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Direct projections from the spinal cord to the trigeminal sympathetic ganglion of the puffer fish, Takifugu niphobles
ELSEVIER
Neuroscience Letters 213 (1996) 115-118
Direct projections from the spinal cord to the trigeminal sympathetic ganglion of the puffer fish, Takifugu niphobles Kengo Funakoshi*,
Toshio Abe, Reiji Kishida
Department of Anatomy, Yamaguchi UniversitySchool of Medicine, 1144 Kogushi, Ube, Yamaguchi, 755 Japan Received 7 May 1996; revised version received 20 June 1996; accepted 21 June 1996
Abstract The sympathetic trunk of teleosts extends into the cranial levels, forming the cranial sympathetic ganglia. When horseradish peroxidase was applied to the trigeminal sympathetic ganglion (a sympathetic ganglion at the level of the trigeminal nerve) of the puffer fish, Takifugu niphobles, retrogradely labeled neurons were found in the central autonomic nucleus (a distinct cell column in the rostral part of the spinal cord). The central autonomic nucleus has been known to contain preganglionie neurons projecting to the sympathetic ganglia at the spinal levels. Thus, the present results indicate that the central autonomic nucleus in the spinal cord of teleosts contains not only preganglionic neurons projecting to the sympathetic ganglia at the spinal levels, but also neurons projecting to the sympathetic ganglia at the cranial levels.
Keywords: Teleost; Spinal cord; Sympathetic ganglion; Preganglionic neuron; Horseradish peroxidase; Takifugu niphobles (Tetraodontiformes)
The organization of the sympathetic nervous system is variable among the classes of vertebrates. The teleost sympathetic nervous system is unique in that the sympathetic trunk extends into the cranial levels and forms cranial sympathetic ganglia which are connected to the cranial nerves [3,4,6-8,11]. In teleosts, preganglionic fibers projeering to the cranial sympathetic ganglia have been thought to originate predominantly from the spinal cord [3,8,11], but this has not been confirmed experimentally. In our previous studies by the retrograde tract-tracing method with horseradish peroxidase (HRP) in the puffer fish (Takifugu niphobles) [2] and the filefish (Stephanolepis cirrhifer) [1], it was shown that sympathetic preganglionic neurons sending axons to the sympathetic trunk at the spinal levels were distributed within the central autonomic nucleus, a cell column in the rostral part of the spinal cord. The present study was primarily attempted to see if sympathetic preganglionic neurons of the puffer fish send axons to the trigeminal sympathetic ganglion. Five young puffer fish, Takifugu niphobles, weighing 10-15 g, captured and maintained in 700-1 aquaria, were * Corresponding author. Tel.: +81 836 222206; fax: +81 836 222205.
Fig. I. Lateral view of the left sympathetic nervous system. An arrow indicates the site of HRP application. T, telencephalon; OT, optic rectum; CC, corpus cerebellum; SGV, trigeminal sympathetic ganglion; SGVII, facial sympathetic ganglion; SGIX, glossopharyngeal sympathetic ganglion; SGX, vagal sympathetic ganglion; CG, celiac ganglion; SG1, first spinal sympathetic gangfion; SG2, second spinal sympathetic ganglion; SG3, third spinal sympathetic ganglion; CAN, central autonomic nucleus.
