Brain Research, 577 (1992) 181-188 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00 BRES 17595
181
Research Reports
Sympathetic preganglionic neurons in rabbit spinal cord that project to the stellate or the superior cervical ganglion Paul Pilowsky, Ida J. Llewellyn-Smith, Jane Minson and John Chalmers Department of Medicine and Centre for Neuroscience Flinders University, Bedford Park, South Australia (Australia) (Accepted 19 November 1991) Key words: Retrograde tracing; Cholera toxin B; Colloidal gold; Immunocytochemistry; Ultrastructure; WGA-apo-HRP
The segmental distribution of sympathetic preganglionic neurons in the rabbit spinal cord that project to the stellate or the superior cervical ganglion was determined using retrograde tracing with cholera toxin B subunit from the steUate ganglion and wheat germ agglutininapo-horseradish peroxidase-gold from the superior cervical ganglion. Sympathetic preganglionic neurons that projected to the stellate ganglion were located in spinal segments T l to T10. Sympathetic preganglionic neurons projecting to the superior cervical ganglion were found in segments T1 to T s. Both types of neuron had somata that were elongated in the rostrocaudal direction, and dendrites that were mainly confined to the intermediolateral cell column. Almost 95% of the neurons supplying the superior cervical ganglion had axons that passed through the stellate ganglion. INTRODUCTION Sympathetic preganglionic neurons that control the heart are known to be mainly located in the u p p e r thoracic spinal cord, and project to the stellate ganglion 6'8' 9,13,15,21,22,29,31-33. Previous studies in the rat have d e m o n s t r a t e d that postganglionic neurons that innervate the heart are located almost exclusively in the stellate ganglion with a contribution from only a small n u m b e r of neurons in the superior cervical ganglion 19'2°. Similarly, in the cat 5'14 and the dog 3'4'11, the stellate ganglion has an i m p o r t a n t role in control o f the heart, although in the dog the middle cervical ganglion is also likely to be imp o r t a n t 3'4'u. The location of sympathetic preganglionic neurons that innervate the steUate ganglion have b e e n m a p p e d in the cat and rat but not in the rabbit 8'9'22'29. Since the rabbit is frequently used in studies on the central control of the h e a r t and b l o o d pressure 23'24'27, we were interested to d e t e r m i n e the location in the spinal cord of sympathetic preganglionic neurons that project to the stellate ganglion in this species. To label sympathetic preganglionic neurons projecting exclusively to the stellate ganglion, we c o m b i n e d injections o f cholera toxin B subunit (CTB) into the stellate ganglion with injections of wheat-germ agglutinin-apo-horseradish peroxidase a d s o r b e d to 7 nm gold ( W G A - a p o - H R P - g o l d ) into the superior cervical ganglion. This protocol labelled neurons that p r o j e c t e d to or through the stellate gan-
glion with CTB, and neurons that p r o j e c t e d to the superior cervical ganglion with gold particles. We a d o p t e d this double injection a p p r o a c h because sympathetic preganglionic neurons that provide an input to the superior cervical ganglion have axons that pass through the stellate ganglion and because CTB is taken up by b o t h axon terminals and fibres of passage. With this protocol we o b t a i n e d a complete picture of the distribution of neurons projecting exclusively to the stellate ganglion and of neurons whose axons passed through the stellate ganglion to terminate in the superior cervical ganglion. MATERIALS AND METHODS Injection of retrograde tracers Experiments were conducted on 9 New Zealand White rabbits. Anaesthesia was induced by injection of sodium thiopentone (40-60 mg i.v.; Abbott), and maintained with haiothane. Two operations were carried out in 7 rabbits. First, the left superior cervical ganglion was located through a ventral incision, and 5-10/A of WGA, apo-HRP-7 nm gold (prepared according to Basbaum and Menetrey7) was injected using a capillary glass micropipette with a tip diameter of 50-100/~m. Ampicillin (Ampicyn; Protea, 100 mg in 1 ml i.v.) was given at induction, and two hours postoperatively. A povidone iodine solution was applied topically to prevent infection. On completion of the procedure, anaesthesia was discontinue d and the animals allowed to recover. The rabbits were monitored postoperatively to ensure that their behaviour was normal, and that there was no evidence of discomfort. At least 3 days later, the rab-~ bits were again anaesthetised, and the left (ipsilateral) stellate ganglion was exposed retropleurally after resection of the head of the second rib. Injections (5-10/A) of a 1% solution of CTB (List) containing 0.5-1.0/~1 of Evans blue were made into the ganglion
Correspondence." P. Pilowsky, Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. FaX: (61 i" 8-2045268.
