Localization of Tritiated Norepinephrine in the Renal Arteriolar Nerves LUCIAN0 BARAJAS AND PATRICIA WANG Department of Pathology, Harbor-UCLA Medical Center, 1000 West Carson Street, Torrance, California 90509
ABSTRACT The innervation of the glomerular arterioles was investigated by light and electron microscopy autoradiography for localization of exogenous tritiated norepinephrine. By light microscopy accumulations of grains were seen associated with afferent arterioles and in lesser numbers with efferent arterioles and neighboring tubules. Accumulations of grains were noted t o be in contact with juxtaglomerular granular cells. Electron microscopy autoradiography revealed that nearly two-thirds of the silver grains were on axons. Most of the label was on varicosities packed with small, clear and dense-cored, vesicles. Most varicosities, including those in contact with smooth muscle, juxtaglomerular granular or tubular cells, were labeled. Some varicosities which appeared unlabeled in a given section were labeled in subsequent sections. These findings are consistent with the notion that the glomerular arterioles are innervated mainly by adrenergic nerves. This view is supported by the previously reported observations of the concomitant virtual disappearance of fluorescent and acetylcholinesterase-positive nerves from the region of the glomerular arterioles after two injections of six-hydroxydopamine (a drug which selectively destroys adrenergic nerves) and the presence of small dense-cored vesicles in all axons of the juxtaglomerular region when examined by serial section electron microscopy.
It is currently accepted that the rat glomerular arterioles have an adrenergic innervation and that stimulation of the renal nerves affects renin secretion by direct action on the juxtaglomerular granular cells present in the arteriolar wall (Davis and Freeman, '76). Fluorescent histochemical methods have demonstrated a delicate neural plexus in close association with the afferent arteriole (Nilsson, '65; McKenna and Angelakos, '68; Wagermark et al., '68) and a less prominent innervation of the efferent arteriole and renal tubules (Muller and Barajas, '72). The specificity of fluorescent methods makes them very useful in demonstrating and assessing the extent of adrenergic innervation. Fluorescent methods are, however, of limited usefulness when applied to the study of the anatomical relationships of the nerve fibers with the neighboring structures. Exogenous tritiated norepinephrine has been localized by electron microscopic autoradiography in the central nervous system and ANAT. REC. (1979) 195: 525-534.
in the adrenergic nerves of many tissues (Wolfe et al., '62; Aghajanian and Bloom, '66; Lenn, '67; Devine and Simpson, '68; Taxi, '68; Budd and Salpeter, '69; Sotelo, '71; Bogart and De Lemos, '72; Gershon et al., '74; Forssmann and Ito, '77; Barajas and Wang, '78). In this study, we used the uptake of tritiated norepinephrine as a specific functional marker for monoaminergic nerves. This approach permits investigation of the monoaminergic innervation of glomerular arterioles and surrounding tubules combining the specificity provided by the uptake of tritiated norepinephrine with the resolution of light and electron microscopic autoradiography. MATERIALS A N D METHODS
Five male Sprague-Dawley rats weighing approximately 100-120 g were injected intraReceived Apr 2, '79 Accepted June 11, '79 'Supported by U S Public Health Service Grant R01 HL 18340 (PTHA) from the National Heart, Lung, and Blood Institute, by American Heart Association Grant 78791 and by a grant from the Professional Staff Association, Harbor UCLA Medical Center
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peritoneally with the monoamine oxidase inhibitor pargyline (100 mg/kg) 2 hours before being given 500 pCi of DL-17-3H-(N)1norepinephrine HI"[( NA; specific activity, 10.3 Ci/ mmole; New England Nuclear, Boston, Massachusetts) via the femoral vein. Thirty minutes after the administration of [3HINAto the animals, their kidneys were fixed in vivo by retrograde perfusion through the aorta with 1% glutaraldehyde in 0.135 M phosphate buffer (pH 7.4) and then immediately removed. Blocks of renal tissue including cortex and medulla were immersed in the same fixative for a total fixation time of 1 hour on ice followed by postfixation in 1%osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4). Some blocks were blockstained with 0.5% uranyl magnesium acetate in the dark for 2 hours a t 4°C. All tissues were dehydrated through graded alcohols and embedded in Epon 812.
Light microscopy autoradiography Serial sections mounted on gelatin coated slides were dipped in a 1:l dilution of Ilford nuclear research emulsion L-4 (Polyscience, Inc., Warrington, Pennsylvania) melted a t 45"C, allowed to dry for 3-5hours in a dust free area a t room temperature, packed in a black slide box containing a package of Drierite (Caro and Van Tubergen, '621, and sealed with black tape. The slide box was placed in a threelayer Kodak plate box and stored a t 4°C for 1-2 weeks, after which the slides were developed in Kodak D-19 for 4 minutes at 20"C, fixed in Kodak rapid fixer (diluted 1:l)for 6 minutes, and stained with 1%toluidine blue or 0.1% crystal violet. The background of the emulsion was always checked on blank slides for each series of slides coated. To minimize artefactual grains, only emulsion with less than 10 developed grains per oil immersion field was used. Electron microscopy autoradiography Electron microscopic autoradiographs were prepared according to the method of Salpeter and Bachmann ('64). Ribbons of uniform pale gold-colored sections were placed on parlodion-coated slides premarked on the back with a diamond pencil, so that the sections were placed a t a point 2.5 cm from one short end of the slide. The sections were stained with 2.5% alcoholic uranyl acetate and Reynold's lead citrate, and vacuum-coated with a layer of carbon approximately 50-75 A thick. The slides were then dipped in a 1:5 dilution of 11-
ford L-4 emulsion maintained a t 45"C, allowed t o air-dry for 2-4 hours a t room temperature, and packaged in the same way as for light microscopy autoradiography . Following exposure for 1 to 2 months, the slides were developed in Microdol-X for 2 minutes at 20°C and fixed in 30% sodium thiosulfate for 5 minutes. The specimen "sandwich" consisting of the emulsion, carbon layer, section, and parlodion film was then stripped onto water, and 75-mesh copper grids were placed over the sections. The "sandwich" was then picked up on filter paper and allowed to dry in a covered petri dish. The grids were removed from the filter paper and the parlodion film thinned by gripping the grid with a pair of fine forceps and touching the surface of the parlodion film to a solution of amyl acetate in a spot plate for 3 minutes. The grids were then dried and the sections examined with a Siemens Elmiskop 1A electron microscope a t 80 kv. The thickness and background of the emulsion were always checked prior to each coating. The interference color of the emulsion was purple over the area of the slide where the sections were located. Only emulsion with a background of less than 1grain per 500 p z was used.