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII S0304-3940(96) 12840-8
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K. Funakosh et al. / Neuroscience 12tters 213 (1996) 115-118
used. The fish were anesthetized with 0.01% ethyl m-aminobenzoate methanesulfate in water, and then their bodies were wrapped in vinyl and buried in crushed ice. An incision was made in the skin at an appropriate location, and a piece of bone was removed. After the trigeminal sympathetic ganglion on the left side was exposed, 10% HRP (Toyobo) in 0.05 M phosphate buffer (PB; pH 7.3) was injected into the ganglion iontophoretically through a glass micropipette. After a survival period of 3 - 4 days, the fish were re-anesthetized and perfused transcardially with heparinized saline, followed by fixative containing 2.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M PB. The brain and spinal cord were removed together with the surrounding bone, cranial nerves, and adjacent cranial ganglia; they were postfixed in the same solution, and then placed into 30% sucrose in 0.1 M PB. Subsequently, the brain and spinal cord were cut serially into cross sections of 40 or 50/xm thick on a cryostat. The sections were
mounted on glass slides and processed for visualization of HRP with 3,3'-diaminobentizidine tetrahydrochloride as a chromogen. The sympathetic trunk of the puffer fish extended along the ventrolateral surface of the skull bone into the orbit (Fig. 1). The sympathetic ganglia were located at the levels of the vagal, glossopharyngeal, facial, and trigeminal nerves. They were connected to the corresponding cranial nerves through the rami communicantes. The trigeminal sympathetic ganglion, the most rostral sympathetic ganglion, was located closely to the ventromedial surface of the trigeminal ganglion. Postganglionic fibers from the trigeminal sympathetic ganglion have been reported to be distributed to chromatophores, blood vessels, and also probably, smooth muscles in the eyeball [3,6,8,11]. Retrogradely labeled neurons were found exclusively in the central autonomic nucleus, a distinct cell column just dorsal and lateral to the central canal of the spinal cord
Fig. 2. (A) Neurons (arrows) in the central autonomic nucleus are labeled with HRP injected into the trigeminal sympathetic ganglion. Counterstained with cresyl violet; C, central canal; V, blood vessels. (B) The trigeminal sympathetic ganglion (SGV) is well demarcated from the trigeminal ganglion (GV). Arrowsindicate HRP-labeledpostganglionicneurons. RV, trigemlnal nerve root. (C) Labeled fibers (arrows) and a labeled neuron (large arrow) are seen in the facial sympathetic ganglion (SGVII). (D) Higher magnification of labeled fibers (arrows) in the facial sympathetic ganglion. (E) Higher magnification of the labeled neuron (arrow) in the facial sympatheticganglion. The dark granule seen at bottom right is an erythrocyte.Scale Bar: (A), 50 #m; (B), 400 p.m; (C), 200 txm; (D,E) 25 t~m.
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K. Funakosh et al. / Neuroscience Letters 213 (1996) 115-118 Table 1 Segmental distribution of labeled cells after HRP application to the trigeminal sympathetic ganglion No. of labeled neuron
Case 1 Case 2 Case 3 Case 4 Case 5
(I) (C) (1) (C) (I) (C) (I) (C) (I) (C)
Sgl
Sg2
Sg3
Sg4
Sg5
Sg6
Sg7
Sg8
Sg9
Sgl0
Sgl 1
Sgl2
Total
4 2 2 1 0 0 0 1 0 0
2 4 1 2 0 0 1 2 2 2
1 4 1 1 3 1 1 0 2 0
3 3 2 2 2 1 0 2 1 0
4 4 2 1 1 1 2 3 0 3
3 2 0 1 3 1 1 0 0 0
1 1 1 0 1 1 2 1 1 0
1 3 0 0 1 0 1 0 0 0
1 2 1 1 2 0 0 1 1 0
0 0 0 0 1 1 2 0 0 0
0 1 0 1 0 1 0 0 0 0
0 0
20 26 10 10 14 7 10 10 8 5
1 0
The central autonomic nucleus is rostrocaudally divided into 11-12 segments (Sg). Each segment is 100/~m (Case 2-5) or 120/zm (case 1) in length. (I), number of ipsilaterally labeled neurons; (C), number of contralaterally labeled neurons.
