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Localization of tyrosine hydroxylase and phenylethanolamine N-methyltransferase immunoreactive cells in the medulla of the dog
Neuroscience Letters, 107 (1989) 12 18 Elsevier Scientific Publishers Ireland Ltd.
12
NSL 06508
Localization of tyrosine hydroxylase and phenylethanolamine N-methyltransferase immunoreactive cells in the medulla of the dog G.A. I w a m o t o l, J.H. Mitchell 2'3, M. Sadeq 2 and G.P. Kozlowski 2 1Department of Veterinary Biosciences, College of Veterinary Medicine, University qf lllinois, Urbana, IL 61801 (U.S.A.) and 2Department o["Physiology and the ~Harry S. Moss Heart Center, University q[' Texas Southwestern Medical Center, Dallas TX 75235 (U.S.A.) (Received 21 April 1989; Revised version received 10 July 1989; Accepted 4 August 1989)
Key words': Catecholamine; Epinephrine: Tyrosine hydroxylase: Phenylethanotamine N-methyltransferase; Ventrolateral medulla: Dog The tyrosine hydroxytase (TH)- and phenylethanolamine N-methyltransferase (PNMT)-immunoreactive cells of the medulla are closely associated with cardiovascular control in both the cat and rat. Although it is often the species of choice for cardiovascular studies, no previous study had characterized these cell groups in the dog. The TH- and PNMT-immunoreactive cells of the dog were distributed much as they are in both cat and rat but with some species variations, which may be indicative of their functional role.
Ventrolateral medullary (VLM) cells containing tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) are thought to be involved with cardiovascular regulation [5, 8, 9, 11, 12, 16-19]. Principally through the work of Reis and colleagues, TH-containing cells of the caudal VLM (cVLM) are associated with depressor responses, while the PNMT containing cells of the rostral VLM (rVLM) are associated with pressor responses [9, 15, 17]. These functional areas correspond respectively to the caudal end of the A 1 catecholamine-containing area described by Dahlstr6m and Fuxe [7] and the C1 epinephrine area described by H6kfelt et al. (see ref. 16). We assume that although the TH-containing cells may synthesize a number of catecholamines, the PNMT-containing cells are able to synthesize epinephrine. A number of previous immunocytochemical (ICC) and physiological studies of these cell groups have been done in the rat and cat. However, the dog has been the animal of choice for much experimentation in cardiovascular control. Connelly et al. [6] recently showed that stimulation of the cVLM in the dog did not produce carCorrespondence." G.A. lwamoto, Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61801, U.S.A. 0304-3940/89/$ 03 50 ~:) 1989 Elsevier Scientific Publishers Ireland Ltd.
13
diovascular changes similar to those of the rat. Therefore, to facilitate species comparisons, we determined the positions of the TH and PNMT immunoreactive (IR) cell groups so that these data might be correlated with the physiological data available for the dog. Nine adult dogs, 10-21 kg, were anesthetized with sodium pentobarbital (35 mg/ kg, i.v.). The dogs were artificially ventilated while perfused through the left ventricle. After the descending aorta was clamped, heparinized Ringer's solution (150 ml/kg) was infused followed by cold (5-10°C) Zamboni's fixative (150 ml/kg) [10]. The head was surrounded with ice. The brain was quickly removed, cut into blocks, and postfixed for 6 h. Although it would seem preferable in view of the dog's brain size, we found that longer post-fixation (4 dogs) impaired PNMT immunoreactivity. The brain was then placed in 30% sucrose in 0.1 M phosphate buffer for 12 h prior to being cut into frozen transverse sections (50 gm). The sections were collected in 4°C 0.1 M phosphate buffer. Three of the dogs were pretreated with colchicine (100/Lg/ kg) (infused into the lateral ventricle) and left anesthetized for a period of 14-16. As colchicine made no difference in the data reported here for TH, it was discontinued. The ICC procedure used both biotinylated protein A and the Vectastain ABC (avidin-biotin-complex) kit [14]. The sections were placed in phosphate-buffered saline with 0.02% bovine serum albumin (PBS-BSA) for two washes of 10 min each at room temperature. They were then placed into 1% H202 in PBS-BSA for 30 min to eliminate endogenous peroxidase. Washes between steps consisted of PBS-BSA for two washes of 10 min each. The sections were then treated with a dilute detergent-0.1% Triton X-100 (Sigma) for 20 min, again washed, and incubated at 4°C overnight in primary antisera. Primary antisera to PNMT (lot no. 3012) and TH (lot no. 1012) were obtained commercially (Eugene Tech). The antisera were initially reconstituted by adding 1 