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Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo

1999; Wiley; Volume: 255; Issue: 4 Linguagem: Inglês

10.1002/(sici)1097-0185(19990801)255

1097-0185

Marlies E. Verberne, Adriana C. Gittenberger–de Groot, Liesbeth van Iperen, Robert E. Poelmann,

Cardiovascular Conditions and Treatments

The Anatomical RecordVolume 255, Issue 4 p. 407-419 ArticleFree Access Contribution of the cervical sympathetic ganglia to the innervation of the pharyngeal arch arteries and the heart in the chick embryo Marlies E. Verberne, Marlies E. Verberne Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorAdriana C. Gittenberger-De Groot, Adriana C. Gittenberger-De Groot Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorLiesbeth Van Iperen, Liesbeth Van Iperen Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorRobert E. Poelmann, Corresponding Author Robert E. Poelmann Poelmann@RULLF2.LeidenUniv.NL Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsDepartment of Anatomy and Embryology, Leiden University Medical Center, PO Box 9602, 2300 RC Leiden, The Netherlands.Search for more papers by this author Marlies E. Verberne, Marlies E. Verberne Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorAdriana C. Gittenberger-De Groot, Adriana C. Gittenberger-De Groot Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorLiesbeth Van Iperen, Liesbeth Van Iperen Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsSearch for more papers by this authorRobert E. Poelmann, Corresponding Author Robert E. Poelmann Poelmann@RULLF2.LeidenUniv.NL Department of Anatomy and Embryology, Leiden University Medical Center, 2300 RC Leiden, The NetherlandsDepartment of Anatomy and Embryology, Leiden University Medical Center, PO Box 9602, 2300 RC Leiden, The Netherlands.Search for more papers by this author First published: 02 December 1999 https://doi.org/10.1002/(SICI)1097-0185(19990801)255:4 3.0.CO;2-4Citations: 20AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract In the chick heart, sympathetic innervation is derived from the sympathetic neural crest (trunk neural crest arising from somite level 10–20). Since the trunk neural crest gives rise to sympathetic ganglia of their corresponding level, it suggests that the sympathetic neural crest develops into cervical ganglia 4–14. We therefore tested the hypothesis that, in addition to the first thoracic ganglia, the cervical ganglia might contribute to cardiac innervation as well. Putative sympathetic nerve connections between the cervical ganglia and the heart were demonstrated using the differentiation markers tyrosine hydroxylase and HNK-1. In addition, heterospecific transplantation (quail to chick) of the cardiac and trunk neural crest was used to study the relation between the sympathetic neural crest and the cervical ganglia. Quail cells were visualized using the quail nuclear antibody QCPN. The results by immunohistochemical study show that the superior and the middle cervical ganglia and possibly the carotid paraganglia contribute to the carotid nerve. This nerve subsequently joins the nodose ganglion of the vagal nerve via which it contributes to nerve fibers in cardiac vagal branches entering the arterial and venous pole of the heart. In addition, the carotid nerve contributes to nerve fibers connected to putative baro- and chemoreceptors in and near the wall of pharyngeal arch arteries suggesting a role of the superior and middle cervical ganglia and the paraganglia of the carotid plexus in sensory afferent innervation. The lower cervical ganglia 13 and 14 contribute predominantly to nerve branches entering the venous pole via the anterior cardinal veins. We did not observe a thoracic contribution. Heterospecific transplantation shows that the cervical ganglia 4–14 as well as the carotid paraganglia are derived from the sympathetic neural crest. The cardiac neural crest does not contribute to the neurons of the cervical ganglia. We conclude that the cervical ganglia contribute to cardiac innervation which explains the contribution