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The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat
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Neuroscience Letters 190 (1995) 109-112
The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat Mekbeb Afework, Vera Ralevic, Geoffrey Burnstock* Department of Anatomy and DevelopmentalBiology and Centrefor Neuroscience, University College London, GowerStreet, London WCIE 6BT, UK
Received 7 January 1995; revisedversion received28 March 1995; accepted28 March 1995
Abstract
The intra-adrenal distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibres was studied in rats subjected to various denervations. Splanchnic nerve section eliminated the NOS-immunoreactive nerve fibres which innervate adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels and zona glomemlosa, which instead were affected by adrenal demedullation. Guanethidine, 6-hydroxydopamine (6-OHDA) and capsaicin treatments, however, did not produce any change. These results suggest that nitrergic nerves which innervate adrenal medullary cells are extrinsic (largely preganglionic sympathetic), whilst those innervating the zona glomemlosa and the majority of adrenal vessels are intrinsic, and that they do not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and 6-OHDA, or the sensory neurotoxin, capsaicin. Keywords: Adrenal gland; Nitric oxide synthase; Innervation; Denervation
Nitric oxide synthase (NOS), the enzyme which synthesizes nitric oxide (NO), has been demonstrated in the adrenal gland [2,4]. Functional studies suggest a role for NO in the control of adrenal blood flow [5,7] and evidence also exists which suggests its involvement in catecholamine [ 10,12] and corticosteroid [ 1,7] secretions from the gland. In the rat, NOS-immunoreactive (NOS-ir) nerve fibres are closely associated with adrenal chromaffin and ganglion cells, and cortical cells of the zona glomerulosa, as well as with intra-adrenal blood vessels [2,4]. Such ample distribution of the nitrergic nerve fibres in the gland suggests the possible presence of an extrinsic input, since the number of local NOS-ir neurons, which would account for intrinsic innervation, is relatively small [2]. In addition, NOS-positive preganglionic neurons found in the intermediolateral cell column of the spinal cord have been shown to have their terminals in the gland [3]. The intra-adrenal distribution of extrinsic with respect to intrinsic NOS-ir nerve fibres is, however, not known. Thus, in the study presented here we examined the intraadrenal distribution of NOS-ir nerve fibres following either section of the splanchnic nerve or demedullation. In * Correspondingauthor,Tel.:+44171 3807053; Fax:+44171 3807349.
addition, to investigate the nature of NOS-ir adrenal nerves, the distribution of NOS-ir nerves in the gland was also studied following chemical denervation of sympathetic nerves with guanethidine or 6-hydroxydopamine (6-OHDA) and denervation of primary sensory neurons with capsaicin. Studies were carried out on male Sprague-Dawley rats. Rats were deeply anaesthetized with injections of fentanyl-fluanisone intramuscularly (3 mg/kg body wt.; Janssen, UK) and diazepam subcutaneously (2 mg/kg body wt.; Phoenix Pharmaceuticals, UK). Six 15-weekold rats were laparotomized and transections of the left splanchnic nerves were performed; rats were allowed to survive for 5 days. In another 6 rats (10-week-old) bilateral adrenal enucleations were performed via the dorsal parasagittal approach and the rats were allowed to survive for 6 weeks. Six other rats for each of guanethidine, 6-OHDA and capsaicin treatments were also studied. For chemical sympathectomy, litter rats were subcutaneously injected with guanethidine sulphate (50 mg/kg body wt.; Ciba Laboratories, UK) from day 8 after birth, for 3 weeks, 5 days per week, and were killed at 14 weeks of age. In addition, rats which were 13 weeks old were subcutaneously injected with 6-OHDA (100 mg/kg body wt. on day
0304-3940/95/$09.50 © 1995 ElsevierScience Ireland Ltd. All rights reserved SSD! 0304-3940(95)11514-T
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M. Afework et aLI Neuroscience Letters 190 (1995) 109-112
