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Localization of substance P immunoreactive nerves in the carotid body
Immunoreactive
Nerves in the Carotid Body
Received 14 December 1979 JACOBOWITZ, D. M. ANI) C. 3. HELKE. Localization of substance P immunoreactive nerves in the carotid body. BRAIN RES. BULL. 32) 195-197, 1980.-Immunofluorescent bistochemical observatian of the rat carotid body revealed the Presence of substance P immunoreactivefibers. This supportsthe hypothesisthat substanceP is a neurotransmitterof
chemoreceptorafferent nerves. Carotid body
Substance P
Chemoreceptor
Immunofluorescence
cut in a cryostat (-iYC) and mounted on chrom&alumcoated slides. The slides were placed into a container w&h substance-P antiserum (diluted 1500 with 0.3% T&on X-100 in phosphate-b~ered saline), and kept in the refrigemtor overnight. They were then washed three times for 5 min each time in 0.2% T&on X-100 in phosphate-biers saline and incubated for 20 min with fluorescein-~~1~ goat ~tis~~ to rabbit IgG (Cap@, Downington, PA) diluted 1:360 with phosphate-buffered saline containing 0.3% Triton X-100. They were washed again three times as above and for a fourth time in phosPhate-bread saline and subsequen~y mounted in glycerine phosphate-buffered saline (3:l). Siides were examined under a Leitz Orthoplan fluorescence microscope equipped with a Ploem illuminator. The substance P antibody (Immune Nuclear Corp., Stillwater, MN) showed negligible cross reactivity with the agents tested (met enkephalin,
THE carotid and aortic bodies contain specialized chemoreceptor cells which are found near the carotid bifurcations and the aortic arch. These chesnoreceptors are an important part of the respiratory and cardiovascular control systems. The sensory innervation of the carotid body is by way of the carotid sinus nerve, a branch of the glossopharyngeal nerve, which projects to the CNS and terminates in the nucleus tractus solitarius [4]. A neurotransmitter contained in the carotid sinus nerve has not been demonstrated. Immunohistochemical studies [3,11] have revealed the presence of substance P nerve terminals in the nucleus tractus solitarius of rats. SP immunoreactive fibers could be seen entering the brainstem via the glosopharyngeal nerve [3]. Furthermore, the nodose ganglion, a sensory ganglion whose fibers innervate the aortic body chemore&eptors and aortic arch baroreceptors, was shown to contain SP-immunoreactive cell bodies [12]. Recent work has provided evidence that SP may be a neurotransmitter at baro- and chemoreceptor tierents in the nucleus tractus solitarius 161. A high level of SP-like immunore~tivity was present in the petrosal and nodose ganglia. The above observation led us to formulate the hypothesis that the carotid body was innervated by SP-containing sensory nerve fibers, The present immunohistochemical study reports that SP-imm~or~ctive fibers are indeed contained within the carotid body.
RESULTS
The carotid body was easily identifiable by the morphologic appearance of the glomus cells which are arranged in loose cords around small blood vessels and capillaries, These cells are ovoid in shape and are autofluorescent which demonstrates their unique resemblance to adrenal medulla catecholamine cells as observed with the ~sto~uo~s~e~~e procedure. Many long substance P ~uno~~tive varicose fibers were observed to be interspersed among the glomus cells (Fig. 1). The carotid sinus nerve containing fasicles of substance P fibers could be seen entering the carotid body (Fig. 2).
METHOD
indirect i~unohis~~hemi~~ procedure of Coons [2] and H&felt et al. f9J was us& with minor mutations. Male Sprague-Hawley rats @O&300 g) were perfused through the ascending aorta with 100 ml of cold phosphatebuffered saline @H 7.4) followed by 200-300 ml of ice-cold 4% p~o~~dehyde in phosPhate-biers saline (pH 7.4). The descending aorta was clamped off. The carotid artery bifurcation region which contained the carotid body and superior cervical ganglion was dissected and washed in 5% sucrose in phosphate-buffered saline (overnight at 4°C). The tissue was frozen on a metal chuck and 20 ,um sections were The
DISCUSSION The presence of SP-innnunoreactive fibers in the carotid body supports the hypothesis that SP is a neu~t~s~tte~ of
‘National Institute of General Medical Sciences, NIH, Bethesda, MD 20205.
195
JACOBOWITZ
FIG. 1. Carotid body. Substance P immunoreactive (arrows) coursing among the glomus cells (X380).
