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Descending phenylethanolamine-N-methyltransferase projections to the monkey spinal cord: an immunohistochemical double labeling study
Neurosciem'e Letters, 76 (1987) 133 139 Elsevier Scientitic Publishers Ireland Ltd.
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Descending phenylethanolamine-N-methyltransferase projections to the monkey spinal cord: an immunohistochemical double labeling study S.M. C a r l t o n 1"2, C . N . H o n d a l, L. D e n o r o y 4 and W . D . Willis Jr. I 3 ~Marim, Biomedical Institute aml 2Departments q/'Anatomy & Neuroscience and o1 ~Physiolo~,,y & Biophysics, University o[ Texas Medical Branch. Galveston. T J( 77550 / U.S.A. ). and. 4Department o/ Medicim' E.vperimenlah', Univer.sitO Claude Bernard, L)'on (France) (Received I November 1986; Revised version received 20 December 1986: Accepted 22 December 1986)
Keywor~ls'." Catccholamine: Phenylethanolamine-N-methyltransferase (PNMT): Cervical cord; Monkey
Descending system;
In the present study, we determined that a population of spinally projecting neurons in the monkey brainstem also contained the enzyme phenylethanolamine-N-methyltransferase (PNMT). Following bilateral placements of horseradish peroxidase (HRP) in the cervical spinal cord, brainstem sections containing retrogradely labeled cells were immunohistochemically stained for P N M T . Single labeled P N M T positive cells were found in a distinctive pattern in the dorsomedial and ventrolateral medulla. A population of double labeled cells was observed in the latter group only. This population was dispersed a m o n g other single labeled H R P and single labeled P N M T neurons. Possible functional roles of descending P N M T cells include involvement in sympathetic control of cardiowlscular mechanisms and:or tonic descending inhibition of dorsal horn neurons.
The enzyme phenylethanolamine-N-methyltransferase (PNMT) converts noradrenaline to adrenaline and thus is used as a cytochemical marker for adrenaline-containing neuronal profiles. Immunohistochemical staining techniques have been used to demonstrate neurons containing P N M T in several species, including the rat [I, 9, 11, 18], cat [12, 19] and man [13, 17]. Subsequently, a subpopulation of P N M T - c o n raining cells in the ventrolateral medulla of the rat has been shown to project to the thoracic spinal cord, specifically to the intermediolateral and intermediomedial cell columns [20, 21]. The areas of the reticular formation (RF) where PNMT-containing neurons are located have been cited as areas important in mechanisms of cardiovascular control [5, 20, 21]. Recently, these same areas in the RF have also been shown to play a role in tonic descending inhibition of dorsal horn neurons [8, 16]. ('orrespondence: W.D. Willis Jr., Marine Biomedical Institute, University of Texas Medical Branch, 200 University Boulewlrd, Galveston, TX 77550 2772, U.S.A. 0 3 0 4 - 3 9 4 0 8 7 5 03.50 (~ 1987 Elsevier Scientific Publishers lrehmd Ltd.
134 In the present investigation, we examined the distribution of PNMT-containing neurons in the monkey medulla including those that project to the spinal cord. Somc possible functional roles of these cells are discussed. This work has been presented in abstract form [4]. Studies were conducted using two macaque monkeys (M. Jascicularis). The animals were initially anesthetized with a mixture of halothane and nitrous oxide gas and subsequently given pentobarbital i.v. (35 mg/kg). Following a cervical laminectomy, the lateral funiculi in segments (-!4and C5 were surgically cut to allow bilateral placement of several pellets of HRP gel [7]. Following 3 days survival, the animals were deeply anesthetized with pentobarbital and perfused through the aorta with warm saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4 C . The brainstem and spinal cord were immediately removed and postfixed in fresh 4% paraformaldehyde for 6-8 h. The tissue was then stored overnight in 10% sucrose in 0.1 M PB. The cervical spinal cord was sectioned on a freezing microtome at 50 #m; the brainstem was sectioned on a vibratome at 25 #m. Cervical cord sections were reacted with diaminobenzidine (DAB) to visualize the extent of the HRP gel implants. Alternate sections through the medulla of each animal were reacted for neurons containing retrogradely transported H R P using either the diaminobenzidine-cobalt chloride (DAB--CoCI:) protocol of Bowker et al. [2] or the tetramethylbenzidine (TMB) protocol of Mesulam et al. [15] with subsequent stabilization [22]. Following the H R P reaction, the tissue was immunostained for P N M T using the peroxidase-anti-peroxidase technique [23]. The enzyme P N M T was purified from bovine adrenal gland according to Joh and Goldstein [10]. The antiserum was raised in rabbit and used at a dilution of 1:2000. A detailed description of the purification and specificity of the antibody can be found elsewhere [6, 13]. P N M T antisera