(Fig. 2A). This cell column extended from the level of the exit point of the posterior rootlet of the ventral root of the first spinal nerve to the level of the exit point of the third spinal nerve. Labeled neurons were distributed bilaterally throughout the central autonomic nucleus, although they were seen more frequently in the rostral half than in the caudal half of the nucleus (Table 1). No labeled neurons were found in any other region in the central nervous system. On the side ipsilateral to the HRP injection, some preganglionic nerve fibers were labeled in the sympathetic trunk at the levels from the trigeminal sympathetic ganglion to the celiac ganglion (Fig. 2B-D). A few retrogradely labeled neurons were also found in the facial sympathetic ganglion (Fig. 2E), but no labeled neurons were seen in the glossopharyngeal sympathetic, vagal sympathetic, or celiac ganglion. The present results indicate that the trigeminal sympathetic ganglion of teleosts receives preganglionic fibers from the spinal cord via the sympathetic trunk. Such innervation of the cranial autonomic ganglia of vertebrates by preganglionic neurons in the spinal cord has not been reported so far. The present results, however, do not necessarily indicate that the trigeminal sympathetic ganglion of teleosts is derived from the neural crest at the spinal levels and then migrates rostrally, because the sympathetic preganglionic fibers do not seem to follow the traits established by migration of ganglion cells [5,10]. In mammals, sympathetic preganglionic neurons are arranged rostrocaudally according to the level of their target ganglia [9]. In the present study, the sympathetic preganglionic neurons projecting to the trigeminal sympathetic ganglion were distributed predominantly in the rostral part of the central autonomic nucleus, although a few sympathetic preganglionic neurons were labeled in the caudalmost part of the central autonomic nucleus. On the other hand, in our previous study, the sympathetic preganglionic neurons projecting to the spinal sympathetic ganglia caudal to the celiac ganglion of the puffer fish (Takifugu
niphobles) [2] were distributed mainly in the middle part of the central autonomic nucleus. Thus, the sympathetic preganglionic neurons in the central autonomic nucleus of the puffer fish also appeared to be arranged rostrocaudally according to the levels of their target ganglia. It is generally thought that sympathetic postganglionic fibers emerging from the sympathetic ganglia of teleosts run distally into the corresponding nerves without traveling in the sympathetic trunk [3,11 ]. However, in the present study, some retrogradely labeled neurons were seen in the facial sympathetic ganglion after HRP application to the trigeminal sympathetic ganglion. Thus, in the puffer fish, some postganglionic neurons appear to send axons into the sympathetic trunk. We are grateful to Dr. R.C. Goris for his help in preparing the manuscript and to Mr. J. Oba and C. Matsuo for technical aid. [1] Funakoshi, K., Abe, T. and Kishida, R., NADPH-diaphorase activity in the sympathetic preganglionic neurons of the filefish Stephanolepis cirrhifer, Neurosci. Lea., 191 (1995) 181-184. [2] Funakoshi, K., Abe, T. and Kishida, R., The spinal sympathetic pmganglionic cell column in the puffer fish Talufugu niphobles, Cell Tissue Res., 284 (1996) 111-116. [3l Gibbins, 1., Comparative anatomy and evolution of the autonomic nervous system. In S. Nilsson and S. Hoimgren (Eds.), Comparative Physiology and Evolution of the Autonomic Nervous System, Harwood Academic, Chur, 1994, pp. 1-67. [4] Holmgren, S. and Nilsson, S., Neuropharmacology of adtenergic neurons in teleost fish, Comp. Biochem. Physiol., 72C (1982) 289302. [5] Newgreen, D.F., Establishment of the form of the peripheral nervous system. In I.A. Hendry and C.E. Hill (Eds.), Development, Regeneration and Plasticity of the Autonomic Nervous System, Harwood Academic, Chur, 1992, pp. 1-94. [6] Nicol, J.A.C., Autonomic nervous systems in lower chordates, Biol. Rev., 27 (1952) 1-49. [7] Nilsson, S., Fluorescent histochemistry and cholinesterase staining of sympathetic ganglia in a teleost Gadus nuJrrhua, Acta Zool., 57 (1976) 69-77. [8] Pick, J., The Autonomic Nervous Systems, J.B. Lippincon, Philadelphia, PA, 1970, 197 pp.
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K. Funakosh et al. / Neuroscience Letters 213 (1996) 115-118
[9] Strack, A.M., Sawyer, W.B., Marubio, L.M. and Loewy, A.D., Spinal origins of sympathetic pregangliouic neurons in the rat, Brain Res., 455 (1988) 187-191. [10] Yip, J.W., Target cues are not required for the guidance of sympathetic preganglionic axons, Dev. Brain Res., 32 (1987) 155-159.
[11] Young, J.Z., On the autonomic nervous system of the teleostean fish Uranoscopus glaber, Q. J. Microsc. Sci., 74 (1931) 491-535.
