The Innervation of the Mouse Adrenal Cortex NABIL MIGALLY Department of Anatomy, Medical College of Wisconsin, Milwaukee, Wisconsin 53233
ABSTRACT The fine structure of the mouse adrenal cortex was examined with the electron microscope for the presence of neural elements. Several axon terminals containing mostly clear vesicles (60 nm) were noted in the vicinity (250 nm) of the capsular fibroblasts. In the subcapsular region, myelinated as well as unmyelinated fibers were commonly found. Preterminal and terminal axons were also found in close relationship to the parenchymal cells in the zona glomerulosa. Nerve bundles were the most common neural elements in the zona fasciculata. In the zona reticularis axon terminals containing both clear (60 nm) and dense core (120 nm) vesicles were seen in close proximity (30 nm) to parenchymal cells. Although this study did not delineate the type of fibers involved, t h e axon terminals resemble those of autonomic nerves. This study demonstrates innervation of the mouse adrenal cortex, thus corroborating similar reports by others in different species. Studies concerning adrenocortical innervation have been varied. At the light microscope level, several investigators observed no cortical innervation (Hollinshead, '36; Swinyard, '37; MacFarland and Davenport, '41) while others noted direct nerve termination upon cortical cells (Lever, '53; Mikhail, '61; Mikhail and Mahran, '65; Robinson e t al., '77; Uno, '77). Several workers using silver axonal degeneration techniques and conventional light microscopy noted a well developed subcapsular nervous plexus in humans (Alpert, '31) cats and dogs (Kiss, '511, guinea pigs (Willard, '36) and rats (Lever, '53; Mikhail and Mahran, '65). In the zona glomerulosa short direct extensions from the subcapsular nerve plexus terminated in boutons on parenchymal cells (Alpert, '31; Willard, '36; Mikhail, '61). Extensions from the subcapsular nerve plexus were also observed to terminate in boutons on the zona fasciculata cells (Alpert, '31; Willard, '36; Lever, '53). In the rat, nerve fibers were particularly abundant in the zona reticularis (Mikhail, '61; Mikhail and Mahran, '65). Recently, investigators using catecholamine fluorescence techniques demonstrated the presence of adrenergic terminals in the adrenal cortex of monkeys (Uno, '77) and sheep (Robinson e t al., '77). Robinson et al. ('77) also reported the presence of acetylchoANAT.
REC. (1979) 194: 105-112.
linesterase-positive fibers forming a plexus in the zona reticularis of the sheep adrenal cortex. Using electron microscopy, myelinated nerves were noted in the subcapsular region of rat adrenals by Nishikawa et al. ('63). In the vicinity of the zona glomerulosa and zona fasciculata of hamster, rat, and pig adrenals, Unsicker ('69, '71) noted structures suggestive of nerve terminals in close (20 nm) proximity to the parenchymal cells. These terminals contained two types of granular vesicles measuring 50 and 100 nm in diameter (Unsicker, '71). Uno ('77) demonstrated the presence of presumptive adrenergic and cholinergic terminals in the monkey adrenal cortex. In view of recent investigations linking altered adrenocortical activity with catecholamine content in the normal (Paul et al., '71; Ciaranello et al., '76) as well as in the diseased state (Henry and Stephens, '77; Iankelevich et al., '781, studies concerning adrenocortical innervation in the mouse have failed to demonstrate the presence of autonomic nerves in each zone. The objective of this investigation was to closely examine the mouse adrenal cortex for neural elements. Received July 18, '78. Accepted Oct. 31, '78. I Present address: Department of Anatomy, Wright State University, School of Medicine, Dayton, Ohio 45435.