preabsorbed with purified bovine P N M T resulted in complete absence of" staining in the monkey medulla in the present study. All immunostained tissue was mounted on subbed slides, cleared in alcohols and xylene and coverslipped with permount. Representative sections from the medulla of each animal were drawn using an X-Y plotter. The positions of single labeled P N M T or H R P and double labeled P N M T - H R P cells were plotted on each section. Placements of the H RP gel resulted in crescent-shaped implants which included the entire lateral funiculi and the lateral two-thirds of the gray matter. The retrograde labeling of PNMT-containing cells in the brainstem projecting to the spinal cord was optimized since fibers of passage (in the lateral funiculi) destined for lower cord levels, as well as terminal fields in the cervical gray matter, were bilaterally exposed to HRP. Therefore, the HRP-labeled cells observed in the medulla reflected that population of neurons projecting to the cord via these pathways. Cells in the monkey medulla that were labeled with HRP had a distribution similar to that previously described [14]. The distribution of PNMT-positive neurons in the monkey brainstem is described elsewhere [3], and will only be briefly discussed here. At the level of the caudal pole of the area postrema (AP), a few PNMT-containing cells were seen in the vicinity of the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMX). Rostrally, the cells in these groups were observed below the
135
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D
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i: Fig. 1. Schematic drawings illustrating the distribution of single labeled PNMT, single labeled }IRP and double labeled cells following HRP gel implants in the cervical cord (see text for abbreviations). Labeled neurons from 3 sections were plotted on each drawing. The representative levels are approximately 0.5 mm apart. f l o o r o f the f o u r t h v e n t r i c l e at the level o f t h e e x i t i n g h y p o g l o s s a l n e r v e s (Fig. I B, C). In the v e n t r o l a t e r a l m e d u l l a , P N M T - c o n t a i n i n g n e u r o n s w e r e also o b s e r v e d at the level o f the c a u d a l p o l e o f the A P , d o r s a l to the lateral r e t i c u l a r n u c l e u s ( L R N , Fig. 1A).
136 M o r e rostrally, two distinct g r o u p s o f P N M T - c o n t a i n i n g cells became a p p a r e n t in the ventrolateral medulla; one g r o u p was on the extreme edge o f the medulla, lateral to the inferior olive (IO), while the o t h e r was located directly dorsal to the IO. in the region o f the L R N . Progressing rostrally t h r o u g h the medulla, these two g r o u p s a p p e a r e d to merge (Fig. I D, E). T o w a r d the rostral pole o f the IO, the n u m b e r o f P N M T - c o n t a i n i n g cells in the v e n t r o l a t e r a l m e d u l l a d i m i n i s h e d until only a few cells were o b s e r v e d d o r s o l a t e r a l to the p y r a m i d s (Fig. 1G, H). Finally, a column or b a n d o f P N M T - c o n t a i n i n g cells was observed oriented d o r s o m e d i a l l y to vent r o l a t e r a l l y at m i d - o l i v a r y levels. The d o r s o m e d i a l pole was in the vicinity o f the N T S while the v e n t r o l a t e r a l pole was in the vicinity o f the L R N (Fig. IC). A t the level o f the rostral two thirds o f the IO, d o u b l e labeled cells were in the small cluster o f cells at the extreme edge o f the m e d u l l a a n d in a larger cell cluster in the vicinity o f the L R N (Fig. IC). Few d o u b l e labeled cells were o b s e r v e d rostral to the rostral pole o f the IO. Spinally p r o j e c t i n g P N M T - c o n t a i n i n g cells were in no w a y segregated from single labeled H R P - or P N M T - c o n t a i n i n g cells but rather positioned r a n d o m l y a m o n g b o t h (Fig. 2C). D e p e n d i n g u p o n which cluster they were in. d o u b l e labeled cells h a d a m o r p h o l o g y similar to single labeled cells (Fig. 2A, B) in that those in the e x t r e m e v e n t r o l a t e r a l edge were small a n d s p i n d l e - s h a p e d while those in the vicinity o f the L R N were larger with r o b u s t processes. N o d o u b l e labeled
4
k, Fig. 2. Photomicrographs of a single labeled HRP neuron (A) and a single labeled PNMT neuron (B) in the ventrolateral medulla (x 576). Notice the similarity in morphology between these two cells, The nucleus is highly visible in the PNMT-stained cell. By contrast, the nucleus in the HRP- labeled cell (outlined with arrows) forms an ovoid shape which is clear and devoid of HRP granules. The photomicrograph m C demonstrates a double labeled cell (filled arrowhead) and 3 PNMT-positive cells (open arrowheads). Both the cytoplasm and nucleus of the double labeled cell demonstrate the brown homogeneous immunoreactive staining for PNMT. Furthermore, large black HRP granules can also be seen in the cytoplasm ( × 576).
