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NADPH diaphorase-positive neurons in the intracardiac plexus of human, monkey and canine right atria
BRAIN RESEARCH ELSEVIER
Brain Research 724 (1996) 256 259
Short communication
NADPH diaphorase-positive neurons in the intracardiac plexus of human, monkey and canine right atria Kazuhide Yoshida *, Noboru Toda Department of Pharmacology, Shiga Unit ersity ~)/'Medical Sciences, Seta, Ohtsu 520-21, Japan
Accepted 12 March 1996
Abstract
Distribution of nitric oxide synthase in intracardiac ganglion cells located in human, monkey and canine right atria was histologically investigated using the reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase method and acetylcholinesterase histochemistry. In the intracardiac ganglion, many large neurons exhibited both positive reactions, whereas some of the NADPH diaphorase-positive small neuronal cells were shown with negative acetylcholinesterase reaction. Keywords: NADPH diaphorase; Acetylcholinesterase:Intracardiac plexus; Human; Monkey; Dog: Right atrium
Localization, histology and function of intracardiac ganglion ceils have been reported mainly in amphibians [6,10], and the study on mammalian intracardiac ganglia is limited. In the dog, the intracardiac ganglia locating in the right atrium are suggested to control sinoatrial nodal function [19]. Intracardiac neurons are considered to be primarily e f f e r e n t postganglionic cholinergic ones [2,3,7,12,16,18]. However, atrial ganglion cells receive physiological inputs from efferent parasympathetic and sympathetic neurons and afferent axons arising from cardiac and pulmonary mechanoreceptors [8]. Recently, it has been suggested that nitric oxide (NO) participates in the neural control of cardiac functions by the use of a dissociated cell culture preparation from newborn guinea-pig atria [11]. Tanaka et al. [21] reported NO synthase (NOS)-immunoreactive neuronal cells in the atrioventricular node of guinea-pig by electron microscopy. There is increasing evidence that NO has an important role as a neurogenic messenger molecule in the central and peripheral nervous systems [4]. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase may be used as a marker for NOS since NOS and N A D P H diaphorase are extensively co-localized [5]. Tissue blocks of human right atria (n = 2) were obtained from subjects with no apparent cardiac disease at 6
* Corresponding author. Fax: (81) (775) 48-2183. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PIIS0006-8993(96)00314-9
h (female; 60 yrs) and 2 h (female; 73 yrs) postmortem. Causes of death were thyroid cancer and angiosarcoma of scalp, respectively. Six Japanese monkeys (Macaca fuscam) of either sex (6 to 11 kg) and six mongrel dogs of either sex, weighing 8 to 13 kg, were killed by bleeding from common carotid arteries under anesthesia. Right atrium was isolated. The tissue blocks were fixed in icecold 0.1 M phosphate-buffered saline (PBS, pH 7.4) containing 0.3% glutaraldehyde and 4% paraformaldehyde for 7 min and then postfixed overnight in 0.1 M PBS with 4% paraformaldehyde, followed by cryoprotection in 15% sucrose. The fixed blocks were cut into sections (20 /xm thick) in a cryostat. For N A D P H diaphorase staining [22], the tissue sections were mounted onto gelatine/chromealum-coated glass slides and incubated with 0.1 M PBS at pH 8.0, containing 1 mM /3-NADPH (reduced form) (Kohjin, Tokyo), 2 mM nitro blue tetrazolium (Sigma Chemical, St. Louise, MO) and 0.3% Triton X-100 at 37°C. After several washes with distilled water, the sections were cover-slipped with xylene and alkylacrylates. Neuronal cells of the ganglia were verified by cresyl violet staining in different sections. For double staining of NADPH diaphorase and acetylcholinesterase, the sections were first stained for acetylcholinesterase histochemistry according to the method of Tago et al. [20]. In brief, the mounted sections were incubated first with a Karnovsky and Roots medium diluted 100-fold; and then with a mixture containing 0.56 mM 3,3'-diaminobenzidine and 1.3 /zM hydrogen perox-
K. Yoshida, N. Toda / Brain Research 724 (1996) 256-259
257
ide. The sections were cover-slipped with 90% glycerol in 0.1 M PBS. After photomicrography, cover-glasses were removed and the mounted sections were stained by NADPH diaphorase method as described above. The doubly stained sections were cover-slipped with glycerol in PBS, and were observed under a light microscope. In the present study, neuronal cells, bundles and fine fibers containing NADPH diaphorase were histochemically determined in the intracardiac plexus of human (n = 2), monkey (n = 6) and dog (n = 6) right atria. At least ten ganglia with NADPH diaphorase-positive neurons were found in the individual atrium. The human ganglia ranged from less than 100 /zm with only a few positively-stained neuronal cells (Fig. 1A), to over 1 mm
"
A,
,¢
~/,
f
.:?