182 as described above. The inclusion of Evans blue allowed spillage of CTB to be easily detected and removed with swabs. Large volumes of injectate were used to ensure complete filling of each ganglion. The remaining two rabbits received only injections of CTB into the stellate ganglion. Spinal cord sections from these two rabbits were processed for electron microscopy to examine the intracellular distribution of retrogradely transported CTB.
Tissue processing Twenty-four hours after injection of CTB into the stellate ganglion, the rabbits were deeply anaesthetised (sodium pentobarbitone 100-150 mg i.v.) and perfused through the left ventricle with one litre of DMEM/Ham's F12 tissue culture medium (Sigma D8900) freshly bubbled with Carbogen (95% Oz, 5% CO2) followed by two litres of a fixative solution containing picric acid (0.2%), formaldehyde (4% from a 40% analytical reagent grade solution stabilized with methanol), and glutaraldehyde (0.5%), in 0.1 M phosphate buffer pH 7.4 (ZAGLU), The spinal cords were carefully removed, cut into spinal segments using the dorsal roots as a guide and split dorsoventrally along the midline. After removing the pia mater, the segments were post-fixed in ZAGLU for 4-24 h. The left-hand side segments were then mounted on chucks with the medial surface down, and parasagittal sections (50-75/~m) were cut using a Vibratome (Oxford). Before visualisation of the retrograde tracers, the sections were exposed to 50% ethanol in distilled water for 30 min to improve the penetration of reagents 16 and then washed in several changes of distilled water. Retrogradely-transported WGA-apo-HRP-gold in sympathetic preganglionic neurons was visualized by silver intensification a6. Sections were incubated for 3 x 5 min in a 1:1 mixture of Silver Enhancer Solution A (Sigma $5020): Silver Enhancer Solution B (Sigma $5145). The mixture was prepared immediately before each incubation. Most silver-intensified sections were fixed for 5 min in 2.5% sodium thiosulphate after a brief wash in distilled water. However, some silver-intensified sections were gold-toned before fixation in thiosulphate. These sections were exposed to 0.05% gold chloride for 10 min at room temperature and then to 0.2% oxalic acid for 2 min at 4°C. The sections were washed in distilled water for 3 × 10 rain before and after gold toning and for 5 min between exposures to gold chloride and oxalic acid. All gold-toning steps were carried out in the dark. After visualization of the WGA-apo-HRP-gold, CTB immunoreactivity was detected immunocytochemically using an avidin-biotin-peroxidase detection system 16. Sections were incubated for 30 min in 10% normal horse serum, for 2-3 days in a 1:50,000 dilution of goat antiserum to CTB (List), for 24 h in a 1:200 dilution of biotinylated donkey anti-sheep immunoglobulin (Sigma B7390) and overnight in a 1:1500 dilution of ExtrAvidin-peroxidase (Sigma E2886). All incubations were done at room temperature. All immunoreagents were diluted with 10 mM phosphate-buffered saline containing 10 mM Tris and 0.05% thimerosal (Sigma T5125), pH 7.4 (TPBS). Sections were washed for 3 x 30 min in TPBS after each incubation. CTB immunoreactivity in sympathetic preganglionic neurons was revealed for light microscopy by an imidazoleintensified diaminobenzidine reaction; and for electron microscopy, by a nickel-intensified reaction 16. In both reactions, peroxide was generated by glucose oxidase TM. Sections for light microscopy were dried onto chrome alumtreated slides, dehydrated and mounted in Depex. Sections for electron microscopy were exposed to 0.5% osmium tetroxide for i h, stained en bloc with uranyl acetate, dehydrated through graded ethanol solutions and propylene oxide and embedded fiat in Durcupan (Fluka). Ultrathin sections were cut on a diamond knife, mounted on mesh grids and stained with Reynold's lead citrate.