Neuroscience Letters 213 (1996) 115-118
Direct projections from the spinal cord to the trigeminal sympathetic ganglion of the puffer fish, Takifugu niphobles Kengo Funakoshi*,
Toshio Abe, Reiji Kishida
Department of Anatomy, Yamaguchi UniversitySchool of Medicine, 1144 Kogushi, Ube, Yamaguchi, 755 Japan Received 7 May 1996; revised version received 20 June 1996; accepted 21 June 1996
Abstract The sympathetic trunk of teleosts extends into the cranial levels, forming the cranial sympathetic ganglia. When horseradish peroxidase was applied to the trigeminal sympathetic ganglion (a sympathetic ganglion at the level of the trigeminal nerve) of the puffer fish, Takifugu niphobles, retrogradely labeled neurons were found in the central autonomic nucleus (a distinct cell column in the rostral part of the spinal cord). The central autonomic nucleus has been known to contain preganglionie neurons projecting to the sympathetic ganglia at the spinal levels. Thus, the present results indicate that the central autonomic nucleus in the spinal cord of teleosts contains not only preganglionic neurons projecting to the sympathetic ganglia at the spinal levels, but also neurons projecting to the sympathetic ganglia at the cranial levels.
Keywords: Teleost; Spinal cord; Sympathetic ganglion; Preganglionic neuron; Horseradish peroxidase; Takifugu niphobles (Tetraodontiformes)
The organization of the sympathetic nervous system is variable among the classes of vertebrates. The teleost sympathetic nervous system is unique in that the sympathetic trunk extends into the cranial levels and forms cranial sympathetic ganglia which are connected to the cranial nerves [3,4,6-8,11]. In teleosts, preganglionic fibers projeering to the cranial sympathetic ganglia have been thought to originate predominantly from the spinal cord [3,8,11], but this has not been confirmed experimentally. In our previous studies by the retrograde tract-tracing method with horseradish peroxidase (HRP) in the puffer fish (Takifugu niphobles) [2] and the filefish (Stephanolepis cirrhifer) [1], it was shown that sympathetic preganglionic neurons sending axons to the sympathetic trunk at the spinal levels were distributed within the central autonomic nucleus, a cell column in the rostral part of the spinal cord. The present study was primarily attempted to see if sympathetic preganglionic neurons of the puffer fish send axons to the trigeminal sympathetic ganglion. Five young puffer fish, Takifugu niphobles, weighing 10-15 g, captured and maintained in 700-1 aquaria, were * Corresponding author. Tel.: +81 836 222206; fax: +81 836 222205.
Fig. I. Lateral view of the left sympathetic nervous system. An arrow indicates the site of HRP application. T, telencephalon; OT, optic rectum; CC, corpus cerebellum; SGV, trigeminal sympathetic ganglion; SGVII, facial sympathetic ganglion; SGIX, glossopharyngeal sympathetic ganglion; SGX, vagal sympathetic ganglion; CG, celiac ganglion; SG1, first spinal sympathetic gangfion; SG2, second spinal sympathetic ganglion; SG3, third spinal sympathetic ganglion; CAN, central autonomic nucleus.