Fig. 2. Histochemistry of ganglionated plexus of monkey (A) and dog (B) right atria. Neuronal cells and fibers containing NADPH diaphorase. Bars = 50 /~m.
Fig. 1. Histochemistry of ganglionated plexus of human right atrium. A: a fiber bundle and a small ganglionated plexus containing NADPH diaphorase (60 years, female). B: neuronal cells and fibers containing NADPH diaphorase (73 years, female). C: histology of ganglion stained with cresyl violet. Bars = 50 /xm.
in size. Few large ganglia were associated with nerve bundles (Fig. 1B). The ganglionated cells with multiple nucleoli were also positively stained (data not shown). There were many positive neurons with a long axon (Fig. I A and B), but some positive neurons possessed short axons (Fig. 1B). There were large, (35-50 /zm in diameter), and small neurons (15-35 /~m). The small type of positive neurons had numerous processes that extended out to form close associations with adjacent large cells. In the monkey (Fig. 2A) and dog (Fig. 2B) intracardiac ganglia, also two types of the positively stained neuronal cells were observed. Acetylcholinesterase-containing neurons and fiber bundles were found abundantly in the human, dog (data not shown) and monkey (Fig. 3A) intracardiac ganglia. Although subsequent NADPH diaphorase histochemistry stained many NADPH diaphorase-positive neurons in the same sections, doubly stained neurons were found in most large neurons (Fig. 3B). With negative acetylcholinesterase reaction, some small neurons exhibited NADPH diaphorase activity only (Fig. 3B). The existence of neurons in the mammalian heart, including the atrium, has long been recognized [13]. It has been considered that acetylcholine is the primary chemical neurotransmitter [7,12]. Recently, it was reported that in
258
K. Yoshida, N. Toda / Brain Research 724 (1996) 256 259
m
-B-
ylase or substance P in the intracardiac ganglion [14], but with vasoactive intestinal peptide in the guinea pig intralingual arteries [15]. To our knowledge, however, there was no report regarding co-localization with acetylcholinesterase in NOS-positive neurons. The present study demonstrated that NOS-positive neurons are co-localized with acetylcholinesterase. Some small neurons were intensely stained for N A D P H diaphorase activity, but not co-localized with acetylcholinesterase. Recently, it has been suggested that when the right atrial ganglionated plexus is electrically stimulated [1,17], cardiodepressor responses are elicited, and also that NO is an obligatory mediator of the indirect effects of acetylcholine in pacemaker tissue, using isolated sinoatrial nodal cells of the rabbit heart [9]. The finding that some intracardiac neurons have both NO synthase and acetylcholinesterase is intriguing, since NO released from these cells may alter functions of the pacemaker and conducting tissues or other intracardiac neurons. However, the exact distribution and functional role of intracardiac neuronal cells containing NOS must await further clarification.
References
Fig. 3. Histochemistry of ganglionated plexus of monkey right atrium. A: acetylcholinesterase staining in a ganglionated plexus. B: NADPH diaphorase staining of the same section as A. Arrowheads indicate NADPH diaphorase-positive neurons that exhibited acetylcholinesterase reaction. Arrows indicate the neurons that contain NADPH diaphorase activity only. Bars = 50 /*m.
