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Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla
Journal of the Autonomic Nervous System, 33 (1991) 247-254
247
~'~ 1991 Elsevier Science Publishers B.V. 0165-1838/91/$03.50
JANS 01150
Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla X i n - F u Z h o u , Brian J. Oldfield 1 and Bruce G. Livett Department of Biochemistry, Universi(~' of Melbourne and J Howard Florev Institute, Parkr2ille, Vtctoria, Australia (Received 10 October 1990) (Revision received 28 November 1990) (Accepted 30 November 1990)
Key words: Substance P, Adrenal gland: Sensory neuron: Immunohistochemistry Abstract The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known, in the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 + 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 ± 19 in T4 to 372 ± 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 _+ 26, distributed normally about a peak at T8 (33.8 ± 6.3 cells) and T9 (33.3 ± 6.8 cells) with the least at T3 (1.5 ± 0.8) and T13 (5.2 ± 2.0). No Fast blue-labelled ceils were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelling in 8 experiments was 23 amounting to 14.0% of the total retrograde labelled cells. Most (67%) of the double labelled cells were distributed from T7 to Tg. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.
Introduction
Catecholamine (CA) secretion from the rat adrenal medulla is mediated by cholinergic and non-cholinergic mechanisms in the isolated perfused adrenal gland [36]. In the rat, acetylcholine (ACh) released from splanchnic nerve terminals brings about CA secretion from chromaffin cells by activating both nicotinic and muscarinic recep-
Correspondence:
B.G. Livett, Department of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia.
tors [7,8], Cholinergic innervation of the adrenal gland is derived from the preganglionic sympathetic neurons in the intermediolateral cell column (IML) of the spinal cord [21]. However, the nature of the non-cholinergic mechanisms for CA secretion from the adrenal medulla is not known. A number of neuropeptides including the opioid peptides [32,34,46], substance P (SP) [27,30], neuropeptide Y (NPY) [28], somatostatin [34], neurotensin [35], calcitonin gene-related peptide (CGRP) [29] and vasoactive intestinal peptide (VIP) [25] are present in nerve terminals in the adrenal medulla of mammals. Some of these are known to
248 activate or modulate CA secretion from chromaffin cells. SP is known to exhibit two distinct actions on the adrenal medulla: (1) inhibition of nicotineevoked secretion of CA [33] and (2) protection against desensitization of this nicotinic response [1,53,55]. In addition, in the anaesthetized rat, adrenal CA secretion in response to stress was reduced or abolished in capsaicin-pretreated rats in which SP in the splanchnic nerve was depleted by 70% [22,23]. These studies suggest that SP-containing nerve fibres in the adrenal medulla act together with cholinergic nerve fibres to maintain CA secretion; however, the origin of SP-containing nerve terminals in the adrenal medulla is not known. Retrograde tracing studies show that, in addition to sympathetic preganglionic neurons in the IML, a large number of sensory neurons in the dorsal root ganglia (DRG) innervate the adrenal medulla [40,49]. It is also known that in mammals a large population of small sensory neurons in the dorsal root ganglia contain SP [15,17]. Based on these studies, it has been assumed that adrenal SP-immunoreactive fibres are derived from the sensory neurons in the D R G [27]. However, to our knowledge, there is no direct evidence showing that SP-containing cells in spinal ganglia project to the adrenal medulla. In the present study we have used retrograde tracing techniques combined with SP immunocytochemistry to establish that SP containing cells in the D R G project to the adrenal medulla and to quantitatively assess the distribution and density of these SP-containing sensory neurons in the DRG.
Materials and Methods
Animal preparation Eight 3-month-old male Buffalo rats were anaesthetized with Equithesin ( a mixture of pentobarbital and chloral hydrate, 2.5 m l / k g i.p., for details see ref. 50) and the left adrenal gland was exposed. Three #1 of a 2% suspension of Fast blue (Dr. Illing, Makromolekulare Chemie, F.R.G., courtesy of Dr. Ian Gibbins, Flinders University,
Adelaide) was injected slowly into the left adrenal medulla via a 5-~1 Hamilton syringe coupled with a glass micropipette. The injection was made over 15 20 rain using an infusion pump, after which the micropipette was left in place for a further 15 rain, then withdrawn slowly. There was no leakage seen after removal of the micropipette. Any rat in which leakage of Fast blue during injection was suspected, was not used in the study. After a survival period of 5 or 6 days, rats were anaesthetized with Equithesin as described above and perfused intracardially with 300 ml 0.9% saline followed by 300-400 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer (4°C, pH 7.2). After perfusion, the left D R G from T3 to T13 were dissected from the rats and fixed overnight in 4% paraformaldehyde containing 20% sucrose. Some of the right D R G were also dissected for control material.
