- Email: [email protected]
Serotonin-like immunoreactivity in rat carotid body
348
Brain Research, 276 (1983) 348--350 Elsevier
Serotonin-like immunoreactivity in rat carotid body M. GRONBLAD l, P. LIESI2and L. RECHARDT I 1Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, SF-00170Helsinki 17 and 2Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29 (Finland)
(Accepted March 15th, 1983) Key words: carotid body - - rat - - glomus cells - - serotonin-like immunoreactivity
Serotonin-like immunofluorescence was demonstrated in the glomus cells of the rat carotid body. Similar immunoreactivity was noted in mast cells in the organ, while no immunoreactive nerve fibers were seen. It is suggested that glomus cell serotonin could participate in the modulation of chemoreceptor activity. Biochemical studies indicate that serotonin is pres'ent in the rat carotid body 4, as well as in the carotid bodies of other species including manS. In the carotid body of the rat, serotonin was proposed to exert an excitatory effect on the sinus nerve activity 7. In the dog, the sinus nerve activity was inhibited by low doses of serotonin, while higher doses caused excitation followed by inhibition 1. Thus, it seems that the monoamine could have an important function as a modulator of chemoreception. The exact compartmental localization of the amine in the carotid body is, however, unknown. We now present serotonin-like immunoreactivity in the glomus cells of the carotid body of the adult rat, adding additional impact to its suggested role I in the carotid body function. Forty carotid bodies from 20 adult Sprague-Dawley albino rats (200-250 g) were used. The rats were injected intraperitoneaily with 150 mg/kg nialamide in 0.9% NaCI (Niamid, Pfizer) 6 h and 100 mg/kg L-tryptophan (Merck) in 0.9% NaC12 h before starting the fixation. In some experiments non-treated animals or animals pretreated with nialamide only were used. The fixation was carried out under Nembutal anesthesia by transcardial perfusion with 3.5% paraformaldehyde in 0.1'M phosphate buffer, pH 7.4 or with paraformaldehyde-glyoxylic acid fixatives, pH 4.5 or 6.6 (ref. 6). The excised carotid bodies were further immersed in the respective fixatives overnight. After rinsing in 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
the buffer for 24 h the carotid bodies were frozen in iso-pentane precooled with liquid nitrogen and cut in 10 ~tm cryostat sections. The sections were then immersed in methanol containing 0.5% H20 2 (Perhydrol, Merck, Darmstadt), rinsed in the buffer and stained with anti-serotonin antibodies diluted 1:50 (Cappel Laboratories, Cochranville, PA) for 1 h at room temperature, rinsed again and incubated with sheep anti-rabbit immunoglobulins (Wellcome, Beckenharn, U.K.) diluted 1:20 for 30 min at room temperature. The sections were mounted in phosphate buffer-glycerol (1:1). In control experiments the primary antiserum was substituted with an antiserum preabsorbed with 10 mg/ml serotonin (sodium salt, Sigma) or omitted. The cross-reactivity of the serotonin antibody with L-tryptophan was excluded by immunocytochemistry with a serotonin antibody preabsorbed with L-tryptophan (10 mg/ml; Merck, Darmstadt). The sections were viewed with a Leitz Dialux 20 EB microscope using an epi-illumination and a filter block suitable for FITC-fluorescence. All the pictures were taken with identical exposure times. Moderate or intense immunofluorescence was demonstrated in cell groups in all parts of the carotid body by using serotonin-antibody on the paraformaldehyde-glyoxylic acid fixed tissue (Fig. la). By using the same fixative at pH 6.6 similar results were obtained but the immunofluorescence intensity increased (Fig. lb). Absorption of the primary antise-
349
Fig. 1. Serotonin-like immunofluorescence in the rat carotid body demonstrated by using the acidic (a) or neutral (b) paraformaldehyde--glyoxylicacid fixed tissue, a shows cell groups of moderate immunofluorescence (arrows). Note the more intense reaction in b. Blood vessels (asterisks). a and b x 330. Fig. 2. Glomus cells fixed as in Fig. la, and stained by using serotonin antibody preabsorbed with the antigen. No positive structures are seen. )<330. rum with 10 mg/ml of serotonin totally abolished the immunofluorescence. Mast cells, occasionally observed in the sections exhibited constantly intense immunofluorescence. No positive serotonin immunoreactive nerve fibres were observed with any of the fixation methods. No serotonin-like immunoreactivity was seen in sections from untreated rats or in sections after nialamide-treatment only. Serotonin-like immunoreactivity has recently been demonstrated in the rat adrenal medullary cells tl and in the small intensely fluorescent (SIF) cells 3 of the rat sympathetic ganglion 12. The combined paraformaldehyde-glyoxylic acid fixation, p H 4.5 or 6.6 proved, in the present study, superior to paraformaldehyde fixation yielding a more intense immunofluorescence reaction. The specificity of this method was also confirmed by demonstrating intense immunoreactivity in the rat mesencephalic nuclei (Liesi, unpublished observations), known to contain serotoninergic neurons2,5, 9.