ml of distilled water and finally diluted to 1:24 and 1:36, respectively, with phosphate buffer. (The supplier does not provide the actual concentration of the antisera). The sections were then washed and transferred to a 1:400 dilution of biotinylated protein A (Vector Labs.) for 45 min at room temperature. They were then washed and incubated in ABC complex diluted 1:1000 for 1 h. After a wash, the tissue was incubated in a mixture of diaminobenzidine (DAB, 15 mg%), fl-o-glucose (200 mg%), ammonium chloride (40 mg%), and glucose oxidase (0.3 mg%) dissolved in PBS buffer. The final reaction product was monitored under a dissecting microscope for 30-45 min on a shaker at room temperature. The sections were then mounted onto subbed slides, dehydrated, cleared, and coverslipped. One set of adjacent sections was stained with Cresyl violet to serve as reference for localizing cell groups. Examples of labelled cells found in the VLM are shown in Fig. 1A-D. The cells were fusiform or multipolar (15-35 gm in diameter) often with two or more branching processes. In some cases, the axons of these cells were clearly visible. The ventrolateral group of TH-IR cells corresponded to the A1 cell group as described for many species. This group was distributed from a level approximately 2 mm caudal to the obex continuing rostrally to the level of the retrofacial nucleus (Fig. 2A-F). Caudally, the cells were found both ventral and dorsal to the caudal lateral
14
Fig. 1. Examples of staining for TH and PNMT showing examples of ventrolateral cell groups. A: lowpower magnification of ventrolateral medulla stained for TH. B: higher power of boxed area indicated in A. (': lower-power magnification of VLM stained for PNMT. D: higher power of boxed area indicated in C. The predominant cell type is that of fusilk)rm neurons. reticular nucleus ( L R N ) . The cells a l o n g the ventral aspect o f the c a u d a l L R N were a d j a c e n t to the ventral surface. The rostral pole o f the L R N (which is d i v i d e d into parts as seen in transverse sections) h a d T H - I R cells i n t e r m i n g l e d within the various p o r t i o n s o f the nucleus. The d i s t r i b u t i o n o f these cells e x t e n d e d d o r s a l l y ( a l t h o u g h in reduced n u m b e r s ) into the lateral tegmental field. T h e r e were also some cells between the medial L R N a n d the lateral aspect o f the inferior olive at this level. T h e d o r s o m e d i a l T H - I R cell g r o u p c o r r e s p o n d i n g to the A2 area was located in an a r e a medial and d o r s o m e d i a l to the solitary tract. Some cells were close to the ventricular surface. This g r o u p extended from the level o f the obex to the rostral pole o f the inferior olive. These cells reside in the s o l i t a r y - c o m p l e x a n d a r e a p o s t r e m a . Similar to the TH-1R cells, the P N M T - I R cells were also f o u n d in v e n t r o l a t e r a l and d o r s o m e d i a l g r o u p s (Fig. 2A F). These cells were less n u m e r o u s than those described for the T H cell groups. The v e n t r o l a t e r a l P N M T - I R cells c o r r e s p o n d i n g to the CI area h a d the same rostral extent as the T H - c o n t a i n i n g cells. There were also fewer P N M T - I R cells f o u n d d o r s a l to the m a i n g r o u p o f v e n t r o l a t e r a l l y located cells c o m p a r e d to those labelled for T H . There were very few cells located a d j a c e n t to the inferior olive. T h e P N M T - I R g r o u p also differed f r o m that o f the T H - I R g r o u p at the level o f the c V L M . A t o r near the level o f the obex, no cells were f o u n d lining
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Fig. 2. Data from a representative animal showing location of TH- and PNMT-containing cells. Each figure shows the data from one representative section taken at the level indicated. The wide variation in dog brain size which varied only approximately with body weight makes it difficult to express 'typical" data. Thus, the size of the brain sections while specific for one dog is provided only to give a general frame of reference. The length of the medulla in these dogs varied between 14 and 18 mm. The sections shown were taken at intervals of approximately 2.5 mm. Abbreviations are adapted from Berman's [2] terminology for the cat: 5SP, spinal trigeminal n.; 12, hypoglossal n.; CE, central canal; CUC, cuneate n.; CX, external cuneate n.; GRR, gracile n.; IOD, dorsal accessory inferior olive; IOM, medial accessory inferior olive; lOP, principal n. of inferior olive; CI, n. centralis inferior; LRN, lateral reticular n.; P, pyramidal tract; PH, n. praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; S, solitary tract.
