Innervation of the male genital tract and kidney in the amphibia, Xenopus laevis daudin, Rana temporaria L., and Bufo bufo L

Cell Tiss. Res. 160, 453--484 (1975) �9 by Springer-Verlag 1975

Innervation of the Male Genital Tract and Kidney in the Amphibia, Xenopus laevis Daudin, Rana temporaria L.,

and Bufo b ufo L.*

K. U n s i e k e r , S. A x e l s s o n , Ch. 0 w m a n , a n d K.-13. S v e n s s o n

Depar tment of Anatomy, Universi ty of Kiel, Germany and Depar tment of Histology, Universi ty of Lund, Sweden

Received October 23, 1974

Summary. The innervat ion of the male genital t rac t and kidney in three anuran species was studied by the fluorescence histochemical method of Falck-Hillarp for the demonstrat ion of monoamines whose ident i ty was secured by thin-layer chromatography, and by electron microscopy including administrat ion of 5- or 6-hydroxydopamine (5- and 6-OHDA). The genital t rac t comprises testis, intra- and extratest icular and intrarenal seminal efferent ducts, Bidder 's canal, renal dorsal transverse ducts, and ureter. In addi t ion--depending on the species s tudied--renal corpuscles and the various portions of uriniferous tubules may be involved in sperm transport .

1. Adrenaline is the main t ransmi t te r in nerves supplying the male genital t rac t and kidney. Only in Xenopus is i t possible to demonstrate the presence of noradrenaline, which was confirmed in the chromatographic analysis. No obvious changes are observed with regard to the distribution, amount , and fluorescence intensi ty of adrenergic fibers and their susceptibility towards 5- and 6-OHDA when comparing animals killed in late au tumn and winter, or in late spring, respectively. Non-adrenergic nerve fibers have not been observed.

2. The adrenergic innervat ion in the testis is only scarce and confined to blood vessels. Neuro-endocrine contacts on Leydig cells are not established. The gonadal ducts and the specific (i.e. non-vascular) intratest icular smooth muscle cells in Xenopus are not innervated.

3. Apar t from the uriniferous tubules (see below), only the ureter receives an adrenergic innervat ion which, however, is scarce even around the t ime of spermiation. Bundles of non- terminal and terminal axons are seen running contiguous to the superficial bundles of smooth muscle or smooth muscle-like cells. Neuromuscular relationships comprise synapses at distances of 2000-5000 •, bu t no close contacts. In the seminal vesicle of Rana the same mode of apposition of adrenergic terminals to muscle cells is observed. In addition, a direct innerva- t ion of the epithelium is seen in a few instances.

4. In the kidney the renal arteries, afferent arterioles, and the main branches of the portal veins are supplied by a dense plexus of adrenergic nerves. Small groups of intensely fluorescent cells are found in the walls of the renal portal veins and veins proper. The density of the arteriolar plexus is more pronounced in Rana and Bu/o t han in Xenopus. In Rana and Bu/o the arteriolar innervat ion comprises terminals at " o r d i n a r y " smooth musculature with membrane-to-membrane appositions, as well as contacts a t a distance of 800 to 4000 A on juxtaglomerular epitheloid cells. In Xenopus the lat ter have not been identified with certainty. Adrenergic nerves, running dorsal to the row of glomeruli and ventral to the muscular branches of the portal vein, may reflect a tubular iImervation because, in t ha t region, muscular vessels do not exist. Terminals par t ly or completely devoid of the Schwann

Send o/]print requests to: Professor Christer Owman, Histologiska Inst i tut ionen, Biskops- ga tan 5, S-22362 Lund, Sweden. Dozent Dr. Klaus Unsicker, Anatomisches Ins t i tu t der Uni- versit/~t, Neue Universit/it , Eingang F 1, D-2300 Kiel, Federal Republic of Germany.

* This work was supported by grants from Deutsche Forschungsgemeinschaft (Un 34/2) and the Swedish Medical Research Council. The skilful and invaluable technical assistance of Mrs. K. Jacob, Mrs. R. Sprang, and Mrs. K. FogelstrSm is gratefully acknowledged.

454 K. Unsicker et al.

cell sheath approach the tubular basal lamina with a distance down to 200 A. An innervation related to a particular portion of the nephron is not observed; however, the connecting tubules and dorsal transverse ducts are also supplied by adrenergic nerve terminals.

Key words: Amphibia - - Male reproductive tract - - Kidney - - Adrenergic innervation - - Fluorescence histochemistry - - Thin-layer chromatography - - Electron microscopy.

Introduction

The arrangement and significance of nervous tissue in the testis, seminal efferent pathways, and kidney of mammals has long been the subject of much morphological and physiological research. A variety of electron microscopic and histochemieal techniques have enhanced our knowledge about the relationship between nerve fibers and various effector cells, as well as the nature of the nerve transmitters in these organs. The adrenergic nerves present in the testis of mammals seem to be predominantly, if not exclusively, of vasomotor nature (cf. Baumgarten and Holstein, 1967, 1971; Baumgar tene ta l . , 1968; Dyan, 1970; Norberg et al., 1967), and on the ultrastructural level only few terminals have been reported to contact Leydig cells (Baumgarten and Holstein, 1968, man; Belt and Cavazos, 1968, boar). On the other hand, large adrenergic plexus and nerve terminals contacting Leydig cells have been described to occur in the testis of various birds (Baumgarten and Holstein, 1968; Haase, 1973) and reptiles (Unsicker, 1973), and Gresik {1973) has recently given fine-structural evidence for the presence of nerve terminals on Leydig cells in the testis of a teleost. The innervatiou of the seminal efferent pathways from several mammalian species has been extensively studied and a rich supply of mainly adrenergie and, to a minor extent, cholinergic nerves has been demonstrated (Baumgarten et al., 1968 ; Baum- garten, Holstein and Rosengren, 1971 ; Bell, 1967 ; E1 Badawi and Schenk, 1967 ; Merrillees, Burnstock, and Holman, 1963; Norberg, Risley, and Ungerstedt, 1967; Owman and SjSstrand, 1965; Richardson, 1962; Robinson, 1969; SjSstrand, 1965; Yamauchi and Burnstock, 1969; and others). In addition, a histochemical and fine-structural study exists on the intrinsic innervation of the reproductive tract of the male fowl (Tingari and Lake, 1972); however, no appropriate in- vestigation on the gonadal ducts of male amphibia seems to be available. For accounts of the anatomy, histology and fine structure of the male genital tract, see Gaupp (1904), yon MSllendorff (1930), and Unsicker (1975a, b). Observations on nerves in the amphibian kidney will also be presented in this paper because this organ, as a mesonephros, forms part of the gonadal ducts. In Xenopus, seminal efferent ducts from the testis enter the kidney at the ventral side and join the canal of Bidder, which gives rise to seminal ducts opening into Bowman's capsules. Thus, in this species uriniferous tubules, at least those in the cranial kidney, are involved in sperm transport. In Rana and Bu/o, which are also in- eluded in this study, the canal of Bidder is directly connected with the dorsal transverse ducts, which empty into the ureter. Thus, "ure ter" and "ductus deferens" may be used as synonyms. The innervation of the amphibian kidney has been only scarcely dealt with, in contrast to the series of investigations which have been undertaken to examine the histochemistry and fine structure of mammalian renal nerves (for references, see Discussion). Gaup (1904) has reviewed the early literature on the nerve supply to the kidney of amphibia, and