guinea pig and rat, very few (36% in guinea pig or 4% in rat) intracardiac neuronal cells exhibited a strong N A D P H diaphorase reaction [14]. However, the type of positive cells could not be precisely stated because there were few positive cells [14,15,21]. The present study revealed that a large number of neuronal cells, bundles and fine fibers contained NADPH diaphorase in intracardiac ganglia of human, monkey and canine right atria. Many of the large neurons with a long axon which exhibited both positive reactions of acetylcholinesterase histochemistry and N A D P H diaphorase method, are presumably principal cells that provide postganglionic fibers innervating the cardiac tissue. Some of the N A D P H diaphorase positively stained neurons of small size with short axons may be those that transmit information to adjacent neurons. The intraganglionic neurons or interneurons may also terminate at other non-neuronal tissues, e.g., fat tissue or connective tissue. These neurons are unlikely to be sensory because of their short processes. It has been reported that in rat and guinea pig, NOSpositive neurons are not co-localized with tyrosine hydrox-
[1] Ali, I.M., Butler, C.K., Armour, J.A. and Murphy, D.A., Modification of supraventricular tachyarrhythmias by stimulating atrial neurons, Ann. Thorac. Surg., 50 (1990) 251-256. [2] Ardell, J.L. and Randall, W.C., Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart, Am. J. Physiol., 251 (1986) H764-H773. [3] Blomquist, T.M., Priola, D.V. and Romero, A.M., Source of intrinsic innervation of canine ventricles: a functional study, Am. J. Physiol., 252 (1987) H638-H644. [4] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. [5] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. [6] Dennis, M.J., Harris, A.J. and Kuffler, S.W.. Synaptic transmission and its duplication by focally applied acetylcholine in parasympathetic neurons in the heart of the frog, Proc. Roy. Soc. Lond. B, 177 (1971) 509-539. [7] Ehinger, B., Falck, B., Persson, H. and Sporrong, B., Adrenergic and cholinesterase-containing neurons of the heart, Histochemie. 16 (1968) 197-205. [8] Gagliardi, M., Randall, W.C., Bieger, D., Wurster, R.D., Hopkins, D.A. and Armour, J.A., Activity of in vivo canine cardiac plexus neurons, Am. J. Physiol., 255 (1988) H789-H800. [9] Ham X., Shimoni, Y. and Giles, W.R., An obligatory role for nitric oxide in autonomic control of mammalian heart rate, J. Physiol., 476 (1994) 309-314. [10] Hartzell, H.C., Kuffler, S.W., Stickgold. R. and Yoshikami, D., Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones, J. Physiol., 271 (1977) 817-846. [11] Hassall, C.J.S., Saffrey, M.J., Belai, A.. Hoyle, C.H.V., Moules. E.W., Moss, J., Schmidt, H.H.H.W., Murad, F., F{Srstermann, U. and Burnstock. G., Nitric oxide synthase immunoreactivity and
K. Yoshida, N. Toda / Brain Research 724 (1996) 256-259
[12]
[13] [14]
[15]
[16]
NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart, Neurosci. Lett., 143 (1992) 65-68. Jacobowitz, D., Histochemical studies of the relationship of chromaffin cells and adrenergic nerve fibers to the cardiac ganglia of several species, J. Pharmacol. Exp. Therap., 158 (1967) 227-240. King, T.S. and Coakley, J.B., The intrinsic nerve cells of the cardiac atria of mammals and man, J. Anat., 92 (1958) 353-376. Klimaschewski, L., Kummer, W., Mayer, B., Courand, J.Y., Preissler, U., Philippin, B. and Heym, C., Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart, Circ. Res., 71 (1992) 1533-1537. Kummer, W., Fischer, A., Mundel, P., Mayer, B., Hoba, B., Philippin, B. and Preissler, U., Nitric oxide synthase in VIP-containing vasodilator nerve fibers in the guinea-pig, Neuroreport, 3 (1992) 653-655. Moravec, M., Courtalon, A. and Moravec, J., Intrinsic neurosecretory neurons of the rat heart atrioventricular junction: Possibility of local neuromuscular feed back loops, J. Mol. Cell. Cardiol., 18 (1986) 357-367.
259
[17] Murphy, D.A. and Armour, J.A., Human cardiac nerve stimulation, Ann. Thorac. Surg., 54 (1992) 502-506. [18] Randall, W.C., Ardell, J.L., Calderwood, D., Milosavljevic, M. and Goyal, S.C., Parasympathetic ganglia innervating the canine atrioventricular nodal region, J. Auton. Nerv. Syst., 16 (1986) 311-323. [19] Randall, W.C., Ardell, J.L., Wurster, R.D. and Milosavljevic, M., Vagal postganglionic innervation of the canine sinoatrial node, J. Auton. Nero,. Syst., 20 (1987) 13-23. [20] Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. [21] Tanaka, K. and Chiba, T., Nitric oxide synthase containing nerves in the atrioventricular node of guinea pig heart, J. Auton. Nert:. Syst., 51 (1995) 245-253. [22] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784.