The cell clusters, which showed serotonin-like immunoreactivity were localized around blood vessels, suggesting that the immunoreactivity was present in the chief cells. Thus co-existence of different monoamines in the same glomus cells is possible. Our immunohistochemical observations confirm the previous biochemical findings by Hellstr6m and Koslow 4 and Steele and HinterbergerS and further suggest that serotonin is not only located in mast cells in the carotid body. Since in untreated rats or in rats treated with nialamide only, no serotonin-like immunoreactivity was seen in the carotid body, the uptake of exogenous serotonin into the glomus cells cannot be excluded. However, in hypothalamic nuclei similar preloading to ours was necessary to demonstrate serotonin-like immunoreactivity i n cell bodies10. If present in the glomus cells, serotonin could be a modulator of chemoreception.
1 Bisgard, G. E., Mitchell, R. A. and Herbert, D. A., Effects of dopamine, norepinephrine and 5-hydroxytryptamine on the carotid body of the dog, Resp. Physiol., 37 (1979) 61-80. 2 DahlstrOm, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Actaphysiol. Scand., 62 (1979) Suppl. 232.
3 Er~ink6, O. and Er/ink6, L., Small, intensely fluorescent granule-containing cells in the sympathetic ganglion of the. rat. In O. Er~ink6 (Ed.), Histochemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 39-51. 4 Hellstr6m, S. and Koslow, S. H., Biogenic amines in carotid body of adult and infant rats - - a gas chromatographic-
350
5
6
7
8
9
mass spectrometric assay, Acta physiol. Scand.. 93 (1975) 540-547 Liesi. P. and Recbardt, L.. Acetylcholinesterase activity coexists with monoamine fluorescence in a subpopulation of mesencephalic raphe neurons, Neurosci. Lett.. submitted. Loren. I.. BjOrklund, A., Falck, B. and Lindvall, O.. An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH, Histochemistry, 49 (1976) 17%192. Sapru. H. N. and Krieger. A. J.. Effect of 5-hydroxytryptamine on the peripheral chemoreceptors in the rat. Res. Comm. Chem. Pathol. Pharrnacol. . 16 (19771245-250. Steele. R. H. and Hinterberger. H.. Catecholamines and 5-hydroxytryptamine in the carotid body in vascular, respiratory and other diseases. J. Lab. clin Med.. 80 (1972) 63-70. Steinbusch. H. W. M., Distribution of serotonin-immuno-
reactivity in the central nervous system of the rat - - cell bodies and terminals, Neuroscience, 6 (1981) 557-619. 10 Steinbusch, H. W. M., Verhofstad, A. A. J., Joosten, H. W. J. and Goldstein, M., Serotonin-immunoreactive cell bodies in the nucleus dorsomedialis hypothalami, in the substantia nigra, and in the area tegmentalis ventralis of tsai: observations after pharmacological manipulations in the rat. In Cytochemical Methods in Neuroanatomy. Alan R. Liss, New York, 1982, pp. 407-421. 11 Verhofstad, A. and Jonsson, G., Immunohistochemical and biochemical evidence for the presence of serotonin in the rat adrenal medulla, Anat. Rec., 202 (1982) 196A. 12 Verhofstad, A. A. J., Steinbusch, H. W. M., Penke, B.. Varga, J. and Joosten, H. W. J., Serotonin-immunoreactive cells in the superior cervical ganglion of the rat: evidence for the existence of separate serotonin- and catccholamine-containing small ganglionic cells, Brain Research, 212 (1981) 39-49.
