Brain Research, 586 (1992) 125-129
125
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25258
Sympathetic preganglionic neurons projecting to the adrenal medulla and aorticorenal ganglion in the rabbit l w o n a J e n s e n , P a u l Pilowsky, Ida Liewellyn-Smith, J a n e M i n s o n and J o h n C h a l m e r s Department of Medicbw and Centrefor Neuroscience, Flinders Unil'ersity, Bedford Park, SA (Australia) (Accepted 7 April 1992)
Key words: Retrograde tracing: Cholera toxin B; Wheatgerm agglutinin-apo-horseradish peroxidase-gold; Spinal cord; Blood pressure
The distribution of sympathetic preganglionic neurons (SPN) projecting to the adrenal medulla and the aorticorenal ganglion in the rabbit was studied using a dual retrograde transport technique. The B subunit of cholera toxin (CTB) was injected into the left adrenal medulla and wheatgerm agglutinin-apo-horseradish peroxidase-7 nm gold (WGA-apo-HRP-gold) was injected into the left aorticorenal ganglion. Retrogradely transported CTB was detected by immunohistochemistry, while gold particles were detected by silver intensification. SPN projecting to the adrenal medulla were observed in segments T2-L 2 of the spinal cord in 5 rabbits, with the majority of cells within segments T~,-TIj (79%). SPN projecting to the aorticorenal ganglion were seen in segments Ta-LI of the spinal cord in 5 rabbits, with the greatest number of the cells within T~-Ttt (81%). Only a small number of doubly labelled cells (1%) were found in two rabbits. The results suggest that despite their similar segmental distribution SPN projecting to the adrenal medulla or the aorticorenal ganglion belong to separate populations and few, if any, individual SPN have axonal projvctions to both locations.
Sympathetic preganglionic neurons (SPN) have an important role in the control of blood pressure, adrenal medullary hormone secretion and many other autonomic functions 'j. This control is achieved through a projection from the spinal cord, where SPN cell bodies are located, to the peripheral autonomic ganglia and to the adrenal gland. Many recent physiological studies have suggested that individual SPN do not innervate more than one type of target neuron 1'4'7, but few I°'t'~ have provided anatomical evidence. In the present study the spinal segmental location of the preganglionic innervation of the adrenal gland and the aorticorenal ganglion in the rabbit was examined using a combination of neuroanatomical tract tracing techniques. These two sites were chosen tbr two reasons. First, both the adrenal gland and the kidney are involved in cardiovascular regulation. Secondly, the adrenal gland and the aorticorenal ganglion are anatomically adjacent structures, and are likely to be innervated by overlapping populations of sympathetic preganglionic neurons. An injection of cholera toxin B subunit (CTB) was made into the left adrenal medulla,
while an injection of wheatgerm agglutinin-apohorseradish peroxidase conjugated to 7 nm gold particles (WGA-apo-HRP-gold) was made into the left aorticorenal ganglion of the same rabbits. Since these two methods produce labels that are easily distinguishable light microscopically, we were able to demonstrate the location of SPN that project to these targets, and at the same time examine the possibility that an indMduai SPN might have an axonal projection to both sites. Six male New Zealand white rabbits (2.8-3.2 kg) were used. Anaesthesia was induced with sodium thiopentone (40-60 mg i.e.; Abbott) injected through the marginal ear vein. An endotracheal tube was inserted and anaesthesia maintained with halothane. Ampicillin (Ampicyn; Protea, 100 mg in 1 nil i.v.) was given at induction and one hour post.operatively to prevent infection. The abdomen was then shaved of fur and swabbed with a povidone-iodine solution. A midline abdominal incision was made and the left adrenal gland and the left aorticorenal ganglion were exposed. The aorticorenal ganglion was found at the junction of the aorta and the renal artery. Several postganglionic
Correspondence: P. Pilowsky, Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Fax: (61) (8) 2045268.
