml 0.02 M phosphate buffer pH 6.1 was run through the column to elute 5-HTP. 5-HT was then eluted with 0.4 ml 1.0 M acetate buffer pH 5.2. Aliquots of the eluate were used for the assay of 5-HT.
85 A ,
'
'
[ ' :
! /
j!
/I/II
ll! Iif
/!!
II! ~,~.! ll t~ IJl
,' '; ~ 'i ~ :il~ !~:: ~!//t
2.4. Drugs L-DOPA (courtesy Brocades, G.B. Ltd.) and 5-HTP were dissolved in warm 0.1 N HC1 as 1% stock solutions. Prior to use the pH was adjusted to 6.5 with solid sodium bicarbonate. Pimozide was dissolved in a drop of glacial acetic acid and then diluted with saline. Reserpine was dissolved in 10% ascorbic acid and injected 18 h prior to use. p-Clorophenylalanine methyl ester (Sigma) was dissolved in 0.9% saline. The dose used was 100 mg/kg/ day i.p. for three days. The animals were used 24 h after the last injection. 5,6-Dihydroxytryptamine was made up in dilute mammalian Ringer. 75 #g in 20/J1 of solution was injected into the left lateral ventricle using an Agla syringe (Ciba) fitted with a 26 G × 4 mm needle. The coordinates for injection were found according to the method of Noble et al. (1967). The animals were used 10 days later.
3. Results
3.1. The initial L-DOPA-induced facilitation blockade by pimozide Fig. 1A shows that L-DOPA (25 mg/kg i.v.) caused an intense facilitation of the TSR in the decerebrate rat. Usually the facilitation persisted for 10--20min. The EMG was almost completely abolished between the stretches, in spite of the fact that the reflex response was so greatly enhanced. Fig. 1B shows that pimozide (0.3 mg/kg i.v.) had no independent effect on the TSR for up to 20 rain after injection. However, pimozide prevented the normal
Pimozide
0.3 mg/kg, i.p.
B
--17~ rain
25 mg/kg, i.v.
250 g
2.0 mV
* 30sec '
c I
i
Fig. 1. The antagonism of L-DOPA-induced facilitation of the TSR by pimozide in t h e decerebrate rat. L-DOPA alone caused an intense facilitation of the TSR in 15 of 17 preparations (A). Pimozide alone had no effect in the TSR (B). L-DOPA injected after pimozide was ineffective in facilitating the TSR in all 5 preparations in which this effect was tested. Upper trace: integrated EMG; lower trace: rnyogram.
L-DOPA-induced facilitation of the TSR, fig. 1C. Indeed, after several minutes, the reflex was transiently completely abolished (arrow, fig. 1C), but partly recovered rapidly. It is therefore clear that pimozide abolishes the initial facilitation of the TSR caused by L-DOPA.
86
J.W. COMMISSIONG, E.M. SEDGWICK
125
3.2. Dose-dependent effect o f L-DOPA on the initial facilitation
100
Fig. 2 shows that the initial L-DOPA induced facilitation of the TSR in the rat is a dose
83
© Elsevier/North-Holland Biomedical Press
MODULATION OF THE TONIC STRETCH REFLEX BY MONOAMINES JOHN W. COMMISSIONG * and E. MICHAEL SEDGWICK **
School of Biochemical and Physiological Sciences, Medical and Biological. Sciences Building, Basset Crescent East, Southampton S09 3TU, U.K. Received 18 December 1978, revised MS received 4 April 1979, accepted 17 April 1979
J.W. COMMISSIONG and E.M. SEDGWICK, Modulation of the tonic stretch reflex by monoamines, European J. Pharmacol. 57 (1979) 83--92. The effects of L-DOPA and 5-HTP on the tonic stretch reflex (TSR) in the decerebrate rat were studied. L-DOPA facilitated the TSR in a dose-dependent manner. The facilitation of the TSR was blocked by pimozide. A sensitive electromygraphic (EMG) technique capable of recording single motor unit discharges was used. The EMG results suggest that gamma m o t o n e u r o n sensitivity was increased to a greater degree than alpha motoneuron sensitivity during the facilitation by L-DOPA. The L-DOPA-induced facilitation persisted in animals partly depleted of 5-hydroxytryptamine (5-HT) by reserpine, p-chlorophenylalanine or 5,6-dihydroxytryptamine. 5-HTP inhibited the TSR in a dose-dependent manner. It is concluded that DA not 5-HT is the amine which normally mediates facilitation of the TSR after L-DOPA and that gamma m o t o n e u r o n activation is more likely to be involved to a greater degree than alpha motoneuron activation in the neural mechanisms of the facilitation. L-DOPA
5-HTP
Pimozide
Tonic stretch reflex
1. Introduction We have reported previously that L-DOPA initially facilitates and then depresses the tonic stretch reflex (TSR) in the decerebrate rat (Commissiong and Sedgwick, 1974a). The present investigation had two objectives. The first was to determine more precisely which amine was responsible for the initial facilitation of the TSR. The second was to get a better understanding of the neural mechanisms associated with the facilitation of the reflex. Our previous results suggested that dopamine (DA) synthesized from L-DOPA was the cause of the facilitation (Commissiong and Sedgwick, 1974a). However, it was unclear whether DA was acting directly to facilitate * Present address: Department of Physiology, McIntyre Medical Sciences Building, 3655 Drummond Street, Montreal, Quebec H3G 1Y6, Canada. ** Present address: Wessex Neurological Center, Southampton General Hospital, Shirley, Southampton S09 4XY, U.K.
