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Evidence for the existence of peripheral dopaminergic neurons
Brain Research, 193 (1980) 289-292 © Elsevier/North-Holland Biomedical Press
289
Evidence for the existence of peripheral dopaminergic neurons
Z. LACKOVI(~ and N. H. NEFF* Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted March 6th, 1980) Key words: dopamine neuron -- spinal nerve -- nerve root -- homovanillic acid
There is now pharmacological evidence for the presence of dopaminergic receptors throughout the cardiovascular system 5. In addition there is histological evidence for the presence of dopamine (DA)-containing neuronal elements in kidney glomerulil, 4. These observations are consistent with the notion that there might be a peripheral dopaminergic neuronal system. Spinal nerve roots are a principle source of peripheral nerves. It is generally accepted that the majority of the neurons leaving the spinal cord (somatic nerves and preganglionic autonomic nerves) via the ventral nerve roots are cholinergic, whereas the nature of the neurotransmitter(s) associated with neurons entering the spinal cord via the dorsal nerve roots is unclear. Substance P may be a neurotransmitter candidate for these neurons 6. In 1965, Dahlstr6m and Fuxe 8 reported that catecholamine histofluorescence was present in the ventral nerve roots of the rat spinal cord. Subsequently, 'adrenergic' nerve fibers were observed in spinal ganglia 8. We have investigated spinal nerve roots for their content of norepinephrine (NE), DA and homovanillic acid (HVA) using specific and sensitive gas chromatography-mass spectrometry (GC-MS) techniques 2,7 to evaluate the possible existence of peripheral DA-containing nerves. Our results suggest that bovine spinal nerve roots may, indeed, contain dopaminergic nerves. Bovine spinal cords with attached nerve roots, were removed from the cervical and lumbo-sacral region of the vertebral column within 10-15 min of killing (J. W. Treuth, Baltimore) frozen on dry-ice and stored at --20 °C. Only a few sensory ganglia, from the cervical portion of the cord, were obtained because of their localization deep within the boney structure. Muscle tissue was removed from around the spinal column for analysis as well. Frozen cord samples were allowed to warm to 0 °C for dissection of the nerve roots. Samples were prepared for mass fragmentographic analysis as described previously 2,7. In brief, tissue was homogenized with 1 N HC1, centrifuged at 20,000 × * To whom correspondence should be addressed.
290 g for 20 rain and 500-800 #1 of the supernatant transferred to a glass Microflex vial (Pierce, Ill). Known quantities of deuterium labeled NE ([2Hz]NE), DA ([2H4]DA) and H V A ([2Hs]HVA) were added as internal standards to each vial and the samples lyophilized. The amines were converted to pentafluoropropionyl (PFP) derivatives while H V A was first converted to a methylester and then to a PFP derivate. Analyses were performed on a Finnigan 3200 gas chromatograph quadrupole mass spectrometer system fitted with a 3 m glass column, 2.5 m m i.d., packed with SE 54 3 o / o n Gas Chrom Q, 100-120 mesh. Absolute identification of DA and HVA in the nerve root samples was accomplished by the technique of multiple ion monitoring as well as by the retention times of the compounds (Table I). Within the mass spectrometer characteristic positively-charged fragments, having a fixed mass to charge (m/e) ratio, are generated for each compound. For the purpose of compound identification the mass spectrometer is focused on specific fragments and their ratio to each other as well as their retention times are compared for authentic and biological samples. As shown on Table I, within experimental limits, the spinal nerve roots do indeed contain DA and HVA. The sensitivity of the instrument for DA on many occasions approached ! fmol injected into the column. A positive identification of DA by multiple ion monitoring could be achieved with about 20 fmol of DA. The instrument was less sensitive for the analysis of HVA and NE. The content of DA was about 3-10 times higher than NE in both the ventral and dorsal nerve roots (Table II). The content of catecholamine in the dorsal and ventral nerve roots were similar; however, the lumbo-sacral roots were significantly higher in D A content than the cervical roots. About 200 pmol/g HVA was found in the ventral and dorsal lumbo-sacral roots. In contrast to the nerve roots, the NE content was significantly higher than D A in a cross-sectional portion of the lumbar spinal cord. Muscle tissue, removed from the region of the vertebral column to serve as a comparison, contained low but about equal quantities of NE and DA. TABLE I Identification o f DA and H V A in bovine spinal nerve roots by G C - M S
DA a n d HVA were prepared for analysis as described in the text. Three fragments were monitored for DA-PFP (m/e), 428, 387 a n d 4 1 5 a n d two fragments for methylester-HVA-PFP, 342 and 283. Fragments 428 and 342 were considered as 100% fragments for comparison. Fragment 342 is the m o l e c u l a r i o n of methylester-HVA-PFP. Relative abundance o f lbagments (%) (m/e) 428 Authentic
DA
387
415
342
GC retention 283
time ( min )
100
16
15
--
--
3.45
Dorsal nerve roots Ventral nerve roots
100 100
15 15
13 14
---
---
3.45 3.44
Authentic
_
---
--
100 100
88 90
3.10 3.11
--
--
100
96
3.10
HVA
Dorsal nerve roots Ventral nerve roots
291 TABLE II DA, H V A , and ArE content of bovine dorsal and ventral nerve roots from cervical and lumbo-sacral spinal cord, muscle tissue and spinal cord Tissue (pmol/g)
Ventral nerve roots Cervical Lumbo-sacral Dorsal nerve roots Cervical Lumbo-sacral Lumbar muscle tissue Spinal cord
DA
HVA
113 + 27 (6) 339 ± 79 (10)
N.D. 186 -+- 21 (3)
88 363 58 70
N.D. 208 4- 54 (5) N.D. 79 + 29 (2)
± 22 + 79 ± 9 -4- 12
(7) (10) (4) (6)
ATE
33 ± 11 (3) 31 ± 4 (4) 26 26 46 225
± I0 ± 8 :t: 10 + 25
(3) (5) (3) (3)
For the analysis of NE the mass spectrometer was focused on m/e 590. Results are presented as mean ± S.E.M. (number of observations). N.D., not determined.
