Neuroscience Letters, 122 (1991)91-95
91
ElsevierScientificPublishers Ireland Ltd. NSL 07471
Projections from the rostral ventrolateral medulla to brainstem monoamine neurons in the rat A n t h o n y P. N i c h o l a s a n d M i c h a e l B. H a n c o c k Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received31 August 1990;Revisedversionreceived4 October 1990;Accepted8 October 1990) Key words: Rostralventrolateralmedulla; Brainstem pathway; Phaseolus vulgaris leucoagglutinin;Immunocytochemistry
Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral ventrolateral medulla (RVL), two-color immunoperoxidase staining was employedto demonstrate contiguity between PHA-L-immunoreactive(PHA-LI) varicosefibers and boutons and brainstem monoaminergicceils. Black-stainedPHA-LI ceils in the deposition site were found to be located among amber-stained phenylethanolamine N-methyl transferase-immunoreactive(PNMT-I) neurons of the C1 cell group. RVL projections were contiguous with PNMT-I neurons of the C 1, C2 and C3 cell groups, with tyrosine hydroxylase-immunoreactive(TH-I) neurons of the A1, A2 and A5 cell groups, and with serotoninimmunoreactive(5-HT-I)neurons of the B1, B2 and B3 cellgroups. Preliminaryfindings of this study have been presentedpreviously(Sot. Neurosci. Abstr., 15 (1989)451).
The adrenergic C1 cell group first described by H6kfelt et al. [9] is located in the rostral ventrolateral medulla (RVL), a well-known pressor region [23]. These adrenergic neurons have been shown to project to cardiovascular-regulatory centers in the spinal cord [7, 19, 20, 22, 26] and hypothalamus [18, 26], but local projections of these cells, especially to monoaminergic neurons in the brainstem that also are thought to be involved with blood pressure regulation [2, 8, 15, 16, 21, 23], have not been well documented. Efferent projections from the C 1 area in RVL have been studied previously using the anterograde transport of tritiated amino acids [14, 22] and have been corroborated by the transport of retrograde tracers from selected sites of termination in the rat [7, 14, 18, 19, 22, 24, 26]. However, in animals in which anterogradely-transported tritiated amino acids were injected into the RVL, the detailed anatomic relationships between radiolabeled terminals of RVL neurons and histochemically-identified brainstem monoaminergic cells were not analyzed. In the present study, projections to monoamine cell groups in the pons and medulla that arise from neurons in the RVL were examined using the anterograde tracer
Requests for reprints: M.B. Hancock, Department of Anatomy and Neurosciences, The University of Texas Medical Branch, 200 University Blvd., Suite 730, Galveston,TX 77550,U.S.A. Correspondence: A. Nicholas, Karolinska Institute, Department of Histology and Neurobiology, Box 60400, S-10401 Stockholm, Sweden. 0304-3940/91/$03.50 © 1991 ElsevierScientificPublishers Ireland Ltd.
vulgaris leucoagglutinin (PHA-L, Vector Labs). PHA-L was iontophoretically deposited in the RVL of 5 male Sprague-Dawley rats using a modified version [17] of the technique originally described by Gerfen and Sawchenko [4]. Briefly, animals were deeply anesthetized with intraperitoneal injections of sodium pentobarbital (37.5 mg/kg) and urethane (900 mg/kg) in Ringer's solution. Atropine (0.054).1 mg) in Ringer's solution was injected subcutaneously to reduce pulmonary secretions. After placing an animal in a stereotaxic frame, the head was tilted downward 33 °, and the dorsal medulla was exposed by surgically removing the atlantooccipital membrane. Fiber-filled glass micropipettes with outside tip diameters of 5-8/~m were