Quantitation A quantitative analysis of the distribution of sympathetic preganglionic neurons in the spinal cord was carried out at the light microscope level by counting labelled neurons from each spinal segment. To avoid double-counting of neurons, only the section that
contained the largest number of labelled cells was used. Neurons that contained at least 5 silver-intensified gold particles (usually many more) were considered to have an axonal projection to the superior cervical ganglion, while neurons that contained an amberbrown reaction product indicative of CTB immunoreactivity were considered to have an axon that projected to or through the stellate ganglion. Neurons containing both markers might have either an axonal projection to both ganglia, or only have an axon that passes through the stellate ganglion on its way to the superior cervical ganglion.
RESULTS
Light microscopic distribution of CTB and WGA-apoHRP-gold within sympathetic preganglionic neurons Sympathetic preganglionic neurons containing either CTB or WGA-apo-HRP-gold
o r b o t h c o u l d be readily
d e t e c t e d by light m i c r o s c o p y (Fig. 1). T h e p a t t e r n of labelling s e e n in n e u r o n s that w e r e i m m u n o r e a c t i v e for C T B at the light m i c r o s c o p e level is s h o w n in Fig. 1 B - D . In c o n t r a s t to the labelling s e e n with W G A - a p o - H R P gold, C T B i m m u n o r e a c t i v i t y was distributed t h r o u g h o u t the n e u r o n s , clearly labelling not only their s o m a but also their d e n d r i t e s and axons. A n a s t o m o s i n g c l u m p s of intense i m m u n o r e a c t i v i t y within the c y t o p l a s m of n e r v e cell b o d i e s s u g g e s t e d that C T B was c o n c e n t r a t e d in and a r o u n d the G o l g i a p p a r a t u s ; the r e m a i n d e r of the cytoplasm c o n t a i n e d h o m o g e n e o u s m o d e r a t e to faint i m m u n o r e a c t i v i t y . T h e characteristic p a t t e r n o f labelling s e e n with the light m i c r o s c o p e in n e u r o n s that c o n t a i n e d retrogradely
transported
WGA-apo-HRP-gold
silver-intensified
particles
of
is s h o w n m o s t clearly in Fig. l B .
T h e particles a p p e a r e d as black p u n c t a in n e u r o n a l som a t a and, in s o m e n e u r o n s , also in p r o x i m a l dendrites. P r e v i o u s e l e c t r o n m i c r o s c o p i c studies h a v e d e m o n s t r a t e d that r e t r o g r a d e l y t r a n s p o r t e d gold particles are r e s t r i c t e d to l y s o s o m e s 7'17.