the ventrolateral border and few cells lined the dorsal border of the LRN. At levels caudal to the obex, no P N M T - I R cells were found. As previously mentioned, the cVLM area had a large number of T H - I R cells in the region just dorsal to the LRN. The dorsomedial P N M T - I R cell group corresponding to the C2 area was very limited compared to the T H - I R group. There were only a few cells which were just medial to the solitary tract at the level of the rostral pole of the L R N , the same level where the inferior olive appears to be most prominent. There were no immunopositive cells
16
in the area postrema or the solitary complex, but there were many P N M T - I R fibers in this region. This report provides the first survey of T H - I R and P N M T - I R cells of the dog. Yhese cell groups and analogous ones in the cat and rat may be compared both in terms of structure and functional correlates. T H - I R cells of the dog were distributed in a manner which resembled that of the A I and A2 areas of the cat, rat and dog as determined by histofluorescence techniques [3, 7]. Their distribution is also very similar to that of T H - I R cells for the rat [9, 16] and DBH (dopamine fl-hydroxylase) T H - I R cells for the cat [5]. There was no evidence of the A3 area described for the rat within the dorsal accessory inferior olive on the basis of histofluorescence techniques [7]. However, a T H - I R cell group was observed around the intramedullary roots of the hypoglossal nerve which was described by Ciriello et al. [5] as D B H - I R in the cat and may likely be analogous to A3 in the rat. The distribution of P N M T - I R cells of the dog appeared to be similar to that found in the cat [5, 12, 16] and rat [9, 16-18], but with some species differences. Similar to the rat, but not the cat, the dog's P N M T - I R cells appear to be arranged in a more column-like manner, with few cells found dorsomediaily in the lateral tegmental field. Like the rat, the dog does not have a large population of P N M T - I R cells on the ventrolateral border of the cVLM, as does the cat. There are no P N M T - I R cells caudal to the obex in the dog, either in the area just dorsal to the L R N or near the ventral margin of LRN. This observation is apparently unlike those made for the cat by Ruggiero et al. [19] who found P N M T - I R cells in these areas. However, Ciriello et al. [5] data appear to indicate otherwise. In the case of the rat, studies have indicated that there are some P N M T - I R cells caudal to the obex but none caudal to the area postrema [9, 18]. The paucity of P N M T - I R cells in the area equivalent to C2 in the dog is an enigma. The ICC procedures are not likely to have been faulty since P N M T - I R cells were very clearly labelled in the VLM. This result is clearly different than that observed by Ruggiero et al., for both the cat [19] and rat [9, 18] in which the C2 areas are much more extensive both in rostro-caudai distribution and overall numbers. However, the recent study in the dog by Barnes et al. [3] using the glyoxylic acid histofluorescence technique appears to support this observation. Our overall impression of the distribution of these cell groups is that the dog most closely resembles the rat rather than the cat. T H - I R and P N M T - I R cells of the V L M may be involved with the cardiovascular regulation with T H cells of the cVLM associated with depressor responses and P N M T cells of the rVLM associated with pressor responses. A population of P N M T IR cells of the rat rVLM has been shown to project to the spinal cord intermediolateral cell column (IML) [14]. The rVLM in and near these rat P N M T - I R cells has been shown to be sympathoexcitatory, necessary for maintenance of resting blood pressure [16, 20], and seems to be responsible for the effects observed on stimulation of Schlaefke's area [16, 19]. The T H - I R cells are identified with areas that are apparently sympathoinhibitory [9, 16]. Observations in the cat on the rVLM areas in and near the PNMT-containing cells have indicated much the same results with electrical