filled with a 2.5% solution of PHA-L in 0.1 M phosphate-buffered saline (pH 7.4). After being clamped to a micromanipulator and angled rostrally 12°, the PHA-L-filled micropipette then was positioned with its tip at the calamus scriptorius, moved laterally 2.0 mm, and lowered into the medulla to a depth of 3.5-3.8 mm. Iontophoresis of PHA-L was performed using electrical current of 3-5/~A passed in 7 s pulses with a 50% duty cycle for 15-20 min. Then the wound was closed and penicillin G (70,000 U) was injected intramuscularily. Following survival periods of 4-8 days, the animals were deeply anesthetized as before and perfused transcardially with Ringer's solution at room temperature, then with 500 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain and spinal cord were postfixed overnight in the same fixative, then serial, 30/tm coronal sections Phaseolus
92 of this material were cut on a Vibratome and stored in multiwell dishes filled with phosphate buffer. The details of the two-color immunoperoxidase staining technique used in the present study have been described previously [6]. Briefly, coronal sections of medulla, caudal pons, hypothalamus and spinal cord were incubated overnight in rabbit anti-PHA-L (1:5,000; D A K O ) and stained with a sequence of peroxidase-antiperoxidase (PAP) immunoreagents and nickel-intensified diaminobenzidine. PHA-L-immunoreactive (PHALI) cells and processes stained black. Since the transition zones between rostral adrenergic (C1, C2) and caudal noradrenergic (A l, A2) cell columns have been localized to the level of the area postrema (AP) [1, 11], medullary sections located rostral and caudal to the AP were incubated in rabbit antisera raised against phenylethanolamine N-methyl transferase (PNMT; 1:500; Eugene Tech) and tyrosine hydroxylase (TH; l:l,000; Eugene
Tech), respectively. Other medullary sections were incubated in rabbit antiserum raised against serotonin (5HT; 1:20,000; I m m u n o Nuclear). Medullary P N M T immunoreactive (PNMT-I), TH-immunoreactive (TH-I) or 5-HT-immunoreactive (5-HT-I) cells then were stained amber using a second sequence of PAP reagents and diaminobenzidine (DAB) alone. The affinity of the PHA-L, P N M T , and 5-HT antisera for their respective antigens was checked by solid-phase immunoassay [12], and the results have been described previously [5, 19]. The antiserum to T H could not be checked by solid-phase assay, since the antigen was not available. However, the distribution of cells immunostained with T H antiserum in the present study is similar to that reported by other investigators [1, 11]. Unilateral P H A - L deposition sites were centered in the RVL at the level of the C1 cell group (Fig. 1A). In dual-stained sections, the location of the black-stained
Fig. 1. Example of a representative Phaseolus vulgaris leucoagglutinin (PHA-L) deposition site in a dual-stained section of the rostral ventrolateral medulla (RVL), showing black-stained (nickel-intensified DAB) PHA-L-immunoreactive(PHA-LI) neurons which totally obscure any lighterstained (DAB alone) phenylethanolamine N-methyl transferase-immunoreactive(PNMT-I) neurons of the adrenergic C1 cell group (A). Blackstained PHA-LI fibers and boutons (arrows) in contiguity with lighter-stained PNMT-I CI neurons found contralateral (B, C) or ipsilateral (D) to the PHA-L deposition site. PHA-LI fibers and boutons originating from neurons in the RVL also were contiguous with PNMT-I C2 neurons in the nucleus tractus solitarius (E-F), and C3 neurons in the dorsomedialmedulla near the dorsal medullary surface (G). IV, fourth ventricle. Dashed line = 1.0 mm, bars = 20/zm.