Brain Research 724 (1996) 256 259
Short communication
NADPH diaphorase-positive neurons in the intracardiac plexus of human, monkey and canine right atria Kazuhide Yoshida *, Noboru Toda Department of Pharmacology, Shiga Unit ersity ~)/'Medical Sciences, Seta, Ohtsu 520-21, Japan
Accepted 12 March 1996
Abstract
Distribution of nitric oxide synthase in intracardiac ganglion cells located in human, monkey and canine right atria was histologically investigated using the reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase method and acetylcholinesterase histochemistry. In the intracardiac ganglion, many large neurons exhibited both positive reactions, whereas some of the NADPH diaphorase-positive small neuronal cells were shown with negative acetylcholinesterase reaction. Keywords: NADPH diaphorase; Acetylcholinesterase:Intracardiac plexus; Human; Monkey; Dog: Right atrium
Localization, histology and function of intracardiac ganglion ceils have been reported mainly in amphibians [6,10], and the study on mammalian intracardiac ganglia is limited. In the dog, the intracardiac ganglia locating in the right atrium are suggested to control sinoatrial nodal function [19]. Intracardiac neurons are considered to be primarily e f f e r e n t postganglionic cholinergic ones [2,3,7,12,16,18]. However, atrial ganglion cells receive physiological inputs from efferent parasympathetic and sympathetic neurons and afferent axons arising from cardiac and pulmonary mechanoreceptors [8]. Recently, it has been suggested that nitric oxide (NO) participates in the neural control of cardiac functions by the use of a dissociated cell culture preparation from newborn guinea-pig atria [11]. Tanaka et al. [21] reported NO synthase (NOS)-immunoreactive neuronal cells in the atrioventricular node of guinea-pig by electron microscopy. There is increasing evidence that NO has an important role as a neurogenic messenger molecule in the central and peripheral nervous systems [4]. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase may be used as a marker for NOS since NOS and N A D P H diaphorase are extensively co-localized [5]. Tissue blocks of human right atria (n = 2) were obtained from subjects with no apparent cardiac disease at 6
* Corresponding author. Fax: (81) (775) 48-2183. 0006-8993/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PIIS0006-8993(96)00314-9
h (female; 60 yrs) and 2 h (female; 73 yrs) postmortem. Causes of death were thyroid cancer and angiosarcoma of scalp, respectively. Six Japanese monkeys (Macaca fuscam) of either sex (6 to 11 kg) and six mongrel dogs of either sex, weighing 8 to 13 kg, were killed by bleeding from common carotid arteries under anesthesia. Right atrium was isolated. The tissue blocks were fixed in icecold 0.1 M phosphate-buffered saline (PBS, pH 7.4) containing 0.3% glutaraldehyde and 4% paraformaldehyde for 7 min and then postfixed overnight in 0.1 M PBS with 4% paraformaldehyde, followed by cryoprotection in 15% sucrose. The fixed blocks were cut into sections (20 /xm thick) in a cryostat. For N A D P H diaphorase staining [22], the tissue sections were mounted onto gelatine/chromealum-coated glass slides and incubated with 0.1 M PBS at pH 8.0, containing 1 mM /3-NADPH (reduced form) (Kohjin, Tokyo), 2 mM nitro blue tetrazolium (Sigma Chemical, St. Louise, MO) and 0.3% Triton X-100 at 37°C. After several washes with distilled water, the sections were cover-slipped with xylene and alkylacrylates. Neuronal cells of the ganglia were verified by cresyl violet staining in different sections. For double staining of NADPH diaphorase and acetylcholinesterase, the sections were first stained for acetylcholinesterase histochemistry according to the method of Tago et al. [20]. In brief, the mounted sections were incubated first with a Karnovsky and Roots medium diluted 100-fold; and then with a mixture containing 0.56 mM 3,3'-diaminobenzidine and 1.3 /zM hydrogen perox-
K. Yoshida, N. Toda / Brain Research 724 (1996) 256-259
257
ide. The sections were cover-slipped with 90% glycerol in 0.1 M PBS. After photomicrography, cover-glasses were removed and the mounted sections were stained by NADPH diaphorase method as described above. The doubly stained sections were cover-slipped with glycerol in PBS, and were observed under a light microscope. In the present study, neuronal cells, bundles and fine fibers containing NADPH diaphorase were histochemically determined in the intracardiac plexus of human (n = 2), monkey (n = 6) and dog (n = 6) right atria. At least ten ganglia with NADPH diaphorase-positive neurons were found in the individual atrium. The human ganglia ranged from less than 100 /zm with only a few positively-stained neuronal cells (Fig. 1A), to over 1 mm
"
A,
,¢
~/,
f
.:?
Fig. 2. Histochemistry of ganglionated plexus of monkey (A) and dog (B) right atria. Neuronal cells and fibers containing NADPH diaphorase. Bars = 50 /~m.