There was a rather large v a r i a t i o n in the c o n t e n t o f D A for s o m e o f the nerve r o o t s as evidenced by the large S.E.M. values (Table II). W e p o s t u l a t e f r o m this o b s e r v a t i o n t h a t n o t all nerve r o o t s c o n t a i n the same content o f catecholamine. I n p r e l i m i n a r y studies using rats, where the nerve r o o t s can be identified m o r e easily a n d collected in consecutive order, we f o u n d t h a t there is a r a t h e r steep g r a d i e n t o f tyrosine h y d r o x y l a s e activity along the spinal c o r d nerve r o o t s which could a c c o u n t for the v a r i a t i o n o f c a t e c h o l a m i n e c o n t e n t a m o n g the samples. In conclusion, o u r results are consistent with the hypothesis t h a t the catecholamine fluorescence 3 observed in the spinal nerve r o o t s represents D A . Clearly the D A in the nerve r o o t s is n o t present as a p r e c u r s o r for NE. M o r e o v e r , the D A is a p p a r e n t l y being m e t a b o l i z e d to H V A within the nerve roots. W h e t h e r the D A - c o n t a i n i n g nerves are afferent o r efferent to the c o r d remains to be studied. The c o n t e n t o f D A a n d N E in the few bovine spinal g a n g l i a we collected was a b o u t 38 a n d 35 p m o l / g respectively, which does n o t p r o v i d e a clear i n d i c a t i o n a b o u t the source o f the D A - c o n t a i n i n g nerves. I n the r a t after c h r o n i c c o r d o t o m y , a b o u t 35 ~ o f the spinal c o r d D A persists b e l o w the t r a n s e c t i o n 2 suggesting the existence o f a n o n - c e r e b r a l d o p a m i n e r g i c p r o j e c t i o n to the spinal cord. A d d i t i o n a l studies are needed to d e t e r m i n e the source, t e r m i n a t i o n a n d function o f this p o t e n t i a l p e r i p h e r a l d o p a m i n e r g i c n e u r o n a l system.
1 Bell, C., Lang, W. T. and Laska, F., Dopamine-containing vasomotor nerves in the dog kidney, J. Neurochem., 31 (1978) 77-83. 2 Commissiong, J. W., Galli, C. L. and Neff, N. H., Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord, J. Neurochem., 30 (1978) 1095-1099. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence ofanoutflow of noradrenaline nerve fibers in the ventral roots of the rat spinal cord, Experientia (Basel), 21 (1965) 409-410. 4 Dinerstein, R. J., Vannice, J., Henderson, R. C., Roth, L. J., Goldberg, L. I. and Hoffmann, P. C., Histofluorescence techniques provide evidence for dopamine-containing neuronal elements in canine kidney, Science, 205 (1979) 497-499. 5 Goidberg, L. I., Cardiovascular and renal actions of dopamine: potential clinical applications, PharmacoL Rev., 24 (1972) 1-29.
292 6 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 232-252. 7 Karoum, F., Gillin, J. C., Wyatt, R. J. and Costa, E., Mass-fragmentography of nanogram quantities of biogenic amine metabolites in human cerebrospinal fluid and whole rat brain, Biomed. Mass Spec., 2 (1975) 183-189. 8 0 w m a n , C. and Santini, M., Adrenergic nerves in spinal ganglia of the cat, Acta physiol, scand., 68 (1966) 127-128.