137 cells were present in the dorsomedial medulla or in that population which formed the column of P N M T cells [4]. The PNMT-spinal pathway in the monkey originated from considerably fewer medullary neurons as compared to the rat. It has been reported that at least one half of the PNMT-containing cells in the rat ventrolateral medulla project to the spinal cord [20, 21]. As demonstrated in Fig. 1, this percentage was much lower in the m o w key. A possible explanation for this discrepancy between species may be due to differences in protocol. Intrinsic granules often appeared in the cytoplasm of the PNMTcontaining cells when the DAB CoCI~ incubation was prolonged, increasing the chances of false-positive labeling. For this reason, alternate sections were reacted with the TMB protocol followed by a stabilization procedure. The latter protocol did not enhance intrinsic granules in the P N M T cells and thus greatly reduced the possibility of false-positive labeling. Previous studies emphasize a spinal projection of PNMT-containing neurons exclusively to the thoracic cord, with PNMT-positive terminals heavily concentrated in the intermediomedial cell column (IMMCC) and intermediolateral cell column (IMLCC). Electrophysiological experiments in rats, in which such parameters as blood pressure (BP) and heart rate (H R) were measured, demonstrated that electrical stimulation in the ventrolateral medulla resulted in an elevation of BP and HR. Subsequently, the functional significance of the PNMT-spinal pathway has been discussed only in the context of cardiovascular control. However, our data indicate that the descending PNMT-spinal pathway is not exclusively to the thoracic cord but in addition projects in a limited fashion to other levels of the monkey spinal cord. Furthermore, PNMT-stained terminal profiles can be found not only in the IMMCC and IMLCC but also in laminae I and V at all cord levels [3]. Although the ventrolateral medulla has been associated with control of cardiovascular function, another body of literature indicates that this same area plays a role in tonic descending inhibition of dorsal horn neurons [8, 16]. Stimulation of the vcntrolateral medulla in the cat selectively inhibits the responses of dorsal horn neurons to noxious input [16]. Conversely, electrolytic lesions of the same area reduce tonic descending inhibition of the responses of dorsal horn cells to noxious input. Thus, this area exerts a considerable control over the transmission of nociceptive information in the spinal cord. The above authors suggest participation of catecholamines in this tonic descending inhibitory system. However, previous double labeling studies indicate that no noradrenergic cells in the ventrolateral medulla project to the spinal cord [24]. Hence it is possible that the PNMT-spinal pathway described here is the source of this descending inhibition. This sites of origin of the spinal pathway controlling cardiovascular function and the spinal pathway exerting tonic inhibition may partially overlap or even be coextensive. Lesions in the ventrolateral medulla reducing tonic inhibition produce persistent hypotension. Conversely, lesions not reducing tonic inhibition do not produce hypotension [16]. There has been described an association between control of central pain and of circulation in hypertensive humans and rats [25, 26]. Thus, the PNMT-spinal pathway may function simultaneously in these two diverse systems.