of the sympathetic neural crest to the innervation of the chick heart. Anat Rec 255:407–419, 1999. © 1999 Wiley-Liss, Inc. Abbreviatioins: AA, aortic arch artery; AD, adrenal; AM, atrial myocardium; Ao, aorta; AoO, aortic orifice; AVS, atrioventricular sulcus; CA, coronary artery; CCA, common carotid artery; CG, cardiac ganglia; 8CSG, 8th cervical SG; CST, cervical sympathetic trunk; 13CSG, 13th cervical SG; CN, carotid nerve; DA, dorsal aorta; GN, glossopharyngeal nerve; H, heart; ICA, internal carotid artery; IVS, interventricular septum; JV, jugular vein; L, lung; LA, left atrium; LCA, left coronary artery; LICA, left internal carotid artery; LV, left ventricle; NG, nodose ganglion; NT, neural tube; PO, pulmonary orifice; PG, carotid paraganglion; RA, right atrium; RACV, right anterior cardinal vein; RCB, right carotid body; RCCA, right common carotid artery; RCSG, right 13th cervical SG; RDAB, right dorsal arterial vagal branch; RDAS, right ductus arteriosus; RICA, right internal carotid artery; RJV, right jugular vein; RL, right lung; RNG, right nodose ganglion; RPA, right pulmonary artery; RSCA, right subclavian artery; RV, right ventricle; S, aorticopulmonary septum; SCG, superior cervical SG; SEP, subepicardium; SN, spinal nerve; SPG, spinal ganglion; T, transplant; T1, first thoracic SG; T7, seventh thoracic SG; VN, vagal nerve. It is reported that in both embryonic (Kirby et al., 1980) and adult chickens (Baumel, 1975) the sympathetic cardiac nerves project from the first pair of thoracic sympathetic ganglia (SG) and reach the heart at day 10–11 of incubation (Higgins and Pappano, 1979; Kirby et al., 1980). These thoracic SG are part of the paired sympathetic trunks. Each trunk consists on average of 37 SG: 14 cervical, 7 thoracic, 13 synsacral, and 3 caudal (Baumel, 1975). These SG originate from the trunk neural crest cells (Le Douarin and Teillet, 1974), migrating from the neural tube caudal from somite level 5 (Kirby and Bockman, 1984). The trunk neural crest from somite level 10–20 (also named the sympathetic neural crest) is shown to be important for the innervation of the heart. Ablation of this area results in a significant depletion of the norepinephrine uptake in the atrium, the absence of sympathetic trunks in the corresponding region and the absence of sympathetic nerves in the atria (Kirby and Stewart, 1984). The trunk neural crest gives rise to the SG of their corresponding somite level (Kirby and Stewart, 1984; Yip, 1983,1986) suggesting that the sympathetic neural crest develops into the cervical SG 4–14. We want to hypothesize that, in addition to the first thoracic SG, the cervical SG might contribute to the cardiac innervation as well. This, as well as reports of a cervical contribution to cardiac innervation in several mammalian species (Randall et al., 1971; Armour and Randall, 1975; Janes et al., 1986), led us to investigate in detail the contribution of the cervical SG to cardiac innervation in the chick embryo. In addition, we studied the neural crest origin of the cervical SG to show their relationship with the sympathetic neural crest. In the present study nerve connections between the cervical SG and the heart are studied by using an antibody against tyrosine hydroxylase (TH), an enzyme involved in catecholamine synthesis and considered to be a marker for sympathetic nerve tissue. This is combined with heterospecific but homotopic transplantation (quail-chick chimera) of different levels of the neural crest, i.e. parts of either the cranial neural crest [this is the neural crest migrating cranial from somite level 6 as described by Kirby and Bockman (1984)], or the trunk neural crest (caudal from somite 5). With these results we discuss the possible function of TH-positive nerve fibers entering the heart and pharyngeal arch arteries (PAA), the specificity of TH-staining for sympathetic nerves and the homology of cardiac innervation between different species. MATERIALS AND METHODS Chimera Technique Fertilized eggs of the White Leghorn chick (Gallus domesticus) and Japanese