Irnmunohistochemistrv Each dorsal root ganglion was sectioned at 30 /,m using a sledge freezing microtome (Leitz) and sections were collected in 0.1 M sodium phosphate buffer (pH 7.2) in cell culture wells. Free-floating sections were transferred to 10% normal horse serum prior to incubation overnight at 4 ° C in phosphate buffer containing a polyclonal antiserum (1:20000) raised in rabbits against substance P [41] (MRSP-1, courtesy of Dr. R. Murphy, Flinders University, Adelaide). The SP antiserum requires both the N-terminal and C-terminal structure of SP for binding. Radioimmunoassay studies have shown that MRSP-1 has no cross-reactivity with N-terminal fragments of SP, and has little cross-reactivity with neurokinin B (NKB) or neuropeptide K, a tachykinin with neurokimn A (NKA) amino acid sequence at its C-terminal (ref. 41, and Basile and Livett, unpublished data). The primary substance P antiserum was subsequently localized with biotinylated anti-rabbit lgG and finally with avidin-fluorescein (Vector). As controls, some sections from the right D R G were incubated with the SP antiserum in the presence of 0.13 m g / m l substance P (Peninsula Laboratories, U.S.A.). No immunoreactive SP neurons were detected when the antiserum was saturated in this way with exogenous SP.
249
Observation and statistics
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All preparations were systematically scanned using a Leitz fluorescence microscope (Leitz Laborlux 12) fitted with filters for the detection of fluorescein-labelled substance P immunoreactivity (I2, Leitz) and Fast blue (D, Leitz). Cell sizes were measured with an M D I microscope digitizer (Minnesota Digital, St, Paul, MN, U.S.A.). All of the substance P immunoreactive cells and retrogradely labelled Fast blue cells in D R G ipsilateral to the injected adrenals were counted. To avoid the possibility of double counting, only those neurons where the nucleus was visible were counted.
Results
Substance P labelled sensory neurons in the D R G
Substance P-labelled cells were characterized by green punctate immunofluorescence in their cytoplasm. These immunoreactive SP cells were distributed in roughly equal numbers in each of the D R G studied between T3 and T13 (Fig. 1). The SP-labelled cells in the D R G were oval or round in shape and occurred both in groups and single throughout the section. The size of these SP-labelled cells ranged from 10 to 40 /~m in diameter, with 85% (111 out of 130 that were measured) in the range of 20 30 /~m in diameter (Fig. 2).
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Fig, 2, F r e q u e n c y h i s t o g r a m s h o w i n g d i a m e t e r of cell bodies of Fast blue-labelled neurons and SP n e u r o n s in the left dorsal root ganglia 6 d a y s after injection of Fast blue in the adrenal medulla. D a t a are plotted as p e r c e n t a g e of the total cells m e a s u r e d ( n = 130).
Fast blue-labelled neurons
Fast blue-labelled neurons in the D R G displayed a bright blue granular fluorescence in the cytoplasm. In some very bright cells, the nuclei were also fluorescent. Neurons labelled with Fast blue ipsilateral to the site of injection were found in the D R G at all levels from T3 to T13. In contrast, no Fast blue-labelled cells were found in D R G on the contralateral side. The average total number of Fast blue-labelled cells in D R G from
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Fig. l. F r e q u e n c y h i s t o g r a m s h o w i n g d i s t r i b u t i o n of cells in the left dorsal root ganglia labelled with SP. D a t a are plotted as means_+ SEM (n = 8).
left dorsal root ganglia
Fig. 3. F r e q u e n c y h i s t o g r a m s h o w i n g the n u m b e r a n d distribution of the cells in the left D R G r e t r o g r a d e l y labelled with Fast blue 6 days after injection of Fast blue into the left a d r e n a l medulla. D a t a are p l o t t e d as m e a n s + SEM ( n = 8).
250
Fig. 4 Dual labelling of neurons in the left DRG (level T8) with Fast blue and SP after injection of Fast blue into the left adrenal medulla. Panel a shows the results with a filter combination selective for Fast blue and panel b with a filter combination selective for SP immunofl.uorescence. Two of the neurons in this field are labelled with Fast blue (single and double arrows in a). The smaller one (single arrow in b) also contains SP. The larger one (double arrow in b) is not labelled with SP. The location of some other SP positive cells can be seen in the Fast blue picture but they are very faint and can be distinguished from the Fast blue-labelled cells
T3 to T13 was 172 _+ 26 (n = 8, Fig. 3). T h e m a x i m u m n u m b e r in o n e rat was 297. T h e m a j o r i t y o f F a s t b l u e - l a b e l l e d cells in the D R G w e r e f o u n d at levels T8 a n d T9 (67% o f the total p o p u l a t i o n ) w i t h the r e m a i n d e r d i s t r i b u t e d n o r m a l l y a m o n g the levels e i t h e r side of the p e a k . T h e size of the l a b e l l e d cells r a n g e d f r o m 10 to 6 5 / ~ m . M o s t F a s t b l u e - l a b e l l e d cells w e r e less t h a n 40 /~m (Fig. 2).
O f these, the m a j o r i t y (67%) w e r e d i s t r i b u t e d bet w e e n T7 a n d T9 (Fig. 5).