138 W e w i s h to t h a n k P h y l l i s W a l d r o p f o r h e r efficient s e c r e t a r i a l a s s i s t a n c e . T h i s s t u d y w a s s u p p o r t e d by g r a n t s N S 09743 ( W . D . W . ) a n d p o s t d o c t o r a l f e l l o w s h i p s N S 07062 ( S . M . C . ) a n d N S 07574 ( C . N . H . ) . 1 Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution ofdopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes, J. Comp. Neurol., 212 (1982) 173 187. 2 Bowker, R.M., Westlund, K.N., Sullivan. M.C. and Coulter, J.D., A combined retrograde transport and immunocytochemical staining method for demonstrating the origins of 5-HT projections, J. Histochem. Cytochem., 30 (1982) 805 810. 3 Carlton, S.M,, Honda, (7.N,, Denoroy, L. and Willis, W.D., Brainstem and spinal cord mapping of PNMT-stained neurons, axons and terminals in the monkey, Soc. Neurosci. Abstr., 12 (1986) 144. 4 Carlton, S.M., Honda, C.N. and Willis, W.D., Identification of spinally projecting adrenaline neurons in the monkey medulla: a double labeling study using HRP and PNMT immunohistochemistry, Anat. Rec., 213 (1985) 195A. 5 Dampney, R.A.L. and Moon, E.A., The role of ventrolateral medulla in vasomotor response to cerebral ischemia. Am. J. Physiol., 239 (1980) H349 H358. 6 Denoroy, L., Utilisation de la Ph6nyl~thanolamine-N-m6thyltransfdrasecomme Marqueur des Structures Adr6nergiques. Application a l'l~tude de I'Hypertension Art6rielle, Th6se de Docteur-lng6nieur, Universitd Claude Bernard, Lyon, 1979. 7 Grillin, G., Watkins, L.R. and Mayer, D.J., HRP pellets and slow release gels: two new techniques for greater localization and sensitivity, Brain Res., 168 (1979) 595 601. 8 Hall, J.G., Duggan, A.W.. Morton, C.R. and Johnson, S.M., The location of brainstem neurones tonically inhibiting dorsal horn neurones of the cat, Brain Res., 244 (1982) 215- 222. 9 H6kfelt, T., Fuxe. K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence of the existence of adrenaline neurons in the rat brain, Brain Res., 66 (1974) 235 251. 10 Joh, T.H. and Goldstein, M., Isolation and characterization of multiple forms of phenylethanolamineN-methyltransferase. MoI. Pharmacol., 9 (1973) 117 129. 11 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. 11I. Adrenergic (CI and C2) neurons. nerve fibers and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 333 349. 12 Kitahama. K., Denoroy, k,, Berod, A. and Jouvet, M., Distribution of PNMT-immunoreactive neurons in the cat medulla oblongata, Brain Res. Bull.. 17 (1986) 197 208. 13 Kitahama, K., Pearson, J., Denoroy. L., Kopp, N., Ulrich, J., Maeda, T. and Jouvet, M., Adrenergic neurons in human brain demonstrated by immunohistochemistry with antibodies to phenylethanolamine-N-methyl transferase (PNMT): discovery of a new group in the nucleus tractus solitarius, Neurosci. Left., 53 (1985) 303 308. 14 Kneisley, L.W., Biber, M.P. and LaVail, J.H., A study of the origin of brainstem projections to monkey spinal cord using the retrograde transport method, Exp. Neurol., 60 (1978) 116 139. 15 Mesulam, M.-M., Tetramethylbenzidine for HRP neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) t06 117. 16 Morton, C.R., Johnson. S.M. and Duggan, A.W., Lateral reticular regions and the descending control of dorsal horn neurones of the cat: selective inhibition by electrical stimulation, Brain Res., 275 (1983) 13 21. 17 Robert, O., Miachon, S., Kopp, N., Denoroy, L., Tommasi, M., Rollet, D. and Pujol, J.F., tmmunohistochemical study of the catecholaminergic systems in the lower brain stem on the human infant, Human Neurobiol., 3 (1984) 229 234. 18 Ruggiero. D.A., Ross, C.A., Anwar, M., Park, D.H., Joh, T.J. and Reis, D.J. Distribution of neurons containing phenylethanolamine-N-methyltransferasein medulla and hypothalamus of rat, J. Comp. Neurol.,239(1985) 127 154. 19 Reiner, P.B. and Vincent, S.R.. The distribution of tyrosine hydroxylase, dopamine-fi-hydroxylaseand
139
2(I
21
22
23 24 25 26
phenylethanolamine-N-methyltransferaseimmunoreactive neurons in the feline medulla oblongata, J. Comp. Neurol.. 248 (1986) 518 531. Ross, ('.A., Ruggiero, D.A., Joh. T.tt., Park. D.tl. and Reis, D.[., Rostral vcntrolatcral medulla: selective projections to the thoracic autonomic cell colunm from the region containing (_71 adrenaline neurons. J. Comp. Neurol., 228 (1984) 168 185. Ross, ('.A., Armstrong, D.M., Ruggiero, D.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Adrenaline neurons in the rostral ventrolateral medulla innervate thoracic spinal cord: a combined immunocytochemical and retrograde transport demonstration, Neurosci. Lett., 25 (1981) 257 262. Rye, D.B., Sapcr, ('.B. and Wainer, B.H., Stabilization of the tetramethylbenzidine (TMB) reaction product: application for retrograde and antcrograde tracing and combination with immunocytochcmislry, J. Histochem. Cytochem., 32 (1984) 1145 1153. Sternbcrger, L.A., lmmunocytochcmistry, 2nd edn., John Wiley. New York, 1979. Westlund, K.N., Bowker, R.M., Ziegler, M.G. and Coulter, J.D.. Origins and terminations of descending noradrenergic projections to the spinal cord of monkey, Brain Res., 292 (1984) I 16. Zamir, N. and Segal, M,, Hypertension-induced analgesia: changes in pain sensitivity m experimental hypertensive rats, Brain Res..160 (1979) lT0 173. Zamir, N. and Shnber, E., Altered pain perception in hypertensive humans, Brain Res., 201 (198{I) 471 474.