AND HELKE
varicose fibers
chemoreceptor afferent nerves [6]. Additional support is provided by the observation that an intracranial section of the IXth and Xth cranial nerves results in a substantial reduction in SP-immunoreactivity in the portions of the nucleus tractus solitarius where chemo- and baroreceptor afferents terminate [6]. The suggestion that SP is a sensory neurotransmitter in the carotid body, however, is not meant to imply that the total afferent neuronal population within the chemoreceptor exclusively contains this peptide. It is of interest that the glomus cells of the carotid body contain large amounts of catecholamines [1,71. In addition, they give a positive chromaffin reaction [lo], and are essentially identical to the chromffi cells of the adrenal medulla. Like the chromafEm cells of the adrenal medulla the carotid bodies are stimulated by anoxia, hypercapnia, and acidemia. Whether the catecholamines in glomus cells function in the initiation of the sensory discharge is not known. The chemoreceptor (chromaflin) cells may release catecholamines that act on the sensory nerve endings which lie in close apposition to them. Low concentrations of epinephrine infused into the carotid body increase the sensitivity of the
FIG. 2. Carotid body with carotid sinus nerve (CSN). Substance P immunoreactive fibers (arrows) are observed in the CSN and withk the carotid body. The autofluorescence contained within the glomus cells make it difficult to view the small delicate quick fading fibers, particularly at low magnification (X180).
chemoreceptors to other stimuli such as acetylcholine; large concentrations decrease it [5]. The close proximity of the sensory endings to the chromaffin cell, shown by electron micrograph studies [lo] suggests that catecholamine release may alter the discharge of sensory nerve impulses. The role of catechohtmine in the initiation of sensory discharge remains obscure, but a unique relationship between catecholamine and SP-containing sensory nerves is a distinct possibility.
SUBSTANCE
P IN THE CAROTID
197
BODY REFERENCES
1. Chiocchio, S. R., A. M. Biscardo and J. H. Tramezzani. S-Hydroxytryptamine in the carotid body of the cat. Science 158: 7%791, 1967. 2. Coons, A. H. In: General Cytochemical Methods, edited by J. Danielli. New York: Academic Press, 1958, pp. 399422. 3. Cuello, A. C. and I. Kanazawa. The distribution of substance P immunoreactive fibers in the rat central nervous sytem. J. camp. Neurol. 178: 129-156, 1978. 4. Davies, R. 0. and M. W. Edwards. Distribution of carotid body chemoreceptor atferents in the medulla of the cat. Brain Res. 64: 451-454, 1973. 5. Eyzaguirre, C., H. Koyano and J. B. Taylor. Presence of acetylcholine and transmitter release from carotid body chemoreceptors. J. Physiol., Lond. 178: 46W76, 1%5. 6. Gillis, R. A., C. J. Helke, B. L. Hamilton, W. P. Norman and D. hf. Jacobowitz. Evidence that substance P is a neurotransmitter of bare- and chemoreceptor afferents in nucleus tractus solitarius. Brain Res. 181: 4?6-I81, 1980. 7. Hamberger, B., M. Ritz& and J. Wersall. Demonstration of catecholamines and 5-hydroxytryptamine in the human carotid body. J. Pharmac. exp. Ther. 152: 197-201, 1966.
8. H&felt, T., J. Kellerth, G. Nilsson and B. Pemow. Experimental immunohistochemical studies on the localization and distribution of substance P in cat mimarv sensorv neurons. _ Brain Res. 100: 235-252, 1975. _ _ 9. Hdkfelt, T., R. Elde, K. Fuxe, 0. Johansson, A. Ljungdahl, hi. Goldstein. R. Luft. S. Efendic. G. Wilsson. L. Terenius, D. Ganten, S. L. Jeffcoate, J. Rehfeld, S. Said, M. Perez de la Mora, L. Possani, R. Tapia, L. Teran and R. Palacios. In: The Hypothalamus, edited by S. Reichlin, R. J. Baldessarini and J. B. Martin. New York: Raven Press. 1978. DD. 69-135. 10. Lever, J. D., P. R. Lewis and J. D. Boyd: bbservations on the fine structure and histochemistry of the carotid body in the cat and rabbit. .I. Anat. 93: 478-490, 1959. Il. Ljungdahl, A., T. H&felt and G. Nilsson. Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3: 861-943, 1978. 12. Lundberg, J. M., T. Hiikfelt, G. Nilsson, L. Terenius, J. Rehfeld, R. Aldle and S. Said. Peptide neurons in the vagus, splanchnic and sciatic nerves. Acta physiol. stand. 104: 499501, 1978.