105
106
NABIL MIGALLY MATERIALS AND METHODS
Animals Adrenal glands were obtained from adult male C,,BL mice (20-30 g) ranging in age from 8 to 12 weeks. All animals were housed in a controlled environment with respect to light, humidity and temperature. The animals were obtained at seven weeks of age from a local breeder and were acclimated to laboratory conditions for one week before the onset of the experiments. The mice were anesthetized with sodium pentobarbital (Abbott 40 mg/kg intraperitoneally), and fixed by intracardiac systemic perfusion with 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.25) a t 0-4°C. During perfusion, in situ fixation was also employed by flooding the glands directly with the glutaraldehyde solution. After perfusion, the adrenals were excised, cut into smaller pieces (1 mm?, and fixed for an additional three hours in a fresh fixative solution a t 0-4OC. The tissues were then washed with 0.1 M phosphate buffer (pH 7.25) several times, and postfixed for one hour in cold 1.0%osmium tetroxide in 0.1 M phosphate buffer. The tissues were rapidly dehydrated in ethanol and embedded in Spurr's epoxy resin and polymerized a t 6570°C (Spurr, '69); 0.5- to 1.0-pm-thick sections were cut with Porter-Blum ultramicrotome and stained with toluidine blue for light microscopic survey. Thin (60-70nm) sections were cut with an LKB ultratome, mounted on uncoated 300-mesh grids and stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63). The sections were examined and photographed with an RCA EMU 4B electron microscope using 100 kv accelerating voltage. RESULTS
In the adrenal capsule occasional nerve terminals were seen in the vicinity (250 nm) of the capsular fibroblasts (fig. 2). The nerve terminals contained mostly clear vesicles measuring 60 nm in diameter, although occasional dense core vesicles were also seen (fig. 2). In the subcapsular region, myelinated as well as unmyelinated axons were observed (fig. 1). In the region of the zona glomerulosa, preterminal axons were occasionally seen in the vicinity (310 nm) of parenchymal cells (fig. 3). Axons containing clear synaptic vesicles measuring 60 nm in diameter were also seen in
close proximity to parenchymal cells (fig. 4). No membrane specialization was seen on either the axon or the cortical cell (fig. 4). In the zona fasciculata, unmyelinated nerve fibers with accompanying basal laminae were the only neural components noted (fig. 5). Such profiles were usually located in the perivascular area. Nerve terminals were present in close proximity (30 nm) to the zona reticularis parenchymal cells (fig. 6). Clear vesicles measuring 60 nm in diameter and vesicles measuring 120 nm in diameter could be distinguished in these terminals (fig. 7). Nerve bundles with accompanying basal laminae were often observed in the perivascular compartment of the zona reticularis (fig. 8). DISCUSSION
In the adrenal capsule of rats and pigs, Unsicker ('71) described small nerve fibers in close proximity to the capsular fibroblasts. These nerves contained neurotubules, however, synaptic vesicles were not seen. In the present study, axons as well as terminals which contained both clear and dense core vesicles were seen near the fibroblasts. Such terminals have not been previously described in the mammalian adrenal capsule. Varicose and non-varicose axons were noted in close relationship to zona glomerulosa cells in the present study. These terminal and preterminal axons met the criteria of Thaemert ('66) for classification as autonomic nerve terminals. Pre-terminal axons in this zone were noted by Unsicker ('71), who postulated that some axons were influencing parenchymal cells, while others were probably in transit to deeper layers of the adrenal cortex or medulla. Although the present study was not designed to identify t h e nerve terminals with regard to vesicle content or origin, a functional relationship may exist between some zona glomerulosa cells and autonomic fibers. Close examination of numerous adjacent sections to the nerve terminals did not reveal the presence of vascular smooth muscle, however the possibility that these terminals were innervating vascular smooth muscle cannot be ruled out. Autonomic nerve terminals were reported in close relationship with parenchymal cells of the zona fasciculata in pigs and rats (Unsicker, '71) and in sheep (Robinson e t al., '77). In the present study axon terminals were not observed in t h e zona fasciculata, although some nerves were observed either associated
MOUSE ADRENAL CORTEX INNERVATION
with small blood vessels or as isolated nerve bundles. It is likely that many of the isolated nerve bundles observed were fibers associated with the adrenal medulla. Examination of the zona reticularis demonstrated autonomic nerve terminals in close apposition to the parenchymal cells. Since the presence of vascular smooth muscle was not encountered in the zona fasciculata or zona reticularis (figs. 5-71, the possibility that these terminals were associated with vascular smooth muscle seemed quite remote. The terminals contained 120-nm vesicles and 60-nm vesicles. The 120-nm vesicles were shown by Ruskell ('67) to be present in sympathetic as well as parasympathetic nerve terminals. The 60-nm vesicles were generally believed to contain acetylcholine or norepinephrine (Burnstock, '70). Although some terminals contained vesicles clustered away from the cortical cell (fig. 61, the vesicles were demonstrably close, so as to strongly suggest an innervation. The exact identification of the transmitter substance within these vesicles remains to be elucidated. The functional ramifications of autonomic innervation of the adrenal cortex are variable. The evidence presented in this study concerning the innervation of the zona glomerulosa corroborates the findings of Henry and Stephens ('77) who implicated neural stimulation of adrenocortical cells as a mechanism for increased corticosterone secretion in essential hypertension. Also from this investigation, the results seem to compliment the work of Ciaranello e t al. ('76) who demonstrated the presence of autonomic regulation of adrenal corticoidogenesis which in turn affects the catecholamine synthesizing enzymes in the adrenal medulla (Wurtman and Axelrod, '66). Although the morphological findings presented in this study seem to be implicated in affecting catecholamine synthesis, as well as in regulation of secretion of vasopressive substances, the exact mechanism of neural control of adrenocortical function remains to be found. LITERATURE CITED Alpert, L. K. 1931 The innervation of t h e suprarenal glands. Anat. Rec., 50: 221-233. Burnstock, G. 1970 Structure of smooth muscle and its innervation. In: Smooth Muscle. E. Bulbring, A. F. Brading, A. W. Jones and T. Tomita, &. The Williams and Wilkins Company, Baltimore, pp. 1-69.