quail (Coturnix coturnix japonica) were incubated at 37°C for 41–48 hr and 37–46 hr respectively, i.e. until the embryos reached stage HH10–13 (Hamburger and Hamilton, 1951). The eggs were opened and the embryos stained lightly with nile blue sulphate. The dorsal part of the neural tube was removed at different levels using sharpened tungsten needles. The homotopic part of a quail embryo at the comparable developmental stage was excised and transplanted into the exposed site in the chick embryo. Transplantation of the cardiac neural crest, between the centre of the otic vesicle (MO) and the third somite (S3), was performed at stage HH10. Likewise, different levels of the trunk neural crest were transplanted: between somite level 4–9 (S4–9) at stage HH11, S10–15 at stage HH12 and S16–20 at stage HH13. Surviving embryos were sacrificed at stage HH28+ (n = 1), HH29 (n = 1), HH29+ (n = 1), HH35 (n = 3), HH36 (n = 4), HH37 (n = 6), HH37+ (n = 1), HH38 (n = 3) and HH39 (n = 1). They were fixed in ethanol-acetic acid for 24–72 hr and embedded in paraffin. Histology Serial 5 μm sections of normal chick embryos (n = 3) (fixed in ethanol-acetic acid for 24–72 hr and embedded in paraffin) and quail-chick chimeras (n = 21) were alternatingly incubated with TH antibody (Boehringer Mannheim, Germany), a marker for sympathetic nerve tissue, and with HNK-1 (American tissue type connection, see Abo and Balch, 1981), a marker for migrating neural crest and nerve tissue (Luider et al., 1993; Poelmann et al., 1994). TH (1:50) and HNK-1 (1:10) were diluted in phosphate-buffered saline (PBS) containing 0.05% Tween-20 (Merck, Schuchardt) and 1% Ovalbumine (Sigma). In addition, quail-chick chimera sections were incubated with QCPN (undiluted), a quail cell nuclear marker (Hybridoma Bank, Iowa City). The incubation with the first antibody lasted overnight. All sections were incubated for 1.5 hr in the second rabbit anti-mouse antibody conjugated to peroxidase and diluted in the same buffer (1:300). The sections incubated for TH were further incubated with a third goat anti-rabbit antibody (1:50; Nordic Immunology, Tilburg), followed by a fourth antibody, the rabbit peroxidase-antiperoxidase complex (1:500; Nordic Immunology, Tilburg) both for 1.5 hr in the same buffer as used for the first and second antibodies. After treatment of all sections with 0.04% diaminobenzidine tetrahydrochloride (DAB)/0.06‰ H2O2 in 0.05 mol/L TRIS-maleic acid (pH 7.6) for 10 min at room temperature, they were briefly counterstained with Mayer's haematoxylin, dehydrated via ethanol, transferred to xylene and coverslipped using Entellan (Merck, Darmstadt). In the quail-chick chimeras, neurons derived from the transplanted neural crest can be identified when they are immunopositive both for the quail marker QCPN and the differentiation markers TH and HNK-1. Normal embryos from stage HH37, HH40 and HH42 were used as controls to check whether the HNK-1 and TH staining pattern found in the quail-chick chimeras can be considered as normal. No differences were observed in HNK-1 and TH staining patterns between quail-chick chimeras and normal chick embryos of similar stages. RESULTS Transverse sections of quail-chick chimeras (HH28+39), incubated with QCPN, were examined to study the origin of the cervical SG. Table 1 summarizes the results of the quail-chick chimeras. The transplant often partially attached to the ablated region and/or showed an ingrowth in the adjacent caudal non-ablated region (Table 1). Table 1. Neural crest origin of cervical sympathetic ganglia Somite level (S) ablation Stage Somite level (S) transplant Somite level (S) QCPN spinal ganglia Somite level (S) QCPN positive ganglia Corresponding sympathetic trunk level MO–S3 HH35 — — S6, 7 1 MO–S3 HH35 — — S6, 7 1 S4–9 HH28+ S5–9 S6–8 S6–10 1–4Caa C, cervical sympathetic ganglion. S4–9 HH36 S6–13 S11 S6–14 1–8C S5–9 HH29+ S6–8 S6–7 S6–8 1, 2C S7–9 HH35 S8–10 S8–10 S6–12 1–6C S10–12 HH37 S10–11 S9–11 S6–12 1–6C S10–12 HH36 S12–13 S11–14 S12–15 6–9C S10–14 HH37+ S10–11 S10#–11 S10#bb #, further cranial not