Ianervation of Amphibian Male Genital Tract 455

Spanner (1928), Hi r t (1930) and Waleewa (1960) have added some light-microscopic descriptions. I n their work on adrenergic nerves in the frog Falck, H~ggen-

dal, and Owman (1963) briefly men t ion a rich ne twork of fluorescent adrenergic fibers a round vessels and certain parts of the t ubu la r system in the frog kidney. The purpose of the present invest igat ion is to ob ta in informat ion concerning the dis t r ibut ion, monoamine histochemistry, and u l t ras t ruc ture of the intr insic nerve fibers and their relationships to the different port ions of the male reproductive t ract in three species of amphibia .

Materials and Methods More than 130 adult male frogs (Xenopus laevis and Rana temporaria) and toads (Bu/o

bu/o) were used for the present study. Xenopus (obtained from Drs. De Rover, Venhorst, Holland) weighing 50-70 g were kept in water at 20~ with 12 hr intervals of light and darkness for usually two weeks before they were killed. Rana and Bu/o (captured in South Germany) weighing 25-50 g were kept under open air conditions.

Fluorescence Histochemistry. Experiments were carried out over two relatively short periods of the year: September to November and April to early May. About half of the animals were pretreated with the monoamine oxidase (MAO) inhibitor, nialamide (Niamid, Pfizer; 400 mg/kg into the drosal lymph sac, 3-6 hrs before killing the rest serving as controls. Tissue from untreated animals was always processed together with that from experimental animals. Injections of a-methyl noradrenaline (Neocobefrin, Sterling; 30 mg/kg) and T.-DOPA (200 mg/kg) into some animals only produced a very high background fluorescence and these experiments are therefore not included in the study.

The animals were killed by pithing. Testes, kidneys (divided into upper and lower half or upper, middle, and lower third), and seminal vesicles (from Rana) were quickly removed and frozen in liquid propane-propylene cooled by liquid nitrogen. The specimens were freeze- dried and processed according to the method of Falck and Hillarp. The formaldehyde treatment was performed at 50 ~ C for 1 hr or at 80 ~ C for 3 hrs. These procedures are adequate for the histochemical localization of noradrenaline (NA) and adrenaline (A), respectively. The paraformaldehyde used had previously been equilibrated in air at 50% or 70% relative humidity. After vacuum embedding in paraffin, specimens were sectioned and the sections mounted in liquid paraffin. They were analyzed in a Zeiss fluorescence microscope with BG 3 or BG 12 (Schott) primary (lamp) filters and Zeiss 47-I-50 secondary (barrier) filters. Autofluorescent structures were identified by omitting the formaldehyde step. For further technical details, see Falck et al. (1962), Falck and Owman (1965), Fuxe et al. (1970); BjSrk- lund, Falck, and Owman (1972). Whole-mount preparations of the ureter and urinary bladder were dried in a desiccator at room temperature over phosphorous pentoxide and treated with formaldehyde or, in some instances, with glyoxylic acid (el. Bj6rklund et al., 1972; Axelsson et al., 1973).

Thin-layer Chromatography. Two series of analyses were carried out on animals (5 each of Xenopus, Rana, and Bu/o) killed during the two above-mentioned periods of the year. After pithing the testes, kidneys (including adrenal gland tissue), ureter, and urinary bladder were removed and homogenized in 5 ml of a cold mixture of acetone and 0.1 M hydrochloric acid (95:5, v/v) for chromatographic analysis (Axelsson, Bj6rklund and Seiler, 1973). After storage for 15 min in a deep-freezer the homogenates were centrifuged for 5 min. To the supernatant were added 1 ml of 1 M sodium carbonate and 10 mg of dansyl chloride (British Drug Houses) in acetone (1 ml). The dansylation took place at room temperature over-night. An equal volume of toluene was then added, the mixture centrifuged and the toluene-acetone phase sucked off. The water phase was washed with 1 ml of toluene and the volume of the combined extracts reduced to about 1 ml. After washing once with water, the volume was further reduced to 10 ~zl and applied to silica gel plates (Kieselgel H, Merck AG, Darmstadt on 20 • cm gIass plates) activated for 1 hr at t00 ~ C. The thin-layer plates were run bi- dimensionally in the following systems: First direction: (a) diisopropylether, (b) n-butyl- acetate-triethylamine 5:1 (twice), second direction: trietbylamine-diisopropylether 5:1 (twice).

30 CellTiss. Res. 160


Appropriate standards and controls were run along with the samples. After evaluation of the plates under UV-light, relevant spots were scraped off the plates, the gel extracted twice with acetone, and the extract applied to new silica gel plates for unidimensional rechromategraphy in chloroform-triethylamine 5 : 1. The fluorescence intensities of the amine spots were estimated visually under UV-light.