'~l-[! o| Sa!ldde qo]q~ g u] mff 0E pup. 'V u! tuff001 = Je[l 'l~lJ~ q][~ PallOqel uoJnou e :3 "~J~ ql!~ Pall~q~l suoJn~u qlI.M p~lSu!uuolu! sic pio[i ql!~ POll~qUl suoznau leql ~iu!~oq,~ sqdeaSoa~!m jas~od qS!q :(3-1] "s~alsnp u! polnq!z;sIp a~e suoz~:~u P~llOqel I~ql llu!~oqs qdejSo~!m zas~od ~ o I :Y "e.llnp~tu le.uazPe aql olu] ~ J ~ pue *UO!lSUe.t~ [nuo~o~Ii~on oq] olu! PlOS-d~lH-ode-VOA~ jo uo!l~fu! ~olje P~llOqel ~lop~Jl~o~lo~ NdS jo ,~ueJe~dde aql 8u!~oq,~ sqdejSo~!moloqd "1 "~E!
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127 nerves arose from the aorticorenal ganglion and travelled with the renal artery towards the kidney. Care was taken not to disturb blood vessels or nerves supplying the gland and the ganglion. Cholera toxin B subunit (5-10 #l; CTB (List) containing 0.5-1.0/~l of a concentrated solution of Evans blue) was slowly pressure injected into the adrenal medulla with a hand-held capillary glass micropipette with a tip diameter of about 50-100/.~m attached to a 10 ml plastic syringe with vinyl tubing. Injections of WGA-apo-HRP-gold were made into the aorticorenal ganglion in the same way. Care was taken to avoid spillage of the tracer. After surgery, all rabbits recovered without incident, as evidenced by resumption of normal feeding, drinking and grooming b,~haviour. Three days later, the animals were deeply anaesthetized with sodium pentobarbitone and perfused through the heart with 1 liter of oxygenated tissue culture medium (Sigma D8900) followed by 2 liters of an aldehyde fixative (4% formaldehyde, 0.5% glutaraldehyde, 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). The left side of the spinal cords from T~ to La were sectioned in the parasaggitai plane at 70 /~m. Retrogradely transported WGA-apoHRP-gold was detected by silver intensification, CTB was detected by immunohistochemistry~. Briefly, sections were incubated in a 1:1 mixture of Silver Enhancer Solution A (Sigma SS020):Silver Enhancer Solution B (Sigma S5145). in addition to silver intensification, the sections were gold toned with 0.05% gold chloride and 0,2% oxalic acid. After visualisation of the WGA-apo-HRP-8old, CTB was detected using immunohistochemist~ and an avidin-biotin-peroxidase detection system. After a 30 rain incubation in 10% normal horse serum, sections were placed in a 1 : 50,000 dilution of goat antiserum to CTB. The sections were then incubated in a 1:200 dilution of biotinylated donkey anti-sheep immunoglobulin (Sigma B7390) and overnight in a 1:1,500 dilution of ExtrAvidin-peroxidase (Sigma E2886). An imidazole intensified di. aminobenzidine reaction was used to reveal the retrogradely transported CTB 12a3aT. The CTB.labelled cells and gold-labelled cells present in each spinal, cord segment were counted under the light microscope at a total magnification of 500 times. Both labels were found in the spinal cords of 4 rabbits. In one rabbit, only CTB was found, in another rabbit, only WGA-apo-HRP-gold was found. Sympathetic preganglionic neurons that were immunoreactive for CTB were distinctively labelled with an amber brown reaction product that was mainly associated with the Golgi apparatus ~3, but was also distributed diffusely throughout the neuron, with good labelling of the soma and dendrites (Fig. 1A-E). The
shape of the cell bodies varied from spherical to fusiform with the long axis running in the rostrocaudal direction. Although dendrites extended in several directions, the majority ran longitudinally within the confines of the intermediolateral cell column. SPN that were labelled with WGA-apo-HRP-gold contained black silver-intensified gold particles within their cytoplasm. In contrast to the labelling observed with CTB, gold particles were mainly seen in the soma and only occasionally in the dendrites (Fig. 1). Within each segment labelled cells were found in clusters within the intermediolateral cell column (Fig. IA). Neurons projecting to the adrenal medulla were intermingled with neurons projecting to the aorticorenal