Nerves in the Carotid Body
Received 14 December 1979 JACOBOWITZ, D. M. ANI) C. 3. HELKE. Localization of substance P immunoreactive nerves in the carotid body. BRAIN RES. BULL. 32) 195-197, 1980.-Immunofluorescent bistochemical observatian of the rat carotid body revealed the Presence of substance P immunoreactivefibers. This supportsthe hypothesisthat substanceP is a neurotransmitterof
chemoreceptorafferent nerves. Carotid body
Substance P
Chemoreceptor
Immunofluorescence
cut in a cryostat (-iYC) and mounted on chrom&alumcoated slides. The slides were placed into a container w&h substance-P antiserum (diluted 1500 with 0.3% T&on X-100 in phosphate-b~ered saline), and kept in the refrigemtor overnight. They were then washed three times for 5 min each time in 0.2% T&on X-100 in phosphate-biers saline and incubated for 20 min with fluorescein-~~1~ goat ~tis~~ to rabbit IgG (Cap@, Downington, PA) diluted 1:360 with phosphate-buffered saline containing 0.3% Triton X-100. They were washed again three times as above and for a fourth time in phosPhate-bread saline and subsequen~y mounted in glycerine phosphate-buffered saline (3:l). Siides were examined under a Leitz Orthoplan fluorescence microscope equipped with a Ploem illuminator. The substance P antibody (Immune Nuclear Corp., Stillwater, MN) showed negligible cross reactivity with the agents tested (met enkephalin,
THE carotid and aortic bodies contain specialized chemoreceptor cells which are found near the carotid bifurcations and the aortic arch. These chesnoreceptors are an important part of the respiratory and cardiovascular control systems. The sensory innervation of the carotid body is by way of the carotid sinus nerve, a branch of the glossopharyngeal nerve, which projects to the CNS and terminates in the nucleus tractus solitarius [4]. A neurotransmitter contained in the carotid sinus nerve has not been demonstrated. Immunohistochemical studies [3,11] have revealed the presence of substance P nerve terminals in the nucleus tractus solitarius of rats. SP immunoreactive fibers could be seen entering the brainstem via the glosopharyngeal nerve [3]. Furthermore, the nodose ganglion, a sensory ganglion whose fibers innervate the aortic body chemore&eptors and aortic arch baroreceptors, was shown to contain SP-immunoreactive cell bodies [12]. Recent work has provided evidence that SP may be a neurotransmitter at baro- and chemoreceptor tierents in the nucleus tractus solitarius 161. A high level of SP-like immunore~tivity was present in the petrosal and nodose ganglia. The above observation led us to formulate the hypothesis that the carotid body was innervated by SP-containing sensory nerve fibers, The present immunohistochemical study reports that SP-imm~or~ctive fibers are indeed contained within the carotid body.
RESULTS
The carotid body was easily identifiable by the morphologic appearance of the glomus cells which are arranged in loose cords around small blood vessels and capillaries, These cells are ovoid in shape and are autofluorescent which demonstrates their unique resemblance to adrenal medulla catecholamine cells as observed with the ~sto~uo~s~e~~e procedure. Many long substance P ~uno~~tive varicose fibers were observed to be interspersed among the glomus cells (Fig. 1). The carotid sinus nerve containing fasicles of substance P fibers could be seen entering the carotid body (Fig. 2).
METHOD
indirect i~unohis~~hemi~~ procedure of Coons [2] and H&felt et al. f9J was us& with minor mutations. Male Sprague-Hawley rats @O&300 g) were perfused through the ascending aorta with 100 ml of cold phosphatebuffered saline @H 7.4) followed by 200-300 ml of ice-cold 4% p~o~~dehyde in phosPhate-biers saline (pH 7.4). The descending aorta was clamped off. The carotid artery bifurcation region which contained the carotid body and superior cervical ganglion was dissected and washed in 5% sucrose in phosphate-buffered saline (overnight at 4°C). The tissue was frozen on a metal chuck and 20 ,um sections were The
DISCUSSION The presence of SP-innnunoreactive fibers in the carotid body supports the hypothesis that SP is a neu~t~s~tte~ of
‘National Institute of General Medical Sciences, NIH, Bethesda, MD 20205.
195
JACOBOWITZ
FIG. 1. Carotid body. Substance P immunoreactive (arrows) coursing among the glomus cells (X380).