Discussion
In the p r e s e n t s t u d y we c o m b i n e d s u b s t a n c e P i m m u n o c y t o c h e m i s t r y with the F a s t b l u e retro-
Double labelled neurons
W i t h the filter s y s t e m s used ( W i l d L e i t z F i l t e r D for F a s t blue- a n d 12 for f l u o r e s c e i n - l a b e l l e d SP) the f l u o r e s c e n c e e m i t t e d by F a s t b l u e - l a b e l l e d cells ( d o u b l e a r r o w s in Fig. 4a) was n o t d e t e c t e d w i t h the filter c o m b i n a t i o n for SP ( d o u b l e a r r o w s in Fig. 4b). T h e c o n v e r s e was n o t the case so t h a t the m o s t b r i g h t f l u o r e s c e n t SP cells w e r e visible w i t h the filters used for F a s t blue. H o w e v e r , w i t h the filter c o m b i n a t i o n for F a s t blue, the SP cells w e r e g r e e n a n d easily d i s t i n g u i s h a b l e f r o m the p a l e F a s t b l u e cells. Fig. 4 ( p a n e l a) s h o w s t w o F a s t b l u e - l a b e l l e d cells (arrows). O n e of these cells (single a r r o w ) was also l a b e l l e d with SP (single a r r o w in p a n e l b) a n d the o t h e r was n o t ( d o u b l e a r r o w in p a n e l b). A p p r o x i m a t e l y 14% o f the F a s t b l u e - l a b e l l e d cells in the D R G o f 8 rats also c o n t a i n e d SP ( t o t a l 184 cells, 23 cells o n a v e r a g e , r a n g e 1 1 - 4 5 cells).
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Fig. 5. Frequency histogram showing the distribution of the cells in the left dorsal root ganglia labelled with both SP and Fast blue 6 days after injection of Fast blue into left adrenal medulla in 8 rats. Data is derived from counts of all double labelled cells from 8 rats.
251
grade tracing technique to quantitate the number of SP-containing neurons in the rat D R G innervating the adrenal medulla. The results show that up to 14% of the neurons in the D R G that innervate the rat adrenal medulla are SP-positive. The D R G between T3 and T13 contained an average of 281 + 31 SP immunoreactive cells per ganglion. These results are in agreement with a previous study in the rat [2] where it was found that there were approximately 250 SP-immunorcactive cells in each sensory ganglion. In accord with previous studies [15,17], 85% of the SP-labelled cells were of small diameter ( < 30 /,m). In the present study an average of 172 + 26 neurons in the ipsilateral D R G were retrogradely labelled with Fast blue following injection of the dye into the left adrenal medulla. Using Fluorogold for retrograde tracing, Strack et al. [49] found that only 55 neurons in the rat D R G innervated the adrenal medulla, Whereas in the guinea pig [40], use of Fast blue to trace the sensory innervation of the adrenal medulla [40] resulted in the labelling of twice the number of cells detected in the present study in the rat (582 vs. 297). The present study showed that 14% of the Fast blue-labelled cells in the D R G projecting to the [eft adrenal medulla were SP-positive. Others have shown that between 2 and 60% of the cells in D R G contributing sensory innervation to peripheral tissues were SP-positive [5,6,18,39]. It is possible that the figure of 14% for dual labelled neurons is an underestimate, due to the the difficulty of SP antibody penetration in 30-/,m tissue sections. The sensory innervation of the adrenal gland is likely to contain putative transmitters other than SP. A number of neuropeptides such as somatostatin [15.17], V1P, cholecystokinin [6], gastrin releasing peptide [44], endorphin [24], dynorphin [11], C G R P [11,45], corticotropin releasing factor [48], galanin [47], oxytocin and arginine vasopressin [19] have been found in sensory neurons of the DRG. Some of these neuropeptides also exist in the nerve terminals innervating the adrenal medulla. For example, SPand CGRP-immunoreactivities co-exist in nerve fibres of the rat adrenal medulla [29]. In addition, somatostatin and VIP have been detected in the
nerve terminals innervating the adrenal medulla [25,34]. It is likely, therefore, that in addition to SP, other neuropeptide-containing neurons in the D R G also innervate the adrenal medulla either alone or in combination with SP. The question arises as to whether or not all the SP fibres in the adrenal medulla originate from sensory neurons. The adrenal medulla receives innervation from supra-adrenal ganglia and sympathetic ganglia [21] in addition to D R G : however, these neurons do not contain SP [4,16,38]. SP has been localized in sympathetic preganglionic neurons in the cat [27] and in parasympathetic neurons in the toad [12] but to date no substance P-containing preganglionic neurons have been identified in the rat. In the present experiments, SP was not found in preganglionic neurons in spinal cord of the rat {data not presented). Nevertheless, our