Quantitative analyses Electron micrographs, a t a final magnification of 30,000, were taken of the glomerular arterioles and of the periarteriolar space within l o p of the arteriolar wall. This area included all the nerves present in the sections. Silver grains over or within 0.2p of axons, smooth muscle cells, juxtaglomerular granular cells, Schwann cells, and those lying over interstitial cells or in the interstitial space were counted, and the density expressed as total number of grains per 100 p2.The area of the different structures was measured by an electronic rolling disc planimeter (Model 1250S1, Lasico, Inc., Los Angeles, California). Electron micrographs with a final magnification of 4,000 were also taken. Montages were constructed and the different structures were measured to insure that adequate repreFig. 1 Light microscopic autoradiograph revealing nerve fibers (arrows) associated with arteries (A). X 400. Fig. 2 Light microscopic autoradiograph demonstrating nerve fibers (arrows) associated with glomerular afferent (a) and efferent (el arterioles. Glomerulus (G). X 600. Fig. 3 Light microscopic autoradiograph showing nerve fibers (arrows) in contact with proximal (PT) and distal (DT)tubules. Glomerulus ( G ) . x 550.
TRITIATED NOREPINEPHRINE IN RENAL NERVES
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TRITIATED NOREPINEPHRINE I N RENAL NERVES
sentation was provided by the 30,000 magnification micrographs. No discrepancies were found. A total of 1,098 silver grains of which 670 grains were lying over axons were counted in 64 electron micrographs. RESULTS
Light microscopy autoradiography : Examination a t low magnification of the Epon embedded thick sections, revealed accumulations; of autoradiographic grains associated with blood vessels (fig. 1).They were consistently observed adjacent to the glomerular arterioles (fig. 2) and on occasion with a neighboring tubule. Accumulations of grains were larger and more frequently seen associated with the afferent than with the efferent arterioles. (One arteriole is readily distinguished from the other by noting in serial sections whether
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ScW INT SMC GC
Fig. 4 Light microscopic autoradiograph revealing nerves (arrow) adjacent to granular cells of the afferent arteriole (a). Glomerulus (GI. x 1,250. Fig. 5 Distribution of silver grains (expressed as grains per l o o p 2 of tissue) over axon8 (AX), Schwanri cells (ScW), interstitial connective tissue (INTI, smooth muscle cells of the arteriolar wall (SMC) and juxtaglomerular granular cell (GC) in electron microscopic autoradiographs. Fig. 6 Electron microscopic autoradiograph showing labeled axons (area between bars) adjacent to granular cells (GC) and smooth muscle cells (S) of the wall of t h e afferent arteriole (a). x 4,000.
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it connects with a larger artery Iafferentl or it breaks into capillaries [efferent]1. In some instances accumulations of grains were seen adjacent to proximal and distal tubules without apparent relation to the glomerular arterioles (fig. 3). Serial sections, however, revealed that these nerves, though in contact with the tubule, were part of the arteriolar plexus. At high magnification the granules of juxtaglomerular cells were clearly visible in t h e plastic sections stained with crystal violet. Autoradiography of these sections revealed accumulations of grains in contact with t h e juxtaglomerular granular cells (fig. 4).
Electron microscopy autoradiography Silver grains were seen over the nerve bundles, smooth muscle and juxtaglomerular granular cells and on the interstitium surrounding the blood vessels. Most of the observed label appeared on the axons with a count of 395 grains per 100 p 2 (fig. 5). At the level of the glomerular arterioles the renal sympathetic axons appeared as dilated varicosities and as narrower intervaricose regions (figs. 6-10). Varicosities contained large numbers of vesicles, some mitochondria and microtubules. Varicosities in contact with smooth muscle (figs. 8, 9), granular (figs. 6, 7), or tubular cells (fig. 10) are referred to as nerve endings and had similar content. At the site of contact a space of 1,500-2,500 A in width separated the plasma membrane of the effector cell from that of the axolemma. Basement membrane material was present in this space. The intervaricose region contained numerous microtubules. The small vesicles which predominate in the varicosities, usually appeared packed and filled most of the axoplasm. Small dense-cored vesicles were scarce and were found intermingled with clear ones (fig. 9). Their cores were of variable density and often were small and eccentric appearing attached to the vesicular membrane. Large dense-cored vesicles were frequently seen. The density of their core was reduced. Accumulations of the vesicles were seen adjacent to the nerve plasma membrane either at the junction with the effector cell in the nerve endings or facing the interstitial space. Mitochondria were seen in the axoplasm of the varicosities adjacent to the accumulation of vesicles. The radioactive label was observed predominantly on varicosities and nerve endings (figs. 6-10). Grains were also seen on intervaricose regions though
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Fig. 7 Heavily labeled varicosities, filled with vesicles and an occasional mitochondria associated with granular cells. Two nerve endings (N, and NJ are in contact with the granular cell (GC). x 26,500. Fig. 8 Group of axons including varicosities and intervaricose regions, adjacent to arteriolar smooth muscle cells 6). Though the grain appears predominantly on the varicosities some of the intervaricose regions, containing microtubules, are also labeled. x 11.500.
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Fig. 9 Nerve ending (N) in contact with smooth muscle cell (S)of the arteriole. The silver grain overlay the region of t h e varicosity filled with small vesicles. Small dense-core vesicles (arrows). x 45,000. Fig. 10 Group of axons associated with a distal tubule (DT). Only the varicosities are labeled. The silver grains lie on t h e region of the axoplasm filled with small vesicles. A nerve ending (N) is in contact with the distal tubule separated from the tubular cell by a thin basement membrane. X 47,500.