Fig. 1. Fluorescence photomicrographs of NOS immunoreactivityin control sections from: (a) adrenal medulla of normal non-denervated rat; (b) adrenal medulla of the eontralateral side of the splanchnic nerve section; and (c) adrenal cortex of normal non-denervatedrats. Note the high density of NOS-ir nerve fibres around large non-immunoreaetive ganglion cells (arrows in (b)). In (a) and (b), arrowheads and arrows point to NOS-immunopositiveand immunonegativeneuronal cell bodies, respectively. V, blood vessel; ZG, zona glomerulosa; ZF, zona fasciculata. Bars = 52/am. 1 and 250 mg/kg body wt. on day 2; Sigma, UK), dissolved in saline containing 1 mg/ml ascorbic acid, and killed on day 8. For sensory denervation, neonatal rats were subcutaneously treated with capsaicin (50mg/kg body wt.; Sigma) dissolved in saline containing 10% Tween 80 and 10% ethanol at 1, 2, 3, 5, 7 and 14 days of age, and were killed at 14 weeks of age. As controls, adrenal glands from three sham-operated or vehicle treated rats for each of the experiments were
studied. Animals were killed by ether asphyxiation and the adrenals were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4, for 2 h at room temperature and left overnight in PBS containing 15% sucrose, at 4°C. Frozen sections were cut at 1 4 ~ m in a cryostat (Reichert Jung, Germany) and thaw-mounted onto poly L-lysine coated glass slides. NOS immunohistochemistry and NADPH-diaphorase histochemistry were carried out as described previously [2]. Briefly, sections were incubated with rabbit antibody against neuronal NOS (1:2500; Euro-Diagnostica, Sweden), at room temperature for 18 h. This was followed by incubation with biotinylated donkey anti-rabbit antibody (1:250; Amersham, UK) and then with streptavidin fluorescein (1:100; Amersham) for 1 h each. NADPH-diaphorase histochemistry was carried out on consecutive sections of the NOS-immunoreacted ones. Sections were incubated in a medium which contained: 1 mg/ml /~-NADPH, 0.2 mg/ml nitroblue tetrazolium, 2.7 mg/ml L-malic acid and 0.1% Triton X-100 in 0.1 M Tris-HC1 at 37°C for 30 min. In the adrenal glands from all groups of control rats, NOS immunoreactivity was observed in several intrinsic neurons located in the medulla and in nerve fibres found in both the medulla and cortex, as described previously [2]. In the medulla, the nerve fibres were closely associated with chromaffin and neuronal cells, and blood vessels (Fig. la). However, in the adrenal medulla of the glands from the contralateral side of the splanchnic nerve section, the NOS-ir nerve fibres associated with the large NOS-immunonegative ganglion cells were found to be increased (Fig. lb). In the adrenal cortex, immunoreactive nerve fibres formed a plexus in the subcapsular region and zona glomerulosa (Fig. lc). In the adrenal glands ipsilateral to the splanchnic nerve section side, only a few immunoreactive nerve fibres were found near the chromaffin ceils (Fig. 2a). The vast majority of the nerve fibres seen innervating the chromaffin and neuronal cells in the medulla of control rats were absent. There were, however, occasional NOS-ir nerve fibres found in association with the blood vessels (Fig. 2a). Some of these nerve fibres were seen projecting from nearby reactive neurons, which did not show any apparent change from that of the controls. Occasional immunoreactive nerve fibres and moderate size nerve bundles were seen running radially across the cortex (Fig. 2b). The immunoreactive nerve fibre plexus in the subcapsular region and the zona glomerulosa of cortex was similar to that in controls (Fig. 2c). In sections of the demedullated adrenal glands, a small number of immunoreactive nerve fibres were seen in the connective tissue which replaced the region of the enucleated medulla of the gland (Fig. 3a). Some of these nerve fibres were closely associated with a few large blood vessels found in the region. A few large immunoreactive nerve bundles were also seen running radially in
M. Afework et al. / Neuroscience Letters 190 (1995) 109-112
111
trois, closely matched their respective NOS immunoreac-
tivity. The increase in NOS-ir nerve fibres observed innervating the large ganglion cells in the adrenal gland, from the contralateral side o f section o f the splanchnic nerve, is intriguing. It may be related to increased secretory activity of the gland, occurring to compensate for a reduced secretion from the denervated gland. However, it is not clear at present as to the significance o f such nitrergic innervation o f the ganglion cells in the glandular activity. Both intrinsic and extrinsic origins for the NOS-ir intra-adrenal nerve fibres could be inferred from the present study. Those that innervate chromaffin and neuronal cells appear to be extrinsic, as shown by the disappearance of these fibres following section of the splanchnic nerve. In contrast, the NOS-ir nerve fibres found as plexus in the subcapsular region and the zona glomerulosa of the cortex seem to arise from the intrinsic neurons. This is implied since these fibres were unaffected by