study cannot rule out the possibility that some SP nerve fibres in the adrenal medulla may come from preganglionic sympathetic neurons. Recently, it was found that the adrenal medulla receives innervation from nodose ganglia and vagal ganglia [3]. In the present study, we also found that some neurons in the nodose ganglia and vagal ganglia were labelled after injection of Fast blue into the ipsilateral adrenal medulla (data not shown). Moreover, a few retrogradely labelled neurons in the vagal ganglia were also labelled with SP. It has been found that a large number of neurons in the nodose and vagal ganglia in the cat contain SP [20]. Taken together, these studies suggest that, in addition to the DRG, nodose ganglia and vagal ganglia may be a source of SP fibres in the adrenal medulla. Substance P is now well established as a neuropeptide marker for a population of sensory neurons in D R G [11,15,17]. The function of the sensory innervation of the adrenal medulla, however, is not known [13,14]. It has been reported that adrenal afferent fibres fire following a change of blood pressure or injection of adrenaline and noradrenaline, suggesting that the sensory neurons innervating the adrenal medulla may convey baroreflex and chemoreflex information [42,43]. In recent years, evidence has accumulated to show that neuropeptides present in nerve terminals innervating the adrenal medulla modulate adrenal
252
medullary catecholamine secretion (for review, see refs. 31,37). For example, SP inhibits the nicotinic secretion of catecholamines from isolated bovine chromaffin cells [33] and protects against nicotinic desensitization [1]. In addition, SP at low concentrations increases CA secretion from perfused rat adrenal glands evoked by electrical stimulation [53,55]. In capsaicin-pretreated rats in which SP levels in the splanchnic nerve are depleted by 70-90%, [9,22], adrenal medullary CA secretion in response to stress under anaesthesia is abolished or reduced [22,23]. Likewise, CA secretion evoked by electrical stimulation of the isolated perfused rat adrenal gland from capsaicin pretreated rats was reduced [53,54]. This indicates that capsaicinsensitive sensory neurons are involved in the local control of CA secretion from the adrenal gland. Although SP release from sensory neurons in the adrenal medulla has not been proven, local release and effects of SP from visceral and skin sensory neurons are well established [10,52]. In addition, SP can be released from the adrenal medulla in vivo in response to selective stressors [51]. These studies suggest that SP-containing neurons innervating adrenal medulla act to maintain CA secretion during stress. The retrograde tracing studies in the present study indicate that some SP-containing nerve terminals in the rat adrenal medulla arise from the ipsilateral D R G . It is likely that this pathway constitutes the normal substrate for SP modulation of adrenal CA secretion.
Acknowledgements We thank Dr. Phil Marley for his advice and comments, Dr. Ian Gibbins (Flinders University, Adelaide) for his generous gift of Fast blue, Dr. Roger Murphy (Flinders University, Adelaide) for his gift of SP antiserum, Mr. Tim Anning, Ms. Daphne K. Hards and Mr. Phillip McGlashan for technical assistance. X.-F.Z. was supported by a University of Melbourne Postgraduate Award. This work was supported by a project grant to B.G.L. from the National Health and Medical Research Council ( N H & M R C ) of Australia.
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254 44 Panula, P.. Hadjiconstantinou, M., Yang, H.-Y.T. and Costa, E., lmmunohistochemical localization of b o m b e s i n / gastrin releasing peptide and substance P in primary sensory neurons, J. Neurosci., 3 (1983) 2021 2029. 45 Rosenfeh, M.G., Mermod, J.-J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W. and Evans, R.M., Production of a novel neuropeptide encoded by the calcitonin gene with tissue specific R N A processing, Nature (Lond.). 304 (1983) 129 135. 46 Schultzberg, M., Lundberg, J.M., H6kfelt, T., Terenius, L., Brandt, J., Elde, R.P. and Goldstein, M., Enkephalin-like immunoreactivity in gland cells and nerve terminals of the adrenal medulla, Neuroscience, 3 (1978) 1169-1186. 47 Skofitsch, G. and Jacobowitz, D.M., Galanin-like immunoreactivity in capsaicin sensitive sensory neurons and ganglia, Brain Res. Bull., 156 (1985) 191-195. 48 Skofitsch, G., Zamir, N., Helke, C.J., Savitt, J.M. and Jacobowitz, D.M., Corticotropin releasing factor like immunoreactivity in sensory ganglia and capsaicin sensory neurons of the rat central nervous system: colocalization with other peptides, Peptides, 6 (1985) 307-318. 49 Strack, A.M., Sawyer, W.B., Marubio, L.M. and Loewy, A.D., Spinal origin of sympathetic preganglionic neurons in the rat, Brain Res., 455 (1988) 187-191.