ganglion throughout the thoracolumbar spinal cord (Fig. 1A-D). In 2 out of 4 rabbits where both labels were present in the spinal cord, a small percentage (1%) of the total number of neurons counted were found to contain both labels. These doubly labelled cells generally contained weak CTB immunoreactivity when compared with neurons labelled with CTB alone. The distribution of SPN in the spinal cord after injection of the WGA-apo-HRP-gold into the aorticoreaal ganglion is shown in the top histogram in Fig. 2. Retrogradely labelled SPN were seen in segments T2-L 2 (mean - 453, S.E.M. - 128, n - 5). The greatest number (81%; 1836/2266 labelled neurons) of the total labelled cells were found within T~-T~. The distribution of the SPN in the spinal cord after injection of CTB into the adrenal medulla is shown in the bottom histogram in Fig. 2. Retrogradely labelled neurons projecting to the adrenal medulla were observed in segments T~-L.~ of the spinal cord (mean 449, S.E.M. = 70, n--5). The greatest number (79%; 1770/2247 labelled neurons) of labelled cells were found within segments 1"6-'1"1I. The findings presented here demonstrate that SPN that provide an input to either the adrenal medulla or the aorticorenal ganglion of the rabbit are widely distributed in the spinal cord. In this study, sympathoadrenal SPN were found from T2 to L 3 and aorticorenal SPN from T 2 to L 2. Other workers have shown H~as a similar but more restricted spinal segmental location of sympathoadrenal SPN in the rabbit. In these experiments neurons labelled with horseradish peroxidase, Dil or Herpes Simplex Virus, were present from T 3 to Ti2. The distribution of SPN projecting to the aorticorenal ganglion has not been previously described in rabbits. The distribution of adreno-medullary SPN in the rabbit is similar to that observed in the rat (T~-LI) TM, more restricted than in the kitten (Tj-L4) s, and more extensive than in the neonatal dog (T4-TI0) 3. Neurons
128
4o~o ]
]
,o
g
o
•aS
of spilt CTB was taken up by fibers projecting to the aorticorenal ganglion. If this was the case, our data support recent electrophysiological studies in the cat demonstrating separate sympathetic pathways to the adrenal medulla and the kidney m,as well as the general hypothesis that autonomic target organs are individually regulated by separate sets of sympathetic
Aorticorenal Ganglion
nerves6-8. ~s.
T1 11'2 T$ 1'4 TS TO 1"/ 1"8 TO T I O T I I T I 2
L1
1.2 L3
I
Z
i~- 40
Adrenal Gland
! 1o
o
T1 T2 1'3 T4 TS TO 1"7 TII 1"9 T 1 0 T l t T 1 2
L1
L2
L3
Spinal Cord Segment Fig, 2. Histograms showing the distribution of sympathetic prcganglionic neurons that project to the aorticorenal ganglion (upper paneh a total of 2266 labelled neurons in 5 rabbits), and to the adrenal ghmd (lower panel: a total of labelled 2247 neurons in 5 rabbits),
projecting to the aorticorenal ganglion in the rabbit show a more widespread distribution than that found in the rat (TT-Tt.0 tf', in this study, there was no apparent topographical segregation of SPN according to their target. Sympathoadrenal SPN were intermingled with SPN projecting to the aorticorenal ganglion in every spinal segment where both types of neuron were found, This finding is in contrast to the cat lumbar spinal cord where SPN with axonal projections to the hindlimb are located more laterally in the spinal cord than SPN that project to the inferior mesenteric ganglion ~. Most of the labelled neurons in this study contained either CTB or gold but not both, although a small number of doubly labelled neurons were found in two rabbits. These doubly labelled neurons may have an axonal projection to both the adrenal medulla and the aorticorenal ganglion, However, since doubly labelled neurons were found in only two rabbits, and since the CTB immunoreactivity in these neurons was relatively weak, a more likely explanation is that a small amount