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Descending phenylethanolamine-N-methyltransferase projections to the monkey spinal cord: an immunohistochemical double labeling study S.M. C a r l t o n 1"2, C . N . H o n d a l, L. D e n o r o y 4 and W . D . Willis Jr. I 3 ~Marim, Biomedical Institute aml 2Departments q/'Anatomy & Neuroscience and o1 ~Physiolo~,,y & Biophysics, University o[ Texas Medical Branch. Galveston. T J( 77550 / U.S.A. ). and. 4Department o/ Medicim' E.vperimenlah', Univer.sitO Claude Bernard, L)'on (France) (Received I November 1986; Revised version received 20 December 1986: Accepted 22 December 1986)
Keywor~ls'." Catccholamine: Phenylethanolamine-N-methyltransferase (PNMT): Cervical cord; Monkey
Descending system;
In the present study, we determined that a population of spinally projecting neurons in the monkey brainstem also contained the enzyme phenylethanolamine-N-methyltransferase (PNMT). Following bilateral placements of horseradish peroxidase (HRP) in the cervical spinal cord, brainstem sections containing retrogradely labeled cells were immunohistochemically stained for P N M T . Single labeled P N M T positive cells were found in a distinctive pattern in the dorsomedial and ventrolateral medulla. A population of double labeled cells was observed in the latter group only. This population was dispersed a m o n g other single labeled H R P and single labeled P N M T neurons. Possible functional roles of descending P N M T cells include involvement in sympathetic control of cardiowlscular mechanisms and:or tonic descending inhibition of dorsal horn neurons.
The enzyme phenylethanolamine-N-methyltransferase (PNMT) converts noradrenaline to adrenaline and thus is used as a cytochemical marker for adrenaline-containing neuronal profiles. Immunohistochemical staining techniques have been used to demonstrate neurons containing P N M T in several species, including the rat [I, 9, 11, 18], cat [12, 19] and man [13, 17]. Subsequently, a subpopulation of P N M T - c o n raining cells in the ventrolateral medulla of the rat has been shown to project to the thoracic spinal cord, specifically to the intermediolateral and intermediomedial cell columns [20, 21]. The areas of the reticular formation (RF) where PNMT-containing neurons are located have been cited as areas important in mechanisms of cardiovascular control [5, 20, 21]. Recently, these same areas in the RF have also been shown to play a role in tonic descending inhibition of dorsal horn neurons [8, 16]. ('orrespondence: W.D. Willis Jr., Marine Biomedical Institute, University of Texas Medical Branch, 200 University Boulewlrd, Galveston, TX 77550 2772, U.S.A. 0 3 0 4 - 3 9 4 0 8 7 5 03.50 (~ 1987 Elsevier Scientific Publishers lrehmd Ltd.