50 Tagerud, S.E.O. and Cuello, A.C., Dopamine release from the rat substantia nigra in vitro. Effect of raphe lesions and veratridine stimulation. Neuroscience. 4 (1979) 2021 2029. 51 Vaupel, R., Jarry, H., Schlomer, H.-T. and Wuttke, W., Differential response of substance P-containing subtypes of adrenomedullary cells to different stressors, Endocrinology, 123 (1988) 2140 2145. 52 White, D.M. and Helme, R.D., Release of substance P from peripheral nerve terminals following electrical stimulation of the sciatic nerve, Brain Res., 336 (1985) 27 31. 53 Zhou, X.-F. and kivett, B.G., Substance P increases catecholamine secretion from perfused rat adrenal glands evoked by prolonged field stimulation, J. Physiol. I kond.), 425 (1990) 321 334. 54 Zhou, X.-F., Marley, M.D. and Livett, B.G., Role of capsaicin-sensitive sensory fibres in catecholaminc secretion from perfused rat adrenal glands, Eur. J. Pharmacol., 186 (1991) 247 255. 55 Zhou, X.-F. and kivett, B.G., Substance P has biphasic effects on catecholaminc secretion evoked by electrical stimulation of perfused rat adrenal glands in vitro. J. Auton. Nerv. Syst., 31 (1990) 31 40.
247
~'~ 1991 Elsevier Science Publishers B.V. 0165-1838/91/$03.50
JANS 01150
Substance P-containing sensory neurons in the rat dorsal root ganglia innervate the adrenal medulla X i n - F u Z h o u , Brian J. Oldfield 1 and Bruce G. Livett Department of Biochemistry, Universi(~' of Melbourne and J Howard Florev Institute, Parkr2ille, Vtctoria, Australia (Received 10 October 1990) (Revision received 28 November 1990) (Accepted 30 November 1990)
Key words: Substance P, Adrenal gland: Sensory neuron: Immunohistochemistry Abstract The adrenal medulla is innervated by both cholinergic and substance P (SP)-containing fibres via the splanchnic nerve. SP has been shown to modulate catecholamine (CA) secretion in isolated chromaffin cells and in the perfused rat adrenal gland, however, the origin of SP-containing fibres is not known, in the present study, we have combined the techniques of SP immunohistochemistry and retrograde tracing with Fast blue injected into the left adrenal medulla of the rat in order to study whether SP-containing sensory neurons in the dorsal root ganglia innervate the adrenal medulla. The results showed that there were on average 281 + 31 SP-like immunoreactive cells in each left dorsal root ganglion, T3-T13 (range, 234 ± 19 in T4 to 372 ± 43 in T13, n = 8). The average total number of Fast blue-labelled cells (T3-T13) in 8 experiments was 172 _+ 26, distributed normally about a peak at T8 (33.8 ± 6.3 cells) and T9 (33.3 ± 6.8 cells) with the least at T3 (1.5 ± 0.8) and T13 (5.2 ± 2.0). No Fast blue-labelled ceils were found in the right DRG. In the left DRG, the average number of cells exhibiting both SP and Fast blue labelling in 8 experiments was 23 amounting to 14.0% of the total retrograde labelled cells. Most (67%) of the double labelled cells were distributed from T7 to Tg. These results demonstrate that SP-containing sensory neurons in the dorsal root ganglia provide an ipsilateral innervation of the adrenal medulla in rats.
Introduction
Catecholamine (CA) secretion from the rat adrenal medulla is mediated by cholinergic and non-cholinergic mechanisms in the isolated perfused adrenal gland [36]. In the rat, acetylcholine (ACh) released from splanchnic nerve terminals brings about CA secretion from chromaffin cells by activating both nicotinic and muscarinic recep-
Correspondence:
B.G. Livett, Department of Biochemistry, University of Melbourne, Parkville, Victoria 3052, Australia.
tors [7,8], Cholinergic innervation of the adrenal gland is derived from the preganglionic sympathetic neurons in the intermediolateral cell column (IML) of the spinal cord [21]. However, the nature of the non-cholinergic mechanisms for CA secretion from the adrenal medulla is not known. A number of neuropeptides including the opioid peptides [32,34,46], substance P (SP) [27,30], neuropeptide Y (NPY) [28], somatostatin [34], neurotensin [35], calcitonin gene-related peptide (CGRP) [29] and vasoactive intestinal peptide (VIP) [25] are present in nerve terminals in the adrenal medulla of mammals. Some of these are known to
248 activate or modulate CA secretion from chromaffin cells. SP is known to exhibit two distinct actions on the adrenal medulla: (1) inhibition of nicotineevoked secretion of CA [33] and (2) protection against desensitization of this nicotinic response [1,53,55]. In addition, in the anaesthetized rat, adrenal CA secretion in response to stress was reduced or abolished in capsaicin-pretreated rats in which SP in the splanchnic nerve was depleted by 70% [22,23]. These studies suggest that SP-containing nerve fibres in the adrenal medulla act together with cholinergic nerve fibres to maintain CA secretion; however, the origin of SP-containing nerve terminals in the adrenal medulla is not known. Retrograde tracing studies show that, in addition to sympathetic preganglionic neurons in the IML, a large number of sensory neurons in the dorsal root ganglia (DRG) innervate the adrenal medulla [40,49]. It is also known that in mammals a large population of small sensory neurons in the dorsal root ganglia contain SP [15,17]. Based on these studies, it has been assumed that adrenal SP-immunoreactive fibres are derived from the sensory neurons in the D R G [27]. However, to our knowledge, there is no direct evidence showing that SP-containing cells in spinal ganglia project to the adrenal medulla. In the present study we have used retrograde tracing techniques combined with SP immunocytochemistry to establish that SP containing cells in the D R G project to the adrenal medulla and to quantitatively assess the distribution and density of these SP-containing sensory neurons in the DRG.