We are grateful to Ms. K. Branch, Ms. R. Coffey, Ms. C. Frisby, Ms. M. McLaren, Ms. D. Molloy and Mr. A. Wright for their expert technical assistance. IJ, was supported by a National Heart Foundation of Australia Vacation Scholarship. This work was supported by grants from the National Health and Medical Research Council, the National Heart Foundation and the National Sudden Infant Death Syndrome Council of Australia. I Backman, S.B.. Sequeira-Martinho, H. and Henry, J.L., Adrenal versus nonadrenal sympathetic preganglionic neurones in the lower thoracic intermediolateral nucleus of the cat: physiological properties, Can. J. Physiol Pharmacol., 68 (1990) 1447-1456. 2 Bacon. S.J. and Smith, A.D., Preganglionic sympathetic neurons innervating the rat adrenal medulla: immunocytochemical evidence of synaptic input from nerve terminals containing substance P, GABA or 5-hydroxytryptamine, 1. Aaron. Nero'. Syst., 24 (1988) 97-122. 3 Cummings. J.F., Thoracolumbar preganglionic neurons and adrenal innervation in the dog, Acta Anat,, 73 (1969) 27-37, 4 ltayes, K. and Weaver, L.C, Selective control of sympathetic pathways to the kidney, spleen and intestine by the ventrolateral medulla in rats, J, Physiol.,, 428 (1990)371-385, 5 Holets, V. and Bide, R., Sympathoadrenal preganglionic neurons: their distribution and relationship to chemically-coded fibers in the kitten intermediolateral cell column, J, A,~mn, Neff, ,~,,vt,, 7 (1983) 149ol63, 6 Jiinig, W., Spinal cord integration of visceral sensory systems and sympathetic nervous system reflexes, i~8, Brain Res,, 67 (1986) 255 -276, 7 Jiinig, W,, Krauspe, R, and Wiedersatz, G., Transmission of impulses from pre- to postganglionic vasoconstrictor and sudomotor neurons, d', Autonom, Net,'. $yst,, 6 (1982) 95-106, 8 Jiinig, W. and McLachlan, E,M,, Identification of distinct topographical distributions of lumbar sympathetic and sensory neurons projecting to end organs with different functions in the cat, J. Comp. Nmrol,, 246 (1986) 104-112, 9 Laskey, W, and Polosa, C,, Characteristics of the sympathetic preganglionic neuron and its synaptic input, Ping, Neurobiol., 31 (1988) 47-84, 10 Li, Y,.W,, Ping, Z,,Q,, Wesselingh, S,L, and Blessing, W.W., Renal and adrenal sympathetic preganglionic neurons in rabbit spinal cord: tracing with tlerpes Simplex Virus, Brain Res,, in press, II Llewellyn-Smith, l,J,, Pilowsky, P,M, and Minson, J.B., Retrograde tracers that can be used for either light or electron microscopy: HRP-based tracers, gold-labelled tracers and unconjugated cholera toxin B subunit, in P, Bolam (Ed.), Experimental Nmroanatomy: a Practical Approach, Oxford University Press, Oxford, 1992, 12 Oldfield, B J,, Hou-Yu, A, and Silverman, A,-J., Technique for the simultaneous ultrastructural demonstration of anterogradely transported horseradish peroxidase and an immunocytochemically identified neuropeptide, J, Histochem, Cytochem., 31 (1983) 1145-1150.
129 13 Pilowsky, P.M., Llewellyn-Smith, l.J., Minson, J.B. and Chalmers, J.P., Sympathetic preganglionic neurons in rabbit spinal cord that project to the stellate or the superior cervical ganglion, Brain Res., in press (1992) 14 Schramm, L.P., Adair, J.R., Stribling, J.M. and Gray, L.P., Preganglionic innervation of the adrenal gland of the rat: a study using horseradish peroxidase, Exp. Neurol., 49 (1975) 540-553. 15 Strack, A.M. and Loewy, A.D., Pseudorabies virus: a highly specific transneuronal cell body marker in the sympathetic nervous system, J. Neurosci., 10 (1990) 2139-2147. 16 Strack, A.M., Sawyer, W.B., Hughes, J.H., Platt, K.B. and Loewy,
A.D., A general pattern of CNS innervation of the sympathetic outflow demonstrated by transneuronal pseudorabies viral infections, Brain Res., 491 (1989) 156-162. 17 Straus, W., Imidazole increases the sensitivity of the cytochemical reaction for peroxidase with diaminobenzidine at neutral pH, J. Histochem. Cytochem., 30 (1982) 491-492. 18 Vera, P.L., Hurwitz, B.E. and Schneiderman, N., Sympathoadrenal preganglionic neurons in the adult rabbit send their dendrites into the contralateral hemicord, Brain Res., 30 (1990) 193-198.