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PHA-LI neurons exactly mirrored the location of amber-stained PNMT-I C1 neurons on the contralateral side of the medulla, and only a few PNMT-I cells occa: sionally could be seen intermingled among PHA-LI neurons in the deposition site. In the majority Of medullary sections at the level of the PHA-L deposition site, the black PHA-L staining in RVL obscured amber-stained PNMT-I cells and processes. Varicose PHA-LI fibers originating from cells in the RVL were observed in contiguity with PNMT-I neurons of the C1 cell group that were located rostral, caudal, ipsilateral and contralateral to the deposition site (Fig. 1B-D). PHA-LI terminal fields in the rostral C1 area were more prominant on the ipsilateral side, but the contralateral C1 area also received a heavy PHA-LI projection. PHA-LI boutons also were seen in contiguity with PNMT-I neurons of the C2 cell group in the nucleus tractus solitarii (Fig. 1E-F) and of the C3 cell group in the dorsomedial medulla (Fig. 1G). In regions of the caudal medulla (caudal to AP), PHA-LI fibers and boutons were seen in contiguity with TH-I neurons of the A1 (Fig. 2A, B) and A2 (Fig. 2C, D) cell groups. In more rostral regions, PHA-LI boutons were seen in contiguity with TH-I neurons in the A5 cell group in the pons (Fig. 2E). Medullary sero-
tonergic nuclei also seem to receive an input from the RVL, since PHA-LI fibers and boutons were observed in contiguity with 5-HT-I neurons of the B 1, B2 and B3 cell groups, which include nucleus raphe pallidus (Fig. 3A), nucleus interfasicularis hypoglossi (Fig. 3B), nucleus raphe obscurus (Fig. 3C, D), nucleus raphe magnus (Fig. 3E) and nucleus reticularis paragigantocellularis (Fig. 3F). PHA-LI terminal arborizations were also observed in the intermediolateral cell column and intermediate grey matter of the rostral thoracic spinal cord, and in the supraoptic and paraventricular nuclei of the hypothalamus. Using the anterograde transport of PHA-L and twocolor immunoperoxidase staining, the results of the present study indicate that the RVL, an important medullary vasopressor region [23], contains neurons with extensive local projections to adrenergic, noradrenergic and serotonergic cell groups in the brainstem. Therefore, the effects on sympathetic vasomotor tone elicited by RVL stimulation could be mediated by direct projections from RVL to the spinal cord, or indirectly by RVL projections to local brainstem monoaminergic neurons, many of which are known to project to the spinal cord [3, 7, 13, 14, 19, 22, 26] or hypothalamus [18, 25, 26]. Since the
Fig. 2. Black-stained PHA-LI boutons and varicose fibers (arrows) originating from the deposition site shown in Fig. 1A and in contiguity with lighter-stained, noradrenergic, tyrosine hydroxylase-immunoreactive (TH-I) cells and dendrites of the A1 cell group in the caudal ventrolateral medulla (A, B) of the A2 cell group in the nucleus commissuralis (C, D), and of the A5 cell group in the ventral pons (E). cc, central canal. Bars = 20 ,um.
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Fig. 3. Black-stained PHA-LI fibers and boutons (arrows) originating from the deposition site shown in Fig. 1A and in contiguity with lighter-stained serotonin-immunoreactive(5-HT-I) neurons of the B I (A, B), B2 (C, D) and B3 (E, F) cell groups in nucleus raphe pallidus (A), nucleus interfasicularis hypoglossi (B), nucleus raphe obscurus (C, D), nucleus raphe magnus (E), and nucleus reticularis paragigantocellularis (F), respectively. XII, hypoglossal nerve rootlet. Bars = 20/~m.
P H A - L I d e p o s i t s were l o c a t e d w i t h i n the C1 area, a n d since the P H A - L I t e r m i n a l fields i n the p r e s e n t s t u d y are in the s a m e l o c a t i o n s as w e l l - d o c u m e n t e d P N M T - I term i n a l a r b o r i z a t i o n s [9, 10], it is likely t h a t s o m e o f these PHA-LI projections include adrenergic pathways from C 1 n e u r o n s . T h i s will, h o w e v e r , h a v e to be verified b y future e x p e r i m e n t s , w h e r e the t r a n s m i t t e r i d e n t i t y o f P H A L - l a b e l e d fibers is e s t a b l i s h e d u s i n g i m m u n o h i s t o c h e m i cal d o u b l e (or triple) s t a i n i n g m e t h o d s . T h e a u t h o r s wish to t h a n k Ms. P. T h a t c h e r for t e c h n i cal assistance. T h i s w o r k was s u p p o r t e d b y the T e x a s Affiliate o f the A m e r i c a n H e a r t A s s o c i a t i o n . 1 Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution of dopamine-, noradrenaline- and adrenalinecontaining cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes. J. Comp. Neurol., 212 (1982) 173-187. 2 Blessing, W.W., Sved, A.F. and Reis, D.J., Destruction of noradrenergic neurons in rabbit brainstem elevated plasma vasopressin, causing hypertension, Science, 217 (1982) 661~62. 3 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Res., 226 (1981) 187-199. 4 Gerfen, C.R. and Sawchenko, P.E., Anterograde neuroanatomical tracing method that shows the detailed morphology of neurons,
their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), Brain Res., 290 (1984) 219-238. 5 Hancock, M.B., Evidence for direct projections from the nucleus of the solitary tract onto medullary adrenaline cells, J. Comp. Neurol., 276 (1988) 460-467. 6 Hancock, M.B., Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements, Am. J. Anat., 175 (1986) 343-352. 7 Hancock, M.B. and Nicholas, A.P., Oxytocin-immunoreactiveprojections onto medullary adrenaline neurons, Brain Res. Bull., 18 (1987) 213 219. 8 Howe, P.R.C., Kuhn, D.M., Minson, J.B., Stead, B.H. and Chalmers, J.P., Evidence for a bulbospinal serotonergic pressor pathway in the rat brain, Brain Res+, 270 (1983) 29-36. 9 Hrkfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res., 66 (1974) 235-251. 10 Hrkfelt, T., Johansson, O. and Goldstein, M., Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Chapter V: Classical Transmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 157-276. 11 H6kfelt, T., Mhrtensson, R., Bjrrklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bjrrklund and T. Hrkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Chapter VI: Classical Transmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 277-379.