17 and chemical stimulation evoking a pressor response (see ref. 5). Cells in the area project to the I M L [I, 5] and are likely to contribute to tonic rhythmogenesis o f sympathetic outflow [1]. In an early report, M c Q u e e n et al. [12] identified the d o g ' s medullary lateral reticular formation as a pressor region, m u c h as one would expect based on the foregoing. Unfortunately, the exact location o f their stimulus sites is uncertain. However, a recent report by D o r m e r and colleagues [8] left no d o u b t that the r V L M is a sympathoexcitatory site in the dog and that stimulus sites near P N M T I R cells could yield pressor effects. The position o f the T H - I R cells not containing P N M T can also be evaluated. Connelly et al. [6] found that, unlike the cat or rat, no apparent cardiovascular responses could be obtained with chemical stimulation o f the c V L M o f the dog, but decreases in lung resistance were observed. One might expect, based on w o r k in other species, that depressor responses would result. Thus, one cannot be assured that T H - I R cells which presumably do not contain P N M T are indicative o f a particular function in the c V L M . While it is important to recognize that the functional roles o f these cell groups remain controversial [20], one cannot help but note that there are also some similarities across species in the position o f these cell groups and the cardiovascular responses evoked on their stimulation. If the P N M T - I R cells o f V L M do not contain the actual neurotransmitter involved with the process o f sympathoexcitation, it must be conceded that their presence highly correlates with medullary pressor sites. M a n y other neurotransmitters are t h o u g h t to be present in this area (based on data from the cat [4, 12]) and while a controversial issue [15] some might be co-localized [11] with T H and P N M T . Finally, if P N M T - I R cells are not sympathoexcitatory, considering the evidence accumulated thus far, one must ask the question: what is their role, if any, in circulatory control? These observations await further elucidation. This research was supported by N I H Grants H L 37400, K04 H L 01910, and A A 06014.
1 Barman, S.M. and Gebber, G.L., Axonal projection patterns of ventrolateral medullospinal sympathoexcitatory neurons, J. Neurophysiol., 53 (1985) 1551 1556. 2 Berman, A.L., The Brain Stem of the Cat, University of Wisconsin Press Madison, WI, 1968. 3 Barnes, K., Chernicky, C.L., Block, C.H. and Ferrario, C.M., Distribution of catecholaminergic neuronal systems in the canine medulla oblongata and pons, J. Comp. Neurol., 274 (1988) 127-141. 4 Ciriello, J., Caverson, M.M., Calaresu, F.R. and Krukoff, T.L., Neuropeptide and serotonin immunoreactive neurons in the cat ventrolateral medulla, Brain Res., 440 (1988) 53-66. 5 Ciriello, J., Caverson, M.M. and Park, D.H., Immunohistochemical identification of noradrenalineand adrenaline synthesizing neurons in the cat ventrolateral medulla, J. Comp. Neurol., 253 (1986) 216-230. 6 Connelly, J.C., McCallister, L.W. and Kaufman, M.P., Stimulation of the caudal ventrolateral medulla decreases total lung resistance in dogs, J. Appl. Physiol., 63(3) (1987) 912-917. 7 Dahlstrom, A. and Fuxe, K., Evidence for the existence of monoamine containing cells in the central nervous system. I. Demonstration of monoamines in the cell bodies of brain stem neurons, Acta Physiol. Scand., 62 Suppl. 232 (1964) 1-55. 8 Dormer, K.J., Ruggiero, D.A., Anwar, M. and Ashlock, S.R., Functional mapping of the canine ros-
L~
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I1