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in much lesser numbers (fig. 8). Much of the interior of the varicosities was filled with small vesicles and therefore most of the grains appeared in portions of the axoplasm almost totally occupied by the vesicles. Grains were seen occasionally on intervaricose regions containing numerous microtubules but no vesicles. Grains were seen on the Schwann cell with a count of 14 grains per 100 p z . A small number of grains, 16 per 100 p 2 ,was observed on the smooth muscle cell, and an even smaller one, eight grains per 100 p z , on the juxtaglomerular granular cells (fig. 5). No pattern of distribution was established in either cell type though on occasion grains were observed on the granules. Autoradiographic grains were observed on cells without nerve contacts present in the section. The grains present on the interstitium were located mainly on the interstitial cells, and with a count of 13 per 100 p z (fig. 5). Very little collagen or elastica was observed in the periarteriolar spaces and no grains were seen on those structures. There was considerable variation in the number of silver grains overlying different varicosities. Rarely, an unlabeled varicosity was observed among a group of labeled ones. No differences, however, were observed in the population of vesicles between the varicosities with overlying silver grains and the rare ones without them. In both instances, absence of small dense-cored vesicles was occasionally found. In addition, it was noted that an unlabeled varicosity, surrounded by labeled ones, might be without overlying grains in one section and be heavily labeled in another. DISCUSSION
The uptake of tritiated norepinephrine in vivo by the sympathetic axons around the intestinal arterioles has been studied by electron microscopy autoradiography by Devine and Simpson ('68). Though our results are in general agreement with theirs, we have recorded much higher counts of label on the renal glomerular arteriolar nerves than they obtained on the sympathetic fibers innervating the intestinal arterioles. This may well be due to our use of higher doses of tritiated norepinephrine and of the monoamine oxidase inhibitor pargyline. This drug was administered to prevent the degradation of norepinephrine and the formation of labeled metabolites. Our results show that most of the label appeared on neurons and very little on the effec-
tor cell (smooth muscle or granular cells). No concentration of grains was observed near the cell membrane of the smooth muscle cells, or of granular cells, at the neuroeffector junction or at other points in the cell surface. The marked difference in grain distribution between axons and effector cells can be explained on the basis of differences between neuronal and extraneuronal uptake of norepinephrine. First, neuronal uptake is followed by vesicular uptake and storage of the neurotransmitter while extraneuronal uptake is followed by metabolic degradation (Iversen, '73; Gillespie, '73). Second, neuronal uptake can occur a t concentrations of norepinephrine 1,000-fold lower than that required for extraneuronal uptake. The respective Michaelis and 2.5 X constants for uptake are 2.7 X M (Lightman and Iversen, '69). In addition, binding of norepinephrine to a-adrenergic receptors has been shown to be rapidly reversible (U'Prichard and Snyder, '77). Under our experimental conditions tritiated norepinephrine was injected 30 minutes before sacrifice and removal of the tissues. Hertting et al. ('61) have shown that this was sufficient time for circulating catecholamines to decline to near zero, thus, allowing metabolism of norepinephrine taken up non-neuronally and dissociation of norepinephrine from adrenergic receptors. The resolution of electron microscopy autoradiography does not permit the localization of the label on structures as small as the axoplasmic vesicles; but the regional distribution of the label on parts of the axon packed with vesicles is consistent with biochemical data indicating that norepinephrine is stored in the sympathetic nerves in a particulate form (see review by Smith, '72) and electron microscopic autoradiographic studies suggesting that the small dense-cored vesicles contain norepinephrine (see reviews by Taxi, '69; Jaim-Etcheverry and Ziher, '71). We confirmed the observations of Devine and Simpson ('68) of the presence of grains not only on varicosities in contact with the effector cell (nerve endings) but also in those apparently separated from them by considerable distance. If uptake of norepinephrine occurs a t the same sites as release, these observations suggest that the discharge of norepinephrine by the axon takes place a t other sites besides the neuroeffector junction. An unexpected finding in our study was the relative scarcity of small dense-cored vesicles.
TRITIATED NOREPINEPHRINE IN RENAL NERVES
The fixative used is considered to be a major factor in the preservation of the core and the loss of the core in the small dense-cored vesicles appears not to be accompanied by loss of radioactivity. Taxi ('68) reported that pineal gland fixed in osmium tetroxide showed a 14% loss of small dense-cored vesicles compared to the same tissue fixed in potassium permanganate. However, the amount of radioactive label observed in the osmium fixed pineal gland was almost double the amount observed when i t was fixed with potassium permanganate. In our studies we used perfusion with glutaraldehyde followed by immersion in osmium tetroxide. This method of fixation is considered optimal for kidney tissue (Maunsbach, '66). It is also thought to be effective in preserving the dense cores of the vesicles (Tranzer and Thoenen, '67; Hokfelt , '66). In our material, however, we have observed fewer small densecored vesicles and their cores appear smaller than those we have previously observed in the renal arteriolar nerves (Barajas, '64; Muller and Barajas, '72; Silverman and Barajas, '74). Our label counts, on the other hand, are more than twice those reported by Devine and Simpson ('68) for the intestinal arteriolar nerves which appeared to have the usual population of small dense-cored vesicles. Although the number of silver grains overlying the different varicosities varied, the vast majority were labeled and, therefore, can be considered monoaminergic in nature. This finding is consistent with the notion that the renin-containing juxtaglomerular cells and smooth muscle cells of glomerular arterioles and the neighboring tubules are innervated mainly by adrenergic nerves. This view is supported by the action of six-hydroxydopamine on the arteriolar nerves. After one injection of this drug, which selectively destroys the adrenergic nerve terminals, concomitant reduction of fluorescent and acetylcholinesterase-positive nerves was observed. Two injections produced the virtual disappearance of the nerves, as observed by both methods, from the region of the glomerular arterioles and surrounding tubules (Barajas and Wang, '75). These results indicate that most, perhaps all nerves in the region of the juxtaglomerular arterioles, previously labeled as cholinergic on the basis of their acetylcholinesterase positivity, were actually adrenergic nerves. Further support of this view is provided by the observation of small dense-cored vesicles in all axons followed through serial sections of the