107
Ciaranello, R. D., G. F. Wooten and J. Axelrod 1976 Kegulation of r a t adrenal dopamine P-hydroxylase. 11. Receptor interaction in the regulation of enzyme synthesis and degradation. Brain Res., 113: 349-362. Henry, J. P., and P. M. Stephens 1977 The social environment and essential hypertension in mice: possible role of the innervation of the mouse adrenal cortex. Prog. Brain Res., 47: 263-276. Hollinshead, W.H. 1936 The innervation of the adrenal glands. J. Comp. Neur., 64: 449-468. Iankelevich, D.E., N. G. Tsarikovskaia, F. S. Tkatch, V. M. Nicolaeva and N. A. Langova 1978 Glucocorticoid and androgenic functions of the adrenal cortex and the s t a t e of t h e sympathetic-adrenal system in thyrotoxicosis with primary organic disease of t h e central nervous system. Probl. Endokrinol. (Moscow), 24(3): 22-27. Kiss, T. 1951 Experimentelle morphologische analyse der nebennieren innervation. Acta Anat., 13: 181-189. Lever, J. D. 1953 Nerve fibers in t h e adrenal cortex of t h e rat. Nature, 171: 882-883. MacFarland, W.E., and H. A. Davenport 1941 Adrenal innervation. J. Comp. Neur., 75: 219-234. Mikhail, Y. 1961 Innervation of the different zones of t h e adrenal cortex. J. Comp. Neur., 11 7: 365-369. Mikhail, Y., and Z. Mahran 1965 Innervation of the cortical and medullary portions of the adrenal gland of the rat during postnatal life. Anat. Rec., 152: 431-438. Nishikawa, M., I. Murone and T. Sat0 1963 Electron microscopic investigations of the adrenal cortex. Endocrinology, 72: 197-209. Paul, M. I., R. Kvetnansky, H. Cramer, S. Silbergeld and I. J. Kopin 1971 Immobilization stress induced changes in adrenocortical and medullary cyclic AMP content in the rat. Endocrinology, 88: 338-343. Reynolds, E. R. 1963 The use of lead citrate a t a high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-213. Robinson, P. M., R. A. Perry, K. J. Hardy, J. P. Coghlan and B. A. Scoggins 1977 The innervation of t h e adrenal cortex in the sheep, Ouzs ouis. J. Anat., 124: 117.129. Ruskell, G. L. 1967 Vasomotor axons of the lacrimal glands of monkeys and the ultrastructural identification of sympathetic terminals. Z. Zellforsch., 83: 321-333. Spurr, A. R. 1969 A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Swinyard, C. A. 1937 The innervation of the suprarenal glands. Anat. Rec., 68: 417-429. Thaemert, J. C. 1966 Ultrastructure of cardiac muscle and nerve continguities. J. Cell Biol., 29: 156-162. Uno, H. 1977 Catecholaminergic terminals in the perisinusoidal spaces of t h e hepatic acini and adrenal cortex of macaques. Anat. Rec., 187(4): 735. Unsicker, K. 1969 Zur Innervation der Nebennierenrinde von Gold Hamster. Z. Zellforsch., 95: 608-619. - 1971 On the innervation of rat and pig adrenal cortex. Z.Zellforsch., 116: 151-156. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Willard, D. M. 1936 The innervation of the adrenal glands of mammals, a contribution to t h e study of nerve endings. Quart. J. Micros. Sci., 78: 475-486. Wurtman, R. J., and J. Axelrod 1966 Control of enzymatic synthesis of adrenaline in t h e adrenal medulla by adrenal cortical steroids. J. Biol. Chem., 241: (10): 2301-2305.