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tral ventrolateral medulla with L-glutamate and phenylethanolamine n-methyltransferase (PNMT) immunohistochemistry, Soc. Neurosci. Abstr., 14 (1988) 191. Granata, A.R., Ruggiero, D.A., Park, D.H., Joh, T.H. and Reis, D.J., Brain stem area with epinephrine neurons mediates baroreflex vasodepressor responses, Am. J. Physiol. (Heart Circ. Physiol., 17), 248 (1985) 547 567. Kozlowski, G.P., and Nilaver, G., lmmunoelectronmicroscopy of neuropeptides: Theoretical and technical considerations. In J.L. Barker and J.F. McKelvey (Eds.), Current Methods in Cellular Neurobiology, Wiley, New York, 1983, pp. 134 174. Lorenz, R,G., Saper, C.B., Wong, D.L., Ciaranello, R.B. and Loewy, A.D., Co-localization of substance P and phenylethanolamine n-methyltransferase like immunoreactivity in neurons of ventrolateral medulla that project to the spinal cord: Potential role in vasomotor tone, Neurosci. Lett., 55 (1985) 255 260. Marson, L. and Loewy, A.D., Topographical organization of substance P and monoamine cells in the ventral medulla of the cat, J. Autonom. Nerv. Syst., 14 (1985) 271 285. McQueen, J.D., Brownne, K.M. and Walker, A.E,, Role of the brainstem in blood pressure regulation in the dog, Neurology, 4 (1954) I 13. Nilaver, G. and Kozlowski, G.P.. Comparison ol" the PAP versus the ABC immunocytochemical technique. In G.R. Bullock and P. Petrusz (Eds.), Techniques in Immunocytochemistry, Vol. 4, Academic Press, New York, in press, Pilowsky, P., Minson. J., Hodgson, A., Howe, P. and Chalmers, J., Does substance P co-exist with adrenaline m neurons of the rostral ventrolateral medulla in the rat'?, Neurosci. Lett., 71 (1986) 293 298. Reis. D.J., Granata, A.R., Joh, T.H., Ross, C.A., Ruggiero, D.A. and Park, D.A., Brain stem catecholamine mechanisms in tonic and reflex control of blood pressure, Hypertension, 6 Suppl. (1984) 117 115. Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Fernandez-Pardal, J., Saavedra, J.M. and Reis, D.J., Tonic vasomotor control by the rostral ventrolateral medulla: effect of electrical or chemical stimulation of the area containing CI adrenaline neurons on arterial blood pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (2) (1984) 474 494. Ruggiero, D.A., Ross, C.A., Anwar, M., Park, D.H., Joh, T.H. and Reis, D.J, Distribution of neurons containing phenylethanolamine N-methyltransferase in medulla and hypothalamus of rat, J. Comp. Neurol., 239 (1985) 127 154. Ruggiero, D.A., Gatti, P., Gillis, R.A., Norman, W.P., Anwar, M. and Reis, D.J., Adrenaline-synthesizing neurons in the medulla of the cat, J. Comp. Neurol., 252 (1986) 532 542. Sun, M.-K., Young, B.S.. Hackett, J.T. and Guyenet, P.G., Rostral ventrolateral medullary neurons with intrinsic pacemaker properties are not catecholaminergic, Brain Res., 451 (1988) 345 349.
12
NSL 06508
Localization of tyrosine hydroxylase and phenylethanolamine N-methyltransferase immunoreactive cells in the medulla of the dog G.A. I w a m o t o l, J.H. Mitchell 2'3, M. Sadeq 2 and G.P. Kozlowski 2 1Department of Veterinary Biosciences, College of Veterinary Medicine, University qf lllinois, Urbana, IL 61801 (U.S.A.) and 2Department o["Physiology and the ~Harry S. Moss Heart Center, University q[' Texas Southwestern Medical Center, Dallas TX 75235 (U.S.A.) (Received 21 April 1989; Revised version received 10 July 1989; Accepted 4 August 1989)
Key words': Catecholamine; Epinephrine: Tyrosine hydroxylase: Phenylethanotamine N-methyltransferase; Ventrolateral medulla: Dog The tyrosine hydroxytase (TH)- and phenylethanolamine N-methyltransferase (PNMT)-immunoreactive cells of the medulla are closely associated with cardiovascular control in both the cat and rat. Although it is often the species of choice for cardiovascular studies, no previous study had characterized these cell groups in the dog. The TH- and PNMT-immunoreactive cells of the dog were distributed much as they are in both cat and rat but with some species variations, which may be indicative of their functional role.