determined. –15 4#–9C S10–14 HH38 S10–11 S13 S12#–19 6#–13C S10–14 HH37 S12–13 S9#–13 S9#–17 3#–12C S10–14 HH38 S12–14 S12#–13 S12#–15 6#–9C S10–14 HH38 S12–16 S11#–17 S11#–20 5#–14C S10–15 HH39 S13–14 — S9–18 3–12C S10–14 HH37 S15–16 S14 S14–17 8–11C S10–14 HH37 S16–17 S15–16 S12–19 6–13C S11–14 HH37 S15–17 — S6, 12, 13, 15–21 1, 6, 7, 9–14C, 1Tcc T, thoracic sympathetic ganglion. S12–13 HH36 S12–13 S11–14 S9–14 3–8C S12–13 HH36 S12–13 S13–15 S12–15 6–9C S13–14 HH37 S14–15 S13–16 S10–21 4–14C, 1T S16–20 HH29 S19–22 S19–21 S20–23 14C, 1–3T a C, cervical sympathetic ganglion. b #, further cranial not determined. c T, thoracic sympathetic ganglion. In general the highest number of QCPN-positive neuronal cells are situated in the SG at the corresponding levels of the transplant. The number of quail-derived cells decreases in SG at greater distance from the transplant (Fig. 1). In some embryos the cranial spread could not be determined due to the removal of a larger part of the neck region. On average, QCPN-positive neuronal cells are observed in SG up to two somite levels cranial and three levels caudal from the attached transplant. A much smaller longitudinal spread of QCPN-positive neurons is present in the spinal ganglia as compared to the SG (Table 1). Figure 1Open in figure viewerPowerPoint a: Transverse section of an HH38 S12–14 quail-chick chimera stained with QCPN (×40). Note that at the level of the transplant (T), located in the dorsal part of the neural tube (NT), the cervical SG (CSG) as well as the spinal ganglia (SPG) are QCPN-positive. b–e: Transverse sections of an HH38 S12-S16 quail-chick chimera stained with QCPN. b: Note that the 12th cervical SG (CSG) two levels caudal from the transplant contains a large number of QCPN-positive cells (×41). c: Magnification of the cervical SG in b (×205). d: At the level of the 14th cervical SG (CSG) four levels caudal from the transplant (×41). e: Magnification of d (×210). Note that the 14th cervical SG (CSG) contains only a few QCPN-positive cells (arrows). In addition, sections incubated with TH of both normal chick embryos (HH37–42) and the quail-chick chimeras (n = 15, HH36–39) were examined for nerve connections between the cervical SG and the heart. Figure 2 gives a schematic overview of the sympathetic trunk and its nerve connections with the heart in a stage HH37 chick embryo. Three main levels in the cervical sympathetic trunk (superior cervical, middle cervical and lower cervical SG) contain TH-positive nerve connections with the heart in the normal embryos and quail-chick chimeras. Figure 2Open in figure viewerPowerPoint Schematic sagittal section of a chicken embryo of about HH37. It presents an overview of three levels of nerve connections (arrows) between the right cervical sympathetic trunk and the heart. The levels of the transverse sections of Figures 3, 4 and 5 are indicated. To the left the corresponding somite levels (MO-S21) of origin are indicated. The most cranial arrow points to the level where the superior cervical SG (SCG) changes into the carotid nerve (CN). The middle arrow points at the nerve connections between the middle cervical SG (CSG) 7–10 and the carotid nerve (CN). The most caudal arrow points at the connection between the 13th cervical sympathetic ganglion (13CSG) and the jugular vein (JV). The Superior Cervical Sympathetic Ganglia At the level of the postotic hindbrain, the internal carotid arteries are in close association with the superior cervical SG (the most cranial part of the paired cervical sympathetic trunks, Fig. 2), the cranial nerves IX and X and the jugular vein (Fig. 3a–c). Tyrosine hydroxylase antibody stains very specifically the SG and their nerve branches while other nerve branches, e.g. the vagal nerve and the glossopharyngeal nerve are TH-negative (Fig. 3b). In the trunk neural crest chimeras, caudal to S5, chimeric SG contain both QCPN-positive neurons and satellite cells (Fig. 3d). In the cardiac neural crest chimeras (MO-S3), QCPN-staining is confined to the satellite and supportive cells of the superior