289
Evidence for the existence of peripheral dopaminergic neurons
Z. LACKOVI(~ and N. H. NEFF* Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032 (U.S.A.)
(Accepted March 6th, 1980) Key words: dopamine neuron -- spinal nerve -- nerve root -- homovanillic acid
There is now pharmacological evidence for the presence of dopaminergic receptors throughout the cardiovascular system 5. In addition there is histological evidence for the presence of dopamine (DA)-containing neuronal elements in kidney glomerulil, 4. These observations are consistent with the notion that there might be a peripheral dopaminergic neuronal system. Spinal nerve roots are a principle source of peripheral nerves. It is generally accepted that the majority of the neurons leaving the spinal cord (somatic nerves and preganglionic autonomic nerves) via the ventral nerve roots are cholinergic, whereas the nature of the neurotransmitter(s) associated with neurons entering the spinal cord via the dorsal nerve roots is unclear. Substance P may be a neurotransmitter candidate for these neurons 6. In 1965, Dahlstr6m and Fuxe 8 reported that catecholamine histofluorescence was present in the ventral nerve roots of the rat spinal cord. Subsequently, 'adrenergic' nerve fibers were observed in spinal ganglia 8. We have investigated spinal nerve roots for their content of norepinephrine (NE), DA and homovanillic acid (HVA) using specific and sensitive gas chromatography-mass spectrometry (GC-MS) techniques 2,7 to evaluate the possible existence of peripheral DA-containing nerves. Our results suggest that bovine spinal nerve roots may, indeed, contain dopaminergic nerves. Bovine spinal cords with attached nerve roots, were removed from the cervical and lumbo-sacral region of the vertebral column within 10-15 min of killing (J. W. Treuth, Baltimore) frozen on dry-ice and stored at --20 °C. Only a few sensory ganglia, from the cervical portion of the cord, were obtained because of their localization deep within the boney structure. Muscle tissue was removed from around the spinal column for analysis as well. Frozen cord samples were allowed to warm to 0 °C for dissection of the nerve roots. Samples were prepared for mass fragmentographic analysis as described previously 2,7. In brief, tissue was homogenized with 1 N HC1, centrifuged at 20,000 × * To whom correspondence should be addressed.
290 g for 20 rain and 500-800 #1 of the supernatant transferred to a glass Microflex vial (Pierce, Ill). Known quantities of deuterium labeled NE ([2Hz]NE), DA ([2H4]DA) and H V A ([2Hs]HVA) were added as internal standards to each vial and the samples lyophilized. The amines were converted to pentafluoropropionyl (PFP) derivatives while H V A was first converted to a methylester and then to a PFP derivate. Analyses were performed on a Finnigan 3200 gas chromatograph quadrupole mass spectrometer system fitted with a 3 m glass column, 2.5 m m i.d., packed with SE 54 3 o / o n Gas Chrom Q, 100-120 mesh. Absolute identification of DA and HVA in the nerve root samples was accomplished by the technique of multiple ion monitoring as well as by the retention times of the compounds (Table I). Within the mass spectrometer characteristic positively-charged fragments, having a fixed mass to charge (m/e) ratio, are generated for each compound. For the purpose of compound identification the mass spectrometer is focused on specific fragments and their ratio to each other as well as their retention times are compared for authentic and biological samples. As shown on Table I, within experimental limits, the spinal nerve roots do indeed contain DA and HVA. The sensitivity of the instrument for DA on many occasions approached ! fmol injected into the column. A positive identification of DA by multiple ion monitoring could be achieved with about 20 fmol of DA. The instrument was less sensitive for the analysis of HVA and NE. The content of DA was about 3-10 times higher than NE in both the ventral and dorsal nerve roots (Table II). The content of catecholamine in the dorsal and ventral nerve roots were similar; however, the lumbo-sacral roots were significantly higher in D A content than the cervical roots. About 200 pmol/g HVA was found in the ventral and dorsal lumbo-sacral roots. In contrast to the nerve roots, the NE content was significantly higher than D A in a cross-sectional portion of the lumbar spinal cord. Muscle tissue, removed from the region of the vertebral column to serve as a comparison, contained low but about equal quantities of NE and DA. TABLE I Identification o f DA and H V A in bovine spinal nerve roots by G C - M S
DA a n d HVA were prepared for analysis as described in the text. Three fragments were monitored for DA-PFP (m/e), 428, 387 a n d 4 1 5 a n d two fragments for methylester-HVA-PFP, 342 and 283. Fragments 428 and 342 were considered as 100% fragments for comparison. Fragment 342 is the m o l e c u l a r i o n of methylester-HVA-PFP. Relative abundance o f lbagments (%) (m/e) 428 Authentic
DA
387
415
342
GC retention 283
time ( min )
100
16
15
--
--
3.45
Dorsal nerve roots Ventral nerve roots
100 100
15 15
13 14
---
---
3.45 3.44
Authentic
_
---
--
100 100
88 90
3.10 3.11
--
--
100
96
3.10
HVA
Dorsal nerve roots Ventral nerve roots
291 TABLE II DA, H V A , and ArE content of bovine dorsal and ventral nerve roots from cervical and lumbo-sacral spinal cord, muscle tissue and spinal cord Tissue (pmol/g)
Ventral nerve roots Cervical Lumbo-sacral Dorsal nerve roots Cervical Lumbo-sacral Lumbar muscle tissue Spinal cord
DA
HVA
113 + 27 (6) 339 ± 79 (10)
N.D. 186 -+- 21 (3)
88 363 58 70
N.D. 208 4- 54 (5) N.D. 79 + 29 (2)
± 22 + 79 ± 9 -4- 12
(7) (10) (4) (6)
ATE
33 ± 11 (3) 31 ± 4 (4) 26 26 46 225
± I0 ± 8 :t: 10 + 25
(3) (5) (3) (3)
For the analysis of NE the mass spectrometer was focused on m/e 590. Results are presented as mean ± S.E.M. (number of observations). N.D., not determined.