PLATE 1 EXPLANATION OF FIGURES
1 Subcapsular nervous plexus showing both myelinated (MY) and unmyelinated axon
(UN).
X
6,480.
2 A nerve terminal (NT) containing mostly clear vesicles (arrow) in close proximity to capsular cell (CP). x 14,400.
3 Pre-terminal fibers containing unmyelinated axons (UN) and clear (arrow) and dense-core vesicles (arrowhead) in proximity to a zona glomerulosa cell (ZG). X 12,960. 4 Several autonomic terminals (T)containing clear 60-nm vesicles (arrow) and 120-nm dense core vesicles (arrowhead) in proximity t o a zona glomerulosa cell (ZG). X 15,120.
108
MOUSE ADRENAL CORTEX INNERVATION Nabil Migally
PLATE 1
109
PLATE 2 EXPLANATION OF FIGURES
5 Nerve bundle (N) located in t h e zona fasciculata in t h e area between the vascular lumen (LU) and t h e parenchymal cell (ZF).x 23,140. 6
Unmyelinated axons (UN) in close proximity to a zona reticularis cell (ZR).Note autonomic terminals (TI found between t h e vascular lumen (LU) and t h e parenchymal cell are also in close (30 nm) proximity to the parenchymal cell. X 15,120.
7 In the zona reticularis, several autonomic terminals (T) are found in the area between the vascular lumen (LU) and t h e parenchymal cell (ZR). These terminals contained both clear (arrow) and dense (arrowhead) vesicles. x 17,280.
8 Nerve trunks (N) associated with the medulla, in t h e zona reticularis between the vascular Iumen (LU) and t h e parenchymal cell (ZR).x 15,870.
110
bll"x!W I!TN NOILVAB3NNI X 3 L 8 0 3 T V N 3 B a V 3SnOW
ABSTRACT The fine structure of the mouse adrenal cortex was examined with the electron microscope for the presence of neural elements. Several axon terminals containing mostly clear vesicles (60 nm) were noted in the vicinity (250 nm) of the capsular fibroblasts. In the subcapsular region, myelinated as well as unmyelinated fibers were commonly found. Preterminal and terminal axons were also found in close relationship to the parenchymal cells in the zona glomerulosa. Nerve bundles were the most common neural elements in the zona fasciculata. In the zona reticularis axon terminals containing both clear (60 nm) and dense core (120 nm) vesicles were seen in close proximity (30 nm) to parenchymal cells. Although this study did not delineate the type of fibers involved, t h e axon terminals resemble those of autonomic nerves. This study demonstrates innervation of the mouse adrenal cortex, thus corroborating similar reports by others in different species. Studies concerning adrenocortical innervation have been varied. At the light microscope level, several investigators observed no cortical innervation (Hollinshead, '36; Swinyard, '37; MacFarland and Davenport, '41) while others noted direct nerve termination upon cortical cells (Lever, '53; Mikhail, '61; Mikhail and Mahran, '65; Robinson e t al., '77; Uno, '77). Several workers using silver axonal degeneration techniques and conventional light microscopy noted a well developed subcapsular nervous plexus in humans (Alpert, '31) cats and dogs (Kiss, '511, guinea pigs (Willard, '36) and rats (Lever, '53; Mikhail and Mahran, '65). In the zona glomerulosa short direct extensions from the subcapsular nerve plexus terminated in boutons on parenchymal cells (Alpert, '31; Willard, '36; Mikhail, '61). Extensions from the subcapsular nerve plexus were also observed to terminate in boutons on the zona fasciculata cells (Alpert, '31; Willard, '36; Lever, '53). In the rat, nerve fibers were particularly abundant in the zona reticularis (Mikhail, '61; Mikhail and Mahran, '65). Recently, investigators using catecholamine fluorescence techniques demonstrated the presence of adrenergic terminals in the adrenal cortex of monkeys (Uno, '77) and sheep (Robinson e t al., '77). Robinson et al. ('77) also reported the presence of acetylchoANAT.
