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Spinal cord dopaminergic neurons: Evidence for an uncrossed nigrospinal pathway
Neuropharmacology Vol.
Fergamon
18, pp. 565 to 568
Press Ltd 1979. Printed
in Great Britain
PRELIMINARY NOTES
SPINAL CORD DOPAMINERGIC
NEURONS:
EVIDENCE
J. W. Commissiong,
FOR AN UNCROSSED
S. Gentleman
Laboratory of Preclinical Pharmacology, Mental Health, Saint Elizabeths Hospital,
(Accepted
5 Mamh
NIGROSPINAL
PATHWAY
and N. H. Neff National Institute of Washington, D.C. 20032
1979)
Dopamine metabolism in the thoracic cord and striatum can be Summary: modified by 6-hydroxydopamine injection into or electrical stimulation Electrothermic destruction of the of the ipsilateral substantia nigra. locus coeruleus lowers the spinal cord content of norepinephrine but not of dopamine. Our results suggest that there is an uncrossed nigrospinal dopaminergic pathway. Introduction The concentration of dopamine (DA) in the spinal cord is rather low when compared with the concentration of norepinephrine (NE). Consequently, DA has been considered to be in the spinal We report cord solely as a precursor for the synthesis of NE, and not as a neurotransmitter. evidence suggesting that dopaminergic neurons are present in the spinal cord, that some of these neurons originate in the substantia nigra and enter the cord uncrossed and that dopaminergic These observations may provide new insight axons are differentially distributed in the cord. into spinal cord physiology, Parkinson's disease pathology, the untoward consequences of treating Parkinson's disease with L-DOPA and the mechanisms underlying the side effects of neuroleptic drugs. Methods NE, DA, homovanillic acid (HVA) and 3,4_dihydroxyphenylacetic acid (DCPAC) were analyzed in the spinal cord of male Sprague Dawley rats by gas chromatography-mass spectrometry (Commissiong, Animals were killed by decapitation and their striata and spinal cords Galli and Neff, 1978). rapidly removed and frozen on dry ice. The cord was then placed on a cold block and divided In one series of studies into left and right halves by cutting at the posterior median sulcus. the cervical, thoracic and lumbar regions of the cord were dissected into dorsal and ventral In the thoracic cord the sona intermedia between the dorsal and ventral horns was horns. dissected out. For this dissection the thoracic spinal cord was removed, placed on a glass It was subsequently warmed to approximately 4'C to allow for easy slide and frozen on dry ice. cutting. For cutting, the cord was laid on its ventral surface. The myelin lateral to the ventrolateral sulcus was removed on either side using a double-edged razor blade. This proceThe cord dure exposes the grey matter of the lateral column nucleus laterally, on either side. was then turned on its left cut side. The dorsal and ventral horns were then removed by making straight longitudinal cuts at either side of the right lateral column nucleus. The remainder is the sona intermedia. The whole procedure requires about 30 seconds. Three types of studies were performed to evaluate the presence of spinal dopaminergic neurons: 1) unilateral injection of 6-hydroxydopamine (6-HDA) into the substantia nigra; 2) unilateral electrical stimulation of the substantia nigra; and 3) bilateral electrolytic lesion of the locus coeruleus to lower the concentration of NE in the cord (Commissiong, Hellstrom and Neff, 1978). Results and Discussion The DA content of striatum and thoracic spinal cord was significantly reduced on the left side when 6-HDA was injected into the left substantia nigra (Table 1). The NE content in the This study suggests that the DA is present in axons that are cord of these rats was unaltered. different from the noradrenergic axons and that these dopaminergic axons originate in substantia In support of this hypothesis, unilateral electrical nigra and enter the spinal cord uncrossed. stimulation of the substantia nigra resulted in the accumulation of HVA and DOPAC in the striaturn and HVA in the thoracic cord on the stimulated side (Table 2). Interestingly, the anesthetic chloral hydra-, During had a differential effect on spinal cord and striatal DOPAC. Additional anesthesia WPAC concentrations increased in the cord but not in the striatum. studies are needed to determine whether this is a pharmacological response to all anesthetics or is a characteristic response to chloral hydrate.
preliminary
566
Table 1.