There was a rather large v a r i a t i o n in the c o n t e n t o f D A for s o m e o f the nerve r o o t s as evidenced by the large S.E.M. values (Table II). W e p o s t u l a t e f r o m this o b s e r v a t i o n t h a t n o t all nerve r o o t s c o n t a i n the same content o f catecholamine. I n p r e l i m i n a r y studies using rats, where the nerve r o o t s can be identified m o r e easily a n d collected in consecutive order, we f o u n d t h a t there is a r a t h e r steep g r a d i e n t o f tyrosine h y d r o x y l a s e activity along the spinal c o r d nerve r o o t s which could a c c o u n t for the v a r i a t i o n o f c a t e c h o l a m i n e c o n t e n t a m o n g the samples. In conclusion, o u r results are consistent with the hypothesis t h a t the catecholamine fluorescence 3 observed in the spinal nerve r o o t s represents D A . Clearly the D A in the nerve r o o t s is n o t present as a p r e c u r s o r for NE. M o r e o v e r , the D A is a p p a r e n t l y being m e t a b o l i z e d to H V A within the nerve roots. W h e t h e r the D A - c o n t a i n i n g nerves are afferent o r efferent to the c o r d remains to be studied. The c o n t e n t o f D A a n d N E in the few bovine spinal g a n g l i a we collected was a b o u t 38 a n d 35 p m o l / g respectively, which does n o t p r o v i d e a clear i n d i c a t i o n a b o u t the source o f the D A - c o n t a i n i n g nerves. I n the r a t after c h r o n i c c o r d o t o m y , a b o u t 35 ~ o f the spinal c o r d D A persists b e l o w the t r a n s e c t i o n 2 suggesting the existence o f a n o n - c e r e b r a l d o p a m i n e r g i c p r o j e c t i o n to the spinal cord. A d d i t i o n a l studies are needed to d e t e r m i n e the source, t e r m i n a t i o n a n d function o f this p o t e n t i a l p e r i p h e r a l d o p a m i n e r g i c n e u r o n a l system.
1 Bell, C., Lang, W. T. and Laska, F., Dopamine-containing vasomotor nerves in the dog kidney, J. Neurochem., 31 (1978) 77-83. 2 Commissiong, J. W., Galli, C. L. and Neff, N. H., Differentiation of dopaminergic and noradrenergic neurons in rat spinal cord, J. Neurochem., 30 (1978) 1095-1099. 3 Dahlstr6m, A. and Fuxe, K., Evidence for the existence ofanoutflow of noradrenaline nerve fibers in the ventral roots of the rat spinal cord, Experientia (Basel), 21 (1965) 409-410. 4 Dinerstein, R. J., Vannice, J., Henderson, R. C., Roth, L. J., Goldberg, L. I. and Hoffmann, P. C., Histofluorescence techniques provide evidence for dopamine-containing neuronal elements in canine kidney, Science, 205 (1979) 497-499. 5 Goidberg, L. I., Cardiovascular and renal actions of dopamine: potential clinical applications, PharmacoL Rev., 24 (1972) 1-29.
292 6 H6kfelt, T., Kellerth, J. O., Nilsson, G. and Pernow, B., Experimental immunohistochemical studies on the localization and distribution of substance P in cat primary sensory neurons, Brain Research, 100 (1975) 232-252. 7 Karoum, F., Gillin, J. C., Wyatt, R. J. and Costa, E., Mass-fragmentography of nanogram quantities of biogenic amine metabolites in human cerebrospinal fluid and whole rat brain, Biomed. Mass Spec., 2 (1975) 183-189. 8 0 w m a n , C. and Santini, M., Adrenergic nerves in spinal ganglia of the cat, Acta physiol, scand., 68 (1966) 127-128.