Electromyography
the reflex. An alternative possibility is that DA mediated the release of 5-hydroxytryptamine (5-HT) from serotonergic terminals in the cord, and that 5-HT was the active agent responsible for the facilitation. It is known that L-DOPA after metabolism to DA mediates the release of 5-HT elsewhere in the central nervous system (CNS) (Bartholini et al., 1968; Maj et al., 1971; Okada et al., 1972). Also, other workers have shown that 5-hydroxytryptophan (5-HTP), the precursor of 5-HT facilitates the stretch reflex in the decerebrate, spinal cat (Ahlman et al., 1971; Ellaway and Trott, 1975). The idea of an indirect effect of DA in the cord has been attractive, because it had previously been thought that dopaminergic neurons were not present in the cord (Osterholm, 1974). However, recent evidence has suggested that there are dopaminergic neurons in the cord (Commissiong et al., 1978a,b). Moreover, Geger and Dupelj (1977) have shown that in the spinal cat the potentiation of the monosynaptic reflex by
84 L-DOPA is blocked by pimozide, a dopamine receptor blocker. Also in the spinal cat apomorphine, a dopaminergic agonist mimics the effect of L-DOPA in potentiating the flexor reflex (Nygren and Olsen, 1976). It is therefore possible t h a t DA in the cord could have a direct effect in facilitating the TSR. Concerning the neural basis of the facilitation of the TSR by L-DOPA, an increased gamma m o t o n e u r o n activation, leading to increased sensitization of the peripheral muscle spindle receptor, and an increased afferent input during stretch would increase the size of the reflex response. Alternatively, if the alpha m o t o n e u r o n s , but n o t the gamma m o t o n e u r o n s became sensitized, the peripheral input from the receptor would remain constant, but be amplified centrally, leading again to an increased o u t p u t . We have tried to distinguish between these two alternatives, by the use of a very sensitive, integrated, electromyographic m e t h o d , capable of detecting single m o t o r unit discharges. L-DOPA is k n o w n to cause a selective activation of gamma m o t o n e u r o n s in the decerebrate, spinal cat (Grillner, 1969a; Ahlman et al., 1971). Moreover our present electromyographic evidence clearly indicates that in the rat, ~-motoneurons are n o t greatly sensitized when the L-DOPA-induced facilitation of the reflex is most intense. Also in the present experiments, we have demonstrated t h a t 5-HTP depresses the TSR. We, therefore, propose as a working hypothesis that in the decerebrate rat, the facilitation of the TSR by L-DOPA is mediated by DA, and results primarily from an activation of ~f-motoneurons.
2. Materials and methods
2.1. Animals Six adult cats of either sex, weighing 2.5-3.0 kg and 59 male Wistar rats, 250--300 g were used for the stretch reflex experiments.
J.W. COMMISSIONG, E.M. SEDGWICK The animals were decerebrated intercollicularly under halothane or ether anesthesia. Following decerebration the anesthesia was discontinued and the animals were cannulated and prepared for recording the stretch reflex as previously described (Commissiong and Sedgwick, 1974a).