cervical SG (Fig. 3e), while the neuronal somata are QCPN-negative. Each superior cervical SG sends a branch, the carotid nerve, along the internal carotids in the neck, which is TH-positive (Fig. 2). Figure 3Open in figure viewerPowerPoint Transverse alternate sections of the upper part of the neck of an HH36 S6–13 quail-chick chimera stained with (a) HNK-1 (×98), (b) TH (×96) and (c) QCPN (×127). The superior cervical SG (SCG) in c is shown in higher magnification in d (×411). Note that the superior cervical SG (SCG) is positive for TH (b) and QCPN (c,d) while the glossopharyngeal (GN) and vagal (VN) nerves are negative for these markers (b,c). All nerves are positive for HNK-1 (a). Note that the superior cervical SG (SCG) contains QCPN-positive neurons (arrows) and satellite cells (arrowheads) (d). e: Transverse section of the superior cervical SG (SCG) (×485) in an HH39 MO-S3 (cardiac neural crest) quail-chick chimera stained with QCPN. Note that only the satellite cells (arrowheads) are QCPN-positive. The much larger neuronal somata are negative (arrows). Cervical Sympathetic Ganglia 7–10 At the level between the seventh and 10th cervical SG, nerve connections are present between the cervical SG and the carotid nerves (Fig. 2, 4a). These connections are less prominent from stage HH40 onwards. The carotid nerves form a carotid plexus at these levels, which contains TH-positive paraganglia (Fig. 2, 4b). These paraganglia of the carotid plexus contain QCPN-positive cells in quail-chick chimeras (n = 10) in which the transplant adhered somewhere between the levels S10-S14 (Fig. 4c). Quail-chick chimeras in which the transplant attached over levels S15-S17 (n = 3) show no QCPN-positive paraganglia (n = 2) or these ganglia contain only a few QCPN-positive cells (n = 1). The cervical SG 2–6 do not contain nerve connections with the carotid nerves (Fig. 2). Figure 4Open in figure viewerPowerPoint a: Transverse section in the upper part of the neck of an HH37 S15–17 quail-chick chimera (×132) showing a TH-positive connection (arrow) between the cervical sympathetic trunk (CST) just caudal from the 8th cervical SG and the carotid nerves (CN) along the right (RICA) and left (LICA) internal carotid arteries. b,c: Transverse alternate sections in the neck of an HH38 S12–16 quail-chick chimera (×220) showing a paraganglion (PG) of the carotid plexus which is both positive for TH (b) and QCPN (c). Cervical Sympathetic Ganglia 13 and 14 At the level of the 12th cervical sympathetic ganglia, the carotid nerve joins the nodose ganglion of the vagal nerve as well as the recurrent nerve (Fig. 2, 5a). Near the nodose ganglia both the carotid bodies and the media of the common carotid arteries contain TH-positive nerve fibers and cells (Fig. 5a–c). The 13th cervical SG (and also the 12th, although less prominent) sends branches via the ventral rami of the spinal nerve along the vertebral vein towards the jugular vein (Fig. 2, 5d). The 14th cervical SG send nerve fiber branches via the ventral rami towards the subclavian veins, joining the nerve branches along the anterior cardinal veins (Fig. 2, 5e). The nerve branches from the thoracic SG travel ventrolateral as well as ventromedial from the lungs but do not reach the heart (Fig. 2). The ventromedial branches join the plexus around the dorsal aorta and to some extent are in contact with the pulmonary plexus and the dorsal part of the plexus around the oesophagus. The branches which move ventrolateral from the lungs bend into a lateral direction towards the bodywall (Fig. 5f). Figure 5Open in figure viewerPowerPoint a: Transverse section (×220) at the level of the right common carotid artery (RCCA) and the right nodose ganglion (RNG) in an HH37 embryo stained with TH. Note the TH-positive branches (arrows) from the carotid nerve and right carotid body (RCB) joining the nodose ganglion (RNG) of the vagal nerve. b,c: Transverse alternate sections of an HH38 embryo at the level of the right common carotid artery (RCCA) stained with HNK-1 (b) and TH (c) (×205). Note that nerve fibers