Ventrolateral medullary (VLM) cells containing tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT) are thought to be involved with cardiovascular regulation [5, 8, 9, 11, 12, 16-19]. Principally through the work of Reis and colleagues, TH-containing cells of the caudal VLM (cVLM) are associated with depressor responses, while the PNMT containing cells of the rostral VLM (rVLM) are associated with pressor responses [9, 15, 17]. These functional areas correspond respectively to the caudal end of the A 1 catecholamine-containing area described by Dahlstr6m and Fuxe [7] and the C1 epinephrine area described by H6kfelt et al. (see ref. 16). We assume that although the TH-containing cells may synthesize a number of catecholamines, the PNMT-containing cells are able to synthesize epinephrine. A number of previous immunocytochemical (ICC) and physiological studies of these cell groups have been done in the rat and cat. However, the dog has been the animal of choice for much experimentation in cardiovascular control. Connelly et al. [6] recently showed that stimulation of the cVLM in the dog did not produce carCorrespondence." G.A. lwamoto, Department of Veterinary Biosciences, College of Veterinary Medicine, University of Illinois, 2001 South Lincoln Ave., Urbana, IL 61801, U.S.A. 0304-3940/89/$ 03 50 ~:) 1989 Elsevier Scientific Publishers Ireland Ltd.
13
diovascular changes similar to those of the rat. Therefore, to facilitate species comparisons, we determined the positions of the TH and PNMT immunoreactive (IR) cell groups so that these data might be correlated with the physiological data available for the dog. Nine adult dogs, 10-21 kg, were anesthetized with sodium pentobarbital (35 mg/ kg, i.v.). The dogs were artificially ventilated while perfused through the left ventricle. After the descending aorta was clamped, heparinized Ringer's solution (150 ml/kg) was infused followed by cold (5-10°C) Zamboni's fixative (150 ml/kg) [10]. The head was surrounded with ice. The brain was quickly removed, cut into blocks, and postfixed for 6 h. Although it would seem preferable in view of the dog's brain size, we found that longer post-fixation (4 dogs) impaired PNMT immunoreactivity. The brain was then placed in 30% sucrose in 0.1 M phosphate buffer for 12 h prior to being cut into frozen transverse sections (50 gm). The sections were collected in 4°C 0.1 M phosphate buffer. Three of the dogs were pretreated with colchicine (100/Lg/ kg) (infused into the lateral ventricle) and left anesthetized for a period of 14-16. As colchicine made no difference in the data reported here for TH, it was discontinued. The ICC procedure used both biotinylated protein A and the Vectastain ABC (avidin-biotin-complex) kit [14]. The sections were placed in phosphate-buffered saline with 0.02% bovine serum albumin (PBS-BSA) for two washes of 10 min each at room temperature. They were then placed into 1% H202 in PBS-BSA for 30 min to eliminate endogenous peroxidase. Washes between steps consisted of PBS-BSA for two washes of 10 min each. The sections were then treated with a dilute detergent-0.1% Triton X-100 (Sigma) for 20 min, again washed, and incubated at 4°C overnight in primary antisera. Primary antisera to PNMT (lot no. 3012) and TH (lot no. 1012) were obtained commercially (Eugene Tech). The antisera were initially reconstituted by adding 1 ml of distilled water and finally diluted to 1:24 and 1:36, respectively, with phosphate buffer. (The supplier does not provide the actual concentration of the antisera). The sections were then washed and transferred to a 1:400 dilution of biotinylated protein A (Vector Labs.) for 45 min at room temperature. They were then washed and incubated in ABC complex diluted 1:1000 for 1 h. After a wash, the tissue was incubated in a mixture of diaminobenzidine (DAB, 15 mg%), fl-o-glucose (200 mg%), ammonium chloride (40 mg%), and glucose oxidase (0.3 mg%) dissolved in PBS buffer. The final reaction product was monitored under a dissecting microscope for 30-45 min on a shaker at room temperature. The sections were then mounted