AND HELKE
varicose fibers
chemoreceptor afferent nerves [6]. Additional support is provided by the observation that an intracranial section of the IXth and Xth cranial nerves results in a substantial reduction in SP-immunoreactivity in the portions of the nucleus tractus solitarius where chemo- and baroreceptor afferents terminate [6]. The suggestion that SP is a sensory neurotransmitter in the carotid body, however, is not meant to imply that the total afferent neuronal population within the chemoreceptor exclusively contains this peptide. It is of interest that the glomus cells of the carotid body contain large amounts of catecholamines [1,71. In addition, they give a positive chromaffin reaction [lo], and are essentially identical to the chromffi cells of the adrenal medulla. Like the chromafEm cells of the adrenal medulla the carotid bodies are stimulated by anoxia, hypercapnia, and acidemia. Whether the catecholamines in glomus cells function in the initiation of the sensory discharge is not known. The chemoreceptor (chromaflin) cells may release catecholamines that act on the sensory nerve endings which lie in close apposition to them. Low concentrations of epinephrine infused into the carotid body increase the sensitivity of the
FIG. 2. Carotid body with carotid sinus nerve (CSN). Substance P immunoreactive fibers (arrows) are observed in the CSN and withk the carotid body. The autofluorescence contained within the glomus cells make it difficult to view the small delicate quick fading fibers, particularly at low magnification (X180).
chemoreceptors to other stimuli such as acetylcholine; large concentrations decrease it [5]. The close proximity of the sensory endings to the chromaffin cell, shown by electron micrograph studies [lo] suggests that catecholamine release may alter the discharge of sensory nerve impulses. The role of catechohtmine in the initiation of sensory discharge remains obscure, but a unique relationship between catecholamine and SP-containing sensory nerves is a distinct possibility.
SUBSTANCE
P IN THE CAROTID
197
BODY REFERENCES
1. Chiocchio, S. R., A. M. Biscardo and J. H. Tramezzani. S-Hydroxytryptamine in the carotid body of the cat. Science 158: 7%791, 1967. 2. Coons, A. H. In: General Cytochemical Methods, edited by J. Danielli. New York: Academic Press, 1958, pp. 399422. 3. Cuello, A. C. and I. Kanazawa. The distribution of substance P immunoreactive fibers in the rat central nervous sytem. J. camp. Neurol. 178: 129-156, 1978. 4. Davies, R. 0. and M. W. Edwards. Distribution of carotid body chemoreceptor atferents in the medulla of the cat. Brain Res. 64: 451-454, 1973. 5. Eyzaguirre, C., H. Koyano and J. B. Taylor. Presence of acetylcholine and transmitter release from carotid body chemoreceptors. J. Physiol., Lond. 178: 46W76, 1%5. 6. Gillis, R. A., C. J. Helke, B. L. Hamilton, W. P. Norman and D. hf. Jacobowitz. Evidence that substance P is a neurotransmitter of bare- and chemoreceptor afferents in nucleus tractus solitarius. Brain Res. 181: 4?6-I81, 1980. 7. Hamberger, B., M. Ritz& and J. Wersall. Demonstration of catecholamines and 5-hydroxytryptamine in the human carotid body. J. Pharmac. exp. Ther. 152: 197-201, 1966.
8. H&felt, T., J. Kellerth, G. Nilsson and B. Pemow. Experimental immunohistochemical studies on the localization and distribution of substance P in cat mimarv sensorv neurons. _ Brain Res. 100: 235-252, 1975. _ _ 9. Hdkfelt, T., R. Elde, K. Fuxe, 0. Johansson, A. Ljungdahl, hi. Goldstein. R. Luft. S. Efendic. G. Wilsson. L. Terenius, D. Ganten, S. L. Jeffcoate, J. Rehfeld, S. Said, M. Perez de la Mora, L. Possani, R. Tapia, L. Teran and R. Palacios. In: The Hypothalamus, edited by S. Reichlin, R. J. Baldessarini and J. B. Martin. New York: Raven Press. 1978. DD. 69-135. 10. Lever, J. D., P. R. Lewis and J. D. Boyd: bbservations on the fine structure and histochemistry of the carotid body in the cat and rabbit. .I. Anat. 93: 478-490, 1959. Il. Ljungdahl, A., T. H&felt and G. Nilsson. Distribution of substance P-like immunoreactivity in the central nervous system of the rat. I. Cell bodies and nerve terminals. Neuroscience 3: 861-943, 1978. 12. Lundberg, J. M., T. Hiikfelt, G. Nilsson, L. Terenius, J. Rehfeld, R. Aldle and S. Said. Peptide neurons in the vagus, splanchnic and sciatic nerves. Acta physiol. stand. 104: 499501, 1978.