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juxtaglomerular regions (Barajas and Muller, '73). The absence of autoradiographic grains on an occasional varicosity was harder to interpret. In some instances, an unlabeled varicosity was observed within a group of labeled ones in a given section. In subsequent sections of the same group, however,that same varicosity, also appeared heavily labeled. It seems, therefore, that a varicosity cannot be construed as not being monoaminergic on the basis of the absence of label alone. ACKNOWLEDGMENTS
The authors wish to thank Drs. M. Lubran, J. Lechago and R. Purdy for their many helpful suggestions regarding the manuscript, and Steve Takeshita for his technical assistance. We want to express our special thanks to Dr. Alfred Weinstock for his advice on the methods of electron microscopic autoradiography. The typing skills of Ms. Astara Mayeda are gratefully acknowledged. LITERATURE CITED Aghajanian, G. K., and F. E. Bloom 1966 Electron-microscopic autoradiography of rat hypothalamus after intraventricular H3-Norepinephrine. Science, 153: 308-310. Barajas, L. 1964 The innervation of the juxtaglomerular apparatus. An electron microscopic study of t h e innervation of the glomerular arterioles. Lab. Invest., 13: 916-929. Barajas, L., and J. Muller 1973 The innervation of the juxtaglomerular apparatus and surrounding tubules: A quantitative analysis by serial section electron microscopy. J. Ultrastruct. Res., 43: 107-132. Barajas, L., and P. Wang 1975 Demonstration of acetylcholinesterase in the adrenergic nerves of the renal glomerular arterioles. J. Ultrastruct. Res., 53: 244-253. 1978 Myelinated nerves of the rat kidney. A light and electron microscopic study. J. Ultrastruct. Res., 65: 135-147. Bogart, B. I., and C. L. De Lemos 1973 Adrenergic innervation of the rat submandibular and parotid glands. An electron microscopic autoradiographic study of the uptake of tritiated norepinephrine. Anat. Rec., 177: 219-224. Budd, G. C., and M. M. Salpeter 1969 The distribution of labeled norepinephrine within sympathetic nerve terminals studied with electron microscopic radioautography. J. Cell Biol., 41: 21-32. Caro, L. G., and R. P. Van Tubergen 1962 High resolution autoradiography. I. Methods. J. Cell Biol., 15: 173-188. Davis, J. O., and R. H. Freeman 1976 Mechanisms regulating renin release. Physiol. Rev., 56: 1-56. Devine, C. E., and F. 0. Simpson 1968 Localization of tritiated norepinephrine in vascular sympathetic axons of the rat intestine and mesentery by electron microscope radioautography. J. Cell Biol., 38: 184-192. Forssmann, W. G., and S. Ito 1977 Hepatocyte innervation in primates. J. Cell Biol., 74: 299-313. Gershon, M. D., M. Hagopian and E. A. Nunez 1974 An electron microscope autoradiographic study of the neuronal and extraneuronal localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine. J. Cell Biol., 62: 610-624.
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Gillespie, J. S. 1973 Uptake of noradrenaline by smooth muscle. Br. Med. Bull., 29: 136-141. Hertting, G., J. Axelrod and L. G. Whitby 1961 Effect of drugs on the uptake and metabolism of H”-norepinephrine. J. Pharmacol. Exp. Ther., 134: 146-153. Hokfelt. T. 1966 Electron microscopic observations on nerve terminals in the intrinsic muscles of the albino rat iris. Acta Physiol. Scand., 67: 255-256. Iversen, L. L. 1973 Catecholamine uptake processes. Br. Med. Bull., 29: 130-135. Jaim-Etcheverry, G., and L. M. Zieher 1971 Ultrastructural aspects of neurotransmitter storage in adrenergic nerves. Adv. Cytopharmacol., I : 343-361. Lenn, N. J . 1967 Localization of uptake of tritiated norepinephrine by rat brain in uiuo and in uitro using electron microscopic autoradiography. Am. J. Anat., 120: 377-390. Lightman, S. L., and L. L. Iversen 1969 The role of uptake, in the extraneuronal metabolism of catecholamines in the isolated rat heart. Br. J. Pharmac., 37: 638-649. Maunsbach, A. G. 1966 The influence of the different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubules. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J. Ultrastruct. Res., 15: 242-282. McKenna, 0. C., and E. T. Angelakos 1968 Adrenergic innervation of the canine kidney. Circ. Res., 22: 345-354. Mdller, J.. and L. Barajas 1972 Electron microscopic and histochemical evidence for a tubular innervation in the renal cortex of the monkey. J. Ultrastruct. Res.. 41: 533-549. Nilsson, 0. 1965 The adrenergic innervation of the kidney. Lab. Intest., 14: 1392-1395. Salpeter, M. M., and L. Bachmann 1964 Autoradiography with the electron microscope. A procedure for improving
resolution, sensitivity, and contrast. J. Cell Biol., 22: 469-477. Salpeter, M. M., and F. A. McHenry 1973 Electron microscope autoradiography. In: Advanced techniques in biological electron microscopy. J. K. Koehler, ed. SpringerVerlag, New York, pp. 113-152. Silverman, A. J., and L. Barajas 1974 Effect of reserpine on the juxtaglomerular granular cells and renal nerves. Lab. Invest., 30: 723-731. Smith, A. D. 1972 Subcellular localization of noradrenaline in sympathetic neurons. Pharmacol. Rev., 24: 435-457. Sotelo, C. 1971 The fine structural localization of norepinephrine-’’Hin the substantia nigra and area postrema of the rat. An autoradiographic study. J. Ultrastruct. Res., 36: 824-841. Taxi, J. 1968 Sur la fixation e t la signification du contenu dense des vesicles des fibres adrenergiques etudikes au microscope electronique. Comptes rendus de 1’Academie Bulgare des Sciences, 21: 1229-1231. 1969 Morphological and cytochemical studies on the synapses in the autonomic nervous system. In: Mechanisms of Synaptic Transmission. K. Akert and P. G. Waser, eds. Elsevier, Amsterdam, pp. 5-20. Tranzer, J. P., and H. Thoenen 1967 Significance of “empty vesicles” in pstganglionic sympathetic nerve terminals. Experientia, 23: 123-124. U’Prichard, D. C., and S. H. Snyder 1977 Binding of W c a t echolamines to a-noradrenergic receptor sites in calf brain. J. Biol. Chem., 252: 6450-6463. Wagermark, J., U. Ungerstedt and A. Ljundqvist 1968 Sympathetic innervation of the juxtaglomerular cells of the kidney. Circ. Res., 22: 149-153. Wolfe, D. E., L. T. Potter, K. C. Richardson and J. Axelrod 1962 Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138: 440-442.