Fig. 1. Histochemistry of ganglionated plexus of human right atrium. A: a fiber bundle and a small ganglionated plexus containing NADPH diaphorase (60 years, female). B: neuronal cells and fibers containing NADPH diaphorase (73 years, female). C: histology of ganglion stained with cresyl violet. Bars = 50 /xm.
in size. Few large ganglia were associated with nerve bundles (Fig. 1B). The ganglionated cells with multiple nucleoli were also positively stained (data not shown). There were many positive neurons with a long axon (Fig. I A and B), but some positive neurons possessed short axons (Fig. 1B). There were large, (35-50 /zm in diameter), and small neurons (15-35 /~m). The small type of positive neurons had numerous processes that extended out to form close associations with adjacent large cells. In the monkey (Fig. 2A) and dog (Fig. 2B) intracardiac ganglia, also two types of the positively stained neuronal cells were observed. Acetylcholinesterase-containing neurons and fiber bundles were found abundantly in the human, dog (data not shown) and monkey (Fig. 3A) intracardiac ganglia. Although subsequent NADPH diaphorase histochemistry stained many NADPH diaphorase-positive neurons in the same sections, doubly stained neurons were found in most large neurons (Fig. 3B). With negative acetylcholinesterase reaction, some small neurons exhibited NADPH diaphorase activity only (Fig. 3B). The existence of neurons in the mammalian heart, including the atrium, has long been recognized [13]. It has been considered that acetylcholine is the primary chemical neurotransmitter [7,12]. Recently, it was reported that in
258
K. Yoshida, N. Toda / Brain Research 724 (1996) 256 259
m
-B-
ylase or substance P in the intracardiac ganglion [14], but with vasoactive intestinal peptide in the guinea pig intralingual arteries [15]. To our knowledge, however, there was no report regarding co-localization with acetylcholinesterase in NOS-positive neurons. The present study demonstrated that NOS-positive neurons are co-localized with acetylcholinesterase. Some small neurons were intensely stained for N A D P H diaphorase activity, but not co-localized with acetylcholinesterase. Recently, it has been suggested that when the right atrial ganglionated plexus is electrically stimulated [1,17], cardiodepressor responses are elicited, and also that NO is an obligatory mediator of the indirect effects of acetylcholine in pacemaker tissue, using isolated sinoatrial nodal cells of the rabbit heart [9]. The finding that some intracardiac neurons have both NO synthase and acetylcholinesterase is intriguing, since NO released from these cells may alter functions of the pacemaker and conducting tissues or other intracardiac neurons. However, the exact distribution and functional role of intracardiac neuronal cells containing NOS must await further clarification.
References
Fig. 3. Histochemistry of ganglionated plexus of monkey right atrium. A: acetylcholinesterase staining in a ganglionated plexus. B: NADPH diaphorase staining of the same section as A. Arrowheads indicate NADPH diaphorase-positive neurons that exhibited acetylcholinesterase reaction. Arrows indicate the neurons that contain NADPH diaphorase activity only. Bars = 50 /*m.
guinea pig and rat, very few (36% in guinea pig or 4% in rat) intracardiac neuronal cells exhibited a strong N A D P H diaphorase reaction [14]. However, the type of positive cells could not be precisely stated because there were few positive cells [14,15,21]. The present study revealed that a large number of neuronal cells, bundles and fine fibers contained NADPH diaphorase in intracardiac ganglia of human, monkey and canine right atria. Many of the large neurons with a long axon which exhibited both positive reactions of acetylcholinesterase histochemistry and N A D P H diaphorase method, are presumably principal cells that provide postganglionic fibers innervating the cardiac tissue. Some of the N A D P H diaphorase positively stained neurons of small size with short axons may be those that transmit information to adjacent neurons. The intraganglionic neurons or interneurons may also terminate at other non-neuronal tissues, e.g., fat tissue or connective tissue. These neurons are unlikely to be sensory because of their short processes. It has been reported that in rat and guinea pig, NOSpositive neurons are not co-localized with tyrosine hydrox-