Concentration the Injection
Notes
of DA and NE in the Striatum and Thoracic of 6-HDA in the Left Substantia Nigra
Spinal Cord of Rat After
LEFT
RIGHT
DA
STRIATUM Control 6-HDA THORACIC SPINAL CORD Control 6-HDA
NE nmol/q
68*2 2723
(4) f4)**
0.29+0.05 0.14+0.02
8048 72+2
---
(4) (4)*
NE
DA * SEM (N)
2.1eO.3 (4) 1.9rf0.2 (4)
(4) (4)
0.32+0.03 0.31+0.03
(4) (4)
---
2.1tO.4 1.8?0.2
(4) (4)
Four uq of 6-HDA in 2 ul of solvent (0.9% saline containing 0.1% ascorbic acid) were injected Ten into the left substantia niqra. Chloral hydrate, 400 mq/kq i.p., was the anesthetic. days later, the animals were killed by decapitation and the striata and spinal cord rapidly *p
Table 2.
The Effect of Electrical Stimulation of the Left Substantia Niqra on the Levels of DA, DOPAC and HVA in the Left and Right Striatum and Thoracic Spinal Cord STRIATIJM DA
DOPhC nmol/g
THORACIC HVA
DA
+ SEM
SPINAL CORD
DOPAC runol/q + SEM
HVA
UNANESTHETIZED CONTROL Left Side Right Side
6223 67f6
5.4tO.5 6.5TO.Q
6.2+0.8 4.6kO.4
0.34+0.04 0.35+0.04
0,44+0.10 0.53+0.01
ANESTHETIZED CONTROL Left Side Right Side
77+5 6723
7.3kO.9 5.6kO.5
7.8tl.7 5.6-t0.8
5.43f0.06 0.43+0.03
1.3 +0.18+ 0.32rtO.03 1.0 to.o7+ 0.31+0.04
ANESTHETIZED STIMULATED Left Side Right Side
78-+8 23.5'2.2*** 70f3 6.8kO.6
10.8?1.0* 5.3kO.8
0.51+0.06 0.50+0.04
0.78+0.02* 0.89+0.04*
0.25+0.03 0.26tO.03
0.82'0.23*** 0.25+0.02
Silver, concentric bipolar electrodes (Shaft diameter 0.5 mm; model NE-100, Rhodes Medical Instruments, California) were stereotaxically placed in the left substantia niqra. The electrodes were connected to a Grass 588 stimulator fitted with a constant current device (Grass Instruments Model CCU 1A). Stimulation was for 1 hr, at 15 Hz, 400 PA, 2.0 msec. After stimulation the animals Animals were anesthetized with chloral hydrate, 400 mq/kq i-p. were decapitated and the striata and spinal cord removed and immediately frozen on dry ice. Analysis of DA, HVA and DOPAC was done by a previously described method fCos%niSSiong et al., 1978). In both the anesthetized control, and the anesthetized stimulated preparations, the DA levels tended to increase on both sides of the cord, in comparison with the unaneslp
567
Preliminary Notes
mimics some of the effects of L-DOPA on spinal reflexes (Nyqren and Olson, 1976). Moreover, it is questionable whether NE synthesis is increased aftet L-DQPA treatment (Freed and Murphy, 19783. In addition, 3,4_dihydroxyphenylserine,from which NE is produced directly, thereby precluding an effect of DA, depresses the stretch reflex (Commissiongand Sedgwick, 1974), while the initial effect of L-DOPA is to enhance the stretch reflex (Commissiongand Sedqwick, 1974). We, therefore, propose that DA, and not NE is the metabolite of L-DOPA which is responsible for mediating many of the effects of this drug on spinal reflexes. Table 3. Levels of DA and NE in Various Regions of the Spinal Cord After Bilateral Lesion of The Locus Coeruleus CERVICAL
NE
DA
c L
4.7 20.2 2.8 r0.3
(6) (9)***
C L
0.56tO.03 (61 0.48kO.04 (9)
THORACIC DORSAL HORN nmol,'g 3.6 to.2 2.8 +0.4
(6: f9)*
0.42-10.04(6) 0.48f0.06 (9)
LUMBAR
3.8 1rO.2 (6) 2.3 fO.2 (9)*** 0.33f0.02 (6) 0.31+0.02 (9)
VENTRAL HORN NE
C L
4.0 +0.4 1.6 f0.3
(6) (9)***
DA
C L
0.22iO.03 (6) 0.18+0.04 (91
3.9 20.2 1.6 f0.3
(6) (9)***
0.07t0.03 (6) 0.09+0.06 (9)
3.9 f0.2 1.6 20.2
(6) (9)***
0.13%.02 (6) 0.12f0.03 (9)
ZONA INTERMEDIA NE
C L
__ __
4.4 to.2 1.9 to.3
DA
C L
-__
0.29ti.04 (6) 0.22ti.05 (9)
(6) c91***
---
-_
Rats were anesthetized with chloral hydrate 400 mg/kg i.p. A unipolar stainless steel electrode (0.3 mm diameter) insulated to within 0.5 mm of the tip was stereotaxically placed in the locus coeruleus using the coordinates of Korf et al. (1973). The electrode was connected to an Electrolytic Lesion Producing Device (StoeltingCo., Chicago, IL). A direct current of 2.5 mA was passed for 45 sec. Ten days later, the animals were killed by decapitation and the spinal cord removed and.stored on dry ice. The lesion of the locus coeruleus was verified histologicallyand also by monitoring NE levels in the cerebellum. There was a highly significant reduction of NE in the cerebellum (data not shown). *p