REC. (1979) 194: 105-112.
linesterase-positive fibers forming a plexus in the zona reticularis of the sheep adrenal cortex. Using electron microscopy, myelinated nerves were noted in the subcapsular region of rat adrenals by Nishikawa et al. ('63). In the vicinity of the zona glomerulosa and zona fasciculata of hamster, rat, and pig adrenals, Unsicker ('69, '71) noted structures suggestive of nerve terminals in close (20 nm) proximity to the parenchymal cells. These terminals contained two types of granular vesicles measuring 50 and 100 nm in diameter (Unsicker, '71). Uno ('77) demonstrated the presence of presumptive adrenergic and cholinergic terminals in the monkey adrenal cortex. In view of recent investigations linking altered adrenocortical activity with catecholamine content in the normal (Paul et al., '71; Ciaranello et al., '76) as well as in the diseased state (Henry and Stephens, '77; Iankelevich et al., '781, studies concerning adrenocortical innervation in the mouse have failed to demonstrate the presence of autonomic nerves in each zone. The objective of this investigation was to closely examine the mouse adrenal cortex for neural elements. Received July 18, '78. Accepted Oct. 31, '78. I Present address: Department of Anatomy, Wright State University, School of Medicine, Dayton, Ohio 45435.
105
106
NABIL MIGALLY MATERIALS AND METHODS
Animals Adrenal glands were obtained from adult male C,,BL mice (20-30 g) ranging in age from 8 to 12 weeks. All animals were housed in a controlled environment with respect to light, humidity and temperature. The animals were obtained at seven weeks of age from a local breeder and were acclimated to laboratory conditions for one week before the onset of the experiments. The mice were anesthetized with sodium pentobarbital (Abbott 40 mg/kg intraperitoneally), and fixed by intracardiac systemic perfusion with 3% (v/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.25) a t 0-4°C. During perfusion, in situ fixation was also employed by flooding the glands directly with the glutaraldehyde solution. After perfusion, the adrenals were excised, cut into smaller pieces (1 mm?, and fixed for an additional three hours in a fresh fixative solution a t 0-4OC. The tissues were then washed with 0.1 M phosphate buffer (pH 7.25) several times, and postfixed for one hour in cold 1.0%osmium tetroxide in 0.1 M phosphate buffer. The tissues were rapidly dehydrated in ethanol and embedded in Spurr's epoxy resin and polymerized a t 6570°C (Spurr, '69); 0.5- to 1.0-pm-thick sections were cut with Porter-Blum ultramicrotome and stained with toluidine blue for light microscopic survey. Thin (60-70nm) sections were cut with an LKB ultratome, mounted on uncoated 300-mesh grids and stained with uranyl acetate (Watson, '58) and lead citrate (Reynolds, '63). The sections were examined and photographed with an RCA EMU 4B electron microscope using 100 kv accelerating voltage. RESULTS
In the adrenal capsule occasional nerve terminals were seen in the vicinity (250 nm) of the capsular fibroblasts (fig. 2). The nerve terminals contained mostly clear vesicles measuring 60 nm in diameter, although occasional dense core vesicles were also seen (fig. 2). In the subcapsular region, myelinated as well as unmyelinated axons were observed (fig. 1). In the region of the zona glomerulosa, preterminal axons were occasionally seen in the vicinity (310 nm) of parenchymal cells (fig. 3). Axons containing clear synaptic vesicles measuring 60 nm in diameter were also seen in
close proximity to parenchymal cells (fig. 4). No membrane specialization was seen on either the axon or the cortical cell (fig. 4). In the zona fasciculata, unmyelinated nerve fibers with accompanying basal laminae were the only neural components noted (fig. 5). Such profiles were usually located in the perivascular area. Nerve terminals were present in close proximity (30 nm) to the zona reticularis parenchymal cells (fig. 6). Clear vesicles measuring 60 nm in diameter and vesicles measuring 120 nm in diameter could be distinguished in these terminals (fig. 7). Nerve bundles with accompanying basal laminae were often observed in the perivascular compartment of the zona reticularis (fig. 8). DISCUSSION