134 In the present investigation, we examined the distribution of PNMT-containing neurons in the monkey medulla including those that project to the spinal cord. Somc possible functional roles of these cells are discussed. This work has been presented in abstract form [4]. Studies were conducted using two macaque monkeys (M. Jascicularis). The animals were initially anesthetized with a mixture of halothane and nitrous oxide gas and subsequently given pentobarbital i.v. (35 mg/kg). Following a cervical laminectomy, the lateral funiculi in segments (-!4and C5 were surgically cut to allow bilateral placement of several pellets of HRP gel [7]. Following 3 days survival, the animals were deeply anesthetized with pentobarbital and perfused through the aorta with warm saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at 4 C . The brainstem and spinal cord were immediately removed and postfixed in fresh 4% paraformaldehyde for 6-8 h. The tissue was then stored overnight in 10% sucrose in 0.1 M PB. The cervical spinal cord was sectioned on a freezing microtome at 50 #m; the brainstem was sectioned on a vibratome at 25 #m. Cervical cord sections were reacted with diaminobenzidine (DAB) to visualize the extent of the HRP gel implants. Alternate sections through the medulla of each animal were reacted for neurons containing retrogradely transported H R P using either the diaminobenzidine-cobalt chloride (DAB--CoCI:) protocol of Bowker et al. [2] or the tetramethylbenzidine (TMB) protocol of Mesulam et al. [15] with subsequent stabilization [22]. Following the H R P reaction, the tissue was immunostained for P N M T using the peroxidase-anti-peroxidase technique [23]. The enzyme P N M T was purified from bovine adrenal gland according to Joh and Goldstein [10]. The antiserum was raised in rabbit and used at a dilution of 1:2000. A detailed description of the purification and specificity of the antibody can be found elsewhere [6, 13]. P N M T antisera preabsorbed with purified bovine P N M T resulted in complete absence of" staining in the monkey medulla in the present study. All immunostained tissue was mounted on subbed slides, cleared in alcohols and xylene and coverslipped with permount. Representative sections from the medulla of each animal were drawn using an X-Y plotter. The positions of single labeled P N M T or H R P and double labeled P N M T - H R P cells were plotted on each section. Placements of the H RP gel resulted in crescent-shaped implants which included the entire lateral funiculi and the lateral two-thirds of the gray matter. The retrograde labeling of PNMT-containing cells in the brainstem projecting to the spinal cord was optimized since fibers of passage (in the lateral funiculi) destined for lower cord levels, as well as terminal fields in the cervical gray matter, were bilaterally exposed to HRP. Therefore, the HRP-labeled cells observed in the medulla reflected that population of neurons projecting to the cord via these pathways. Cells in the monkey medulla that were labeled with HRP had a distribution similar to that previously described [14]. The distribution of PNMT-positive neurons in the monkey brainstem is described elsewhere [3], and will only be briefly discussed here. At the level of the caudal pole of the area postrema (AP), a few PNMT-containing cells were seen in the vicinity of the nucleus of the solitary tract (NTS) and the dorsal motor nucleus of the vagus (DMX). Rostrally, the cells in these groups were observed below the
135
A
E
"DOUBLE LABEL
B //
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i: Fig. 1. Schematic drawings illustrating the distribution of single labeled PNMT, single labeled }IRP and double labeled cells following HRP gel implants in the cervical cord (see text for abbreviations). Labeled neurons from 3 sections were plotted on each drawing. The representative levels are approximately 0.5 mm apart. f l o o r o f the f o u r t h v e n t r i c l e at the level o f t h e e x i t i n g h y p o g l o s s a l n e r v e s (Fig. I B, C). In the v e n t r o l a t e r a l m e d u l l a , P N M T - c o n t a i n i n g n e u r o n s w e r e also o b s e r v e d at the level o f the c a u d a l p o l e o f the A P , d o r s a l to the lateral r e t i c u l a r n u c l e u s ( L R N , Fig. 1A).
136 M o r e rostrally, two distinct g r o u p s o f P N M T - c o n t a i n i n g cells became a p p a r e n t in the ventrolateral medulla; one g r o u p was on the extreme edge o f the medulla, lateral to the inferior olive (IO), while the o t h e r was located directly dorsal to the IO. in the region o f the L R N . Progressing rostrally t h r o u g h the medulla, these two g r o u p s a p p e a r e d to merge (Fig. I D, E). T o w a r d the rostral pole o f the IO, the n u m b e r o f P N M T - c o n t a i n i n g cells in the v e n t r o l a t e r a l m e d u l l a d i m i n i s h e d until only a few cells were o b s e r v e d d o r s o l a t e r a l to the p y r a m i d s (Fig. 1G, H). Finally, a column or b a n d o f P N M T - c o n t a i n i n g cells was observed oriented d o r s o m e d i a l l y to vent r o l a t e r a l l y at m i d - o l i v a r y levels. The d o r s o m e d i a l pole was in the vicinity o f the N T S while the v e n t r o l a t e r a l pole was in the vicinity o f the L R N (Fig. IC). A t the level o f the rostral two thirds o f the IO, d o u b l e labeled cells were in the small cluster o f cells at the extreme edge o f the m e d u l l a a n d in a larger cell cluster in the vicinity o f the L R N (Fig. IC). Few d o u b l e labeled cells were o b s e r v e d rostral to the rostral pole o f the IO. Spinally p r o j e c t i n g P N M T - c o n t a i n i n g cells were in no w a y segregated from single labeled H R P - or P N M T - c o n t a i n i n g cells but rather positioned r a n d o m l y a m o n g b o t h (Fig. 2C). D e p e n d i n g u p o n which cluster they were in. d o u b l e labeled cells h a d a m o r p h o l o g y similar to single labeled cells (Fig. 2A, B) in that those in the e x t r e m e v e n t r o l a t e r a l edge were small a n d s p i n d l e - s h a p e d while those in the vicinity o f the L R N were larger with r o b u s t processes. N o d o u b l e labeled
4
k, Fig. 2. Photomicrographs of a single labeled HRP neuron (A) and a single labeled PNMT neuron (B) in the ventrolateral medulla (x 576). Notice the similarity in morphology between these two cells, The nucleus is highly visible in the PNMT-stained cell. By contrast, the nucleus in the HRP- labeled cell (outlined with arrows) forms an ovoid shape which is clear and devoid of HRP granules. The photomicrograph m C demonstrates a double labeled cell (filled arrowhead) and 3 PNMT-positive cells (open arrowheads). Both the cytoplasm and nucleus of the double labeled cell demonstrate the brown homogeneous immunoreactive staining for PNMT. Furthermore, large black HRP granules can also be seen in the cytoplasm ( × 576).