Light Microscopy. Testes and kidneys from four specimens of Xenopus were fixed with buffered glutaraldehyde or in Bouin's mixture, embedded in paraffin, and stained according to Bodian (silver impregnation with 1% protargol).

Electron Microscopy. 50 adult male frogs and toads were killed during the first half of December and the first days of May, and divided into three groups: Group I received three injections of 200mg/kg 5-hydroxydopamine 1 (5-OHDA; =267 mg/kg 5-hydroxydopamine- hydrobromide) at 12 hr intervals. Group l I received three injections of 100-150 mg/kg 6-OHDA (AB Biotec, Stockholm) at 24 hr intervals. The animals were sacrificed within 6 hrs after the last injection. Group I I I served as controls. The animals were anesthetized with MS 222 (Sandoz) and perfused via the heart with 3.5% ice-cold glutaraldehyde in phosphate buffer (pH 7.7) and in phosphate buffer for 15 and 10 min, respectively. Slices of testes, kidneys (divided into upper, middle, and lower third) and seminal vesicles (from Rana; 1 mm thick slices) were postfixed in unbnffered 2% aqueous OsOt for 2 hrs dehydrated with ethanol, and embedded in Araldite. For orientation, 0.5-2 tzm thick sections stained with azur II-methylen blue according to Richardson et al. (1962) were used. Ultrathin sections were mounted on naked grids and stained with uranyl acetate and lead citrate each for 5 rain. Zeiss EM 9A and Siemens 101 electron microscopes were used.

Results

Fluorescence Histochemistry

The observat ions descr ibed are confined to nervous e lements and cells wi th a specific fluorescence. F o r autof luorescent s t ructures , cf. Uns icker (1975b).

Testis. The tes t icu la r adrenergic inne rva t ion of the th ree amph ib i an species s tud ied is only scarce. Arter ies in the hilus region, however, possess considerable plexus of varicose nerves exhib i t ing a s t rong yel low-green fluorescence. These plexus follow the ma in a r te r ia l t runks deeper in to the tes t i s (Fig. 1 a). Only the largest in t ra tes t i cu la r veins exhibi t an adrenergic innerva t ion . Del icate varicose fibers running independen t ly from vessels can be observed ve ry except iona l ly (Fig. 1 b) among groups of Leydig cells, which are ident i f ied b y the i r f ine-granular brownish autofluorescence. Adrenergic f ibers do not exhib i t any def ini te re la t ion- ships to e i ther in t ra tes t i cu la r seminal efferent ducts , to the specific i n t e r tubu la r smooth muscle cells in Xenopus (cf. Unsicker , 1975a), or to the l amina p ropr i a of seminiferous tubules . I n Rana and Bu/o these ma in ly consists of e longated Leydig cells. No f luorescent f ibers occur wi th in the seminiferous tubules . The capsular t issue of the tes t is is not innerva ted . Groups of in tense ly yellow- f luorescent cells sca t te red in the capsular t issue a t the hilus region or in the meso- tes t is do no t issue f luorescent processes. Ex t r a t e s t i cu l a r seminal efferent ducts are not accompanied by adrenergic fibers. There are no obvious differences in the p a t t e r n of d i s t r ibu t ion or in the number of nerves be tween the different species s tudied or the same species inves t iga ted at different periods of the seasonal cycle.

Kidney. The bulk of f luorescent nerve fibers and cells is concen t ra ted in the vent ra l half of the k idney. Most renal nerves are found associa ted with blood

1 5-hydroxydopamine was kindly supplied by Prof. Dr. H. G. Baumgarten, Institute of Anatomy, University of Hamburg.

Innervation of Amphibian Male Genital Tract 457

Fig. 1. (a) Bu]o, testis. Strongly fluorescent adrenergic plexus (arrows) surrounding an artery in the hilus region, x 120. (b) Xenopus, testis. Adrenergie nerve fibers (arrows), without obvious relationship to vessels, running among Leydig cells which are recognized by their

fine granular brown-yellow autofluorescence. Seminiferous tubules (s). x 120

vessels. The adrenergic innervation of the larger arterial vessels is made up of an adventitial fiber plexus containing strongly fluorescent preterminal and terminal axons. This plexus is contiguous to the outer perimeter of the tunica media, but does not penetrate into the smooth muscle cell layer. The plexus continue from arteries to the afferent arterioles around which they form a narrow-meshed, two- dimensional fiber network. The amount of fibers and their pat tern of arrangement are not equal in the different species studied. In Xenopus the fibers are remarkably delicate, and often the two-dimensional network is replaced by a single bundle composed of a few or even only one varicose fiber (Fig. 2a), whereas in Rana and particularly in Bu/o true arteriolar plexus formations can be regarded as the rule (Fig. 2b). Arteriolar adrenergic nerves form a prominent brightly fluorescent network at the vascular glomerular pole (Fig. 2 c) surrounding both the afferent and efferent vessels. Frequently, individual fibers separate the vascular glomerular plexus and run along Bowman's capsule and the uriniferous tubules (Fig. 3a) towards the dorsal surface of the kidney, without joining the adrenergic nerves which emerge from the portal venous plexus (see below). Occasionally, it looks as if these fibers accompany the efferent vessels which

30*

Fig. 2 a - -c . Arterial (al) and arteriolar (at) adrenergie nerves in the kidney of Xenopus (a and c) and Bu/o (b). Renal arteries and arterioles in Bu[o possess a true plexus of adrenergic nerves, whereas in Xenopus the perivascular network is often replaced by a single bundle of smooth or varicosed fluorescent fibers. The arteriolar nerves terminate at the glomerular vascular pole (vp). Chromaffin celia in the ~cb'e~xal gland (c). Adrenergie nerves associated with uriniferous tubules (tn). Connecting tubules in Xenopus (ct) show autofluorescent flask-shaped cells. Proximal tubules (pt), distal tubules (dr). Adrenergic nerves closely adjoined to tubules (arrows).