onto subbed slides, dehydrated, cleared, and coverslipped. One set of adjacent sections was stained with Cresyl violet to serve as reference for localizing cell groups. Examples of labelled cells found in the VLM are shown in Fig. 1A-D. The cells were fusiform or multipolar (15-35 gm in diameter) often with two or more branching processes. In some cases, the axons of these cells were clearly visible. The ventrolateral group of TH-IR cells corresponded to the A1 cell group as described for many species. This group was distributed from a level approximately 2 mm caudal to the obex continuing rostrally to the level of the retrofacial nucleus (Fig. 2A-F). Caudally, the cells were found both ventral and dorsal to the caudal lateral
14
Fig. 1. Examples of staining for TH and PNMT showing examples of ventrolateral cell groups. A: lowpower magnification of ventrolateral medulla stained for TH. B: higher power of boxed area indicated in A. (': lower-power magnification of VLM stained for PNMT. D: higher power of boxed area indicated in C. The predominant cell type is that of fusilk)rm neurons. reticular nucleus ( L R N ) . The cells a l o n g the ventral aspect o f the c a u d a l L R N were a d j a c e n t to the ventral surface. The rostral pole o f the L R N (which is d i v i d e d into parts as seen in transverse sections) h a d T H - I R cells i n t e r m i n g l e d within the various p o r t i o n s o f the nucleus. The d i s t r i b u t i o n o f these cells e x t e n d e d d o r s a l l y ( a l t h o u g h in reduced n u m b e r s ) into the lateral tegmental field. T h e r e were also some cells between the medial L R N a n d the lateral aspect o f the inferior olive at this level. T h e d o r s o m e d i a l T H - I R cell g r o u p c o r r e s p o n d i n g to the A2 area was located in an a r e a medial and d o r s o m e d i a l to the solitary tract. Some cells were close to the ventricular surface. This g r o u p extended from the level o f the obex to the rostral pole o f the inferior olive. These cells reside in the s o l i t a r y - c o m p l e x a n d a r e a p o s t r e m a . Similar to the TH-1R cells, the P N M T - I R cells were also f o u n d in v e n t r o l a t e r a l and d o r s o m e d i a l g r o u p s (Fig. 2A F). These cells were less n u m e r o u s than those described for the T H cell groups. The v e n t r o l a t e r a l P N M T - I R cells c o r r e s p o n d i n g to the CI area h a d the same rostral extent as the T H - c o n t a i n i n g cells. There were also fewer P N M T - I R cells f o u n d d o r s a l to the m a i n g r o u p o f v e n t r o l a t e r a l l y located cells c o m p a r e d to those labelled for T H . There were very few cells located a d j a c e n t to the inferior olive. T h e P N M T - I R g r o u p also differed f r o m that o f the T H - I R g r o u p at the level o f the c V L M . A t o r near the level o f the obex, no cells were f o u n d lining
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TH
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Fig. 2. Data from a representative animal showing location of TH- and PNMT-containing cells. Each figure shows the data from one representative section taken at the level indicated. The wide variation in dog brain size which varied only approximately with body weight makes it difficult to express 'typical" data. Thus, the size of the brain sections while specific for one dog is provided only to give a general frame of reference. The length of the medulla in these dogs varied between 14 and 18 mm. The sections shown were taken at intervals of approximately 2.5 mm. Abbreviations are adapted from Berman's [2] terminology for the cat: 5SP, spinal trigeminal n.; 12, hypoglossal n.; CE, central canal; CUC, cuneate n.; CX, external cuneate n.; GRR, gracile n.; IOD, dorsal accessory inferior olive; IOM, medial accessory inferior olive; lOP, principal n. of inferior olive; CI, n. centralis inferior; LRN, lateral reticular n.; P, pyramidal tract; PH, n. praepositus hypoglossi; RB, restiform body; RFN, retrofacial nucleus; S, solitary tract.