95 12 Larsson, L.I., A novel immunocytochemical model system for specificity and sensitivity screening of antisera against multiple antigens, J. Histochem. Cytochem., 24 (198 I) 408-410. 13 Lowey, A.D., McKellar, S. and Saper, C.B., Direct projections from the A5 catecholamine cell group to the intermediolateral cell column, Brain Res., 174 (1979) 309-314. 14 Lowey, A.D., Wallach, J.H. and McKeUar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 15 McCall, R.B., Evidence for a serotonergically mediated sympathoexcitatory response to stimulation of medullary raphe nuclei. Brain Res., 311 (1984) 131-139. 16 Neil, J.J. and Loewy, A.D., Decreases in blood pressure in response to L-glutamate microinjections into the A5 catecholamine cell group, Brain Res., 241 (1982) 271-278. 17 Nicholas, A.P. and Hancock, M.B., Evidence for projections from the rostral medullary raphe onto medullary catecholamine neurons in the rat, Neurosci. Lett., 108 (1990) 22-28. 18 Nicholas, A.P. and Hancock, M.B., Evidence for substance P, serotonin and oxytocin input to medullary catecholamine neurons with diencephalic projections, Brain Res. Bull., 22 (1989) 213-223. 19 Nicholas, A.P. and Hancock, M.B., Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons, Synapse 2 (1988) 569-576. 20 Nicholas, A.P. and Hancock, M.B., Projections from the medullary C1 area onto brainstem monoaminergic neurons, Soc. Neurosci. Abstr., 15 (1989) 451.
21 Pilowsky, P.M., Kapoor, V., Minson, J.B., West, M.J. and Chalmers, J.P., Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection, Brain Res., 366 (1986) 354-357. 22 Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J., Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons, J. Comp. Neurol., 228 (1984) 168-185. 23 Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Pardal, J.F., 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 C 1 adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 24 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla, J. Comp. Neurol., 242 (1985) 511-534. 25 Sawchenko, P.E. and Swanson, L.W., Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses, Science, 214 (1981) 685-687. 26 Tucker, D,C., Saper, C.B., Ruggiero, D.A. and Reis, D.J., Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections of the hypothalamus and spinal cord, J. Comp. Neurol., 259 (1987) 591-603.
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ElsevierScientificPublishers Ireland Ltd. NSL 07471
Projections from the rostral ventrolateral medulla to brainstem monoamine neurons in the rat A n t h o n y P. N i c h o l a s a n d M i c h a e l B. H a n c o c k Department of Anatomy and Neurosciences, University of Texas Medical Branch, Galveston, TX 77550 (U.S.A.)
(Received31 August 1990;Revisedversionreceived4 October 1990;Accepted8 October 1990) Key words: Rostralventrolateralmedulla; Brainstem pathway; Phaseolus vulgaris leucoagglutinin;Immunocytochemistry
Following the iontophoretic deposition of Phaseolus vulgaris leucoagglutinin (PHA-L) into the rostral ventrolateral medulla (RVL), two-color immunoperoxidase staining was employedto demonstrate contiguity between PHA-L-immunoreactive(PHA-LI) varicosefibers and boutons and brainstem monoaminergicceils. Black-stainedPHA-LI ceils in the deposition site were found to be located among amber-stained phenylethanolamine N-methyl transferase-immunoreactive(PNMT-I) neurons of the C1 cell group. RVL projections were contiguous with PNMT-I neurons of the C 1, C2 and C3 cell groups, with tyrosine hydroxylase-immunoreactive(TH-I) neurons of the A1, A2 and A5 cell groups, and with serotoninimmunoreactive(5-HT-I)neurons of the B1, B2 and B3 cellgroups. Preliminaryfindings of this study have been presentedpreviously(Sot. Neurosci. Abstr., 15 (1989)451).