• 120


Fig. 3. (a) Xenopus, kidney. Fluorescent varicose fibers running along Bowman capsules. Glomeruli (g) appear as large dark areas. Adrenal chromaffin cells (c) exhibit a strong fluorescence and show close relationships to adrenergic nerves. • 150. (b) Rana, kidney. Adrenergic nerve fibers (arrows) close to proximal and distal tubules (pt and dr, respectively). • 120

empty into the wide capillaries of the portal vein. However, these fibers usually seem to establish neurotubular relationship (Fig. 3b). A preferential localization of tubular nerves at certain parts of the nephron cannot be established. The nerves m a y also be observed in close approach to connecting tubules, which extend from the ventral ly located distal convoluted tubules to the dorsal transverse ducts. Adrenergic fibers are never seen to enter the glomerulus, though they are common in close proximi ty to the glomerular capsule, which often appears to be ensheathed by varicose axons (Fig. 3 a). Concerning species differences in tubular innervat ion it is notable tha t the supply is most prominent in Rana, followed by Xenopus and Bu]o, which exhibits the lowest amount of tubular nerves.

Renal portal veins and their main branches, which are embedded into the dorsal surface of the kidney, receive a rich adrenergic nerve supply in accordance with the thickness of the muscular wall. The main trunks, s i tuated at the lateral edge of the k idney close to the ureter, display a wide-meshed network of fluorescent fibers which a t tenuates both in caudo-cranial and latcro-medial directions (Fig. 4b and c). The vascular innervat ion stops abrupt ly at the point where the

Fig. 4. (a) Xenopus, kidney. Adrenal chromaffin cell (c) which seems to give rise to a fluorescent fiber. Glomeruli (g) proximal (pt) and connecting (ct) tubules, x 120. (b) Xenopus, kidney. Main branch of a portal vein (pv) supplied by an adrenergic plexus penetrat ing into the smooth musculature. Proximal tubules (pt). x 120. (c) Rana, kidney. A smaller branch of a portal vein (sv) encircled by one layer of adrenergic fibers which do not continue along the wide capillary (c). A few nerves (arrows) are also seen in the myeloid tissue (m). Dorsal

surface of the kidney (ds). x 120


capillaries leave the vessels (Fig. 4@ However, a considerable number of varicose fibers from the portal venous plexus can be traced towards the ventral surface of the kidney surrounding proximal convoluted tubules, dorsal transverse ducts and extending within the renal myeloid tissue (Fig. 4c).

A large branched sinusoid vessel at the ventral surface of the kidney serves as a confluence for the wide renal capillaries. I t is found in close proximity to the adrenal gland, but has no innervation of its own. Intra- and extrarenal veins, covered by a thin muscular sheath, are accompanied by very wide-meshed adrenergic plexus with a considerably less pronounced supply of terminal fibers than found in arterioles and branches of the portal veins. In the walls of the large intra- and extrarenal veins, cells emitting a bright yellow fluorescence are visible, some of which give rise to short undulating processes. The fluorophore in the adrenal ehromaffin cells appears intensively yellow after formaldehyde t reatment for three hours at 80 ~ C. When the freeze-dried preparations are treated for one hour at 50 ~ C it is possible to distinguish between green-fluorescent NA- containing and the more yellow-fluorescent A-containing adrenal cells which exist in almost equal proportions. Smoothly outlined and strongly fluorescent processes emanate from some adrenal cells and can be followed along the course of arteriolar afferent vessels or uriniferous tubules (Fig. 4a).

Intrarenal seminal efferent duets and the canal of Bidder receive no adrenergic innervation. After incubation for 3 hours at 80~ in formaldehyde vapor, all fibers emit a bright yellow-green fluorescence while after exposure for one hour at 50 ~ C, only a faint green fluorescence can be observed. However, in Xenopus it appears to be stronger than in the two other species studied.

Ureter. The adrenergic innervation of the ureter is very scarce. In the region depicted in Fig. 5a the nerves are particularly numerous and are seen to be confined to the outer perimeter of the fibromuseular wall. As a rule, only a few fluorescent fibers are seen throughout the ureter (Fig. 5b); the amount of nerves is higher in Xenopus than in Rana and Bu[o. No seasonal fluctuations in the number of nerve fibers or their fluorescence intensity can be observed. That part of the ureter wall which is contiguous with the portal vein receives a few aberrant fibers from the vascular plexus.

Whole-mounts from the ureter (Fig. 5 c) t reated with formaldehyde or glyoxylic acid vapor confirm the paucity of innervation observed in paraffin sections.

Seminal Vesicle. The seminal vesicle in Rana can be described as a dilated part of the caudal ureter and consists of several cavities, which empty into the ductus deferens. The interstitial tissue is formed by connective tissue and large branching smooth muscle cells (ef. Gaupp, 1904; Unsicker, 1975b). Smoothly outlined strands of intensely green-fluorescent nerves with only few varicosities are spread among smooth muscle cells and occasionally approach the basis of epithelial cells (Fig. 6), but never enter the epithelial duets and cavities. Typical varicose adrenergic plexus surround small arteries and arterioles (Fig. 6). No significant differences can be found with regard to the amount and distribution of adrenergie nerves in frogs killed in autumn or late spring, respectively.

Sections from Xenopus testes and kidney stained according to Bodian do not reveal the amount of nerves demonstrable by the Falek-Hillarp technique nor do they show nerve fibers at places where only few or no adrenergic nerves are visible (e.g. ureter).

Fig. 5a- -c . Xenopus, ureter, lower half, cross-sectioned (a and b) and as a stretch-preparation (c). (a) The richest adrenergic nerve supply to the ureter ever found in the present study. Fibers are contiguous to the outer perimeter of the fibromuscular wall, which contains a few autofluorescent cells (a/). Fluorescence on the epithelial surface is due to the autofluorescent contents of flask-shaped cells. Portal vein (pv), proximal tubules (pt). • 120. (b) Adrenergic nerves outside the wall of the ureter. Portal vein (pv), proximal tubules (pt). • 120. (c) Stretch-preparation confirms the paucity of innervation observed in the sectioned material.