Materials and Methods
Animal preparation Eight 3-month-old male Buffalo rats were anaesthetized with Equithesin ( a mixture of pentobarbital and chloral hydrate, 2.5 m l / k g i.p., for details see ref. 50) and the left adrenal gland was exposed. Three #1 of a 2% suspension of Fast blue (Dr. Illing, Makromolekulare Chemie, F.R.G., courtesy of Dr. Ian Gibbins, Flinders University,
Adelaide) was injected slowly into the left adrenal medulla via a 5-~1 Hamilton syringe coupled with a glass micropipette. The injection was made over 15 20 rain using an infusion pump, after which the micropipette was left in place for a further 15 rain, then withdrawn slowly. There was no leakage seen after removal of the micropipette. Any rat in which leakage of Fast blue during injection was suspected, was not used in the study. After a survival period of 5 or 6 days, rats were anaesthetized with Equithesin as described above and perfused intracardially with 300 ml 0.9% saline followed by 300-400 ml 4% paraformaldehyde in 0.1 M sodium phosphate buffer (4°C, pH 7.2). After perfusion, the left D R G from T3 to T13 were dissected from the rats and fixed overnight in 4% paraformaldehyde containing 20% sucrose. Some of the right D R G were also dissected for control material.
Irnmunohistochemistrv Each dorsal root ganglion was sectioned at 30 /,m using a sledge freezing microtome (Leitz) and sections were collected in 0.1 M sodium phosphate buffer (pH 7.2) in cell culture wells. Free-floating sections were transferred to 10% normal horse serum prior to incubation overnight at 4 ° C in phosphate buffer containing a polyclonal antiserum (1:20000) raised in rabbits against substance P [41] (MRSP-1, courtesy of Dr. R. Murphy, Flinders University, Adelaide). The SP antiserum requires both the N-terminal and C-terminal structure of SP for binding. Radioimmunoassay studies have shown that MRSP-1 has no cross-reactivity with N-terminal fragments of SP, and has little cross-reactivity with neurokinin B (NKB) or neuropeptide K, a tachykinin with neurokimn A (NKA) amino acid sequence at its C-terminal (ref. 41, and Basile and Livett, unpublished data). The primary substance P antiserum was subsequently localized with biotinylated anti-rabbit lgG and finally with avidin-fluorescein (Vector). As controls, some sections from the right D R G were incubated with the SP antiserum in the presence of 0.13 m g / m l substance P (Peninsula Laboratories, U.S.A.). No immunoreactive SP neurons were detected when the antiserum was saturated in this way with exogenous SP.
249
Observation and statistics
10o
All preparations were systematically scanned using a Leitz fluorescence microscope (Leitz Laborlux 12) fitted with filters for the detection of fluorescein-labelled substance P immunoreactivity (I2, Leitz) and Fast blue (D, Leitz). Cell sizes were measured with an M D I microscope digitizer (Minnesota Digital, St, Paul, MN, U.S.A.). All of the substance P immunoreactive cells and retrogradely labelled Fast blue cells in D R G ipsilateral to the injected adrenals were counted. To avoid the possibility of double counting, only those neurons where the nucleus was visible were counted.
Results
Substance P labelled sensory neurons in the D R G
Substance P-labelled cells were characterized by green punctate immunofluorescence in their cytoplasm. These immunoreactive SP cells were distributed in roughly equal numbers in each of the D R G studied between T3 and T13 (Fig. 1). The SP-labelled cells in the D R G were oval or round in shape and occurred both in groups and single throughout the section. The size of these SP-labelled cells ranged from 10 to 40 /~m in diameter, with 85% (111 out of 130 that were measured) in the range of 20 30 /~m in diameter (Fig. 2).
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Fig, 2, F r e q u e n c y h i s t o g r a m s h o w i n g d i a m e t e r of cell bodies of Fast blue-labelled neurons and SP n e u r o n s in the left dorsal root ganglia 6 d a y s after injection of Fast blue in the adrenal medulla. D a t a are plotted as p e r c e n t a g e of the total cells m e a s u r e d ( n = 130).