0304-3940/96/$12.00 © 1996 Elsevier Science Ireland Ltd. All fights reserved PII S0304-3940(96) 12840-8
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K. Funakosh et al. / Neuroscience 12tters 213 (1996) 115-118
used. The fish were anesthetized with 0.01% ethyl m-aminobenzoate methanesulfate in water, and then their bodies were wrapped in vinyl and buried in crushed ice. An incision was made in the skin at an appropriate location, and a piece of bone was removed. After the trigeminal sympathetic ganglion on the left side was exposed, 10% HRP (Toyobo) in 0.05 M phosphate buffer (PB; pH 7.3) was injected into the ganglion iontophoretically through a glass micropipette. After a survival period of 3 - 4 days, the fish were re-anesthetized and perfused transcardially with heparinized saline, followed by fixative containing 2.5% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M PB. The brain and spinal cord were removed together with the surrounding bone, cranial nerves, and adjacent cranial ganglia; they were postfixed in the same solution, and then placed into 30% sucrose in 0.1 M PB. Subsequently, the brain and spinal cord were cut serially into cross sections of 40 or 50/xm thick on a cryostat. The sections were
mounted on glass slides and processed for visualization of HRP with 3,3'-diaminobentizidine tetrahydrochloride as a chromogen. The sympathetic trunk of the puffer fish extended along the ventrolateral surface of the skull bone into the orbit (Fig. 1). The sympathetic ganglia were located at the levels of the vagal, glossopharyngeal, facial, and trigeminal nerves. They were connected to the corresponding cranial nerves through the rami communicantes. The trigeminal sympathetic ganglion, the most rostral sympathetic ganglion, was located closely to the ventromedial surface of the trigeminal ganglion. Postganglionic fibers from the trigeminal sympathetic ganglion have been reported to be distributed to chromatophores, blood vessels, and also probably, smooth muscles in the eyeball [3,6,8,11]. Retrogradely labeled neurons were found exclusively in the central autonomic nucleus, a distinct cell column just dorsal and lateral to the central canal of the spinal cord
Fig. 2. (A) Neurons (arrows) in the central autonomic nucleus are labeled with HRP injected into the trigeminal sympathetic ganglion. Counterstained with cresyl violet; C, central canal; V, blood vessels. (B) The trigeminal sympathetic ganglion (SGV) is well demarcated from the trigeminal ganglion (GV). Arrowsindicate HRP-labeledpostganglionicneurons. RV, trigemlnal nerve root. (C) Labeled fibers (arrows) and a labeled neuron (large arrow) are seen in the facial sympathetic ganglion (SGVII). (D) Higher magnification of labeled fibers (arrows) in the facial sympathetic ganglion. (E) Higher magnification of the labeled neuron (arrow) in the facial sympatheticganglion. The dark granule seen at bottom right is an erythrocyte.Scale Bar: (A), 50 #m; (B), 400 p.m; (C), 200 txm; (D,E) 25 t~m.
117
K. Funakosh et al. / Neuroscience Letters 213 (1996) 115-118 Table 1 Segmental distribution of labeled cells after HRP application to the trigeminal sympathetic ganglion No. of labeled neuron
Case 1 Case 2 Case 3 Case 4 Case 5
(I) (C) (1) (C) (I) (C) (I) (C) (I) (C)
Sgl
Sg2
Sg3
Sg4
Sg5
Sg6
Sg7
Sg8
Sg9
Sgl0
Sgl 1
Sgl2
Total
4 2 2 1 0 0 0 1 0 0
2 4 1 2 0 0 1 2 2 2
1 4 1 1 3 1 1 0 2 0
3 3 2 2 2 1 0 2 1 0
4 4 2 1 1 1 2 3 0 3
3 2 0 1 3 1 1 0 0 0
1 1 1 0 1 1 2 1 1 0
1 3 0 0 1 0 1 0 0 0
1 2 1 1 2 0 0 1 1 0
0 0 0 0 1 1 2 0 0 0
0 1 0 1 0 1 0 0 0 0
0 0
20 26 10 10 14 7 10 10 8 5
1 0
The central autonomic nucleus is rostrocaudally divided into 11-12 segments (Sg). Each segment is 100/~m (Case 2-5) or 120/zm (case 1) in length. (I), number of ipsilaterally labeled neurons; (C), number of contralaterally labeled neurons.