Melanocytes (m). • 100


Fig. 6. Rana, seminal vesicle. Smoothly outlined strands (s/) and a few varicose fibers (v/) are spread in the interstitial areas and may approach the basis of the epithelium (arrows). Ducts (d) contain autofluorescent cells within the epithelium and occasionally autofluorescent

contents (c). Adrenergic vascular plexus (vp). • 120

Thin-layer Chromatography Dopamine is not present in measurable amounts in any of the uro-genital pre-

parations, except in the specimens comprising kidney and adrenal. In the latter specimens it is a constant finding in the different amphibians studied. NA can be demonstrated in the testicular tissue from all species and, in addition, in the ureter of Xenopus. A is present in considerable quantities in bladder and ureter from Bufo and Rana; it does not occur in detectable amounts in Xenopus. Thin-layer chromatography, however, does not reveal any A in the testes from any of the animals. The kidney-adrenal preparations from all three species of animals contain very high levels of A, and also NA is present. There is no clear evidence for seasonal variability in the amount or identity of the amine in any of the tissues from the various animals.

Electron Microscopy Testis. At the ultrastructure level it is extremely difficult to find nerves in the

testis, thus confirming the scarce innervation seen in the fluorescence microscope. Nevertheless, a few groups of preterminal and terminal axons, sharing individual infoldings of Schwann cells, may be detected facing the outer surface of the muscle cell layer of arteries. In many instances bundles of axons are embedded in groves provided by two adjacent smooth muscle cells. The diameter of axons varies from 0. t5-2 ~zm. Non-terminal portions display neurotubules and -filaments in varying proportions and occasional mitochondria and dense bodies. Terminals are frequently dilated, thus corresponding to varicose swellings of adrenergic fibers

Fig. 7. (a) Bu/o, testis. Bundle of axons (ba), some of which are devoid of a complete Schwann cell sheath , bordering on bo th smooth muscle cells (sm) of a vessel (v) and a Leydig cell (L). Due to t he 5 -OHDA t r e a t m e n t a lmost all of the vesicles possess an e lect ron-dense core. • 24000. (b) Rana, test is , 5-OHDA. Bundle of axons (ba) s i tua ted be tween a seminiferous tubule (st), and Leydig (L) and smooth muscle cells (sin), respectively. • 12000. (c) Rana, testis. Af ter admin is t ra t ion of 5 -OHDA only a few large granular vesicles re ta in a core of med ium electron dens i ty (arrows), whereas mos t of bo th small and large granular

vesicles are ent i re ly filled wi th a dense osmiophil ic material . •


observed in the fluorescence microscope. However, non-dilated terminal portions of axons also exist. In all instances, terminals lack a complete Schwann cell sheath and contain a typical population of granular and agranular vesicles. The latter are usually small and measure 350-500 A; they account for only a few per cent of the total vesicle population. Granular vesicles with diameters of 400-700 or 800-1400 A, are generally spheric and contain a homogeneously dense core separated from the vesicle membrane by a light halo of about 100 width. Granular vesicles filled with an exeentrically located, strongly electron- dense spot are few. Large vesicles with granules of medium electron density rarely occur. Varying amounts of the types of vesicles described may be observed already more proximally in axons which are surrounded by an uninterrupted layer of Schwann cell cytoplasm. Glycogen particles are only found in lower amounts in testicular nerves. Most terminals are covered by basal lamina material. Axon terminals approach muscle cells of arteries, arterioles, and larger veins, as well as contractile cells of smaller veins (of. Unsicker, 1975a) to distances of about 1200-3000 A; close contacts of naked axons with smooth muscle cells are not found. Very occasionally, vascular axon terminals border on both smooth muscle and Leydig cells (Fig. 7a) thus providing the possibility of a neuro-endocrine relationship. However, unequivocal relations between adrenergic terminals and endocrine cells are never seen. Sometimes, bundles of terminal and non- terminal axons occupy a position close to seminiferous tubules (Fig. 7b), but in all cases observed serial sectioning undoubtedly reveals their perivascular nature. Inter tubular non-vascular smooth muscle cells which are characteristic for Xeno- pus testes (cf. Unsieker, 1975a) as well as intratesticular seminal efferent ducts, including their periductal contractile cell layer, receive no nerve supply.

After t reatment with 5-OHDA, almost all the vesicles of nerve endings are filled with a dense osmiophilic material corroborating the view that the testicular innervation of the amphibian species investigated is exclusively adrenergic. However, some of the larger granular vesicles, which possess a core of medium density, remain unaffected by this t reatment {Fig. 7 c).

Changes in preterminal and terminal axons induced by 6-OHDA comprise various stages of degeneration, i.e. swelling of mitochondria and axons, aggre- gation of vesicles, and formation of myelin and dense bodies.

No apparent discrepandes can be recognized concerning the morphology of adrenergic nerve fibers and their susceptibility towards 5- and 6-OHDA in winter and spring, respectively.

Kidney: Vascular Innervation. The electron microscopic observations on the innervation of the renal vascular system generally confirm and extend the results obtained by fluorescence microscopy. Arteries and afferent arterioles receive the richest nerve supply in all species studied, followed by the portal vein and its main branches, and the renal veins proper. The main trunks of these vessels exhibit large nerve fascicles containing a few myelinated and several groups of unmyelinated axons, which run in the outer adventitia separated by masses of collagen and surrounded by one or two layers of highly vesiculated neurolemmal ceils. When these bundles approach the media they lose their myelinated axons and neurolemmal sheath. Individual axons which, when running in the adventitia contained only neurotubules and -filaments and a few mite-


Fig. 8. Rana, kidney, 5-OHDA. Relationship between adrenergic terminal (t) and smooth muscle cell (sm) which is typical of renal arteries. The cleft separating the muscle cell and the axonal membrane is about 2000 A wide and partly filled with collagen fibers and basal

lamina material. Bundle of axons (ba). •

chondria, now show increasing amounts of clear and dense-cored vesicles. F rom larger axon bundles, comprising up to 10 individual axons, smaller entities with no t more than about 5 axons separate and come in close proximity to the outer surface of medial smooth muscle cells, and become par t ly devoid of their Schwann cell sheath. There are characteristic differences among the various types of renal vessels with respect to the types of relationships established between axon terminals and vascular smooth muscle cells. I n arteries, portal veins, and veins proper (including their muscular branches) the min imum distances observed between muscle cell membrane and axolemma va ry from about 4000-1500 (Fig. 8). The cleft between nerve and muscle membranes is usually filled with connective tissue fibers and basal lamina material. Occasionally, membrane-to- membrane apposition occurs in arteries (Fig. 9a), but the cells contacted by the terminals are no true smooth muscle cells, even though they are rich in 60 A filaments. Terminal varicose and non-varicose axons at smooth muscle cells of afferent arterioles make the closest contacts (Fig. 9b), with about 150 A wide clefts lacking interposed basal laminae. In contrast to the prominent innervat ion