137 cells were present in the dorsomedial medulla or in that population which formed the column of P N M T cells [4]. The PNMT-spinal pathway in the monkey originated from considerably fewer medullary neurons as compared to the rat. It has been reported that at least one half of the PNMT-containing cells in the rat ventrolateral medulla project to the spinal cord [20, 21]. As demonstrated in Fig. 1, this percentage was much lower in the m o w key. A possible explanation for this discrepancy between species may be due to differences in protocol. Intrinsic granules often appeared in the cytoplasm of the PNMTcontaining cells when the DAB CoCI~ incubation was prolonged, increasing the chances of false-positive labeling. For this reason, alternate sections were reacted with the TMB protocol followed by a stabilization procedure. The latter protocol did not enhance intrinsic granules in the P N M T cells and thus greatly reduced the possibility of false-positive labeling. Previous studies emphasize a spinal projection of PNMT-containing neurons exclusively to the thoracic cord, with PNMT-positive terminals heavily concentrated in the intermediomedial cell column (IMMCC) and intermediolateral cell column (IMLCC). Electrophysiological experiments in rats, in which such parameters as blood pressure (BP) and heart rate (H R) were measured, demonstrated that electrical stimulation in the ventrolateral medulla resulted in an elevation of BP and HR. Subsequently, the functional significance of the PNMT-spinal pathway has been discussed only in the context of cardiovascular control. However, our data indicate that the descending PNMT-spinal pathway is not exclusively to the thoracic cord but in addition projects in a limited fashion to other levels of the monkey spinal cord. Furthermore, PNMT-stained terminal profiles can be found not only in the IMMCC and IMLCC but also in laminae I and V at all cord levels [3]. Although the ventrolateral medulla has been associated with control of cardiovascular function, another body of literature indicates that this same area plays a role in tonic descending inhibition of dorsal horn neurons [8, 16]. Stimulation of the vcntrolateral medulla in the cat selectively inhibits the responses of dorsal horn neurons to noxious input [16]. Conversely, electrolytic lesions of the same area reduce tonic descending inhibition of the responses of dorsal horn cells to noxious input. Thus, this area exerts a considerable control over the transmission of nociceptive information in the spinal cord. The above authors suggest participation of catecholamines in this tonic descending inhibitory system. However, previous double labeling studies indicate that no noradrenergic cells in the ventrolateral medulla project to the spinal cord [24]. Hence it is possible that the PNMT-spinal pathway described here is the source of this descending inhibition. This sites of origin of the spinal pathway controlling cardiovascular function and the spinal pathway exerting tonic inhibition may partially overlap or even be coextensive. Lesions in the ventrolateral medulla reducing tonic inhibition produce persistent hypotension. Conversely, lesions not reducing tonic inhibition do not produce hypotension [16]. There has been described an association between control of central pain and of circulation in hypertensive humans and rats [25, 26]. Thus, the PNMT-spinal pathway may function simultaneously in these two diverse systems.