The adrenergic C1 cell group first described by H6kfelt et al. [9] is located in the rostral ventrolateral medulla (RVL), a well-known pressor region [23]. These adrenergic neurons have been shown to project to cardiovascular-regulatory centers in the spinal cord [7, 19, 20, 22, 26] and hypothalamus [18, 26], but local projections of these cells, especially to monoaminergic neurons in the brainstem that also are thought to be involved with blood pressure regulation [2, 8, 15, 16, 21, 23], have not been well documented. Efferent projections from the C 1 area in RVL have been studied previously using the anterograde transport of tritiated amino acids [14, 22] and have been corroborated by the transport of retrograde tracers from selected sites of termination in the rat [7, 14, 18, 19, 22, 24, 26]. However, in animals in which anterogradely-transported tritiated amino acids were injected into the RVL, the detailed anatomic relationships between radiolabeled terminals of RVL neurons and histochemically-identified brainstem monoaminergic cells were not analyzed. In the present study, projections to monoamine cell groups in the pons and medulla that arise from neurons in the RVL were examined using the anterograde tracer
Requests for reprints: M.B. Hancock, Department of Anatomy and Neurosciences, The University of Texas Medical Branch, 200 University Blvd., Suite 730, Galveston,TX 77550,U.S.A. Correspondence: A. Nicholas, Karolinska Institute, Department of Histology and Neurobiology, Box 60400, S-10401 Stockholm, Sweden. 0304-3940/91/$03.50 © 1991 ElsevierScientificPublishers Ireland Ltd.
vulgaris leucoagglutinin (PHA-L, Vector Labs). PHA-L was iontophoretically deposited in the RVL of 5 male Sprague-Dawley rats using a modified version [17] of the technique originally described by Gerfen and Sawchenko [4]. Briefly, animals were deeply anesthetized with intraperitoneal injections of sodium pentobarbital (37.5 mg/kg) and urethane (900 mg/kg) in Ringer's solution. Atropine (0.054).1 mg) in Ringer's solution was injected subcutaneously to reduce pulmonary secretions. After placing an animal in a stereotaxic frame, the head was tilted downward 33 °, and the dorsal medulla was exposed by surgically removing the atlantooccipital membrane. Fiber-filled glass micropipettes with outside tip diameters of 5-8/~m were filled with a 2.5% solution of PHA-L in 0.1 M phosphate-buffered saline (pH 7.4). After being clamped to a micromanipulator and angled rostrally 12°, the PHA-L-filled micropipette then was positioned with its tip at the calamus scriptorius, moved laterally 2.0 mm, and lowered into the medulla to a depth of 3.5-3.8 mm. Iontophoresis of PHA-L was performed using electrical current of 3-5/~A passed in 7 s pulses with a 50% duty cycle for 15-20 min. Then the wound was closed and penicillin G (70,000 U) was injected intramuscularily. Following survival periods of 4-8 days, the animals were deeply anesthetized as before and perfused transcardially with Ringer's solution at room temperature, then with 500 ml of ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain and spinal cord were postfixed overnight in the same fixative, then serial, 30/tm coronal sections Phaseolus
92 of this material were cut on a Vibratome and stored in multiwell dishes filled with phosphate buffer. The details of the two-color immunoperoxidase staining technique used in the present study have been described previously [6]. Briefly, coronal sections of medulla, caudal pons, hypothalamus and spinal cord were incubated overnight in rabbit anti-PHA-L (1:5,000; D A K O ) and stained with a sequence of peroxidase-antiperoxidase (PAP) immunoreagents and nickel-intensified diaminobenzidine. PHA-L-immunoreactive (PHALI) cells and processes stained black. Since the transition zones between rostral adrenergic (C1, C2) and caudal noradrenergic (A l, A2) cell columns have been localized to the level of the area postrema (AP) [1, 11], medullary sections located rostral and caudal to the AP were incubated in rabbit antisera raised against phenylethanolamine N-methyl transferase (PNMT; 1:500; Eugene Tech) and tyrosine hydroxylase (TH; l:l,000; Eugene