Fast blue-labelled neurons
Fast blue-labelled neurons in the D R G displayed a bright blue granular fluorescence in the cytoplasm. In some very bright cells, the nuclei were also fluorescent. Neurons labelled with Fast blue ipsilateral to the site of injection were found in the D R G at all levels from T3 to T13. In contrast, no Fast blue-labelled cells were found in D R G on the contralateral side. The average total number of Fast blue-labelled cells in D R G from
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Fig. l. F r e q u e n c y h i s t o g r a m s h o w i n g d i s t r i b u t i o n of cells in the left dorsal root ganglia labelled with SP. D a t a are plotted as means_+ SEM (n = 8).
left dorsal root ganglia
Fig. 3. F r e q u e n c y h i s t o g r a m s h o w i n g the n u m b e r a n d distribution of the cells in the left D R G r e t r o g r a d e l y labelled with Fast blue 6 days after injection of Fast blue into the left a d r e n a l medulla. D a t a are p l o t t e d as m e a n s + SEM ( n = 8).
250
Fig. 4 Dual labelling of neurons in the left DRG (level T8) with Fast blue and SP after injection of Fast blue into the left adrenal medulla. Panel a shows the results with a filter combination selective for Fast blue and panel b with a filter combination selective for SP immunofl.uorescence. Two of the neurons in this field are labelled with Fast blue (single and double arrows in a). The smaller one (single arrow in b) also contains SP. The larger one (double arrow in b) is not labelled with SP. The location of some other SP positive cells can be seen in the Fast blue picture but they are very faint and can be distinguished from the Fast blue-labelled cells
T3 to T13 was 172 _+ 26 (n = 8, Fig. 3). T h e m a x i m u m n u m b e r in o n e rat was 297. T h e m a j o r i t y o f F a s t b l u e - l a b e l l e d cells in the D R G w e r e f o u n d at levels T8 a n d T9 (67% o f the total p o p u l a t i o n ) w i t h the r e m a i n d e r d i s t r i b u t e d n o r m a l l y a m o n g the levels e i t h e r side of the p e a k . T h e size of the l a b e l l e d cells r a n g e d f r o m 10 to 6 5 / ~ m . M o s t F a s t b l u e - l a b e l l e d cells w e r e less t h a n 40 /~m (Fig. 2).
O f these, the m a j o r i t y (67%) w e r e d i s t r i b u t e d bet w e e n T7 a n d T9 (Fig. 5).
Discussion
In the p r e s e n t s t u d y we c o m b i n e d s u b s t a n c e P i m m u n o c y t o c h e m i s t r y with the F a s t b l u e retro-
Double labelled neurons
W i t h the filter s y s t e m s used ( W i l d L e i t z F i l t e r D for F a s t blue- a n d 12 for f l u o r e s c e i n - l a b e l l e d SP) the f l u o r e s c e n c e e m i t t e d by F a s t b l u e - l a b e l l e d cells ( d o u b l e a r r o w s in Fig. 4a) was n o t d e t e c t e d w i t h the filter c o m b i n a t i o n for SP ( d o u b l e a r r o w s in Fig. 4b). T h e c o n v e r s e was n o t the case so t h a t the m o s t b r i g h t f l u o r e s c e n t SP cells w e r e visible w i t h the filters used for F a s t blue. H o w e v e r , w i t h the filter c o m b i n a t i o n for F a s t blue, the SP cells w e r e g r e e n a n d easily d i s t i n g u i s h a b l e f r o m the p a l e F a s t b l u e cells. Fig. 4 ( p a n e l a) s h o w s t w o F a s t b l u e - l a b e l l e d cells (arrows). O n e of these cells (single a r r o w ) was also l a b e l l e d with SP (single a r r o w in p a n e l b) a n d the o t h e r was n o t ( d o u b l e a r r o w in p a n e l b). A p p r o x i m a t e l y 14% o f the F a s t b l u e - l a b e l l e d cells in the D R G o f 8 rats also c o n t a i n e d SP ( t o t a l 184 cells, 23 cells o n a v e r a g e , r a n g e 1 1 - 4 5 cells).
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Fig. 5. Frequency histogram showing the distribution of the cells in the left dorsal root ganglia labelled with both SP and Fast blue 6 days after injection of Fast blue into left adrenal medulla in 8 rats. Data is derived from counts of all double labelled cells from 8 rats.