ABSTRACT The innervation of the glomerular arterioles was investigated by light and electron microscopy autoradiography for localization of exogenous tritiated norepinephrine. By light microscopy accumulations of grains were seen associated with afferent arterioles and in lesser numbers with efferent arterioles and neighboring tubules. Accumulations of grains were noted t o be in contact with juxtaglomerular granular cells. Electron microscopy autoradiography revealed that nearly two-thirds of the silver grains were on axons. Most of the label was on varicosities packed with small, clear and dense-cored, vesicles. Most varicosities, including those in contact with smooth muscle, juxtaglomerular granular or tubular cells, were labeled. Some varicosities which appeared unlabeled in a given section were labeled in subsequent sections. These findings are consistent with the notion that the glomerular arterioles are innervated mainly by adrenergic nerves. This view is supported by the previously reported observations of the concomitant virtual disappearance of fluorescent and acetylcholinesterase-positive nerves from the region of the glomerular arterioles after two injections of six-hydroxydopamine (a drug which selectively destroys adrenergic nerves) and the presence of small dense-cored vesicles in all axons of the juxtaglomerular region when examined by serial section electron microscopy.
It is currently accepted that the rat glomerular arterioles have an adrenergic innervation and that stimulation of the renal nerves affects renin secretion by direct action on the juxtaglomerular granular cells present in the arteriolar wall (Davis and Freeman, '76). Fluorescent histochemical methods have demonstrated a delicate neural plexus in close association with the afferent arteriole (Nilsson, '65; McKenna and Angelakos, '68; Wagermark et al., '68) and a less prominent innervation of the efferent arteriole and renal tubules (Muller and Barajas, '72). The specificity of fluorescent methods makes them very useful in demonstrating and assessing the extent of adrenergic innervation. Fluorescent methods are, however, of limited usefulness when applied to the study of the anatomical relationships of the nerve fibers with the neighboring structures. Exogenous tritiated norepinephrine has been localized by electron microscopic autoradiography in the central nervous system and ANAT. REC. (1979) 195: 525-534.
in the adrenergic nerves of many tissues (Wolfe et al., '62; Aghajanian and Bloom, '66; Lenn, '67; Devine and Simpson, '68; Taxi, '68; Budd and Salpeter, '69; Sotelo, '71; Bogart and De Lemos, '72; Gershon et al., '74; Forssmann and Ito, '77; Barajas and Wang, '78). In this study, we used the uptake of tritiated norepinephrine as a specific functional marker for monoaminergic nerves. This approach permits investigation of the monoaminergic innervation of glomerular arterioles and surrounding tubules combining the specificity provided by the uptake of tritiated norepinephrine with the resolution of light and electron microscopic autoradiography. MATERIALS A N D METHODS
Five male Sprague-Dawley rats weighing approximately 100-120 g were injected intraReceived Apr 2, '79 Accepted June 11, '79 'Supported by U S Public Health Service Grant R01 HL 18340 (PTHA) from the National Heart, Lung, and Blood Institute, by American Heart Association Grant 78791 and by a grant from the Professional Staff Association, Harbor UCLA Medical Center
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peritoneally with the monoamine oxidase inhibitor pargyline (100 mg/kg) 2 hours before being given 500 pCi of DL-17-3H-(N)1norepinephrine HI"[( NA; specific activity, 10.3 Ci/ mmole; New England Nuclear, Boston, Massachusetts) via the femoral vein. Thirty minutes after the administration of [3HINAto the animals, their kidneys were fixed in vivo by retrograde perfusion through the aorta with 1% glutaraldehyde in 0.135 M phosphate buffer (pH 7.4) and then immediately removed. Blocks of renal tissue including cortex and medulla were immersed in the same fixative for a total fixation time of 1 hour on ice followed by postfixation in 1%osmium tetroxide in 0.1 M cacodylate buffer (pH 7.4). Some blocks were blockstained with 0.5% uranyl magnesium acetate in the dark for 2 hours a t 4°C. All tissues were dehydrated through graded alcohols and embedded in Epon 812.
Light microscopy autoradiography Serial sections mounted on gelatin coated slides were dipped in a 1:l dilution of Ilford nuclear research emulsion L-4 (Polyscience, Inc., Warrington, Pennsylvania) melted a t 45"C, allowed to dry for 3-5hours in a dust free area a t room temperature, packed in a black slide box containing a package of Drierite (Caro and Van Tubergen, '621, and sealed with black tape. The slide box was placed in a threelayer Kodak plate box and stored a t 4°C for 1-2 weeks, after which the slides were developed in Kodak D-19 for 4 minutes at 20"C, fixed in Kodak rapid fixer (diluted 1:l)for 6 minutes, and stained with 1%toluidine blue or 0.1% crystal violet. The background of the emulsion was always checked on blank slides for each series of slides coated. To minimize artefactual grains, only emulsion with less than 10 developed grains per oil immersion field was used. Electron microscopy autoradiography Electron microscopic autoradiographs were prepared according to the method of Salpeter and Bachmann ('64). Ribbons of uniform pale gold-colored sections were placed on parlodion-coated slides premarked on the back with a diamond pencil, so that the sections were placed a t a point 2.5 cm from one short end of the slide. The sections were stained with 2.5% alcoholic uranyl acetate and Reynold's lead citrate, and vacuum-coated with a layer of carbon approximately 50-75 A thick. The slides were then dipped in a 1:5 dilution of 11-
ford L-4 emulsion maintained a t 45"C, allowed t o air-dry for 2-4 hours a t room temperature, and packaged in the same way as for light microscopy autoradiography . Following exposure for 1 to 2 months, the slides were developed in Microdol-X for 2 minutes at 20°C and fixed in 30% sodium thiosulfate for 5 minutes. The specimen "sandwich" consisting of the emulsion, carbon layer, section, and parlodion film was then stripped onto water, and 75-mesh copper grids were placed over the sections. The "sandwich" was then picked up on filter paper and allowed to dry in a covered petri dish. The grids were removed from the filter paper and the parlodion film thinned by gripping the grid with a pair of fine forceps and touching the surface of the parlodion film to a solution of amyl acetate in a spot plate for 3 minutes. The grids were then dried and the sections examined with a Siemens Elmiskop 1A electron microscope a t 80 kv. The thickness and background of the emulsion were always checked prior to each coating. The interference color of the emulsion was purple over the area of the slide where the sections were located. Only emulsion with a background of less than 1grain per 500 p z was used.