Tech), respectively. Other medullary sections were incubated in rabbit antiserum raised against serotonin (5HT; 1:20,000; I m m u n o Nuclear). Medullary P N M T immunoreactive (PNMT-I), TH-immunoreactive (TH-I) or 5-HT-immunoreactive (5-HT-I) cells then were stained amber using a second sequence of PAP reagents and diaminobenzidine (DAB) alone. The affinity of the PHA-L, P N M T , and 5-HT antisera for their respective antigens was checked by solid-phase immunoassay [12], and the results have been described previously [5, 19]. The antiserum to T H could not be checked by solid-phase assay, since the antigen was not available. However, the distribution of cells immunostained with T H antiserum in the present study is similar to that reported by other investigators [1, 11]. Unilateral P H A - L deposition sites were centered in the RVL at the level of the C1 cell group (Fig. 1A). In dual-stained sections, the location of the black-stained
Fig. 1. Example of a representative Phaseolus vulgaris leucoagglutinin (PHA-L) deposition site in a dual-stained section of the rostral ventrolateral medulla (RVL), showing black-stained (nickel-intensified DAB) PHA-L-immunoreactive(PHA-LI) neurons which totally obscure any lighterstained (DAB alone) phenylethanolamine N-methyl transferase-immunoreactive(PNMT-I) neurons of the adrenergic C1 cell group (A). Blackstained PHA-LI fibers and boutons (arrows) in contiguity with lighter-stained PNMT-I CI neurons found contralateral (B, C) or ipsilateral (D) to the PHA-L deposition site. PHA-LI fibers and boutons originating from neurons in the RVL also were contiguous with PNMT-I C2 neurons in the nucleus tractus solitarius (E-F), and C3 neurons in the dorsomedialmedulla near the dorsal medullary surface (G). IV, fourth ventricle. Dashed line = 1.0 mm, bars = 20/zm.
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PHA-LI neurons exactly mirrored the location of amber-stained PNMT-I C1 neurons on the contralateral side of the medulla, and only a few PNMT-I cells occa: sionally could be seen intermingled among PHA-LI neurons in the deposition site. In the majority Of medullary sections at the level of the PHA-L deposition site, the black PHA-L staining in RVL obscured amber-stained PNMT-I cells and processes. Varicose PHA-LI fibers originating from cells in the RVL were observed in contiguity with PNMT-I neurons of the C1 cell group that were located rostral, caudal, ipsilateral and contralateral to the deposition site (Fig. 1B-D). PHA-LI terminal fields in the rostral C1 area were more prominant on the ipsilateral side, but the contralateral C1 area also received a heavy PHA-LI projection. PHA-LI boutons also were seen in contiguity with PNMT-I neurons of the C2 cell group in the nucleus tractus solitarii (Fig. 1E-F) and of the C3 cell group in the dorsomedial medulla (Fig. 1G). In regions of the caudal medulla (caudal to AP), PHA-LI fibers and boutons were seen in contiguity with TH-I neurons of the A1 (Fig. 2A, B) and A2 (Fig. 2C, D) cell groups. In more rostral regions, PHA-LI boutons were seen in contiguity with TH-I neurons in the A5 cell group in the pons (Fig. 2E). Medullary sero-
tonergic nuclei also seem to receive an input from the RVL, since PHA-LI fibers and boutons were observed in contiguity with 5-HT-I neurons of the B 1, B2 and B3 cell groups, which include nucleus raphe pallidus (Fig. 3A), nucleus interfasicularis hypoglossi (Fig. 3B), nucleus raphe obscurus (Fig. 3C, D), nucleus raphe magnus (Fig. 3E) and nucleus reticularis paragigantocellularis (Fig. 3F). PHA-LI terminal arborizations were also observed in the intermediolateral cell column and intermediate grey matter of the rostral thoracic spinal cord, and in the supraoptic and paraventricular nuclei of the hypothalamus. Using the anterograde transport of PHA-L and twocolor immunoperoxidase staining, the results of the present study indicate that the RVL, an important medullary vasopressor region [23], contains neurons with extensive local projections to adrenergic, noradrenergic and serotonergic cell groups in the brainstem. Therefore, the effects on sympathetic vasomotor tone elicited by RVL stimulation could be mediated by direct projections from RVL to the spinal cord, or indirectly by RVL projections to local brainstem monoaminergic neurons, many of which are known to project to the spinal cord [3, 7, 13, 14, 19, 22, 26] or hypothalamus [18, 25, 26]. Since the
Fig. 2. Black-stained PHA-LI boutons and varicose fibers (arrows) originating from the deposition site shown in Fig. 1A and in contiguity with lighter-stained, noradrenergic, tyrosine hydroxylase-immunoreactive (TH-I) cells and dendrites of the A1 cell group in the caudal ventrolateral medulla (A, B) of the A2 cell group in the nucleus commissuralis (C, D), and of the A5 cell group in the ventral pons (E). cc, central canal. Bars = 20 ,um.