In the adrenal capsule of rats and pigs, Unsicker ('71) described small nerve fibers in close proximity to the capsular fibroblasts. These nerves contained neurotubules, however, synaptic vesicles were not seen. In the present study, axons as well as terminals which contained both clear and dense core vesicles were seen near the fibroblasts. Such terminals have not been previously described in the mammalian adrenal capsule. Varicose and non-varicose axons were noted in close relationship to zona glomerulosa cells in the present study. These terminal and preterminal axons met the criteria of Thaemert ('66) for classification as autonomic nerve terminals. Pre-terminal axons in this zone were noted by Unsicker ('71), who postulated that some axons were influencing parenchymal cells, while others were probably in transit to deeper layers of the adrenal cortex or medulla. Although the present study was not designed to identify t h e nerve terminals with regard to vesicle content or origin, a functional relationship may exist between some zona glomerulosa cells and autonomic fibers. Close examination of numerous adjacent sections to the nerve terminals did not reveal the presence of vascular smooth muscle, however the possibility that these terminals were innervating vascular smooth muscle cannot be ruled out. Autonomic nerve terminals were reported in close relationship with parenchymal cells of the zona fasciculata in pigs and rats (Unsicker, '71) and in sheep (Robinson e t al., '77). In the present study axon terminals were not observed in t h e zona fasciculata, although some nerves were observed either associated
MOUSE ADRENAL CORTEX INNERVATION
with small blood vessels or as isolated nerve bundles. It is likely that many of the isolated nerve bundles observed were fibers associated with the adrenal medulla. Examination of the zona reticularis demonstrated autonomic nerve terminals in close apposition to the parenchymal cells. Since the presence of vascular smooth muscle was not encountered in the zona fasciculata or zona reticularis (figs. 5-71, the possibility that these terminals were associated with vascular smooth muscle seemed quite remote. The terminals contained 120-nm vesicles and 60-nm vesicles. The 120-nm vesicles were shown by Ruskell ('67) to be present in sympathetic as well as parasympathetic nerve terminals. The 60-nm vesicles were generally believed to contain acetylcholine or norepinephrine (Burnstock, '70). Although some terminals contained vesicles clustered away from the cortical cell (fig. 61, the vesicles were demonstrably close, so as to strongly suggest an innervation. The exact identification of the transmitter substance within these vesicles remains to be elucidated. The functional ramifications of autonomic innervation of the adrenal cortex are variable. The evidence presented in this study concerning the innervation of the zona glomerulosa corroborates the findings of Henry and Stephens ('77) who implicated neural stimulation of adrenocortical cells as a mechanism for increased corticosterone secretion in essential hypertension. Also from this investigation, the results seem to compliment the work of Ciaranello e t al. ('76) who demonstrated the presence of autonomic regulation of adrenal corticoidogenesis which in turn affects the catecholamine synthesizing enzymes in the adrenal medulla (Wurtman and Axelrod, '66). Although the morphological findings presented in this study seem to be implicated in affecting catecholamine synthesis, as well as in regulation of secretion of vasopressive substances, the exact mechanism of neural control of adrenocortical function remains to be found. LITERATURE CITED Alpert, L. K. 1931 The innervation of t h e suprarenal glands. Anat. Rec., 50: 221-233. Burnstock, G. 1970 Structure of smooth muscle and its innervation. In: Smooth Muscle. E. Bulbring, A. F. Brading, A. W. Jones and T. Tomita, &. The Williams and Wilkins Company, Baltimore, pp. 1-69.