2.2. Electromyographic recording Fine wire silver electrodes were used. One electrode was inserted into the muscle near to the m o t o r point and the other near to the Achilles tendon. The signal was externally amplified using a Tektronix 122 Differential Amplifier and then fed into an integrator. True integration followed half wave rectification. The integrator was designed to reset itself either when the integrated voltage reached 80% of the power supply voltage of the integrator (+15 V) or when the input signal ceased for 200 msec. Both the raw and integrated EMG signals were monitored on a Tektronix 502A oscilloscope and by an audio amplifier. Permanent records of the EMG were made on a Grass Model 7 Polygraph. A more intense EMG is indicated by (1) greater slope of the integrated voltage curve and (2) a greater total number of displacements of the curve.
2.3. Assay of 5-HT 5-HT was assayed by the m e t h o d of Snyder et al. (1965), with an exception that a cation exchange column was used to separate any 5-HTP present from 5-HT. Following preparation of the tissues as described by Snyder et al. (1965) the supernatant was introduced on to an Amberlite CG (50) Type 2, Mesh 200400 (BDH) column. The resin was previously cycled through the H ÷ and Na ÷ forms as described by Hirs et al. (1953), and finally used in a N d form. 15 × 0.6 cm Glass columns were used. The height of the resin packing was 1.5 cm. Each column was first eluted with 30 ml 0.02 M phosphate buffer, pH 6.1 to facilitate settling. Operation was at atmos-
MONOAMINES AND THE TONIC STRETCH REFLEX
pheric pressure. After adding the solution (0.8 ml) containing 5-HT and 5-HTP to the column, 1.0 ml 0.02 M phosphate buffer pH 6.1 was run through the column to elute 5-HTP. 5-HT was then eluted with 0.4 ml 1.0 M acetate buffer pH 5.2. Aliquots of the eluate were used for the assay of 5-HT.
85 A ,
'
'
[ ' :
! /
j!
/I/II
ll! Iif
/!!
II! ~,~.! ll t~ IJl
,' '; ~ 'i ~ :il~ !~:: ~!//t
2.4. Drugs L-DOPA (courtesy Brocades, G.B. Ltd.) and 5-HTP were dissolved in warm 0.1 N HC1 as 1% stock solutions. Prior to use the pH was adjusted to 6.5 with solid sodium bicarbonate. Pimozide was dissolved in a drop of glacial acetic acid and then diluted with saline. Reserpine was dissolved in 10% ascorbic acid and injected 18 h prior to use. p-Clorophenylalanine methyl ester (Sigma) was dissolved in 0.9% saline. The dose used was 100 mg/kg/ day i.p. for three days. The animals were used 24 h after the last injection. 5,6-Dihydroxytryptamine was made up in dilute mammalian Ringer. 75 #g in 20/J1 of solution was injected into the left lateral ventricle using an Agla syringe (Ciba) fitted with a 26 G × 4 mm needle. The coordinates for injection were found according to the method of Noble et al. (1967). The animals were used 10 days later.
3. Results
3.1. The initial L-DOPA-induced facilitation blockade by pimozide Fig. 1A shows that L-DOPA (25 mg/kg i.v.) caused an intense facilitation of the TSR in the decerebrate rat. Usually the facilitation persisted for 10--20min. The EMG was almost completely abolished between the stretches, in spite of the fact that the reflex response was so greatly enhanced. Fig. 1B shows that pimozide (0.3 mg/kg i.v.) had no independent effect on the TSR for up to 20 rain after injection. However, pimozide prevented the normal
Pimozide
0.3 mg/kg, i.p.
B
--17~ rain
25 mg/kg, i.v.
250 g
2.0 mV
* 30sec '
c I
i
Fig. 1. The antagonism of L-DOPA-induced facilitation of the TSR by pimozide in t h e decerebrate rat. L-DOPA alone caused an intense facilitation of the TSR in 15 of 17 preparations (A). Pimozide alone had no effect in the TSR (B). L-DOPA injected after pimozide was ineffective in facilitating the TSR in all 5 preparations in which this effect was tested. Upper trace: integrated EMG; lower trace: rnyogram.
L-DOPA-induced facilitation of the TSR, fig. 1C. Indeed, after several minutes, the reflex was transiently completely abolished (arrow, fig. 1C), but partly recovered rapidly. It is therefore clear that pimozide abolishes the initial facilitation of the TSR caused by L-DOPA.
86
J.W. COMMISSIONG, E.M. SEDGWICK
125
3.2. Dose-dependent effect o f L-DOPA on the initial facilitation
100
Fig. 2 shows that the initial L-DOPA induced facilitation of the TSR in the rat is a dose