Fig. 2. Fluorescence photomicrographs of NOS immunoreactivity in sections of adrenal glands ipsilateral to the splanchnic nerve section side showing NOS-ir nerve fibres are: (a) absent from medulla (M), but present in relation to a blood vessel (V); (b) radially traversing the adrenal cortex; (c) present as plexus in the zona glomerulosa (ZG) of the cortex. Small arrows in (a) and (b) point to immunoreactive nerve fibres, while arrowheads and the large arrow in (a) indicate NOSimmunopositive and immunonegative neurons, respectively. Co, cortex; ZF, zona fasciculata; ZR, zona reticularis. Bars = 52/zm. the superficial region of the cortex, with some crossing the capsule (Fig. 3b). However, the type of the nerve fibre plexus found in the subcapsular region and zona glomerulosa of the control rat adrenals was not present. No apparent change, with respect to the controls, was observed in NOS immunoreactivity in adrenal gland sections from rats treated with guanethidine, 6-OHDA or capsaicin. NADPH-diaphorase-staining of the adrenal gland sections from all groups of rats, both experimental and con-
i
Fig. 3. Fluorescence photomicrographs of NOS immunoreactivity in sections of demedullated adrenal glands. (a) NOS-ir nerve fibres (small arrows) are seen in the connective tissue occupying the region of enucleated medulla; some of such fibres are associated with large blood vessels (V). (b) NOS-ir nerve bundles (small arrows) are seen in the peri-adrenal connective tissue; one such bundle is seen crossing the adrenal capsule (Ca). Note the absence of immunoreactive nerve plexus from the subcapsular region and the zona glomerulosa (ZG) of the cortex. Open arrows in (a) point to adrenal cortical cells. ZF, zona fasciculata. Bars = 52/.tm.
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M. Afework et al. I Neuroscience Letters 190 (1995) 109-112
splanchnic nerve section, but disappeared upon demedullation. The nitrergic innervation of the adrenal vasculature also appears to originate largely from the intrinsic NOS-ir neurons; indeed some immunoreactive neurons have been seen to project to nearby vessels. This is also in agreement with the finding that no apparent change in the vascular nitrergic innervation occurred following section of the splanchnic nerve. In contrast, the existence of a small number of immunoreactive nerve fibres associated with the vasculature in the fibrous connective tissue occupying the central region of the demedullated gland suggests that there may be an additional minor extrinsic innervation as well. It is possible that the loss of such minor extrinsic nerve fibres may not be detected by splanchnic nerve section alone. However, it is also possible that these fibres may not pass via the splanchnic nerve. Instead, they may use an alternative route, probably running along with the adrenal blood vessels, and therefore may have been missed during splanchnic nerve section. Alternatively, such innervations of the blood vessels observed in the demedullated gland may be a compensatory development for the loss of innervation following removal of the intrinsic nerves. The absence of any detectable change in the distribution of NOS immunoreaetivity in the adrenal gland of rats treated with guanethidine, 6-OHDA or capsaicin indicates that the NOS-ir nerves in the gland are not components of the sympathetic or primary sensory nerves which are affected by the above drugs [6,8,9]. It is also evident from the present study that the loss of nerves sensitive to these drugs and found within or outside the gland, do not appear to have any secondary effect on the distribution of NO synthesizing nerves in the gland. The majority of the extrinsic NOS-ir nerve fibres may be sympathetic preganglionic fibres as shown previously with tracer injection into the gland and colocalizing NOS in the retrogradely labelled neurons located in the intermediolateral cell column of the spinal cord [3]. Further similar studies which also include the other neural projection sites, such as the sympathetic chain ganglia, dorsal root ganglia and vagal ganglia [11], may be needed to fully elucidate the origin and relative proportions of the extrinsic nitrergic nerve fibres to the gland.