Fig. 9. Rana, kidney, 5-OHDA. Varicose axon (a) completely devoid of a Schwann cell sheath in close contact with a smooth muscle-like cell (lc) of an artery. • Insert: Similar relationship between a terminal (t) and a smooth muscle cell of an arteriole which, however,

in the region of contact lacks plasmalemmal vesicles and filaments. • 42 000

of afferent arterioles, the efferent vessels from the glomerul i (whose endothe l ium is only covered b y one or two layers of per icyte- l ike cells) have only a scarce nerve supply . Usual ly , i t is impossible to make a clear-cut separa t ion be tween nerves to afferent and efferent vessels and t ubu l a r nerves (see below) in t h a t region. The r e m a r k a b l y wide capi l lar ies in the amph ib i an k idney are p a r t l y sur rounded by cells shar ing character is t ic fea tures of bo th smooth muscle cells and f ibrocytes (cf. Unsicker , 1975b). Axon t e rmina l s come in more or less close contac t wi th these elements , forming t igh t au tonomic neuroeffector junct ions in some ins tances (Fig. 10). The following types of vesicles are found in axon


Fig. 10. Xenopus, kidney, non-treated animal. Two axon terminals (at) in close contact with an interstitial, pericyte-like (pc), cell of a capillary (c). Arteriole with smooth muscle (sin) and

endothelial cell (ec). x 10000

te rmina l s a t rena l vessels: 1. Small clear vesicles, 350-500 A in size. On the average, t hey are more numerous t han in tes t icu la r nerves (see above). They often conta in a t i ny e lectron-dense spot closely ad jacen t to the vesicle membrane . 2. Small , dense-cored vesicles of a p p r o x i m a t i v e l y the same size are less f requent . 3. Medium-sized (400-700 A) and large (900-1800 A) vesicles wi th usual ly opaque dense cores. These vesicles m a y somet imes be devoid of the i r content or re ta in i r regular ly ou t l ined r emnan t s of the cores. The propor t ions of the different t ypes of vesicles var ies f rom one por t ion of an axon to the o ther ; as a rule, large granular vesicles are more f requent in p re te rmina l port ions, whereas a b u n d a n t small clear and granu la r vesicles appea r to be the character is t ic fea ture of funct ional te rminals . Dispersed chromaffin cells, mos t ly of the A-conta in ing type , are loca ted in the walls of the largest renal vessels, of ten closely a t t a c h e d to the


media. Their processes appear like axons; they are ensheathed by Schwann cells. However, the termination of these fibers has not been possible to reveal.

Innervation of the Juxtaglomerular Apparatus. Like in the mammals, a juxtaglomerular apparatus composed of granular epitheloid smooth muscle cells in the media of afferent arterioles and an occasionally present macula densa, exists in amphibia (Bellocci et al., 1971 ; van Dongen and van der Heijden, 1971 ; Lammers and van Dongen, 1972; Sokabe and Ogawa, 1974; Unsicker, 1975 b). Non-terminal and terminal axons, which are usually enveloped by an incomplete Schwann cell sheath though they may also run as naked terminals (Fig. 11), come in close proximity of the epitheloid granular cells and distal tubular cells. Their relation to the arteriolar media as well as their vesicle population resemble the axons innervating non-granular smooth muscle cells. Occasionally, direct contacts between nerve terminals and epitheloid cells are observed, but usually the membrane-to-membrane distance is 800 to about 4000 A, and the basal lamina is not reduced (Fig. 12a). Axons containing small clear and granular vesicles as well as large granular vesicles also approach the distal tubular portion of the nephron adjacent to the afferent arteriole, the minimum distance being about 2000 A.

Tubular Innervation. A clear-cut distinction between tubular and vascular nerves is difficult or sometimes impossible to make. When sections are taken from the region dorsal to the row of glomeruli and ventral to the muscular branches of the portal vein it is highly probable that the nerves seen are of true tubular nature. Bundles of a x o n s ~ i s t i n c t from arteriolar plexus as revealed by fluorescence microscopy--running either alongside efferent vessels or Bowman's capsules, are often observed in between two renal corpuscles (Fig. 12b). These bundles obviously correspond to the above-mentioned fluorescent nerve envelope surrounding numerous glomeruli. Terminal axons devoid of Schwann cells are occasionally seen at a 8000 A distance from the capsular epithelium. Groups of axons pass on the urinary poles and run along the neck piece and the proximal tubule, either in pericapillary or intertubular spaces. They also cross over to join distal tubules and connecting tubules. The dorsal termination of these bundles could not be determined. The adrenergic portal venous plexus constitutes a second source for tubular nerves. Bundles of axons derived from these plexus preferentially accompany the transverse ducts and the dorsal portions of connecting and proximal convoluted tubules. In addition, a few groups of non-terminal and terminal axons are dispersed among myeloid cells. The following observations can be made with regard to the relation between terminal axons and tubular cells: Bundles of nerves, rarely comprising more than half a dozen individual axons infolded in Schwann cell cytoplasm, show terminals filled with vesicles of the same types as described above at a distance of 5000 to 2000 A away from the tubular basal lamina (Fig. 13). Terminals, partly or completely devoid of the Schwann cell sheath (Fig. 13), approach the tubular basal lamina to about 200 A, but never penetrate the lamina. At the same distance from the nerve terminal one may occasionally detect an interstitial cell or a pericyte. Varicosities of the same nerve bundle may be in close association with both tubular and interstitial cells. The tubular nerves are far less numerous than the nerve fibers at afferent arterioles. However, terminals related to tubular ceils in all portions of the nephron, including connecting tubules and transverse ducts, are a common feature


Fig. 11. Bu/o, kidney, 5-OHDA. Terminal axons (at) in p rox imi ty to bo th an epi theloid juxtaglomerular cell (]gc) in the wall of an afferent arteriole and a distal tubular cell (dr).