(Fig. 2A). This cell column extended from the level of the exit point of the posterior rootlet of the ventral root of the first spinal nerve to the level of the exit point of the third spinal nerve. Labeled neurons were distributed bilaterally throughout the central autonomic nucleus, although they were seen more frequently in the rostral half than in the caudal half of the nucleus (Table 1). No labeled neurons were found in any other region in the central nervous system. On the side ipsilateral to the HRP injection, some preganglionic nerve fibers were labeled in the sympathetic trunk at the levels from the trigeminal sympathetic ganglion to the celiac ganglion (Fig. 2B-D). A few retrogradely labeled neurons were also found in the facial sympathetic ganglion (Fig. 2E), but no labeled neurons were seen in the glossopharyngeal sympathetic, vagal sympathetic, or celiac ganglion. The present results indicate that the trigeminal sympathetic ganglion of teleosts receives preganglionic fibers from the spinal cord via the sympathetic trunk. Such innervation of the cranial autonomic ganglia of vertebrates by preganglionic neurons in the spinal cord has not been reported so far. The present results, however, do not necessarily indicate that the trigeminal sympathetic ganglion of teleosts is derived from the neural crest at the spinal levels and then migrates rostrally, because the sympathetic preganglionic fibers do not seem to follow the traits established by migration of ganglion cells [5,10]. In mammals, sympathetic preganglionic neurons are arranged rostrocaudally according to the level of their target ganglia [9]. In the present study, the sympathetic preganglionic neurons projecting to the trigeminal sympathetic ganglion were distributed predominantly in the rostral part of the central autonomic nucleus, although a few sympathetic preganglionic neurons were labeled in the caudalmost part of the central autonomic nucleus. On the other hand, in our previous study, the sympathetic preganglionic neurons projecting to the spinal sympathetic ganglia caudal to the celiac ganglion of the puffer fish (Takifugu
niphobles) [2] were distributed mainly in the middle part of the central autonomic nucleus. Thus, the sympathetic preganglionic neurons in the central autonomic nucleus of the puffer fish also appeared to be arranged rostrocaudally according to the levels of their target ganglia. It is generally thought that sympathetic postganglionic fibers emerging from the sympathetic ganglia of teleosts run distally into the corresponding nerves without traveling in the sympathetic trunk [3,11 ]. However, in the present study, some retrogradely labeled neurons were seen in the facial sympathetic ganglion after HRP application to the trigeminal sympathetic ganglion. Thus, in the puffer fish, some postganglionic neurons appear to send axons into the sympathetic trunk. We are grateful to Dr. R.C. Goris for his help in preparing the manuscript and to Mr. J. Oba and C. Matsuo for technical aid. [1] Funakoshi, K., Abe, T. and Kishida, R., NADPH-diaphorase activity in the sympathetic preganglionic neurons of the filefish Stephanolepis cirrhifer, Neurosci. Lea., 191 (1995) 181-184. [2] Funakoshi, K., Abe, T. and Kishida, R., The spinal sympathetic pmganglionic cell column in the puffer fish Talufugu niphobles, Cell Tissue Res., 284 (1996) 111-116. [3l Gibbins, 1., Comparative anatomy and evolution of the autonomic nervous system. In S. Nilsson and S. Hoimgren (Eds.), Comparative Physiology and Evolution of the Autonomic Nervous System, Harwood Academic, Chur, 1994, pp. 1-67. [4] Holmgren, S. and Nilsson, S., Neuropharmacology of adtenergic neurons in teleost fish, Comp. Biochem. Physiol., 72C (1982) 289302. [5] Newgreen, D.F., Establishment of the form of the peripheral nervous system. In I.A. Hendry and C.E. Hill (Eds.), Development, Regeneration and Plasticity of the Autonomic Nervous System, Harwood Academic, Chur, 1992, pp. 1-94. [6] Nicol, J.A.C., Autonomic nervous systems in lower chordates, Biol. Rev., 27 (1952) 1-49. [7] Nilsson, S., Fluorescent histochemistry and cholinesterase staining of sympathetic ganglia in a teleost Gadus nuJrrhua, Acta Zool., 57 (1976) 69-77. [8] Pick, J., The Autonomic Nervous Systems, J.B. Lippincon, Philadelphia, PA, 1970, 197 pp.
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K. Funakosh et al. / Neuroscience Letters 213 (1996) 115-118
[9] Strack, A.M., Sawyer, W.B., Marubio, L.M. and Loewy, A.D., Spinal origins of sympathetic pregangliouic neurons in the rat, Brain Res., 455 (1988) 187-191. [10] Yip, J.W., Target cues are not required for the guidance of sympathetic preganglionic axons, Dev. Brain Res., 32 (1987) 155-159.
[11] Young, J.Z., On the autonomic nervous system of the teleostean fish Uranoscopus glaber, Q. J. Microsc. Sci., 74 (1931) 491-535.