251
grade tracing technique to quantitate the number of SP-containing neurons in the rat D R G innervating the adrenal medulla. The results show that up to 14% of the neurons in the D R G that innervate the rat adrenal medulla are SP-positive. The D R G between T3 and T13 contained an average of 281 + 31 SP immunoreactive cells per ganglion. These results are in agreement with a previous study in the rat [2] where it was found that there were approximately 250 SP-immunorcactive cells in each sensory ganglion. In accord with previous studies [15,17], 85% of the SP-labelled cells were of small diameter ( < 30 /,m). In the present study an average of 172 + 26 neurons in the ipsilateral D R G were retrogradely labelled with Fast blue following injection of the dye into the left adrenal medulla. Using Fluorogold for retrograde tracing, Strack et al. [49] found that only 55 neurons in the rat D R G innervated the adrenal medulla, Whereas in the guinea pig [40], use of Fast blue to trace the sensory innervation of the adrenal medulla [40] resulted in the labelling of twice the number of cells detected in the present study in the rat (582 vs. 297). The present study showed that 14% of the Fast blue-labelled cells in the D R G projecting to the [eft adrenal medulla were SP-positive. Others have shown that between 2 and 60% of the cells in D R G contributing sensory innervation to peripheral tissues were SP-positive [5,6,18,39]. It is possible that the figure of 14% for dual labelled neurons is an underestimate, due to the the difficulty of SP antibody penetration in 30-/,m tissue sections. The sensory innervation of the adrenal gland is likely to contain putative transmitters other than SP. A number of neuropeptides such as somatostatin [15.17], V1P, cholecystokinin [6], gastrin releasing peptide [44], endorphin [24], dynorphin [11], C G R P [11,45], corticotropin releasing factor [48], galanin [47], oxytocin and arginine vasopressin [19] have been found in sensory neurons of the DRG. Some of these neuropeptides also exist in the nerve terminals innervating the adrenal medulla. For example, SPand CGRP-immunoreactivities co-exist in nerve fibres of the rat adrenal medulla [29]. In addition, somatostatin and VIP have been detected in the
nerve terminals innervating the adrenal medulla [25,34]. It is likely, therefore, that in addition to SP, other neuropeptide-containing neurons in the D R G also innervate the adrenal medulla either alone or in combination with SP. The question arises as to whether or not all the SP fibres in the adrenal medulla originate from sensory neurons. The adrenal medulla receives innervation from supra-adrenal ganglia and sympathetic ganglia [21] in addition to D R G : however, these neurons do not contain SP [4,16,38]. SP has been localized in sympathetic preganglionic neurons in the cat [27] and in parasympathetic neurons in the toad [12] but to date no substance P-containing preganglionic neurons have been identified in the rat. In the present experiments, SP was not found in preganglionic neurons in spinal cord of the rat {data not presented). Nevertheless, our study cannot rule out the possibility that some SP nerve fibres in the adrenal medulla may come from preganglionic sympathetic neurons. Recently, it was found that the adrenal medulla receives innervation from nodose ganglia and vagal ganglia [3]. In the present study, we also found that some neurons in the nodose ganglia and vagal ganglia were labelled after injection of Fast blue into the ipsilateral adrenal medulla (data not shown). Moreover, a few retrogradely labelled neurons in the vagal ganglia were also labelled with SP. It has been found that a large number of neurons in the nodose and vagal ganglia in the cat contain SP [20]. Taken together, these studies suggest that, in addition to the DRG, nodose ganglia and vagal ganglia may be a source of SP fibres in the adrenal medulla. Substance P is now well established as a neuropeptide marker for a population of sensory neurons in D R G [11,15,17]. The function of the sensory innervation of the adrenal medulla, however, is not known [13,14]. It has been reported that adrenal afferent fibres fire following a change of blood pressure or injection of adrenaline and noradrenaline, suggesting that the sensory neurons innervating the adrenal medulla may convey baroreflex and chemoreflex information [42,43]. In recent years, evidence has accumulated to show that neuropeptides present in nerve terminals innervating the adrenal medulla modulate adrenal
252
medullary catecholamine secretion (for review, see refs. 31,37). For example, SP inhibits the nicotinic secretion of catecholamines from isolated bovine chromaffin cells [33] and protects against nicotinic desensitization [1]. In addition, SP at low concentrations increases CA secretion from perfused rat adrenal glands evoked by electrical stimulation [53,55]. In capsaicin-pretreated rats in which SP levels in the splanchnic nerve are depleted by 70-90%, [9,22], adrenal medullary CA secretion in response to stress under anaesthesia is abolished or reduced [22,23]. Likewise, CA secretion evoked by electrical stimulation of the isolated perfused rat adrenal gland from capsaicin pretreated rats was reduced [53,54]. This indicates that capsaicinsensitive sensory neurons are involved in the local control of CA secretion from the adrenal gland. Although SP release from sensory neurons in the adrenal medulla has not been proven, local release and effects of SP from visceral and skin sensory neurons are well established [10,52]. In addition, SP can be released from the adrenal medulla in vivo in response to selective stressors [51]. These studies suggest that SP-containing neurons innervating adrenal medulla act to maintain CA secretion during stress. The retrograde tracing studies in the present study indicate that some SP-containing nerve terminals in the rat adrenal medulla arise from the ipsilateral D R G . It is likely that this pathway constitutes the normal substrate for SP modulation of adrenal CA secretion.
Acknowledgements We thank Dr. Phil Marley for his advice and comments, Dr. Ian Gibbins (Flinders University, Adelaide) for his generous gift of Fast blue, Dr. Roger Murphy (Flinders University, Adelaide) for his gift of SP antiserum, Mr. Tim Anning, Ms. Daphne K. Hards and Mr. Phillip McGlashan for technical assistance. X.-F.Z. was supported by a University of Melbourne Postgraduate Award. This work was supported by a project grant to B.G.L. from the National Health and Medical Research Council ( N H & M R C ) of Australia.
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