and eventually cells (arrows) are positive for TH in the media of the common carotid artery (RCCA) as well as in the carotid body (RCB) (c). These fibers and cells are also positive for HNK-1 (arrows in b). d,e: Transverse sections of an HH38 embryo stained with TH (×44). Note a TH-positive nerve branch (arrow in d) from the right 13th cervical SG (RCSG) running along the spinal nerve (SN) towards the right jugular vein (RJV) (d). e: Note a TH-positive branch (arrows) from the 14th cervical SG running towards the right anterior cardinal vein (RACV) at the level where the right subclavian artery (RSCA) joins the common carotid artery (RCCA). f: Transverse section of an HH37 embryo stained with HNK-1 (×44). Note the nerve branch from the first thoracic SG, traveling ventrolateral from the left lung (L) and bending in a lateral direction towards the bodywall (arrowheads). Arrows show the directions where the neural tube (NT) and heart (H) are located. Arterial and Venous Pole At the level where the vagal nerve crosses the fourth PAA, TH-positive nerve branches and cells are present in the media of the dorsal part of the fourth PAA in eight of 12 embryos (Fig. 6a,b). At the level of the sixth PAA, TH-positive nerve branches from the vagal and recurrent nerves join the arterial cardiac vagal branches which travel along the pulmonary arteries (sixth PAA) towards the arterial pole of the heart (Fig. 6c,d). At the arterial pole TH-positive nerve fibers and cells are located at the cranial part of the aortico-pulmonary septum where the arterial vagal cardiac branches connect (Fig. 6e,f). In the thorax ventral to the lungs near the venous pole of the heart, nerve branches along the anterior cardinal veins are in contact with the TH-positive branches along the vagal nerves (Fig. 6g). Figure 6Open in figure viewerPowerPoint a,b: Transverse alternate sections of the dorsal part of the 4th PAA of an HH37 embryo stained for HNK-1 (×106) (a) and TH (×121) (b). Note that some nerve fibers and/or cells (arrows) in the media of the 4th PAA (AA) are both positive for HNK-1 (a) and TH (b). c: Transverse section of an HH42 embryo stained with TH (×41). Note TH-positive nerve fibers (arrows) in the right dorsal arterial vagal branch (RDAB) traveling along the right pulmonary artery (RPA) towards the arterial pole of the heart. d: Magnification of the area indicated in c (×103). Note that only fibers are stained with TH. e,f: Transverse alternate sections of the aorticopulmonary septum (S) of an HH37 embryo stained with HNK-1 (×91) (e) and TH (×83) (f). Note that a few HNK-1-positive fibers and/or cells (arrows in e) are also positive for TH (arrows in f). g: Transverse section at the level of the arterial pole of an HH42 embryo stained with TH (×44). Note that nerve branches along the right anterior cardinal vein (RACV) are in contact with TH-positive nerve branches of the vagal nerve (VN) (see arrows). Intracardiac Nerves and Ganglia At the arterial pole and along the ventral side of the heart between stage HH37–40, TH-positive nerve fibers are situated close to the aortic and pulmonary orifices. At the venous pole and dorsal side of the heart nerve fibers are present in the sinal cardiac vagal branch and branches along the anterior cardinal veins close to the orifices of the large veins near the sinus venosus. At stage HH42, at the ventral side of the heart nerve fibers are located deeper into the heart, i.e. in cardiac ganglia and nerve fiber bundles reaching the atrioventricular sulcus, especially around coronary arteries (Fig. 7a). At the dorsal side of the stage HH42 heart, TH-positive nerve fibers are present in nerve bundles and cardiac ganglia in the subepicardium of the atria unto the dorsal upper part of the ventricles and just caudal from the sinus venosus (Fig. 7b). The ventricular myocardium near the base of the heart including the upper part of the ventricular septum as well as the atrial myocardium, including the ventral part of the interatrial septum bordering the left ventricular outflow tract, contain a few nerve fibers (Fig. 7c,d). The neurons in the cardiac ganglia at both the dorsal and ventral side of the heart are TH-negative in all trunk neural crest chimeras as well as the normal embryos. The cardiac ganglia are QCPN-negative in the trunk neural crest chimeras (n = 19), in contrast with the cardiac neural crest chimeras (n = 2) which contain QCPN-positive cardiac ganglia. Figure 7Open in figure viewerPowerPoint a,b,c,d: Transverse sections in the heart of an HH42 embryo. Note TH-positive nerve fibers (arrows) around the left coronary artery (LCA) in the atrioventricular sulcus (AVS) as depicted in a (×108). Panel b (×215) shows TH-positive nerve fibers in cardiac ganglia (CG) and nerve bundles in the subepicardium (SEP) caudal from the sinus venosus. Note that the neurons are TH-negative (arrows). Panel c (×197) shows TH-positive nerve fibers (arrows) in the interventricular septum (IVS). d: A higher magnification of the indicated area in c (×440). Panel e (×207) shows TH-positive nerve fibers (arrows) ventrocaudal from the interatrial septum near the atrioventricular node region. f: A higher magnification of the area indicated in e (×440). DISCUSSION Tyrosine Hydroxylase Specificity Earlier morphological studies used catecholamine histofluorescence to detect sympathetic cardiac nerves (Higgins and Pappano, 1979; Kirby et al., 1980), while TH activity has only been measured quantitatively in the chick embryonic heart (Stewart and Kirby, 1985). From the present study it appears that TH can be used to detect cardiac sympathetic nerves as well, since our results show that TH-immunohistochemistry stains very specifically the sympathetic trunk and its branches. However, it is still uncertain whether all TH-positive fibers near the embryonic heart can be considered as sympathetic efferent nerve fibers. Baumel (1975) has described in adult birds that the nerves around the internal carotids also contain branches from the glossopharyngeal nerve and fine variable rami from the vagus which contribute to the innervation of the parathryroid, thyroid, the ultimobranchial glands and the carotid body. In the cat, sensory afferent branches from the glossopharyngeal nerve which join the carotid nerve to the carotid body and the carotid sinus have been shown to be TH-positive and are involved in relaying baro- and chemoreceptor information to the medulla (Massari et al., 1996). In this study TH-positive nerve fibers and cells are present in the carotid body, in the media of the dorsal part of the aortic arch artery, in the aorticopulmonary septum, and the media of the common carotid artery. These are probably putative chemo- and baroreceptors. The media of the mammalian aortic arch are known to contain a similar structure as the carotid body (Ham and Cormack, 1979), which might also apply to the chick. The catecholamine histofluorescence technique shows staining of these receptor areas as Le Lievre and Le Douarin (1975) report the presence of fluorogenic monoamines-containing cells (using the formol-induced-fluorescence technique) in the wall of the common carotid arteries. This shows that catecholamines and TH are located in the same areas. Tyrosine hydroxylase staining in the upper part of the aorticopulmonary septum might also implicate chemoreceptors as aortic bodies are described to be localized near the roots of the aorta and pulmonary trunk (Baumel, 1975). We conclude, therefore, that TH stains not only sympathetic postganglionic efferent nerve fibers but also chemo- and baroreceptor cells and their sensory afferent innervation. Since the glossopharyngeal nerve and its petrosal ganglion as well as the proximal part of the vagal nerve in the neck did not show any TH-positivity, we assume that TH-positive nerve fibers in the carotid nerve are derived from the cervical SG and possibly the paraganglia of the carotid plexus. This might implicate that in addition to sympathetic efferent neurons, the SG and paraganglia contain sensory afferent neurons as well. This means that the (para)sympathetic efferent and sensory afferent nervous system are