107
Ciaranello, R. D., G. F. Wooten and J. Axelrod 1976 Kegulation of r a t adrenal dopamine P-hydroxylase. 11. Receptor interaction in the regulation of enzyme synthesis and degradation. Brain Res., 113: 349-362. Henry, J. P., and P. M. Stephens 1977 The social environment and essential hypertension in mice: possible role of the innervation of the mouse adrenal cortex. Prog. Brain Res., 47: 263-276. Hollinshead, W.H. 1936 The innervation of the adrenal glands. J. Comp. Neur., 64: 449-468. Iankelevich, D.E., N. G. Tsarikovskaia, F. S. Tkatch, V. M. Nicolaeva and N. A. Langova 1978 Glucocorticoid and androgenic functions of the adrenal cortex and the s t a t e of t h e sympathetic-adrenal system in thyrotoxicosis with primary organic disease of t h e central nervous system. Probl. Endokrinol. (Moscow), 24(3): 22-27. Kiss, T. 1951 Experimentelle morphologische analyse der nebennieren innervation. Acta Anat., 13: 181-189. Lever, J. D. 1953 Nerve fibers in t h e adrenal cortex of t h e rat. Nature, 171: 882-883. MacFarland, W.E., and H. A. Davenport 1941 Adrenal innervation. J. Comp. Neur., 75: 219-234. Mikhail, Y. 1961 Innervation of the different zones of t h e adrenal cortex. J. Comp. Neur., 11 7: 365-369. Mikhail, Y., and Z. Mahran 1965 Innervation of the cortical and medullary portions of the adrenal gland of the rat during postnatal life. Anat. Rec., 152: 431-438. Nishikawa, M., I. Murone and T. Sat0 1963 Electron microscopic investigations of the adrenal cortex. Endocrinology, 72: 197-209. Paul, M. I., R. Kvetnansky, H. Cramer, S. Silbergeld and I. J. Kopin 1971 Immobilization stress induced changes in adrenocortical and medullary cyclic AMP content in the rat. Endocrinology, 88: 338-343. Reynolds, E. R. 1963 The use of lead citrate a t a high pH as a n electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-213. Robinson, P. M., R. A. Perry, K. J. Hardy, J. P. Coghlan and B. A. Scoggins 1977 The innervation of t h e adrenal cortex in the sheep, Ouzs ouis. J. Anat., 124: 117.129. Ruskell, G. L. 1967 Vasomotor axons of the lacrimal glands of monkeys and the ultrastructural identification of sympathetic terminals. Z. Zellforsch., 83: 321-333. Spurr, A. R. 1969 A low viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res., 26: 31-43. Swinyard, C. A. 1937 The innervation of the suprarenal glands. Anat. Rec., 68: 417-429. Thaemert, J. C. 1966 Ultrastructure of cardiac muscle and nerve continguities. J. Cell Biol., 29: 156-162. Uno, H. 1977 Catecholaminergic terminals in the perisinusoidal spaces of t h e hepatic acini and adrenal cortex of macaques. Anat. Rec., 187(4): 735. Unsicker, K. 1969 Zur Innervation der Nebennierenrinde von Gold Hamster. Z. Zellforsch., 95: 608-619. - 1971 On the innervation of rat and pig adrenal cortex. Z.Zellforsch., 116: 151-156. Watson, M. L. 1958 Staining of tissue sections for electron microscopy with heavy metals. J. Biophys. Biochem. Cytol., 4: 475-478. Willard, D. M. 1936 The innervation of the adrenal glands of mammals, a contribution to t h e study of nerve endings. Quart. J. Micros. Sci., 78: 475-486. Wurtman, R. J., and J. Axelrod 1966 Control of enzymatic synthesis of adrenaline in t h e adrenal medulla by adrenal cortical steroids. J. Biol. Chem., 241: (10): 2301-2305.
PLATE 1 EXPLANATION OF FIGURES
1 Subcapsular nervous plexus showing both myelinated (MY) and unmyelinated axon
(UN).
X
6,480.
2 A nerve terminal (NT) containing mostly clear vesicles (arrow) in close proximity to capsular cell (CP). x 14,400.
3 Pre-terminal fibers containing unmyelinated axons (UN) and clear (arrow) and dense-core vesicles (arrowhead) in proximity to a zona glomerulosa cell (ZG). X 12,960. 4 Several autonomic terminals (T)containing clear 60-nm vesicles (arrow) and 120-nm dense core vesicles (arrowhead) in proximity t o a zona glomerulosa cell (ZG). X 15,120.
108
MOUSE ADRENAL CORTEX INNERVATION Nabil Migally
PLATE 1
109
PLATE 2 EXPLANATION OF FIGURES
5 Nerve bundle (N) located in t h e zona fasciculata in t h e area between the vascular lumen (LU) and t h e parenchymal cell (ZF).x 23,140. 6
Unmyelinated axons (UN) in close proximity to a zona reticularis cell (ZR).Note autonomic terminals (TI found between t h e vascular lumen (LU) and t h e parenchymal cell are also in close (30 nm) proximity to the parenchymal cell. X 15,120.
7 In the zona reticularis, several autonomic terminals (T) are found in the area between the vascular lumen (LU) and t h e parenchymal cell (ZR). These terminals contained both clear (arrow) and dense (arrowhead) vesicles. x 17,280.
8 Nerve trunks (N) associated with the medulla, in t h e zona reticularis between the vascular Iumen (LU) and t h e parenchymal cell (ZR).x 15,870.
110
bll"x!W I!TN NOILVAB3NNI X 3 L 8 0 3 T V N 3 B a V 3SnOW