The editorial assistance of Dr D. Christie is gratefully acknowledged. [1] Adams, M.L., Nock, B., Truong, R. and Cicero, T.J., Nitric oxide control of steroidogenesis: endocrine effects of NG-nilro-Larginine and comparisons to alcohol, Life Sci., 50 (1992) PL35PL40. [2] Afework, M., Tomlinson, A. and Burnstock, G., Distribution and coloealization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats, Cell Tissue Res., 276 (1994) 133-141. [3] Blottner, D. and Banmagarten, H.G., Nitric oxide synthase (NOS)-containing sympathoadrenal eholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry, and retrograde labelling, J. Comp. Neurol., 316 (1992) 45-55. [4] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768-770. [5] Breslow, M.J., Tobin, J.R., Bredt, D.S., Ferris, C.D., Snyder, S.H. and Traystman, R.J., Role of nitric oxide in adrenal medullary vasodilatation during cateeholamine secretion, Eur. J. Pharmacol., 210 (1992) 105-106. [6] Bumstock, G., Evans, B., Gannon, B.J., Heath, J.W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Br. J. Pharmacol., 43 (1970) 295-301. [7] Cameron, L.A. and Hinson, J.P., The role of nitric oxide derived from L-arginine in the control of steroidogenesis, and perfusion medium flow rate in the isolated perfused rat adrenal gland, J. Endocrinol., 139 (1993) 415-423. [8] Fumess, J.B., Campbell, G.R., Gillard, S.M., Malmfors, T., Cobb, J.L.S. and Burnstock, G., Cellular studies of sympathetic denervafion produced by 6-hydroxydopamine in the vas deferens, J. Pharmacol. Exp. Tber., 174 (1970) 111-122. [9] Jancs6, G., Kiraly, E. and Jancs6-G~lbor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons, Nature, 270 (1977) 741-743. [10] Oset-Gasque, M.J., Parramtn, M., Hortelano, S., Bosc& L. and Gonz(dez, M.P., Nitric oxide implication in the control of neurosecretion by chromaffin cells, J. Neurocbem., 63 (1994) 16931700. [11] Parker, T.L., Kesse, W.K., Mohamed, A.A. and Afework, M., The innervation of the mammalian adrenal gland, J. Anat., 183 (1993) 265-276. [12] Uchiyama, Y., Morita, K., Kitayama, S., Suemitsu, T., Minami, N., Miyasako, T. and Dohi, T., Possible involvement of nitric oxide in acetylcholine-induced increase of intracellular Ca 2+ concentration and catecholamine release in bovine adrenal chromaffin cells, Jpn. J. Pharmacol., 65 (1994) 73-77.
,
:!