Morphology of sympathetic preganglionic neurons projecting to the stellate ganglion Since n e u r o n s that w e r e l a b e l l e d after injection of C T B into the stellate g a n g l i o n , but did n o t c o n t a i n gold particles, s h o w e d e x t e n s i v e filling of s o m a t a , d e n d r i t e s and axons, t h e e x p e r i m e n t s g a v e a clear picture of the m o r p h o l o g y of s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n s projecting to this ganglion. In all s e g m e n t s , the s o m a t a of n e u r o n s p r o j e c t i n g to the stellate g a n g l i o n w e r e elongated in the r o s t r o c a u d a l direction, with the m e a n rost r o c a u d a l axis (38.0 +_ 1.6 ~ m ; m e a n +_ S . E . M . ) b e i n g significantly l o n g e r t h a n the m e a n d o r s o v e n t r a l axis (20.2 + 1.1/~m). S o m e n e u r o n s had small s p i n d l e - s h a p e d som a t a with d e n d r i t e s travelling r o s t r o c a u d a l l y within the confines of the i n t e r m e d i o l a t e r a l cell c o l u m n (Fig. 1 B E). T h e s o m a t a of o t h e r n e u r o n s w e r e larger, m o r e r o u n d e d and had d e n d r i t e s that p r o j e c t e d dorsally or
183
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iii i iiiiiii !i!¸iiiiil i ! iiiiiiiiiiiiiiiii '¸if!i'Iif!i i !!ii i i!!!ii!iii!ii!i!!!jili!!!~iii!iii!iii !i!!il!! !!!i~¸¸
i
Fig. 1. Light micrographs of retrogradely labelled sympathetic preganglionic neurons in rabbit spinal cord. A: many neurons in the T 3 segment are labelled after injection of CTB into the stellate ganglion combined with injection of WGA-apo-HRP-gold into the superior cervical ganglion. The arrow indicates a group of neurons that are shown at higher power in B. B - E : sympathetic preganglionic neurons retrogradely labelled with CTB, WGA-apo-HRP-gold or both. A sympathetic preganglionic neuron with gold only is seen in D (star). Neurons labelled with CTB only are seen in B, C and E (asterisks). Arrows indicate deposits of silver-intensified gold in double labelled neurons. The 3 types of labelled neuron are intermingled. Bar in A, 100/~m, and in E, which applies to B - E , 20 pro.
4~
185 Fig. 2. Electron micrographs of a sympathetic preganglionic neuron retrogradely labelled with CTB from the stellate ganglion. A: at low power, large deposits of CTB immunoreactivity occur mainly around the nucleus (Nu) and are associated with Golgi apparatus (box B and *). Bar, 5 pm. B: higher power micrograph of the area indicated by box B in A. Intense CTB immunoreactivity (asterisk) covers most of the Golgi apparatus. Bar, 250 nm. C: higher power micrograph of the area indicated by box C in A. The cytoplasmic matrix is faintly immunbreactive compared to the surrounding neuropil and contains small deposits of intense immunoreactivity. Arrowheads indicate a synapse. Nu, nucleus. Bar, 250 nm.
181
Research Reports
Sympathetic preganglionic neurons in rabbit spinal cord that project to the stellate or the superior cervical ganglion Paul Pilowsky, Ida J. Llewellyn-Smith, Jane Minson and John Chalmers Department of Medicine and Centre for Neuroscience Flinders University, Bedford Park, South Australia (Australia) (Accepted 19 November 1991) Key words: Retrograde tracing; Cholera toxin B; Colloidal gold; Immunocytochemistry; Ultrastructure; WGA-apo-HRP
The segmental distribution of sympathetic preganglionic neurons in the rabbit spinal cord that project to the stellate or the superior cervical ganglion was determined using retrograde tracing with cholera toxin B subunit from the steUate ganglion and wheat germ agglutininapo-horseradish peroxidase-gold from the superior cervical ganglion. Sympathetic preganglionic neurons that projected to the stellate ganglion were located in spinal segments T l to T10. Sympathetic preganglionic neurons projecting to the superior cervical ganglion were found in segments T1 to T s. Both types of neuron had somata that were elongated in the rostrocaudal direction, and dendrites that were mainly confined to the intermediolateral cell column. Almost 95% of the neurons