St8
Preliminary
Notes
(1978). Mass spectrometric measurement of catecholamine Freed, C. R. and Murphy, R. C. turnover in rat hypothalamus after long-term L-DOPA infusion. J. Phannacol. Exp. Ther. 205: 702-709. (1969). The influence of DOPA on static and dynamic fusimotor activity to the Grillner, S. triceps surae of the spinal cat. Acta Physiol. Stand. 77: 490-509. (1968). Jurna, I. and Lundberg, A. The influence of an inhibitor of dopamine-8-hydroxylase on the effect of WPA on transmission in the spinal cord. In: Structure and Functions of Inhibitory Neuronal Mechanisms (van Euler, C., Ed.) pp. 469-472. Pergamon Press, Oxford. Kori, J., Aghajanian, G. K. and Roth, R. B. (1973). Stimulation and destruction of locus coeruleus: Opposite effects on 3-methoxy-4-hydroxyphenylglycol sulfate levels in the rat cerebral cortex. Eur. J. Pharmacol. 21: 305-310. (1977). A new major projection from locus coeruleus: The main Nygren, L. and Olson, L. source of noradrenergic terminals in the ventral and dorsal columns of the spinal cord. Brain Res. 132: 85-93. (1976). On spinal noradrenaline receptor supersensitivity: CorrelaNygren, L. and Olson, L. tion between nerve terminal densities and flexor reflexes various times after intracisternal Brain Res. 116: 455-470. 6-hydroxydopamirie. (1967). Motoneurone depression by noradrenaline. Weight, F. F. and Salmoiraghi, G. C. Nature 213: 1129-1130.
Fergamon
18, pp. 565 to 568
Press Ltd 1979. Printed
in Great Britain
PRELIMINARY NOTES
SPINAL CORD DOPAMINERGIC
NEURONS:
EVIDENCE
J. W. Commissiong,
FOR AN UNCROSSED
S. Gentleman
Laboratory of Preclinical Pharmacology, Mental Health, Saint Elizabeths Hospital,
(Accepted
5 Mamh
NIGROSPINAL
PATHWAY
and N. H. Neff National Institute of Washington, D.C. 20032
1979)
Dopamine metabolism in the thoracic cord and striatum can be Summary: modified by 6-hydroxydopamine injection into or electrical stimulation Electrothermic destruction of the of the ipsilateral substantia nigra. locus coeruleus lowers the spinal cord content of norepinephrine but not of dopamine. Our results suggest that there is an uncrossed nigrospinal dopaminergic pathway. Introduction The concentration of dopamine (DA) in the spinal cord is rather low when compared with the concentration of norepinephrine (NE). Consequently, DA has been considered to be in the spinal We report cord solely as a precursor for the synthesis of NE, and not as a neurotransmitter. evidence suggesting that dopaminergic neurons are present in the spinal cord, that some of these neurons originate in the substantia nigra and enter the cord uncrossed and that dopaminergic These observations may provide new insight axons are differentially distributed in the cord. into spinal cord physiology, Parkinson's disease pathology, the untoward consequences of treating Parkinson's disease with L-DOPA and the mechanisms underlying the side effects of neuroleptic drugs. Methods NE, DA, homovanillic acid (HVA) and 3,4_dihydroxyphenylacetic acid (DCPAC) were analyzed in the spinal cord of male Sprague Dawley rats by gas chromatography-mass spectrometry (Commissiong, Animals were killed by decapitation and their striata and spinal cords Galli and Neff, 1978). rapidly removed and frozen on dry ice. The cord was then placed on a cold block and divided In one series of studies into left and right halves by cutting at the posterior median sulcus. the cervical, thoracic and lumbar regions of the cord were dissected into dorsal and ventral In the thoracic cord the sona intermedia between the dorsal and