ELSEVIER
Neuroscience Letters 190 (1995) 109-112
The intra-adrenal distribution of intrinsic and extrinsic nitrergic nerve fibres in the rat Mekbeb Afework, Vera Ralevic, Geoffrey Burnstock* Department of Anatomy and DevelopmentalBiology and Centrefor Neuroscience, University College London, GowerStreet, London WCIE 6BT, UK
Received 7 January 1995; revisedversion received28 March 1995; accepted28 March 1995
Abstract
The intra-adrenal distribution of nitric oxide synthase (NOS)-immunoreactive nerve fibres was studied in rats subjected to various denervations. Splanchnic nerve section eliminated the NOS-immunoreactive nerve fibres which innervate adrenal chromaffin and neuronal cells. It did not affect those innervating blood vessels and zona glomemlosa, which instead were affected by adrenal demedullation. Guanethidine, 6-hydroxydopamine (6-OHDA) and capsaicin treatments, however, did not produce any change. These results suggest that nitrergic nerves which innervate adrenal medullary cells are extrinsic (largely preganglionic sympathetic), whilst those innervating the zona glomemlosa and the majority of adrenal vessels are intrinsic, and that they do not belong to nerves sensitive to the sympathetic nerve neurotoxins, guanethidine and 6-OHDA, or the sensory neurotoxin, capsaicin. Keywords: Adrenal gland; Nitric oxide synthase; Innervation; Denervation
Nitric oxide synthase (NOS), the enzyme which synthesizes nitric oxide (NO), has been demonstrated in the adrenal gland [2,4]. Functional studies suggest a role for NO in the control of adrenal blood flow [5,7] and evidence also exists which suggests its involvement in catecholamine [ 10,12] and corticosteroid [ 1,7] secretions from the gland. In the rat, NOS-immunoreactive (NOS-ir) nerve fibres are closely associated with adrenal chromaffin and ganglion cells, and cortical cells of the zona glomerulosa, as well as with intra-adrenal blood vessels [2,4]. Such ample distribution of the nitrergic nerve fibres in the gland suggests the possible presence of an extrinsic input, since the number of local NOS-ir neurons, which would account for intrinsic innervation, is relatively small [2]. In addition, NOS-positive preganglionic neurons found in the intermediolateral cell column of the spinal cord have been shown to have their terminals in the gland [3]. The intra-adrenal distribution of extrinsic with respect to intrinsic NOS-ir nerve fibres is, however, not known. Thus, in the study presented here we examined the intraadrenal distribution of NOS-ir nerve fibres following either section of the splanchnic nerve or demedullation. In * Correspondingauthor,Tel.:+44171 3807053; Fax:+44171 3807349.
addition, to investigate the nature of NOS-ir adrenal nerves, the distribution of NOS-ir nerves in the gland was also studied following chemical denervation of sympathetic nerves with guanethidine or 6-hydroxydopamine (6-OHDA) and denervation of primary sensory neurons with capsaicin. Studies were carried out on male Sprague-Dawley rats. Rats were deeply anaesthetized with injections of fentanyl-fluanisone intramuscularly (3 mg/kg body wt.; Janssen, UK) and diazepam subcutaneously (2 mg/kg body wt.; Phoenix Pharmaceuticals, UK). Six 15-weekold rats were laparotomized and transections of the left splanchnic nerves were performed; rats were allowed to survive for 5 days. In another 6 rats (10-week-old) bilateral adrenal enucleations were performed via the dorsal parasagittal approach and the rats were allowed to survive for 6 weeks. Six other rats for each of guanethidine, 6-OHDA and capsaicin treatments were also studied. For chemical sympathectomy, litter rats were subcutaneously injected with guanethidine sulphate (50 mg/kg body wt.; Ciba Laboratories, UK) from day 8 after birth, for 3 weeks, 5 days per week, and were killed at 14 weeks of age. In addition, rats which were 13 weeks old were subcutaneously injected with 6-OHDA (100 mg/kg body wt. on day
0304-3940/95/$09.50 © 1995 ElsevierScience Ireland Ltd. All rights reserved SSD! 0304-3940(95)11514-T
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M. Afework et aLI Neuroscience Letters 190 (1995) 109-112