Quantitative analyses Electron micrographs, a t a final magnification of 30,000, were taken of the glomerular arterioles and of the periarteriolar space within l o p of the arteriolar wall. This area included all the nerves present in the sections. Silver grains over or within 0.2p of axons, smooth muscle cells, juxtaglomerular granular cells, Schwann cells, and those lying over interstitial cells or in the interstitial space were counted, and the density expressed as total number of grains per 100 p2.The area of the different structures was measured by an electronic rolling disc planimeter (Model 1250S1, Lasico, Inc., Los Angeles, California). Electron micrographs with a final magnification of 4,000 were also taken. Montages were constructed and the different structures were measured to insure that adequate repreFig. 1 Light microscopic autoradiograph revealing nerve fibers (arrows) associated with arteries (A). X 400. Fig. 2 Light microscopic autoradiograph demonstrating nerve fibers (arrows) associated with glomerular afferent (a) and efferent (el arterioles. Glomerulus (G). X 600. Fig. 3 Light microscopic autoradiograph showing nerve fibers (arrows) in contact with proximal (PT) and distal (DT)tubules. Glomerulus ( G ) . x 550.
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sentation was provided by the 30,000 magnification micrographs. No discrepancies were found. A total of 1,098 silver grains of which 670 grains were lying over axons were counted in 64 electron micrographs. RESULTS
Light microscopy autoradiography : Examination a t low magnification of the Epon embedded thick sections, revealed accumulations; of autoradiographic grains associated with blood vessels (fig. 1).They were consistently observed adjacent to the glomerular arterioles (fig. 2) and on occasion with a neighboring tubule. Accumulations of grains were larger and more frequently seen associated with the afferent than with the efferent arterioles. (One arteriole is readily distinguished from the other by noting in serial sections whether
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Fig. 4 Light microscopic autoradiograph revealing nerves (arrow) adjacent to granular cells of the afferent arteriole (a). Glomerulus (GI. x 1,250. Fig. 5 Distribution of silver grains (expressed as grains per l o o p 2 of tissue) over axon8 (AX), Schwanri cells (ScW), interstitial connective tissue (INTI, smooth muscle cells of the arteriolar wall (SMC) and juxtaglomerular granular cell (GC) in electron microscopic autoradiographs. Fig. 6 Electron microscopic autoradiograph showing labeled axons (area between bars) adjacent to granular cells (GC) and smooth muscle cells (S) of the wall of t h e afferent arteriole (a). x 4,000.
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it connects with a larger artery Iafferentl or it breaks into capillaries [efferent]1. In some instances accumulations of grains were seen adjacent to proximal and distal tubules without apparent relation to the glomerular arterioles (fig. 3). Serial sections, however, revealed that these nerves, though in contact with the tubule, were part of the arteriolar plexus. At high magnification the granules of juxtaglomerular cells were clearly visible in t h e plastic sections stained with crystal violet. Autoradiography of these sections revealed accumulations of grains in contact with t h e juxtaglomerular granular cells (fig. 4).
Electron microscopy autoradiography Silver grains were seen over the nerve bundles, smooth muscle and juxtaglomerular granular cells and on the interstitium surrounding the blood vessels. Most of the observed label appeared on the axons with a count of 395 grains per 100 p 2 (fig. 5). At the level of the glomerular arterioles the renal sympathetic axons appeared as dilated varicosities and as narrower intervaricose regions (figs. 6-10). Varicosities contained large numbers of vesicles, some mitochondria and microtubules. Varicosities in contact with smooth muscle (figs. 8, 9), granular (figs. 6, 7), or tubular cells (fig. 10) are referred to as nerve endings and had similar content. At the site of contact a space of 1,500-2,500 A in width separated the plasma membrane of the effector cell from that of the axolemma. Basement membrane material was present in this space. The intervaricose region contained numerous microtubules. The small vesicles which predominate in the varicosities, usually appeared packed and filled most of the axoplasm. Small dense-cored vesicles were scarce and were found intermingled with clear ones (fig. 9). Their cores were of variable density and often were small and eccentric appearing attached to the vesicular membrane. Large dense-cored vesicles were frequently seen. The density of their core was reduced. Accumulations of the vesicles were seen adjacent to the nerve plasma membrane either at the junction with the effector cell in the nerve endings or facing the interstitial space. Mitochondria were seen in the axoplasm of the varicosities adjacent to the accumulation of vesicles. The radioactive label was observed predominantly on varicosities and nerve endings (figs. 6-10). Grains were also seen on intervaricose regions though
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Fig. 7 Heavily labeled varicosities, filled with vesicles and an occasional mitochondria associated with granular cells. Two nerve endings (N, and NJ are in contact with the granular cell (GC). x 26,500. Fig. 8 Group of axons including varicosities and intervaricose regions, adjacent to arteriolar smooth muscle cells 6). Though the grain appears predominantly on the varicosities some of the intervaricose regions, containing microtubules, are also labeled. x 11.500.
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Fig. 9 Nerve ending (N) in contact with smooth muscle cell (S)of the arteriole. The silver grain overlay the region of t h e varicosity filled with small vesicles. Small dense-core vesicles (arrows). x 45,000. Fig. 10 Group of axons associated with a distal tubule (DT). Only the varicosities are labeled. The silver grains lie on t h e region of the axoplasm filled with small vesicles. A nerve ending (N) is in contact with the distal tubule separated from the tubular cell by a thin basement membrane. X 47,500.