ventral horns was horns. dissected out. For this dissection the thoracic spinal cord was removed, placed on a glass It was subsequently warmed to approximately 4'C to allow for easy slide and frozen on dry ice. cutting. For cutting, the cord was laid on its ventral surface. The myelin lateral to the ventrolateral sulcus was removed on either side using a double-edged razor blade. This proceThe cord dure exposes the grey matter of the lateral column nucleus laterally, on either side. was then turned on its left cut side. The dorsal and ventral horns were then removed by making straight longitudinal cuts at either side of the right lateral column nucleus. The remainder is the sona intermedia. The whole procedure requires about 30 seconds. Three types of studies were performed to evaluate the presence of spinal dopaminergic neurons: 1) unilateral injection of 6-hydroxydopamine (6-HDA) into the substantia nigra; 2) unilateral electrical stimulation of the substantia nigra; and 3) bilateral electrolytic lesion of the locus coeruleus to lower the concentration of NE in the cord (Commissiong, Hellstrom and Neff, 1978). Results and Discussion The DA content of striatum and thoracic spinal cord was significantly reduced on the left side when 6-HDA was injected into the left substantia nigra (Table 1). The NE content in the This study suggests that the DA is present in axons that are cord of these rats was unaltered. different from the noradrenergic axons and that these dopaminergic axons originate in substantia In support of this hypothesis, unilateral electrical nigra and enter the spinal cord uncrossed. stimulation of the substantia nigra resulted in the accumulation of HVA and DOPAC in the striaturn and HVA in the thoracic cord on the stimulated side (Table 2). Interestingly, the anesthetic chloral hydra-, During had a differential effect on spinal cord and striatal DOPAC. Additional anesthesia WPAC concentrations increased in the cord but not in the striatum. studies are needed to determine whether this is a pharmacological response to all anesthetics or is a characteristic response to chloral hydrate.
preliminary
566
Table 1.
Concentration the Injection
Notes
of DA and NE in the Striatum and Thoracic of 6-HDA in the Left Substantia Nigra
Spinal Cord of Rat After
LEFT
RIGHT
DA
STRIATUM Control 6-HDA THORACIC SPINAL CORD Control 6-HDA
NE nmol/q
68*2 2723
(4) f4)**
0.29+0.05 0.14+0.02
8048 72+2
---
(4) (4)*
NE
DA * SEM (N)
2.1eO.3 (4) 1.9rf0.2 (4)
(4) (4)
0.32+0.03 0.31+0.03
(4) (4)
---
2.1tO.4 1.8?0.2
(4) (4)
Four uq of 6-HDA in 2 ul of solvent (0.9% saline containing 0.1% ascorbic acid) were injected Ten into the left substantia niqra. Chloral hydrate, 400 mq/kq i.p., was the anesthetic. days later, the animals were killed by decapitation and the striata and spinal cord rapidly *p
Table 2.
The Effect of Electrical Stimulation of the Left Substantia Niqra on the Levels of DA, DOPAC and HVA in the Left and Right Striatum and Thoracic Spinal Cord STRIATIJM DA
DOPhC nmol/g
THORACIC HVA
DA
+ SEM
SPINAL CORD
DOPAC runol/q + SEM
HVA
UNANESTHETIZED CONTROL Left Side Right Side
6223 67f6
5.4tO.5 6.5TO.Q
6.2+0.8 4.6kO.4
0.34+0.04 0.35+0.04
0,44+0.10 0.53+0.01
ANESTHETIZED CONTROL Left Side Right Side
77+5 6723
7.3kO.9 5.6kO.5
7.8tl.7 5.6-t0.8
5.43f0.06 0.43+0.03
1.3 +0.18+ 0.32rtO.03 1.0 to.o7+ 0.31+0.04
ANESTHETIZED STIMULATED Left Side Right Side
78-+8 23.5'2.2*** 70f3 6.8kO.6
10.8?1.0* 5.3kO.8
0.51+0.06 0.50+0.04
0.78+0.02* 0.89+0.04*
0.25+0.03 0.26tO.03
0.82'0.23*** 0.25+0.02