Fig. 1. Fluorescence photomicrographs of NOS immunoreactivityin control sections from: (a) adrenal medulla of normal non-denervated rat; (b) adrenal medulla of the eontralateral side of the splanchnic nerve section; and (c) adrenal cortex of normal non-denervatedrats. Note the high density of NOS-ir nerve fibres around large non-immunoreaetive ganglion cells (arrows in (b)). In (a) and (b), arrowheads and arrows point to NOS-immunopositiveand immunonegativeneuronal cell bodies, respectively. V, blood vessel; ZG, zona glomerulosa; ZF, zona fasciculata. Bars = 52/am. 1 and 250 mg/kg body wt. on day 2; Sigma, UK), dissolved in saline containing 1 mg/ml ascorbic acid, and killed on day 8. For sensory denervation, neonatal rats were subcutaneously treated with capsaicin (50mg/kg body wt.; Sigma) dissolved in saline containing 10% Tween 80 and 10% ethanol at 1, 2, 3, 5, 7 and 14 days of age, and were killed at 14 weeks of age. As controls, adrenal glands from three sham-operated or vehicle treated rats for each of the experiments were
studied. Animals were killed by ether asphyxiation and the adrenals were immersion fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS), pH 7.4, for 2 h at room temperature and left overnight in PBS containing 15% sucrose, at 4°C. Frozen sections were cut at 1 4 ~ m in a cryostat (Reichert Jung, Germany) and thaw-mounted onto poly L-lysine coated glass slides. NOS immunohistochemistry and NADPH-diaphorase histochemistry were carried out as described previously [2]. Briefly, sections were incubated with rabbit antibody against neuronal NOS (1:2500; Euro-Diagnostica, Sweden), at room temperature for 18 h. This was followed by incubation with biotinylated donkey anti-rabbit antibody (1:250; Amersham, UK) and then with streptavidin fluorescein (1:100; Amersham) for 1 h each. NADPH-diaphorase histochemistry was carried out on consecutive sections of the NOS-immunoreacted ones. Sections were incubated in a medium which contained: 1 mg/ml /~-NADPH, 0.2 mg/ml nitroblue tetrazolium, 2.7 mg/ml L-malic acid and 0.1% Triton X-100 in 0.1 M Tris-HC1 at 37°C for 30 min. In the adrenal glands from all groups of control rats, NOS immunoreactivity was observed in several intrinsic neurons located in the medulla and in nerve fibres found in both the medulla and cortex, as described previously [2]. In the medulla, the nerve fibres were closely associated with chromaffin and neuronal cells, and blood vessels (Fig. la). However, in the adrenal medulla of the glands from the contralateral side of the splanchnic nerve section, the NOS-ir nerve fibres associated with the large NOS-immunonegative ganglion cells were found to be increased (Fig. lb). In the adrenal cortex, immunoreactive nerve fibres formed a plexus in the subcapsular region and zona glomerulosa (Fig. lc). In the adrenal glands ipsilateral to the splanchnic nerve section side, only a few immunoreactive nerve fibres were found near the chromaffin ceils (Fig. 2a). The vast majority of the nerve fibres seen innervating the chromaffin and neuronal cells in the medulla of control rats were absent. There were, however, occasional NOS-ir nerve fibres found in association with the blood vessels (Fig. 2a). Some of these nerve fibres were seen projecting from nearby reactive neurons, which did not show any apparent change from that of the controls. Occasional immunoreactive nerve fibres and moderate size nerve bundles were seen running radially across the cortex (Fig. 2b). The immunoreactive nerve fibre plexus in the subcapsular region and the zona glomerulosa of cortex was similar to that in controls (Fig. 2c). In sections of the demedullated adrenal glands, a small number of immunoreactive nerve fibres were seen in the connective tissue which replaced the region of the enucleated medulla of the gland (Fig. 3a). Some of these nerve fibres were closely associated with a few large blood vessels found in the region. A few large immunoreactive nerve bundles were also seen running radially in
M. Afework et al. / Neuroscience Letters 190 (1995) 109-112
111
trois, closely matched their respective NOS immunoreac-
tivity. The increase in NOS-ir nerve fibres observed innervating the large ganglion cells in the adrenal gland, from the contralateral side o f section o f the splanchnic nerve, is intriguing. It may be related to increased secretory activity of the gland, occurring to compensate for a reduced secretion from the denervated gland. However, it is not clear at present as to the significance o f such nitrergic innervation o f the ganglion cells in the glandular activity. Both intrinsic and extrinsic origins for the NOS-ir intra-adrenal nerve fibres could be inferred from the present study. Those that innervate chromaffin and neuronal cells appear to be extrinsic, as shown by the disappearance of these fibres following section of the splanchnic nerve. In contrast, the NOS-ir nerve fibres found as plexus in the subcapsular region and the zona glomerulosa of the cortex seem to arise from the intrinsic neurons. This is implied since these fibres were unaffected by