supplying the superior cervical ganglion had axons that passed through the stellate ganglion. INTRODUCTION Sympathetic preganglionic neurons that control the heart are known to be mainly located in the u p p e r thoracic spinal cord, and project to the stellate ganglion 6'8' 9,13,15,21,22,29,31-33. Previous studies in the rat have d e m o n s t r a t e d that postganglionic neurons that innervate the heart are located almost exclusively in the stellate ganglion with a contribution from only a small n u m b e r of neurons in the superior cervical ganglion 19'2°. Similarly, in the cat 5'14 and the dog 3'4'11, the stellate ganglion has an i m p o r t a n t role in control o f the heart, although in the dog the middle cervical ganglion is also likely to be imp o r t a n t 3'4'u. The location of sympathetic preganglionic neurons that innervate the steUate ganglion have b e e n m a p p e d in the cat and rat but not in the rabbit 8'9'22'29. Since the rabbit is frequently used in studies on the central control of the h e a r t and b l o o d pressure 23'24'27, we were interested to d e t e r m i n e the location in the spinal cord of sympathetic preganglionic neurons that project to the stellate ganglion in this species. To label sympathetic preganglionic neurons projecting exclusively to the stellate ganglion, we c o m b i n e d injections o f cholera toxin B subunit (CTB) into the stellate ganglion with injections of wheat-germ agglutinin-apo-horseradish peroxidase a d s o r b e d to 7 nm gold ( W G A - a p o - H R P - g o l d ) into the superior cervical ganglion. This protocol labelled neurons that p r o j e c t e d to or through the stellate gan-
glion with CTB, and neurons that p r o j e c t e d to the superior cervical ganglion with gold particles. We a d o p t e d this double injection a p p r o a c h because sympathetic preganglionic neurons that provide an input to the superior cervical ganglion have axons that pass through the stellate ganglion and because CTB is taken up by b o t h axon terminals and fibres of passage. With this protocol we o b t a i n e d a complete picture of the distribution of neurons projecting exclusively to the stellate ganglion and of neurons whose axons passed through the stellate ganglion to terminate in the superior cervical ganglion. MATERIALS AND METHODS Injection of retrograde tracers Experiments were conducted on 9 New Zealand White rabbits. Anaesthesia was induced by injection of sodium thiopentone (40-60 mg i.v.; Abbott), and maintained with haiothane. Two operations were carried out in 7 rabbits. First, the left superior cervical ganglion was located through a ventral incision, and 5-10/A of WGA, apo-HRP-7 nm gold (prepared according to Basbaum and Menetrey7) was injected using a capillary glass micropipette with a tip diameter of 50-100/~m. Ampicillin (Ampicyn; Protea, 100 mg in 1 ml i.v.) was given at induction, and two hours postoperatively. A povidone iodine solution was applied topically to prevent infection. On completion of the procedure, anaesthesia was discontinue d and the animals allowed to recover. The rabbits were monitored postoperatively to ensure that their behaviour was normal, and that there was no evidence of discomfort. At least 3 days later, the rab-~ bits were again anaesthetised, and the left (ipsilateral) stellate ganglion was exposed retropleurally after resection of the head of the second rib. Injections (5-10/A) of a 1% solution of CTB (List) containing 0.5-1.0/~1 of Evans blue were made into the ganglion
Correspondence." P. Pilowsky, Department of Medicine, Flinders Medical Centre, Bedford Park, South Australia 5042, Australia. FaX: (61 i" 8-2045268.
182 as described above. The inclusion of Evans blue allowed spillage of CTB to be easily detected and removed with swabs. Large volumes of injectate were used to ensure complete filling of each ganglion. The remaining two rabbits received only injections of CTB into the stellate ganglion. Spinal cord sections from these two rabbits were processed for electron microscopy to examine the intracellular distribution of retrogradely transported CTB.