Silver, concentric bipolar electrodes (Shaft diameter 0.5 mm; model NE-100, Rhodes Medical Instruments, California) were stereotaxically placed in the left substantia niqra. The electrodes were connected to a Grass 588 stimulator fitted with a constant current device (Grass Instruments Model CCU 1A). Stimulation was for 1 hr, at 15 Hz, 400 PA, 2.0 msec. After stimulation the animals Animals were anesthetized with chloral hydrate, 400 mq/kq i-p. were decapitated and the striata and spinal cord removed and immediately frozen on dry ice. Analysis of DA, HVA and DOPAC was done by a previously described method fCos%niSSiong et al., 1978). In both the anesthetized control, and the anesthetized stimulated preparations, the DA levels tended to increase on both sides of the cord, in comparison with the unaneslp
567
Preliminary Notes
mimics some of the effects of L-DOPA on spinal reflexes (Nyqren and Olson, 1976). Moreover, it is questionable whether NE synthesis is increased aftet L-DQPA treatment (Freed and Murphy, 19783. In addition, 3,4_dihydroxyphenylserine,from which NE is produced directly, thereby precluding an effect of DA, depresses the stretch reflex (Commissiongand Sedgwick, 1974), while the initial effect of L-DOPA is to enhance the stretch reflex (Commissiongand Sedqwick, 1974). We, therefore, propose that DA, and not NE is the metabolite of L-DOPA which is responsible for mediating many of the effects of this drug on spinal reflexes. Table 3. Levels of DA and NE in Various Regions of the Spinal Cord After Bilateral Lesion of The Locus Coeruleus CERVICAL
NE
DA
c L
4.7 20.2 2.8 r0.3
(6) (9)***
C L
0.56tO.03 (61 0.48kO.04 (9)
THORACIC DORSAL HORN nmol,'g 3.6 to.2 2.8 +0.4
(6: f9)*
0.42-10.04(6) 0.48f0.06 (9)
LUMBAR
3.8 1rO.2 (6) 2.3 fO.2 (9)*** 0.33f0.02 (6) 0.31+0.02 (9)
VENTRAL HORN NE
C L
4.0 +0.4 1.6 f0.3
(6) (9)***
DA
C L
0.22iO.03 (6) 0.18+0.04 (91
3.9 20.2 1.6 f0.3
(6) (9)***
0.07t0.03 (6) 0.09+0.06 (9)
3.9 f0.2 1.6 20.2
(6) (9)***
0.13%.02 (6) 0.12f0.03 (9)
ZONA INTERMEDIA NE
C L
__ __
4.4 to.2 1.9 to.3
DA
C L
-__
0.29ti.04 (6) 0.22ti.05 (9)
(6) c91***
---
-_
Rats were anesthetized with chloral hydrate 400 mg/kg i.p. A unipolar stainless steel electrode (0.3 mm diameter) insulated to within 0.5 mm of the tip was stereotaxically placed in the locus coeruleus using the coordinates of Korf et al. (1973). The electrode was connected to an Electrolytic Lesion Producing Device (StoeltingCo., Chicago, IL). A direct current of 2.5 mA was passed for 45 sec. Ten days later, the animals were killed by decapitation and the spinal cord removed and.stored on dry ice. The lesion of the locus coeruleus was verified histologicallyand also by monitoring NE levels in the cerebellum. There was a highly significant reduction of NE in the cerebellum (data not shown). *p
St8
Preliminary
Notes
(1978). Mass spectrometric measurement of catecholamine Freed, C. R. and Murphy, R. C. turnover in rat hypothalamus after long-term L-DOPA infusion. J. Phannacol. Exp. Ther. 205: 702-709. (1969). The influence of DOPA on static and dynamic fusimotor activity to the Grillner, S. triceps surae of the spinal cat. Acta Physiol. Stand. 77: 490-509. (1968). Jurna, I. and Lundberg, A. The influence of an inhibitor of dopamine-8-hydroxylase on the effect of WPA on transmission in the spinal cord. In: Structure and Functions of Inhibitory Neuronal Mechanisms (van Euler, C., Ed.) pp. 469-472. Pergamon Press, Oxford. Kori, J., Aghajanian, G. K. and Roth, R. B. (1973). Stimulation and destruction of locus coeruleus: Opposite effects on 3-methoxy-4-hydroxyphenylglycol sulfate levels in the rat cerebral cortex. Eur. J. Pharmacol. 21: 305-310. (1977). A new major projection from locus coeruleus: The main Nygren, L. and Olson, L. source of noradrenergic terminals in the ventral and dorsal columns of the spinal cord. Brain Res. 132: 85-93. (1976). On spinal noradrenaline receptor supersensitivity: CorrelaNygren, L. and Olson, L. tion between nerve terminal densities and flexor reflexes various times after intracisternal Brain Res. 116: 455-470. 6-hydroxydopamirie. (1967). Motoneurone depression by noradrenaline. Weight, F. F. and Salmoiraghi, G. C. Nature 213: 1129-1130.