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in much lesser numbers (fig. 8). Much of the interior of the varicosities was filled with small vesicles and therefore most of the grains appeared in portions of the axoplasm almost totally occupied by the vesicles. Grains were seen occasionally on intervaricose regions containing numerous microtubules but no vesicles. Grains were seen on the Schwann cell with a count of 14 grains per 100 p z . A small number of grains, 16 per 100 p 2 ,was observed on the smooth muscle cell, and an even smaller one, eight grains per 100 p z , on the juxtaglomerular granular cells (fig. 5). No pattern of distribution was established in either cell type though on occasion grains were observed on the granules. Autoradiographic grains were observed on cells without nerve contacts present in the section. The grains present on the interstitium were located mainly on the interstitial cells, and with a count of 13 per 100 p z (fig. 5). Very little collagen or elastica was observed in the periarteriolar spaces and no grains were seen on those structures. There was considerable variation in the number of silver grains overlying different varicosities. Rarely, an unlabeled varicosity was observed among a group of labeled ones. No differences, however, were observed in the population of vesicles between the varicosities with overlying silver grains and the rare ones without them. In both instances, absence of small dense-cored vesicles was occasionally found. In addition, it was noted that an unlabeled varicosity, surrounded by labeled ones, might be without overlying grains in one section and be heavily labeled in another. DISCUSSION
The uptake of tritiated norepinephrine in vivo by the sympathetic axons around the intestinal arterioles has been studied by electron microscopy autoradiography by Devine and Simpson ('68). Though our results are in general agreement with theirs, we have recorded much higher counts of label on the renal glomerular arteriolar nerves than they obtained on the sympathetic fibers innervating the intestinal arterioles. This may well be due to our use of higher doses of tritiated norepinephrine and of the monoamine oxidase inhibitor pargyline. This drug was administered to prevent the degradation of norepinephrine and the formation of labeled metabolites. Our results show that most of the label appeared on neurons and very little on the effec-
tor cell (smooth muscle or granular cells). No concentration of grains was observed near the cell membrane of the smooth muscle cells, or of granular cells, at the neuroeffector junction or at other points in the cell surface. The marked difference in grain distribution between axons and effector cells can be explained on the basis of differences between neuronal and extraneuronal uptake of norepinephrine. First, neuronal uptake is followed by vesicular uptake and storage of the neurotransmitter while extraneuronal uptake is followed by metabolic degradation (Iversen, '73; Gillespie, '73). Second, neuronal uptake can occur a t concentrations of norepinephrine 1,000-fold lower than that required for extraneuronal uptake. The respective Michaelis and 2.5 X constants for uptake are 2.7 X M (Lightman and Iversen, '69). In addition, binding of norepinephrine to a-adrenergic receptors has been shown to be rapidly reversible (U'Prichard and Snyder, '77). Under our experimental conditions tritiated norepinephrine was injected 30 minutes before sacrifice and removal of the tissues. Hertting et al. ('61) have shown that this was sufficient time for circulating catecholamines to decline to near zero, thus, allowing metabolism of norepinephrine taken up non-neuronally and dissociation of norepinephrine from adrenergic receptors. The resolution of electron microscopy autoradiography does not permit the localization of the label on structures as small as the axoplasmic vesicles; but the regional distribution of the label on parts of the axon packed with vesicles is consistent with biochemical data indicating that norepinephrine is stored in the sympathetic nerves in a particulate form (see review by Smith, '72) and electron microscopic autoradiographic studies suggesting that the small dense-cored vesicles contain norepinephrine (see reviews by Taxi, '69; Jaim-Etcheverry and Ziher, '71). We confirmed the observations of Devine and Simpson ('68) of the presence of grains not only on varicosities in contact with the effector cell (nerve endings) but also in those apparently separated from them by considerable distance. If uptake of norepinephrine occurs a t the same sites as release, these observations suggest that the discharge of norepinephrine by the axon takes place a t other sites besides the neuroeffector junction. An unexpected finding in our study was the relative scarcity of small dense-cored vesicles.
TRITIATED NOREPINEPHRINE IN RENAL NERVES
The fixative used is considered to be a major factor in the preservation of the core and the loss of the core in the small dense-cored vesicles appears not to be accompanied by loss of radioactivity. Taxi ('68) reported that pineal gland fixed in osmium tetroxide showed a 14% loss of small dense-cored vesicles compared to the same tissue fixed in potassium permanganate. However, the amount of radioactive label observed in the osmium fixed pineal gland was almost double the amount observed when i t was fixed with potassium permanganate. In our studies we used perfusion with glutaraldehyde followed by immersion in osmium tetroxide. This method of fixation is considered optimal for kidney tissue (Maunsbach, '66). It is also thought to be effective in preserving the dense cores of the vesicles (Tranzer and Thoenen, '67; Hokfelt , '66). In our material, however, we have observed fewer small densecored vesicles and their cores appear smaller than those we have previously observed in the renal arteriolar nerves (Barajas, '64; Muller and Barajas, '72; Silverman and Barajas, '74). Our label counts, on the other hand, are more than twice those reported by Devine and Simpson ('68) for the intestinal arteriolar nerves which appeared to have the usual population of small dense-cored vesicles. Although the number of silver grains overlying the different varicosities varied, the vast majority were labeled and, therefore, can be considered monoaminergic in nature. This finding is consistent with the notion that the renin-containing juxtaglomerular cells and smooth muscle cells of glomerular arterioles and the neighboring tubules are innervated mainly by adrenergic nerves. This view is supported by the action of six-hydroxydopamine on the arteriolar nerves. After one injection of this drug, which selectively destroys the adrenergic nerve terminals, concomitant reduction of fluorescent and acetylcholinesterase-positive nerves was observed. Two injections produced the virtual disappearance of the nerves, as observed by both methods, from the region of the glomerular arterioles and surrounding tubules (Barajas and Wang, '75). These results indicate that most, perhaps all nerves in the region of the juxtaglomerular arterioles, previously labeled as cholinergic on the basis of their acetylcholinesterase positivity, were actually adrenergic nerves. Further support of this view is provided by the observation of small dense-cored vesicles in all axons followed through serial sections of the