Tissue processing Twenty-four hours after injection of CTB into the stellate ganglion, the rabbits were deeply anaesthetised (sodium pentobarbitone 100-150 mg i.v.) and perfused through the left ventricle with one litre of DMEM/Ham's F12 tissue culture medium (Sigma D8900) freshly bubbled with Carbogen (95% Oz, 5% CO2) followed by two litres of a fixative solution containing picric acid (0.2%), formaldehyde (4% from a 40% analytical reagent grade solution stabilized with methanol), and glutaraldehyde (0.5%), in 0.1 M phosphate buffer pH 7.4 (ZAGLU), The spinal cords were carefully removed, cut into spinal segments using the dorsal roots as a guide and split dorsoventrally along the midline. After removing the pia mater, the segments were post-fixed in ZAGLU for 4-24 h. The left-hand side segments were then mounted on chucks with the medial surface down, and parasagittal sections (50-75/~m) were cut using a Vibratome (Oxford). Before visualisation of the retrograde tracers, the sections were exposed to 50% ethanol in distilled water for 30 min to improve the penetration of reagents 16 and then washed in several changes of distilled water. Retrogradely-transported WGA-apo-HRP-gold in sympathetic preganglionic neurons was visualized by silver intensification a6. Sections were incubated for 3 x 5 min in a 1:1 mixture of Silver Enhancer Solution A (Sigma $5020): Silver Enhancer Solution B (Sigma $5145). The mixture was prepared immediately before each incubation. Most silver-intensified sections were fixed for 5 min in 2.5% sodium thiosulphate after a brief wash in distilled water. However, some silver-intensified sections were gold-toned before fixation in thiosulphate. These sections were exposed to 0.05% gold chloride for 10 min at room temperature and then to 0.2% oxalic acid for 2 min at 4°C. The sections were washed in distilled water for 3 × 10 rain before and after gold toning and for 5 min between exposures to gold chloride and oxalic acid. All gold-toning steps were carried out in the dark. After visualization of the WGA-apo-HRP-gold, CTB immunoreactivity was detected immunocytochemically using an avidin-biotin-peroxidase detection system 16. Sections were incubated for 30 min in 10% normal horse serum, for 2-3 days in a 1:50,000 dilution of goat antiserum to CTB (List), for 24 h in a 1:200 dilution of biotinylated donkey anti-sheep immunoglobulin (Sigma B7390) and overnight in a 1:1500 dilution of ExtrAvidin-peroxidase (Sigma E2886). All incubations were done at room temperature. All immunoreagents were diluted with 10 mM phosphate-buffered saline containing 10 mM Tris and 0.05% thimerosal (Sigma T5125), pH 7.4 (TPBS). Sections were washed for 3 x 30 min in TPBS after each incubation. CTB immunoreactivity in sympathetic preganglionic neurons was revealed for light microscopy by an imidazoleintensified diaminobenzidine reaction; and for electron microscopy, by a nickel-intensified reaction 16. In both reactions, peroxide was generated by glucose oxidase TM. Sections for light microscopy were dried onto chrome alumtreated slides, dehydrated and mounted in Depex. Sections for electron microscopy were exposed to 0.5% osmium tetroxide for i h, stained en bloc with uranyl acetate, dehydrated through graded ethanol solutions and propylene oxide and embedded fiat in Durcupan (Fluka). Ultrathin sections were cut on a diamond knife, mounted on mesh grids and stained with Reynold's lead citrate.
Quantitation A quantitative analysis of the distribution of sympathetic preganglionic neurons in the spinal cord was carried out at the light microscope level by counting labelled neurons from each spinal segment. To avoid double-counting of neurons, only the section that
contained the largest number of labelled cells was used. Neurons that contained at least 5 silver-intensified gold particles (usually many more) were considered to have an axonal projection to the superior cervical ganglion, while neurons that contained an amberbrown reaction product indicative of CTB immunoreactivity were considered to have an axon that projected to or through the stellate ganglion. Neurons containing both markers might have either an axonal projection to both ganglia, or only have an axon that passes through the stellate ganglion on its way to the superior cervical ganglion.
RESULTS
Light microscopic distribution of CTB and WGA-apoHRP-gold within sympathetic preganglionic neurons Sympathetic preganglionic neurons containing either CTB or WGA-apo-HRP-gold
o r b o t h c o u l d be readily
d e t e c t e d by light m i c r o s c o p y (Fig. 1). T h e p a t t e r n of labelling s e e n in n e u r o n s that w e r e i m m u n o r e a c t i v e for C T B at the light m i c r o s c o p e level is s h o w n in Fig. 1 B - D . In c o n t r a s t to the labelling s e e n with W G A - a p o - H R P gold, C T B i m m u n o r e a c t i v i t y was distributed t h r o u g h o u t the n e u r o n s , clearly labelling not only their s o m a but also their d e n d r i t e s and axons. A n a s t o m o s i n g c l u m p s of intense i m m u n o r e a c t i v i t y within the c y t o p l a s m of n e r v e cell b o d i e s s u g g e s t e d that C T B was c o n c e n t r a t e d in and a r o u n d the G o l g i a p p a r a t u s ; the r e m a i n d e r of the cytoplasm c o n t a i n e d h o m o g e n e o u s m o d e r a t e to faint i m m u n o r e a c t i v i t y . T h e characteristic p a t t e r n o f labelling s e e n with the light m i c r o s c o p e in n e u r o n s that c o n t a i n e d retrogradely
transported
WGA-apo-HRP-gold
silver-intensified
particles
of
is s h o w n m o s t clearly in Fig. l B .