the ventrolateral border and few cells lined the dorsal border of the LRN. At levels caudal to the obex, no P N M T - I R cells were found. As previously mentioned, the cVLM area had a large number of T H - I R cells in the region just dorsal to the LRN. The dorsomedial P N M T - I R cell group corresponding to the C2 area was very limited compared to the T H - I R group. There were only a few cells which were just medial to the solitary tract at the level of the rostral pole of the L R N , the same level where the inferior olive appears to be most prominent. There were no immunopositive cells
16
in the area postrema or the solitary complex, but there were many P N M T - I R fibers in this region. This report provides the first survey of T H - I R and P N M T - I R cells of the dog. Yhese cell groups and analogous ones in the cat and rat may be compared both in terms of structure and functional correlates. T H - I R cells of the dog were distributed in a manner which resembled that of the A I and A2 areas of the cat, rat and dog as determined by histofluorescence techniques [3, 7]. Their distribution is also very similar to that of T H - I R cells for the rat [9, 16] and DBH (dopamine fl-hydroxylase) T H - I R cells for the cat [5]. There was no evidence of the A3 area described for the rat within the dorsal accessory inferior olive on the basis of histofluorescence techniques [7]. However, a T H - I R cell group was observed around the intramedullary roots of the hypoglossal nerve which was described by Ciriello et al. [5] as D B H - I R in the cat and may likely be analogous to A3 in the rat. The distribution of P N M T - I R cells of the dog appeared to be similar to that found in the cat [5, 12, 16] and rat [9, 16-18], but with some species differences. Similar to the rat, but not the cat, the dog's P N M T - I R cells appear to be arranged in a more column-like manner, with few cells found dorsomediaily in the lateral tegmental field. Like the rat, the dog does not have a large population of P N M T - I R cells on the ventrolateral border of the cVLM, as does the cat. There are no P N M T - I R cells caudal to the obex in the dog, either in the area just dorsal to the L R N or near the ventral margin of LRN. This observation is apparently unlike those made for the cat by Ruggiero et al. [19] who found P N M T - I R cells in these areas. However, Ciriello et al. [5] data appear to indicate otherwise. In the case of the rat, studies have indicated that there are some P N M T - I R cells caudal to the obex but none caudal to the area postrema [9, 18]. The paucity of P N M T - I R cells in the area equivalent to C2 in the dog is an enigma. The ICC procedures are not likely to have been faulty since P N M T - I R cells were very clearly labelled in the VLM. This result is clearly different than that observed by Ruggiero et al., for both the cat [19] and rat [9, 18] in which the C2 areas are much more extensive both in rostro-caudai distribution and overall numbers. However, the recent study in the dog by Barnes et al. [3] using the glyoxylic acid histofluorescence technique appears to support this observation. Our overall impression of the distribution of these cell groups is that the dog most closely resembles the rat rather than the cat. T H - I R and P N M T - I R cells of the V L M may be involved with the cardiovascular regulation with T H cells of the cVLM associated with depressor responses and P N M T cells of the rVLM associated with pressor responses. A population of P N M T IR cells of the rat rVLM has been shown to project to the spinal cord intermediolateral cell column (IML) [14]. The rVLM in and near these rat P N M T - I R cells has been shown to be sympathoexcitatory, necessary for maintenance of resting blood pressure [16, 20], and seems to be responsible for the effects observed on stimulation of Schlaefke's area [16, 19]. The T H - I R cells are identified with areas that are apparently sympathoinhibitory [9, 16]. Observations in the cat on the rVLM areas in and near the PNMT-containing cells have indicated much the same results with electrical
17 and chemical stimulation evoking a pressor response (see ref. 5). Cells in the area project to the I M L [I, 5] and are likely to contribute to tonic rhythmogenesis o f sympathetic outflow [1]. In an early report, M c Q u e e n et al. [12] identified the d o g ' s medullary lateral reticular formation as a pressor region, m u c h as one would expect based on the foregoing. Unfortunately, the exact location o f their stimulus sites is uncertain. However, a recent report by D o r m e r and colleagues [8] left no d o u b t that the r V L M is a sympathoexcitatory site in the dog and that stimulus sites near P N M T I R cells could yield pressor effects. The position o f the T H - I R cells not containing P N M T can also be evaluated. Connelly et al. [6] found that, unlike the cat or rat, no apparent cardiovascular responses could be obtained with chemical stimulation o f the c V L M o f the dog, but decreases in lung resistance were observed. One might expect, based on w o r k in other species, that depressor responses would result. Thus, one cannot be assured that T H - I R cells which presumably do not contain P N M T are indicative o f a particular function in the c V L M . While it is important to recognize that the functional roles o f these cell groups remain controversial [20], one cannot help but note that there are also some similarities across species in the position o f these cell groups and the cardiovascular responses evoked on their stimulation. If the P N M T - I R cells o f V L M do not contain the actual neurotransmitter involved with the process o f sympathoexcitation, it must be conceded that their presence highly correlates with medullary pressor sites. M a n y other neurotransmitters are t h o u g h t to be present in this area (based on data from the cat [4, 12]) and while a controversial issue [15] some might be co-localized [11] with T H and P N M T . Finally, if P N M T - I R cells are not sympathoexcitatory, considering the evidence accumulated thus far, one must ask the question: what is their role, if any, in circulatory control? These observations await further elucidation. This research was supported by N I H Grants H L 37400, K04 H L 01910, and A A 06014.
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