Fig. 2. Fluorescence photomicrographs of NOS immunoreactivity in sections of adrenal glands ipsilateral to the splanchnic nerve section side showing NOS-ir nerve fibres are: (a) absent from medulla (M), but present in relation to a blood vessel (V); (b) radially traversing the adrenal cortex; (c) present as plexus in the zona glomerulosa (ZG) of the cortex. Small arrows in (a) and (b) point to immunoreactive nerve fibres, while arrowheads and the large arrow in (a) indicate NOSimmunopositive and immunonegative neurons, respectively. Co, cortex; ZF, zona fasciculata; ZR, zona reticularis. Bars = 52/zm. the superficial region of the cortex, with some crossing the capsule (Fig. 3b). However, the type of the nerve fibre plexus found in the subcapsular region and zona glomerulosa of the control rat adrenals was not present. No apparent change, with respect to the controls, was observed in NOS immunoreactivity in adrenal gland sections from rats treated with guanethidine, 6-OHDA or capsaicin. NADPH-diaphorase-staining of the adrenal gland sections from all groups of rats, both experimental and con-
i
Fig. 3. Fluorescence photomicrographs of NOS immunoreactivity in sections of demedullated adrenal glands. (a) NOS-ir nerve fibres (small arrows) are seen in the connective tissue occupying the region of enucleated medulla; some of such fibres are associated with large blood vessels (V). (b) NOS-ir nerve bundles (small arrows) are seen in the peri-adrenal connective tissue; one such bundle is seen crossing the adrenal capsule (Ca). Note the absence of immunoreactive nerve plexus from the subcapsular region and the zona glomerulosa (ZG) of the cortex. Open arrows in (a) point to adrenal cortical cells. ZF, zona fasciculata. Bars = 52/.tm.
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M. Afework et al. I Neuroscience Letters 190 (1995) 109-112
splanchnic nerve section, but disappeared upon demedullation. The nitrergic innervation of the adrenal vasculature also appears to originate largely from the intrinsic NOS-ir neurons; indeed some immunoreactive neurons have been seen to project to nearby vessels. This is also in agreement with the finding that no apparent change in the vascular nitrergic innervation occurred following section of the splanchnic nerve. In contrast, the existence of a small number of immunoreactive nerve fibres associated with the vasculature in the fibrous connective tissue occupying the central region of the demedullated gland suggests that there may be an additional minor extrinsic innervation as well. It is possible that the loss of such minor extrinsic nerve fibres may not be detected by splanchnic nerve section alone. However, it is also possible that these fibres may not pass via the splanchnic nerve. Instead, they may use an alternative route, probably running along with the adrenal blood vessels, and therefore may have been missed during splanchnic nerve section. Alternatively, such innervations of the blood vessels observed in the demedullated gland may be a compensatory development for the loss of innervation following removal of the intrinsic nerves. The absence of any detectable change in the distribution of NOS immunoreaetivity in the adrenal gland of rats treated with guanethidine, 6-OHDA or capsaicin indicates that the NOS-ir nerves in the gland are not components of the sympathetic or primary sensory nerves which are affected by the above drugs [6,8,9]. It is also evident from the present study that the loss of nerves sensitive to these drugs and found within or outside the gland, do not appear to have any secondary effect on the distribution of NO synthesizing nerves in the gland. The majority of the extrinsic NOS-ir nerve fibres may be sympathetic preganglionic fibres as shown previously with tracer injection into the gland and colocalizing NOS in the retrogradely labelled neurons located in the intermediolateral cell column of the spinal cord [3]. Further similar studies which also include the other neural projection sites, such as the sympathetic chain ganglia, dorsal root ganglia and vagal ganglia [11], may be needed to fully elucidate the origin and relative proportions of the extrinsic nitrergic nerve fibres to the gland.