125
© 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00
BRES 25258
Sympathetic preganglionic neurons projecting to the adrenal medulla and aorticorenal ganglion in the rabbit l w o n a J e n s e n , P a u l Pilowsky, Ida Liewellyn-Smith, J a n e M i n s o n and J o h n C h a l m e r s Department of Medicbw and Centrefor Neuroscience, Flinders Unil'ersity, Bedford Park, SA (Australia) (Accepted 7 April 1992)
Key words: Retrograde tracing: Cholera toxin B; Wheatgerm agglutinin-apo-horseradish peroxidase-gold; Spinal cord; Blood pressure
The distribution of sympathetic preganglionic neurons (SPN) projecting to the adrenal medulla and the aorticorenal ganglion in the rabbit was studied using a dual retrograde transport technique. The B subunit of cholera toxin (CTB) was injected into the left adrenal medulla and wheatgerm agglutinin-apo-horseradish peroxidase-7 nm gold (WGA-apo-HRP-gold) was injected into the left aorticorenal ganglion. Retrogradely transported CTB was detected by immunohistochemistry, while gold particles were detected by silver intensification. SPN projecting to the adrenal medulla were observed in segments T2-L 2 of the spinal cord in 5 rabbits, with the majority of cells within segments T~,-TIj (79%). SPN projecting to the aorticorenal ganglion were seen in segments Ta-LI of the spinal cord in 5 rabbits, with the greatest number of the cells within T~-Ttt (81%). Only a small number of doubly labelled cells (1%) were found in two rabbits. The results suggest that despite their similar segmental distribution SPN projecting to the adrenal medulla or the aorticorenal ganglion belong to separate populations and few, if any, individual SPN have axonal projvctions to both locations.
Sympathetic preganglionic neurons (SPN) have an important role in the control of blood pressure, adrenal medullary hormone secretion and many other autonomic functions 'j. This control is achieved through a projection from the spinal cord, where SPN cell bodies are located, to the peripheral autonomic ganglia and to the adrenal gland. Many recent physiological studies have suggested that individual SPN do not innervate more than one type of target neuron 1'4'7, but few I°'t'~ have provided anatomical evidence. In the present study the spinal segmental location of the preganglionic innervation of the adrenal gland and the aorticorenal ganglion in the rabbit was examined using a combination of neuroanatomical tract tracing techniques. These two sites were chosen tbr two reasons. First, both the adrenal gland and the kidney are involved in cardiovascular regulation. Secondly, the adrenal gland and the aorticorenal ganglion are anatomically adjacent structures, and are likely to be innervated by overlapping populations of sympathetic preganglionic neurons. An injection of cholera toxin B subunit (CTB) was made into the left adrenal medulla,
while an injection of wheatgerm agglutinin-apohorseradish peroxidase conjugated to 7 nm gold particles (WGA-apo-HRP-gold) was made into the left aorticorenal ganglion of the same rabbits. Since these two methods produce labels that are easily distinguishable light microscopically, we were able to demonstrate the location of SPN that project to these targets, and at the same time examine the possibility that an indMduai SPN might have an axonal projection to both sites. Six male New Zealand white rabbits (2.8-3.2 kg) were used. Anaesthesia was induced with sodium thiopentone (40-60 mg i.e.; Abbott) injected through the marginal ear vein. An endotracheal tube was inserted and anaesthesia maintained with halothane. Ampicillin (Ampicyn; Protea, 100 mg in 1 nil i.v.) was given at induction and one hour post.operatively to prevent infection. The abdomen was then shaved of fur and swabbed with a povidone-iodine solution. A midline abdominal incision was made and the left adrenal gland and the left aorticorenal ganglion were exposed. The aorticorenal ganglion was found at the junction of the aorta and the renal artery. Several postganglionic
Correspondence: P. Pilowsky, Department of Medicine, Flinders Medical Centre, Bedford Park, SA 5042, Australia. Fax: (61) (8) 2045268.