Endothe l ium (e). x 18000


Fig. 12. (a) Rana, kidney, 6-OHDA. Terminal axon (at), showing alterations due to the 6-OHDA treatment, in juxtaposition to a degranulated epitheloid smooth muscle cell (sm) in the media of an afferent arteriole (aa). The membrane-to-membrane distance is approxi- mately 2500 A. • 18000. (b) Xenopus, kidney, untreated animal. Terminal axon (at) containing small clear and one large dense-cored vesicle situated between two Bowman capsules (be). Such axons obviously account for the fluorescent nerve envelope of glomeruli (el. Fig. 3 a).

• 24000

of the three amphib ian species studied. As could be judged already from the fluorescence microscopic observations they are more easily ~ound in Rana and Xenopus t h a n in Bu/o. Axon terminals were no t seen a t in~rarenal seminal efferent ducts, Bidder 's canal, or nephrostomal ducts. Moreover, in the connect ing

31 Cell Tiss. P~es. 160

Fig. 13. Rana, kidney, untreated animal. Axons (a) part ly devoid of Schwann cell sheath and filled with small clear and large dense-cored vesicles in close proximity of proximal tubular cells (pt). • 42000. Inser t a: Axon with clear and one granular vesicle approaching the basal lamina of a connecting tubular cell (ct). Note filaments (h facing the basal plasmalemmal Non-terminal axons (nt). Sehwann cell (sc). • 36000. Inser t b: Naked terminal axon (at) labeled with 5-OHDA coursing between two distal tubules (dt). Basal lamina (bl). • 18000


tubules and transverse ducts there seems to be no innervation of the specialized cells, i.e. the flask-shaped and canaliculi cells (cf. Bargmann etal., 1955; Barg- mann and Welsch, 1972; Jonas and Spannhof, 1971; Spannhof and Jonas, 1969; Unsicker, 1975b).

After administration of 5- and 6-OHDA, the vascular and tubular nerves react in the same way. Unlabeled terminals are not visible. Three injections of 200 mg/kg 5-OHDA is satisfactory to induce highly electron-dense cores in almost all of the small vesicles, frequently causing the light halo inside the vesicle membrane to disappear. The vast majori ty of large cored vesicles becomes denser. Degenerative changes in axon terminals after application of 5-OHDA are exceptional. 6-OHDA, given three times at doses varying from 100 to 160 mg/kg, causes previously mentioned changes. A remarkable number of Schwann cells contain dense bodies as well as myelin figures. Frequently, damaged terminals seem to be engulfed by Schwann cells. While vascular and tubular nerve terminals undergo these alterations, cholinergie nerve endings at adrenal chromaffin cells and the cell bodies themselves appear unchanged. In addition, it should be mentioned that 6-OHDA causes an almost complete degranulation of juxtaglomerular epitheloid cells. The alterations occurring in the different portions of the nephron are described elsewhere {Unsicker, 1975b). No seasonal or species- dependent differences are observed in the ultrastructure of the renal nerves.

Ureter. In the electron microscope, nerves are not easy to detect in the ureter, not even in Xenopus where a relatively pronounced nerve supply is seen in the fluorescence microscope. Usually, bundles of non-terminal axons of various sizes run in the periphery of the ureter wall. Axonal distances of less than 2 ~zm from the epithelium are rather exceptional. Free terminals are sometimes observed, but direct contacts at smooth muscle cells do not seem to occur. Contacts en passant at distances of 2000-5000/~ is the rule (Fig. 14). The adrcnergic character of the terminals is established by their typical vesicle population and their susceptibility towards 5- and 6-OHDA. Non-adrenergie fibers do not occur. Ne overt increase in the amount of nerves and terminals is observed in late spring.

Seminal Vesicle. The density of innervation is more pronounced in the seminal vesicle than in the ureter wall. The terminals belong to the adrenergic type, their mode of apposition to smooth muscle cells being the same as in the ureter. Very occasionally a direct innervation of the epithelium may be seen (Fig. 15). Bundles of terminal axons penetrate the basal lamina and lie adjacent to the bases of epithelial cells. The distance between nerves and epithelial cell membrane corresponds to a normal intercellular cleft. The nerve endings contain small clear and dense-cored as well as large granular vesicles. After administration of 5- and 6-OHDA, all nerve terminals running in the interstitial spaces become labeled. Since interstitial spaces are remarkably widened and smooth muscle cells hypertrophied in vernal animals, the distances between individual nerves arc larger. Hence, nerves are more easily found in the seminal vesicle of winter than of late spring frogs.

I)iseussion

General remarks. In a variety of amphibian tissues adrenergic nerves have been shown to contain predominantly adrenaline (A), in contrast to those of

31"


Fig. 14. Rana, ureter, distal third, 6-OHDA. Dilated adrenergic varicosity (av) partly exposed from Schwann cell investment (so) in the ureter wall. The nearest smooth muscle cell (sin) is

about 3000 A away. Ureter epithelium (uc). x 18000


Fig. 15. Rana, seminal vesicle, untreated animal. Terminal axons (at) lying adjacent to the base of epithelial cells (ec), which are identified by their numerous plasmalemmal vesicles and the complete basal lamina cover (bl). Other epithelial cells are seen below the neuroepithelial

complex and in the upper corner. Smooth muscle cells (sin). X 18000

mammal s which are known to have noradrena l ine (NA) as the p r edominan t ea teeholamine (yon Euler , 1956). Quan t i t a t i ve de t e rmina t ions of ca teeholamines in hear t , brain, l iver, spleen, in tes t ine and b ladder of var ious anuran species have suppor t ed th is view (cf. Ho lzbaue r and Sharman, 1972 for ref.), bu t have also shown the somewhat surpr is ing resul t t h a t N A predomina tes in the per iphera l organs of cer ta in newts and sa lamanders (Brodie et al., 1964; Angelakos et al.,


1964). The cellular store of dopamine found in the kidney-adrenal tissue by chromatography is difficult to establish; it may be present in the adrenal chromaffin cells as a precursor in the NA synthesis.