The editorial assistance of Dr D. Christie is gratefully acknowledged. [1] Adams, M.L., Nock, B., Truong, R. and Cicero, T.J., Nitric oxide control of steroidogenesis: endocrine effects of NG-nilro-Larginine and comparisons to alcohol, Life Sci., 50 (1992) PL35PL40. [2] Afework, M., Tomlinson, A. and Burnstock, G., Distribution and coloealization of nitric oxide synthase and NADPH-diaphorase in adrenal gland of developing, adult and aging Sprague-Dawley rats, Cell Tissue Res., 276 (1994) 133-141. [3] Blottner, D. and Banmagarten, H.G., Nitric oxide synthase (NOS)-containing sympathoadrenal eholinergic neurons of the rat IML-cell column: evidence from histochemistry, immunohistochemistry, and retrograde labelling, J. Comp. Neurol., 316 (1992) 45-55. [4] Bredt, D.S., Hwang, P.M. and Snyder, S.H., Localization of nitric oxide synthase indicating a neuronal role for nitric oxide, Nature, 347 (1990) 768-770. [5] Breslow, M.J., Tobin, J.R., Bredt, D.S., Ferris, C.D., Snyder, S.H. and Traystman, R.J., Role of nitric oxide in adrenal medullary vasodilatation during cateeholamine secretion, Eur. J. Pharmacol., 210 (1992) 105-106. [6] Bumstock, G., Evans, B., Gannon, B.J., Heath, J.W. and James, V., A new method of destroying adrenergic nerves in adult animals using guanethidine, Br. J. Pharmacol., 43 (1970) 295-301. [7] Cameron, L.A. and Hinson, J.P., The role of nitric oxide derived from L-arginine in the control of steroidogenesis, and perfusion medium flow rate in the isolated perfused rat adrenal gland, J. Endocrinol., 139 (1993) 415-423. [8] Fumess, J.B., Campbell, G.R., Gillard, S.M., Malmfors, T., Cobb, J.L.S. and Burnstock, G., Cellular studies of sympathetic denervafion produced by 6-hydroxydopamine in the vas deferens, J. Pharmacol. Exp. Tber., 174 (1970) 111-122. [9] Jancs6, G., Kiraly, E. and Jancs6-G~lbor, A., Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons, Nature, 270 (1977) 741-743. [10] Oset-Gasque, M.J., Parramtn, M., Hortelano, S., Bosc& L. and Gonz(dez, M.P., Nitric oxide implication in the control of neurosecretion by chromaffin cells, J. Neurocbem., 63 (1994) 16931700. [11] Parker, T.L., Kesse, W.K., Mohamed, A.A. and Afework, M., The innervation of the mammalian adrenal gland, J. Anat., 183 (1993) 265-276. [12] Uchiyama, Y., Morita, K., Kitayama, S., Suemitsu, T., Minami, N., Miyasako, T. and Dohi, T., Possible involvement of nitric oxide in acetylcholine-induced increase of intracellular Ca 2+ concentration and catecholamine release in bovine adrenal chromaffin cells, Jpn. J. Pharmacol., 65 (1994) 73-77.