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Fig. 3. Black-stained PHA-LI fibers and boutons (arrows) originating from the deposition site shown in Fig. 1A and in contiguity with lighter-stained serotonin-immunoreactive(5-HT-I) neurons of the B I (A, B), B2 (C, D) and B3 (E, F) cell groups in nucleus raphe pallidus (A), nucleus interfasicularis hypoglossi (B), nucleus raphe obscurus (C, D), nucleus raphe magnus (E), and nucleus reticularis paragigantocellularis (F), respectively. XII, hypoglossal nerve rootlet. Bars = 20/~m.
P H A - L I d e p o s i t s were l o c a t e d w i t h i n the C1 area, a n d since the P H A - L I t e r m i n a l fields i n the p r e s e n t s t u d y are in the s a m e l o c a t i o n s as w e l l - d o c u m e n t e d P N M T - I term i n a l a r b o r i z a t i o n s [9, 10], it is likely t h a t s o m e o f these PHA-LI projections include adrenergic pathways from C 1 n e u r o n s . T h i s will, h o w e v e r , h a v e to be verified b y future e x p e r i m e n t s , w h e r e the t r a n s m i t t e r i d e n t i t y o f P H A L - l a b e l e d fibers is e s t a b l i s h e d u s i n g i m m u n o h i s t o c h e m i cal d o u b l e (or triple) s t a i n i n g m e t h o d s . T h e a u t h o r s wish to t h a n k Ms. P. T h a t c h e r for t e c h n i cal assistance. T h i s w o r k was s u p p o r t e d b y the T e x a s Affiliate o f the A m e r i c a n H e a r t A s s o c i a t i o n . 1 Armstrong, D.M., Ross, C.A., Pickel, V.M., Joh, T.H. and Reis, D.J., Distribution of dopamine-, noradrenaline- and adrenalinecontaining cell bodies in the rat medulla oblongata: demonstrated by the immunocytochemical localization of catecholamine biosynthetic enzymes. J. Comp. Neurol., 212 (1982) 173-187. 2 Blessing, W.W., Sved, A.F. and Reis, D.J., Destruction of noradrenergic neurons in rabbit brainstem elevated plasma vasopressin, causing hypertension, Science, 217 (1982) 661~62. 3 Bowker, R.M., Westlund, K.N. and Coulter, J.D., Origins of serotonergic projections to the spinal cord in rat: an immunocytochemical-retrograde transport study, Brain Res., 226 (1981) 187-199. 4 Gerfen, C.R. and Sawchenko, P.E., Anterograde neuroanatomical tracing method that shows the detailed morphology of neurons,
their axons and terminals: immunohistochemical localization of an axonally transported plant lectin, Phaseolus vulgaris leucoagglutinin (PHA-L), Brain Res., 290 (1984) 219-238. 5 Hancock, M.B., Evidence for direct projections from the nucleus of the solitary tract onto medullary adrenaline cells, J. Comp. Neurol., 276 (1988) 460-467. 6 Hancock, M.B., Two-color immunoperoxidase staining: visualization of anatomic relationships between immunoreactive neural elements, Am. J. Anat., 175 (1986) 343-352. 7 Hancock, M.B. and Nicholas, A.P., Oxytocin-immunoreactiveprojections onto medullary adrenaline neurons, Brain Res. Bull., 18 (1987) 213 219. 8 Howe, P.R.C., Kuhn, D.M., Minson, J.B., Stead, B.H. and Chalmers, J.P., Evidence for a bulbospinal serotonergic pressor pathway in the rat brain, Brain Res+, 270 (1983) 29-36. 9 Hrkfelt, T., Fuxe, K., Goldstein, M. and Johansson, O., Immunohistochemical evidence for the existence of adrenaline neurons in the rat brain, Brain Res., 66 (1974) 235-251. 10 Hrkfelt, T., Johansson, O. and Goldstein, M., Central catecholamine neurons as revealed by immunohistochemistry with special reference to adrenaline neurons. In A. Bj6rklund and T. H6kfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Chapter V: Classical Transmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 157-276. 11 H6kfelt, T., Mhrtensson, R., Bjrrklund, A., Kleinau, S. and Goldstein, M., Distributional maps of tyrosine-hydroxylase-immunoreactive neurons in the rat brain. In A. Bjrrklund and T. Hrkfelt (Eds.), Handbook of Chemical Neuroanatomy, Vol. 2, Chapter VI: Classical Transmitters in the CNS, Part I, Elsevier, Amsterdam, 1984, pp. 277-379.