138 W e w i s h to t h a n k P h y l l i s W a l d r o p f o r h e r efficient s e c r e t a r i a l a s s i s t a n c e . T h i s s t u d y w a s s u p p o r t e d by g r a n t s N S 09743 ( W . D . W . ) a n d p o s t d o c t o r a l f e l l o w s h i p s N S 07062 ( S . M . C . ) a n d N S 07574 ( C . N . H . ) . 1 Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution ofdopamine-, noradrenaline-, and adrenaline-containing cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes, J. Comp. Neurol., 212 (1982) 173 187. 2 Bowker, R.M., Westlund, K.N., Sullivan. M.C. and Coulter, J.D., A combined retrograde transport and immunocytochemical staining method for demonstrating the origins of 5-HT projections, J. Histochem. Cytochem., 30 (1982) 805 810. 3 Carlton, S.M,, Honda, (7.N,, Denoroy, L. and Willis, W.D., Brainstem and spinal cord mapping of PNMT-stained neurons, axons and terminals in the monkey, Soc. Neurosci. Abstr., 12 (1986) 144. 4 Carlton, S.M., Honda, C.N. and Willis, W.D., Identification of spinally projecting adrenaline neurons in the monkey medulla: a double labeling study using HRP and PNMT immunohistochemistry, Anat. Rec., 213 (1985) 195A. 5 Dampney, R.A.L. and Moon, E.A., The role of ventrolateral medulla in vasomotor response to cerebral ischemia. Am. J. Physiol., 239 (1980) H349 H358. 6 Denoroy, L., Utilisation de la Ph6nyl~thanolamine-N-m6thyltransfdrasecomme Marqueur des Structures Adr6nergiques. Application a l'l~tude de I'Hypertension Art6rielle, Th6se de Docteur-lng6nieur, Universitd Claude Bernard, Lyon, 1979. 7 Grillin, G., Watkins, L.R. and Mayer, D.J., HRP pellets and slow release gels: two new techniques for greater localization and sensitivity, Brain Res., 168 (1979) 595 601. 8 Hall, J.G., Duggan, A.W.. Morton, C.R. and Johnson, S.M., The location of brainstem neurones tonically inhibiting dorsal horn neurones of the cat, Brain Res., 244 (1982) 215- 222. 9 H6kfelt, T., Fuxe. K., Goldstein, M. and Johansson, O., lmmunohistochemical evidence of the existence of adrenaline neurons in the rat brain, Brain Res., 66 (1974) 235 251. 10 Joh, T.H. and Goldstein, M., Isolation and characterization of multiple forms of phenylethanolamineN-methyltransferase. MoI. Pharmacol., 9 (1973) 117 129. 11 Kalia, M., Fuxe, K. and Goldstein, M., Rat medulla oblongata. 11I. Adrenergic (CI and C2) neurons. nerve fibers and presumptive terminal processes, J. Comp. Neurol., 233 (1985) 333 349. 12 Kitahama. K., Denoroy, k,, Berod, A. and Jouvet, M., Distribution of PNMT-immunoreactive neurons in the cat medulla oblongata, Brain Res. Bull.. 17 (1986) 197 208. 13 Kitahama, K., Pearson, J., Denoroy. L., Kopp, N., Ulrich, J., Maeda, T. and Jouvet, M., Adrenergic neurons in human brain demonstrated by immunohistochemistry with antibodies to phenylethanolamine-N-methyl transferase (PNMT): discovery of a new group in the nucleus tractus solitarius, Neurosci. Left., 53 (1985) 303 308. 14 Kneisley, L.W., Biber, M.P. and LaVail, J.H., A study of the origin of brainstem projections to monkey spinal cord using the retrograde transport method, Exp. Neurol., 60 (1978) 116 139. 15 Mesulam, M.-M., Tetramethylbenzidine for HRP neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents, J. Histochem. Cytochem., 26 (1978) t06 117. 16 Morton, C.R., Johnson. S.M. and Duggan, A.W., Lateral reticular regions and the descending control of dorsal horn neurones of the cat: selective inhibition by electrical stimulation, Brain Res., 275 (1983) 13 21. 17 Robert, O., Miachon, S., Kopp, N., Denoroy, L., Tommasi, M., Rollet, D. and Pujol, J.F., tmmunohistochemical study of the catecholaminergic systems in the lower brain stem on the human infant, Human Neurobiol., 3 (1984) 229 234. 18 Ruggiero. D.A., Ross, C.A., Anwar, M., Park, D.H., Joh, T.J. and Reis, D.J. Distribution of neurons containing phenylethanolamine-N-methyltransferasein medulla and hypothalamus of rat, J. Comp. Neurol.,239(1985) 127 154. 19 Reiner, P.B. and Vincent, S.R.. The distribution of tyrosine hydroxylase, dopamine-fi-hydroxylaseand
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