Brain Research, 276 (1983) 348--350 Elsevier
Serotonin-like immunoreactivity in rat carotid body M. GRONBLAD l, P. LIESI2and L. RECHARDT I 1Department of Anatomy, University of Helsinki, Siltavuorenpenger 20, SF-00170Helsinki 17 and 2Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki 29 (Finland)
(Accepted March 15th, 1983) Key words: carotid body - - rat - - glomus cells - - serotonin-like immunoreactivity
Serotonin-like immunofluorescence was demonstrated in the glomus cells of the rat carotid body. Similar immunoreactivity was noted in mast cells in the organ, while no immunoreactive nerve fibers were seen. It is suggested that glomus cell serotonin could participate in the modulation of chemoreceptor activity. Biochemical studies indicate that serotonin is pres'ent in the rat carotid body 4, as well as in the carotid bodies of other species including manS. In the carotid body of the rat, serotonin was proposed to exert an excitatory effect on the sinus nerve activity 7. In the dog, the sinus nerve activity was inhibited by low doses of serotonin, while higher doses caused excitation followed by inhibition 1. Thus, it seems that the monoamine could have an important function as a modulator of chemoreception. The exact compartmental localization of the amine in the carotid body is, however, unknown. We now present serotonin-like immunoreactivity in the glomus cells of the carotid body of the adult rat, adding additional impact to its suggested role I in the carotid body function. Forty carotid bodies from 20 adult Sprague-Dawley albino rats (200-250 g) were used. The rats were injected intraperitoneaily with 150 mg/kg nialamide in 0.9% NaCI (Niamid, Pfizer) 6 h and 100 mg/kg L-tryptophan (Merck) in 0.9% NaC12 h before starting the fixation. In some experiments non-treated animals or animals pretreated with nialamide only were used. The fixation was carried out under Nembutal anesthesia by transcardial perfusion with 3.5% paraformaldehyde in 0.1'M phosphate buffer, pH 7.4 or with paraformaldehyde-glyoxylic acid fixatives, pH 4.5 or 6.6 (ref. 6). The excised carotid bodies were further immersed in the respective fixatives overnight. After rinsing in 0006-8993/83/$03.00 © 1983 Elsevier Science Publishers B.V.
the buffer for 24 h the carotid bodies were frozen in iso-pentane precooled with liquid nitrogen and cut in 10 ~tm cryostat sections. The sections were then immersed in methanol containing 0.5% H20 2 (Perhydrol, Merck, Darmstadt), rinsed in the buffer and stained with anti-serotonin antibodies diluted 1:50 (Cappel Laboratories, Cochranville, PA) for 1 h at room temperature, rinsed again and incubated with sheep anti-rabbit immunoglobulins (Wellcome, Beckenharn, U.K.) diluted 1:20 for 30 min at room temperature. The sections were mounted in phosphate buffer-glycerol (1:1). In control experiments the primary antiserum was substituted with an antiserum preabsorbed with 10 mg/ml serotonin (sodium salt, Sigma) or omitted. The cross-reactivity of the serotonin antibody with L-tryptophan was excluded by immunocytochemistry with a serotonin antibody preabsorbed with L-tryptophan (10 mg/ml; Merck, Darmstadt). The sections were viewed with a Leitz Dialux 20 EB microscope using an epi-illumination and a filter block suitable for FITC-fluorescence. All the pictures were taken with identical exposure times. Moderate or intense immunofluorescence was demonstrated in cell groups in all parts of the carotid body by using serotonin-antibody on the paraformaldehyde-glyoxylic acid fixed tissue (Fig. la). By using the same fixative at pH 6.6 similar results were obtained but the immunofluorescence intensity increased (Fig. lb). Absorption of the primary antise-
349
Fig. 1. Serotonin-like immunofluorescence in the rat carotid body demonstrated by using the acidic (a) or neutral (b) paraformaldehyde--glyoxylicacid fixed tissue, a shows cell groups of moderate immunofluorescence (arrows). Note the more intense reaction in b. Blood vessels (asterisks). a and b x 330. Fig. 2. Glomus cells fixed as in Fig. la, and stained by using serotonin antibody preabsorbed with the antigen. No positive structures are seen. )<330. rum with 10 mg/ml of serotonin totally abolished the immunofluorescence. Mast cells, occasionally observed in the sections exhibited constantly intense immunofluorescence. No positive serotonin immunoreactive nerve fibres were observed with any of the fixation methods. No serotonin-like immunoreactivity was seen in sections from untreated rats or in sections after nialamide-treatment only. Serotonin-like immunoreactivity has recently been demonstrated in the rat adrenal medullary cells tl and in the small intensely fluorescent (SIF) cells 3 of the rat sympathetic ganglion 12. The combined paraformaldehyde-glyoxylic acid fixation, p H 4.5 or 6.6 proved, in the present study, superior to paraformaldehyde fixation yielding a more intense immunofluorescence reaction. The specificity of this method was also confirmed by demonstrating intense immunoreactivity in the rat mesencephalic nuclei (Liesi, unpublished observations), known to contain serotoninergic neurons2,5, 9.