T h e particles a p p e a r e d as black p u n c t a in n e u r o n a l som a t a and, in s o m e n e u r o n s , also in p r o x i m a l dendrites. P r e v i o u s e l e c t r o n m i c r o s c o p i c studies h a v e d e m o n s t r a t e d that r e t r o g r a d e l y t r a n s p o r t e d gold particles are r e s t r i c t e d to l y s o s o m e s 7'17.
Morphology of sympathetic preganglionic neurons projecting to the stellate ganglion Since n e u r o n s that w e r e l a b e l l e d after injection of C T B into the stellate g a n g l i o n , but did n o t c o n t a i n gold particles, s h o w e d e x t e n s i v e filling of s o m a t a , d e n d r i t e s and axons, t h e e x p e r i m e n t s g a v e a clear picture of the m o r p h o l o g y of s y m p a t h e t i c p r e g a n g l i o n i c n e u r o n s projecting to this ganglion. In all s e g m e n t s , the s o m a t a of n e u r o n s p r o j e c t i n g to the stellate g a n g l i o n w e r e elongated in the r o s t r o c a u d a l direction, with the m e a n rost r o c a u d a l axis (38.0 +_ 1.6 ~ m ; m e a n +_ S . E . M . ) b e i n g significantly l o n g e r t h a n the m e a n d o r s o v e n t r a l axis (20.2 + 1.1/~m). S o m e n e u r o n s had small s p i n d l e - s h a p e d som a t a with d e n d r i t e s travelling r o s t r o c a u d a l l y within the confines of the i n t e r m e d i o l a t e r a l cell c o l u m n (Fig. 1 B E). T h e s o m a t a of o t h e r n e u r o n s w e r e larger, m o r e r o u n d e d and had d e n d r i t e s that p r o j e c t e d dorsally or
183
~iili¸ ~!ill¸ ill!ill!~ ~ii!i
iii i iiiiiii !i!¸iiiiil i ! iiiiiiiiiiiiiiiii '¸if!i'Iif!i i !!ii i i!!!ii!iii!ii!i!!!jili!!!~iii!iii!iii !i!!il!! !!!i~¸¸
i
Fig. 1. Light micrographs of retrogradely labelled sympathetic preganglionic neurons in rabbit spinal cord. A: many neurons in the T 3 segment are labelled after injection of CTB into the stellate ganglion combined with injection of WGA-apo-HRP-gold into the superior cervical ganglion. The arrow indicates a group of neurons that are shown at higher power in B. B - E : sympathetic preganglionic neurons retrogradely labelled with CTB, WGA-apo-HRP-gold or both. A sympathetic preganglionic neuron with gold only is seen in D (star). Neurons labelled with CTB only are seen in B, C and E (asterisks). Arrows indicate deposits of silver-intensified gold in double labelled neurons. The 3 types of labelled neuron are intermingled. Bar in A, 100/~m, and in E, which applies to B - E , 20 pro.
4~
185 Fig. 2. Electron micrographs of a sympathetic preganglionic neuron retrogradely labelled with CTB from the stellate ganglion. A: at low power, large deposits of CTB immunoreactivity occur mainly around the nucleus (Nu) and are associated with Golgi apparatus (box B and *). Bar, 5 pm. B: higher power micrograph of the area indicated by box B in A. Intense CTB immunoreactivity (asterisk) covers most of the Golgi apparatus. Bar, 250 nm. C: higher power micrograph of the area indicated by box C in A. The cytoplasmic matrix is faintly immunbreactive compared to the surrounding neuropil and contains small deposits of intense immunoreactivity. Arrowheads indicate a synapse. Nu, nucleus. Bar, 250 nm.