'~l-[! o| Sa!ldde qo]q~ g u] mff 0E pup. 'V u! tuff001 = Je[l 'l~lJ~ q][~ PallOqel uoJnou e :3 "~J~ ql!~ Pall~q~l suoJn~u qlI.M p~lSu!uuolu! sic pio[i ql!~ POll~qUl suoznau leql ~iu!~oq,~ sqdeaSoa~!m jas~od qS!q :(3-1] "s~alsnp u! polnq!z;sIp a~e suoz~:~u P~llOqel I~ql llu!~oqs qdejSo~!m zas~od ~ o I :Y "e.llnp~tu le.uazPe aql olu] ~ J ~ pue *UO!lSUe.t~ [nuo~o~Ii~on oq] olu! PlOS-d~lH-ode-VOA~ jo uo!l~fu! ~olje P~llOqel ~lop~Jl~o~lo~ NdS jo ,~ueJe~dde aql 8u!~oq,~ sqdejSo~!moloqd "1 "~E!
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127 nerves arose from the aorticorenal ganglion and travelled with the renal artery towards the kidney. Care was taken not to disturb blood vessels or nerves supplying the gland and the ganglion. Cholera toxin B subunit (5-10 #l; CTB (List) containing 0.5-1.0/~l of a concentrated solution of Evans blue) was slowly pressure injected into the adrenal medulla with a hand-held capillary glass micropipette with a tip diameter of about 50-100/.~m attached to a 10 ml plastic syringe with vinyl tubing. Injections of WGA-apo-HRP-gold were made into the aorticorenal ganglion in the same way. Care was taken to avoid spillage of the tracer. After surgery, all rabbits recovered without incident, as evidenced by resumption of normal feeding, drinking and grooming b,~haviour. Three days later, the animals were deeply anaesthetized with sodium pentobarbitone and perfused through the heart with 1 liter of oxygenated tissue culture medium (Sigma D8900) followed by 2 liters of an aldehyde fixative (4% formaldehyde, 0.5% glutaraldehyde, 0.2% picric acid in 0.1 M phosphate buffer, pH 7.4). The left side of the spinal cords from T~ to La were sectioned in the parasaggitai plane at 70 /~m. Retrogradely transported WGA-apoHRP-gold was detected by silver intensification, CTB was detected by immunohistochemistry~. Briefly, sections were incubated in a 1:1 mixture of Silver Enhancer Solution A (Sigma SS020):Silver Enhancer Solution B (Sigma S5145). in addition to silver intensification, the sections were gold toned with 0.05% gold chloride and 0,2% oxalic acid. After visualisation of the WGA-apo-HRP-8old, CTB was detected using immunohistochemist~ and an avidin-biotin-peroxidase detection system. After a 30 rain incubation in 10% normal horse serum, sections were placed in a 1 : 50,000 dilution of goat antiserum to CTB. The sections were then incubated in a 1:200 dilution of biotinylated donkey anti-sheep immunoglobulin (Sigma B7390) and overnight in a 1:1,500 dilution of ExtrAvidin-peroxidase (Sigma E2886). An imidazole intensified di. aminobenzidine reaction was used to reveal the retrogradely transported CTB 12a3aT. The CTB.labelled cells and gold-labelled cells present in each spinal, cord segment were counted under the light microscope at a total magnification of 500 times. Both labels were found in the spinal cords of 4 rabbits. In one rabbit, only CTB was found, in another rabbit, only WGA-apo-HRP-gold was found. Sympathetic preganglionic neurons that were immunoreactive for CTB were distinctively labelled with an amber brown reaction product that was mainly associated with the Golgi apparatus ~3, but was also distributed diffusely throughout the neuron, with good labelling of the soma and dendrites (Fig. 1A-E). The
shape of the cell bodies varied from spherical to fusiform with the long axis running in the rostrocaudal direction. Although dendrites extended in several directions, the majority ran longitudinally within the confines of the intermediolateral cell column. SPN that were labelled with WGA-apo-HRP-gold contained black silver-intensified gold particles within their cytoplasm. In contrast to the labelling observed with CTB, gold particles were mainly seen in the soma and only occasionally in the dendrites (Fig. 1). Within each segment labelled cells were found in clusters within the intermediolateral cell column (Fig. IA). Neurons projecting to the adrenal medulla were intermingled with neurons