95 12 Larsson, L.I., A novel immunocytochemical model system for specificity and sensitivity screening of antisera against multiple antigens, J. Histochem. Cytochem., 24 (198 I) 408-410. 13 Lowey, A.D., McKellar, S. and Saper, C.B., Direct projections from the A5 catecholamine cell group to the intermediolateral cell column, Brain Res., 174 (1979) 309-314. 14 Lowey, A.D., Wallach, J.H. and McKeUar, S., Efferent connections of the ventral medulla oblongata in the rat, Brain Res. Rev., 3 (1981) 63-80. 15 McCall, R.B., Evidence for a serotonergically mediated sympathoexcitatory response to stimulation of medullary raphe nuclei. Brain Res., 311 (1984) 131-139. 16 Neil, J.J. and Loewy, A.D., Decreases in blood pressure in response to L-glutamate microinjections into the A5 catecholamine cell group, Brain Res., 241 (1982) 271-278. 17 Nicholas, A.P. and Hancock, M.B., Evidence for projections from the rostral medullary raphe onto medullary catecholamine neurons in the rat, Neurosci. Lett., 108 (1990) 22-28. 18 Nicholas, A.P. and Hancock, M.B., Evidence for substance P, serotonin and oxytocin input to medullary catecholamine neurons with diencephalic projections, Brain Res. Bull., 22 (1989) 213-223. 19 Nicholas, A.P. and Hancock, M.B., Immunocytochemical evidence for substance P and serotonin input to medullary bulbospinal adrenergic neurons, Synapse 2 (1988) 569-576. 20 Nicholas, A.P. and Hancock, M.B., Projections from the medullary C1 area onto brainstem monoaminergic neurons, Soc. Neurosci. Abstr., 15 (1989) 451.
21 Pilowsky, P.M., Kapoor, V., Minson, J.B., West, M.J. and Chalmers, J.P., Spinal cord serotonin release and raised blood pressure after brainstem kainic acid injection, Brain Res., 366 (1986) 354-357. 22 Ross, C.A., Ruggiero, D.A., Joh, T.H., Park, D.H. and Reis, D.J., Rostral ventrolateral medulla: selective projections to the thoracic autonomic cell column from the region containing C1 adrenaline neurons, J. Comp. Neurol., 228 (1984) 168-185. 23 Ross, C.A., Ruggiero, D.A., Park, D.H., Joh, T.H., Sved, A.F., Pardal, J.F., 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 C 1 adrenaline neurons on arterial pressure, heart rate and plasma catecholamines and vasopressin, J. Neurosci., 4 (1984) 474-494. 24 Ross, C.A., Ruggiero, D.A. and Reis, D.J., Projections from the nucleus tractus solitarii to the rostral ventrolateral medulla, J. Comp. Neurol., 242 (1985) 511-534. 25 Sawchenko, P.E. and Swanson, L.W., Central noradrenergic pathways for the integration of hypothalamic neuroendocrine and autonomic responses, Science, 214 (1981) 685-687. 26 Tucker, D,C., Saper, C.B., Ruggiero, D.A. and Reis, D.J., Organization of central adrenergic pathways: I. Relationships of ventrolateral medullary projections of the hypothalamus and spinal cord, J. Comp. Neurol., 259 (1987) 591-603.