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juxtaglomerular regions (Barajas and Muller, '73). The absence of autoradiographic grains on an occasional varicosity was harder to interpret. In some instances, an unlabeled varicosity was observed within a group of labeled ones in a given section. In subsequent sections of the same group, however,that same varicosity, also appeared heavily labeled. It seems, therefore, that a varicosity cannot be construed as not being monoaminergic on the basis of the absence of label alone. ACKNOWLEDGMENTS
The authors wish to thank Drs. M. Lubran, J. Lechago and R. Purdy for their many helpful suggestions regarding the manuscript, and Steve Takeshita for his technical assistance. We want to express our special thanks to Dr. Alfred Weinstock for his advice on the methods of electron microscopic autoradiography. The typing skills of Ms. Astara Mayeda are gratefully acknowledged. LITERATURE CITED Aghajanian, G. K., and F. E. Bloom 1966 Electron-microscopic autoradiography of rat hypothalamus after intraventricular H3-Norepinephrine. Science, 153: 308-310. Barajas, L. 1964 The innervation of the juxtaglomerular apparatus. An electron microscopic study of t h e innervation of the glomerular arterioles. Lab. Invest., 13: 916-929. Barajas, L., and J. Muller 1973 The innervation of the juxtaglomerular apparatus and surrounding tubules: A quantitative analysis by serial section electron microscopy. J. Ultrastruct. Res., 43: 107-132. Barajas, L., and P. Wang 1975 Demonstration of acetylcholinesterase in the adrenergic nerves of the renal glomerular arterioles. J. Ultrastruct. Res., 53: 244-253. 1978 Myelinated nerves of the rat kidney. A light and electron microscopic study. J. Ultrastruct. Res., 65: 135-147. Bogart, B. I., and C. L. De Lemos 1973 Adrenergic innervation of the rat submandibular and parotid glands. An electron microscopic autoradiographic study of the uptake of tritiated norepinephrine. Anat. Rec., 177: 219-224. Budd, G. C., and M. M. Salpeter 1969 The distribution of labeled norepinephrine within sympathetic nerve terminals studied with electron microscopic radioautography. J. Cell Biol., 41: 21-32. Caro, L. G., and R. P. Van Tubergen 1962 High resolution autoradiography. I. Methods. J. Cell Biol., 15: 173-188. Davis, J. O., and R. H. Freeman 1976 Mechanisms regulating renin release. Physiol. Rev., 56: 1-56. Devine, C. E., and F. 0. Simpson 1968 Localization of tritiated norepinephrine in vascular sympathetic axons of the rat intestine and mesentery by electron microscope radioautography. J. Cell Biol., 38: 184-192. Forssmann, W. G., and S. Ito 1977 Hepatocyte innervation in primates. J. Cell Biol., 74: 299-313. Gershon, M. D., M. Hagopian and E. A. Nunez 1974 An electron microscope autoradiographic study of the neuronal and extraneuronal localization of labeled amine in the heart of the bat after administration of tritiated norepinephrine. J. Cell Biol., 62: 610-624.
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Gillespie, J. S. 1973 Uptake of noradrenaline by smooth muscle. Br. Med. Bull., 29: 136-141. Hertting, G., J. Axelrod and L. G. Whitby 1961 Effect of drugs on the uptake and metabolism of H”-norepinephrine. J. Pharmacol. Exp. Ther., 134: 146-153. Hokfelt. T. 1966 Electron microscopic observations on nerve terminals in the intrinsic muscles of the albino rat iris. Acta Physiol. Scand., 67: 255-256. Iversen, L. L. 1973 Catecholamine uptake processes. Br. Med. Bull., 29: 130-135. Jaim-Etcheverry, G., and L. M. Zieher 1971 Ultrastructural aspects of neurotransmitter storage in adrenergic nerves. Adv. Cytopharmacol., I : 343-361. Lenn, N. J . 1967 Localization of uptake of tritiated norepinephrine by rat brain in uiuo and in uitro using electron microscopic autoradiography. Am. J. Anat., 120: 377-390. Lightman, S. L., and L. L. Iversen 1969 The role of uptake, in the extraneuronal metabolism of catecholamines in the isolated rat heart. Br. J. Pharmac., 37: 638-649. Maunsbach, A. G. 1966 The influence of the different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubules. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium tetroxide fixatives. J. Ultrastruct. Res., 15: 242-282. McKenna, 0. C., and E. T. Angelakos 1968 Adrenergic innervation of the canine kidney. Circ. Res., 22: 345-354. Mdller, J.. and L. Barajas 1972 Electron microscopic and histochemical evidence for a tubular innervation in the renal cortex of the monkey. J. Ultrastruct. Res.. 41: 533-549. Nilsson, 0. 1965 The adrenergic innervation of the kidney. Lab. Intest., 14: 1392-1395. Salpeter, M. M., and L. Bachmann 1964 Autoradiography with the electron microscope. A procedure for improving
resolution, sensitivity, and contrast. J. Cell Biol., 22: 469-477. Salpeter, M. M., and F. A. McHenry 1973 Electron microscope autoradiography. In: Advanced techniques in biological electron microscopy. J. K. Koehler, ed. SpringerVerlag, New York, pp. 113-152. Silverman, A. J., and L. Barajas 1974 Effect of reserpine on the juxtaglomerular granular cells and renal nerves. Lab. Invest., 30: 723-731. Smith, A. D. 1972 Subcellular localization of noradrenaline in sympathetic neurons. Pharmacol. Rev., 24: 435-457. Sotelo, C. 1971 The fine structural localization of norepinephrine-’’Hin the substantia nigra and area postrema of the rat. An autoradiographic study. J. Ultrastruct. Res., 36: 824-841. Taxi, J. 1968 Sur la fixation e t la signification du contenu dense des vesicles des fibres adrenergiques etudikes au microscope electronique. Comptes rendus de 1’Academie Bulgare des Sciences, 21: 1229-1231. 1969 Morphological and cytochemical studies on the synapses in the autonomic nervous system. In: Mechanisms of Synaptic Transmission. K. Akert and P. G. Waser, eds. Elsevier, Amsterdam, pp. 5-20. Tranzer, J. P., and H. Thoenen 1967 Significance of “empty vesicles” in pstganglionic sympathetic nerve terminals. Experientia, 23: 123-124. U’Prichard, D. C., and S. H. Snyder 1977 Binding of W c a t echolamines to a-noradrenergic receptor sites in calf brain. J. Biol. Chem., 252: 6450-6463. Wagermark, J., U. Ungerstedt and A. Ljundqvist 1968 Sympathetic innervation of the juxtaglomerular cells of the kidney. Circ. Res., 22: 149-153. Wolfe, D. E., L. T. Potter, K. C. Richardson and J. Axelrod 1962 Localizing tritiated norepinephrine in sympathetic axons by electron microscopic autoradiography. Science, 138: 440-442.