projecting to the aorticorenal ganglion throughout the thoracolumbar spinal cord (Fig. 1A-D). In 2 out of 4 rabbits where both labels were present in the spinal cord, a small percentage (1%) of the total number of neurons counted were found to contain both labels. These doubly labelled cells generally contained weak CTB immunoreactivity when compared with neurons labelled with CTB alone. The distribution of SPN in the spinal cord after injection of the WGA-apo-HRP-gold into the aorticoreaal ganglion is shown in the top histogram in Fig. 2. Retrogradely labelled SPN were seen in segments T2-L 2 (mean - 453, S.E.M. - 128, n - 5). The greatest number (81%; 1836/2266 labelled neurons) of the total labelled cells were found within T~-T~. The distribution of the SPN in the spinal cord after injection of CTB into the adrenal medulla is shown in the bottom histogram in Fig. 2. Retrogradely labelled neurons projecting to the adrenal medulla were observed in segments T~-L.~ of the spinal cord (mean 449, S.E.M. = 70, n--5). The greatest number (79%; 1770/2247 labelled neurons) of labelled cells were found within segments 1"6-'1"1I. The findings presented here demonstrate that SPN that provide an input to either the adrenal medulla or the aorticorenal ganglion of the rabbit are widely distributed in the spinal cord. In this study, sympathoadrenal SPN were found from T2 to L 3 and aorticorenal SPN from T 2 to L 2. Other workers have shown H~as a similar but more restricted spinal segmental location of sympathoadrenal SPN in the rabbit. In these experiments neurons labelled with horseradish peroxidase, Dil or Herpes Simplex Virus, were present from T 3 to Ti2. The distribution of SPN projecting to the aorticorenal ganglion has not been previously described in rabbits. The distribution of adreno-medullary SPN in the rabbit is similar to that observed in the rat (T~-LI) TM, more restricted than in the kitten (Tj-L4) s, and more extensive than in the neonatal dog (T4-TI0) 3. Neurons
128
4o~o ]
]
,o
g
o
•aS
of spilt CTB was taken up by fibers projecting to the aorticorenal ganglion. If this was the case, our data support recent electrophysiological studies in the cat demonstrating separate sympathetic pathways to the adrenal medulla and the kidney m,as well as the general hypothesis that autonomic target organs are individually regulated by separate sets of sympathetic
Aorticorenal Ganglion
nerves6-8. ~s.
T1 11'2 T$ 1'4 TS TO 1"/ 1"8 TO T I O T I I T I 2
L1
1.2 L3
I
Z
i~- 40
Adrenal Gland
! 1o
o
T1 T2 1'3 T4 TS TO 1"7 TII 1"9 T 1 0 T l t T 1 2
L1
L2
L3
Spinal Cord Segment Fig, 2. Histograms showing the distribution of sympathetic prcganglionic neurons that project to the aorticorenal ganglion (upper paneh a total of 2266 labelled neurons in 5 rabbits), and to the adrenal ghmd (lower panel: a total of labelled 2247 neurons in 5 rabbits),
projecting to the aorticorenal ganglion in the rabbit show a more widespread distribution than that found in the rat (TT-Tt.0 tf', in this study, there was no apparent topographical segregation of SPN according to their target. Sympathoadrenal SPN were intermingled with SPN projecting to the aorticorenal ganglion in every spinal segment where both types of neuron were found, This finding is in contrast to the cat lumbar spinal cord where SPN with axonal projections to the hindlimb are located more laterally in the spinal cord than SPN that project to the inferior mesenteric ganglion ~. Most of the labelled neurons in this study contained either CTB or gold but not both, although a small number of doubly labelled neurons were found in two rabbits. These doubly labelled neurons may have an axonal projection to both the adrenal medulla and the aorticorenal ganglion, However, since doubly labelled neurons were found in only two rabbits, and since the CTB immunoreactivity in these neurons was relatively weak, a more likely explanation is that a small amount
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