The cell clusters, which showed serotonin-like immunoreactivity were localized around blood vessels, suggesting that the immunoreactivity was present in the chief cells. Thus co-existence of different monoamines in the same glomus cells is possible. Our immunohistochemical observations confirm the previous biochemical findings by Hellstr6m and Koslow 4 and Steele and HinterbergerS and further suggest that serotonin is not only located in mast cells in the carotid body. Since in untreated rats or in rats treated with nialamide only, no serotonin-like immunoreactivity was seen in the carotid body, the uptake of exogenous serotonin into the glomus cells cannot be excluded. However, in hypothalamic nuclei similar preloading to ours was necessary to demonstrate serotonin-like immunoreactivity i n cell bodies10. If present in the glomus cells, serotonin could be a modulator of chemoreception.
1 Bisgard, G. E., Mitchell, R. A. and Herbert, D. A., Effects of dopamine, norepinephrine and 5-hydroxytryptamine on the carotid body of the dog, Resp. Physiol., 37 (1979) 61-80. 2 DahlstrOm, A. and Fuxe, K., Evidence for the existence of monoamine-containing neurons in the central nervous system, Actaphysiol. Scand., 62 (1979) Suppl. 232.
3 Er~ink6, O. and Er/ink6, L., Small, intensely fluorescent granule-containing cells in the sympathetic ganglion of the. rat. In O. Er~ink6 (Ed.), Histochemistry of Nervous Transmission, Progress in Brain Research, Vol. 34, Elsevier, Amsterdam, 1971, pp. 39-51. 4 Hellstr6m, S. and Koslow, S. H., Biogenic amines in carotid body of adult and infant rats - - a gas chromatographic-
350
5
6
7
8
9
mass spectrometric assay, Acta physiol. Scand.. 93 (1975) 540-547 Liesi. P. and Recbardt, L.. Acetylcholinesterase activity coexists with monoamine fluorescence in a subpopulation of mesencephalic raphe neurons, Neurosci. Lett.. submitted. Loren. I.. BjOrklund, A., Falck, B. and Lindvall, O.. An improved histofluorescence procedure for freeze-dried paraffin-embedded tissue based on combined formaldehyde-glyoxylic acid perfusion with high magnesium content and acid pH, Histochemistry, 49 (1976) 17%192. Sapru. H. N. and Krieger. A. J.. Effect of 5-hydroxytryptamine on the peripheral chemoreceptors in the rat. Res. Comm. Chem. Pathol. Pharrnacol. . 16 (19771245-250. Steele. R. H. and Hinterberger. H.. Catecholamines and 5-hydroxytryptamine in the carotid body in vascular, respiratory and other diseases. J. Lab. clin Med.. 80 (1972) 63-70. Steinbusch. H. W. M., Distribution of serotonin-immuno-
reactivity in the central nervous system of the rat - - cell bodies and terminals, Neuroscience, 6 (1981) 557-619. 10 Steinbusch, H. W. M., Verhofstad, A. A. J., Joosten, H. W. J. and Goldstein, M., Serotonin-immunoreactive cell bodies in the nucleus dorsomedialis hypothalami, in the substantia nigra, and in the area tegmentalis ventralis of tsai: observations after pharmacological manipulations in the rat. In Cytochemical Methods in Neuroanatomy. Alan R. Liss, New York, 1982, pp. 407-421. 11 Verhofstad, A. and Jonsson, G., Immunohistochemical and biochemical evidence for the presence of serotonin in the rat adrenal medulla, Anat. Rec., 202 (1982) 196A. 12 Verhofstad, A. A. J., Steinbusch, H. W. M., Penke, B.. Varga, J. and Joosten, H. W. J., Serotonin-immunoreactive cells in the superior cervical ganglion of the rat: evidence for the existence of separate serotonin- and catccholamine-containing small ganglionic cells, Brain Research, 212 (1981) 39-49.