[1] Ali, I.M., Butler, C.K., Armour, J.A. and Murphy, D.A., Modification of supraventricular tachyarrhythmias by stimulating atrial neurons, Ann. Thorac. Surg., 50 (1990) 251-256. [2] Ardell, J.L. and Randall, W.C., Selective vagal innervation of sinoatrial and atrioventricular nodes in canine heart, Am. J. Physiol., 251 (1986) H764-H773. [3] Blomquist, T.M., Priola, D.V. and Romero, A.M., Source of intrinsic innervation of canine ventricles: a functional study, Am. J. Physiol., 252 (1987) H638-H644. [4] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neural role for nitric oxide, Nature, 347 (1990) 768-770. [5] Dawson, T.M., Bredt, D.S., Fotuhi, M., Hwang, P.M. and Snyder, S.H., Nitric oxide synthase and neuronal NADPH diaphorase are identical in brain and peripheral tissues, Proc. Natl. Acad. Sci. USA, 88 (1991) 7797-7801. [6] Dennis, M.J., Harris, A.J. and Kuffler, S.W.. Synaptic transmission and its duplication by focally applied acetylcholine in parasympathetic neurons in the heart of the frog, Proc. Roy. Soc. Lond. B, 177 (1971) 509-539. [7] Ehinger, B., Falck, B., Persson, H. and Sporrong, B., Adrenergic and cholinesterase-containing neurons of the heart, Histochemie. 16 (1968) 197-205. [8] Gagliardi, M., Randall, W.C., Bieger, D., Wurster, R.D., Hopkins, D.A. and Armour, J.A., Activity of in vivo canine cardiac plexus neurons, Am. J. Physiol., 255 (1988) H789-H800. [9] Ham X., Shimoni, Y. and Giles, W.R., An obligatory role for nitric oxide in autonomic control of mammalian heart rate, J. Physiol., 476 (1994) 309-314. [10] Hartzell, H.C., Kuffler, S.W., Stickgold. R. and Yoshikami, D., Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones, J. Physiol., 271 (1977) 817-846. [11] Hassall, C.J.S., Saffrey, M.J., Belai, A.. Hoyle, C.H.V., Moules. E.W., Moss, J., Schmidt, H.H.H.W., Murad, F., F{Srstermann, U. and Burnstock. G., Nitric oxide synthase immunoreactivity and
K. Yoshida, N. Toda / Brain Research 724 (1996) 256-259
[12]
[13] [14]
[15]
[16]
NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart, Neurosci. Lett., 143 (1992) 65-68. Jacobowitz, D., Histochemical studies of the relationship of chromaffin cells and adrenergic nerve fibers to the cardiac ganglia of several species, J. Pharmacol. Exp. Therap., 158 (1967) 227-240. King, T.S. and Coakley, J.B., The intrinsic nerve cells of the cardiac atria of mammals and man, J. Anat., 92 (1958) 353-376. Klimaschewski, L., Kummer, W., Mayer, B., Courand, J.Y., Preissler, U., Philippin, B. and Heym, C., Nitric oxide synthase in cardiac nerve fibers and neurons of rat and guinea pig heart, Circ. Res., 71 (1992) 1533-1537. Kummer, W., Fischer, A., Mundel, P., Mayer, B., Hoba, B., Philippin, B. and Preissler, U., Nitric oxide synthase in VIP-containing vasodilator nerve fibers in the guinea-pig, Neuroreport, 3 (1992) 653-655. Moravec, M., Courtalon, A. and Moravec, J., Intrinsic neurosecretory neurons of the rat heart atrioventricular junction: Possibility of local neuromuscular feed back loops, J. Mol. Cell. Cardiol., 18 (1986) 357-367.
259
[17] Murphy, D.A. and Armour, J.A., Human cardiac nerve stimulation, Ann. Thorac. Surg., 54 (1992) 502-506. [18] Randall, W.C., Ardell, J.L., Calderwood, D., Milosavljevic, M. and Goyal, S.C., Parasympathetic ganglia innervating the canine atrioventricular nodal region, J. Auton. Nerv. Syst., 16 (1986) 311-323. [19] Randall, W.C., Ardell, J.L., Wurster, R.D. and Milosavljevic, M., Vagal postganglionic innervation of the canine sinoatrial node, J. Auton. Nero,. Syst., 20 (1987) 13-23. [20] Tago, H., Kimura, H. and Maeda, T., Visualization of detailed acetylcholinesterase fiber and neuron staining in rat brain by a sensitive histochemical procedure, J. Histochem. Cytochem., 34 (1986) 1431-1438. [21] Tanaka, K. and Chiba, T., Nitric oxide synthase containing nerves in the atrioventricular node of guinea pig heart, J. Auton. Nert:. Syst., 51 (1995) 245-253. [22] Vincent, S.R. and Kimura, H., Histochemical mapping of nitric oxide synthase in the rat brain, Neuroscience, 46 (1992) 755-784.