The different yields of fluorescence in testicular and renal adrenergic nerves of Rana and Bu/o after formaldehyde t reatment for 1 hr at 50~ or 3 hrs at 80 ~ C, respectively, are in accordance with the quantitative results from other peripheral organs in anura quoted above and underline the general presence of A as an adrenergic transmitter in these species. This is supported by the present analysis using thin-layer chromatography. The results indicated the existence also of NA in testicular tissue. I t is possible tha t this amine is stored in some non-neuronal element, for example, in the groups of intensely fluorescent cells located in the capsular tissue at the hilus region and in the mesotestis. In this context it should be recalled that SjSstrand (1969) found small, intensely fluorescent NA-cells to constitute the only catecholamine-containing element in the vas deferens of the tortoise. The comparably high fluorescence intensity of adrenergic nerves in Xenopus after exposure for 1 hr at 50 ~ C, and the failure to observe larger amounts of fibers after prolonged t rea tment - -con t ra ry to Rana and Bu/o-- suggest that NA is present in higher concentrations and may tentat ively be proposed not only as a precursor of A, but also as a nervous t ransmit ter in this species. Accordingly, it was not possible to demonstrate A by thin-layer chromatography in tissues (except kidney-adrenal) from Xenopus, whereas significant amounts of this amine occurred in the uro-genital t ract of Rana and Bu/o. Only a limited number of studied has been carried out on the distribution and pharmacology of catecholamines in Xenopus. Burgers et al. (1953) have shown, for example, that Xenopus differs from other amphibia in the response of melanophores towards A. The fluorescence microscopic appearance of the adrenergie nerves and the chromatographic analyses did not give any indications for a seasonal variation in the t ransmit ter levels. However, in plasma, Donoso and Segura (1965) have found a lower concentration of A in the summer, and Segura et al. (1967) have described a lower brain A concentration during hibernation of Bu/o arenarum. On the other hand, according to Azuma et al. (1965) environmental temperature does not alter bullfrog tissue catecholamine levels. I t is possible that any involvement of adrenergic nerves in the seasonal changes of reproductive activity is not necessarily revealed by histochemical or quantitative determina- tions of such a fairly crude parameter as the level or neuronal transmitter.

The presence of small granular vesicles in axons has been used as a criterion for the identification of adrenergic nerves in this study, analogous to the situation in mammals where identical vesicles have been reported to occur in a variety of sympathetically innervated tissues (cf. Fillenz, 1971; Geffen and Livett, 1971; ttSkfelt, 1971). The varying number of small clear vesicles, which are often erroneously interpreted as cholinergic, may be due to a washout effect during the fixation procedure. Furthermore, the present study has shown that 5- and 6-OHDA are useful markers for amphibian as well as for mammalian adrenergic terminals. The chemical sympathectomizing effect of 6-OHDA on toad viscera has been described by Gillard and Read (1971) in a fluorescence histochemical study. The ultrastructural features of degeneration induced by 6-OHDA in amphibia are essentially the same as those seen in the peripheral sympathetic


nervous system of mammals (Furness et al., 1970; HSkfelt, Jonsson and Sachs, 1972; Tranzer et al., 1969; Tranzer and Thoenen, 1967, 1968; and others), birds (Bennet et al., 1970), and reptiles (Unsicker, 1973). The high doses and the prolonged t rea tment applied in this s tudy account for the severe destructions comprising swelling of the axoplasm, formation of bodies with a high electron density and digestion of the debris by Schwann cells. After administration of 5-OHDA, empty vesicles of amphibian adrenergic terminals become filled with a dense osmiophilic material, whereas the cores in the granular vesicles increase in density, particularly in the large ones with medium electron-density. These findings are in good agreement with the observations made in mammals {Chiba, 1973 ; Ehinger, Falck and Sporrong, 1970; Richards and Tranzer, 1969; Tranzer and Thoenen, 1967). Thus, our results give evidence for a similar morphological and, as far as 5- and 6-OHDA are concerned, pharmacological feature of A-containing nerves in amphibia and NA-containing nerves in higher vertebrates.

Testis. Fluorescence and electron microscopic observations indicate that the adrenergic innervation of the amphibian testis is scarce and exclusively confined to blood vessels. This agrees with the difficulty to demonstrate A by thin-layer chromatography in extracts from testicular tissue. Comparison of testes taken in autumn and late spring, respectively, shows no overt difference in the amount or distribution of adrenergic fibers. I t must be admitted that a conclusive answer with regard to seasonal changes in the innervation pat tern cannot be given. How- ever, though only two periods of the seasonal cycle were investigated, the probabili ty that such differences do exist can be considered to be rather low because testes of, for example, Rana taken in autumn and late spring already reflect clear differences in functional activity both with regard to spermatogenesis and Leydig cells (cf. Unsicker, 1975a).

In contrast to the situation in various avian and reptilian species, the amphibia used in this s tudy lack neuroendocrine contacts at Leydig cells. Also the characteristic smooth muscle cells of Xenopus testes are devoid of nerves. I t therefore has to be assumed that they respond to circulating catecholamines, which are known to play an important role in smooth muscle functions of lower vertebrates (cf. Burnstock, 1969). Scattered, intensely fluorescent cells resembling chromaffin cells have been observed in the hilar region of the testis. The chemical analysis indicates tha t they may contain NA. Non-adrenergic neurons, of the cholinergic or purinergic (Burnstock, 1972) types, have not been detected in the testis of Xenopus, Rana, or Bu]o.

Seminal Excretory Duct System. The seminal efferent ducts of amphibia comprise the intra- and extratesticular and the intrarenal seminal ducts, which empty into the canal of Bidder. Depending on species, nephrons may also be involved in the sperm transport. In all the species presently studied, the dorsal transverse ducts and the ureter belong to the seminal excretory duct system.

Intra- and extratesticular ducts part ly possess a thin sheath of possibly contractile cells (Unsicker, 1975a). Adrenergic terminals do not approach this layer. Whether the few adrenergic fibers found in relation to the transverse ducts play a role in sperm transport is doubtful. The scarce adrenergic innervation of the ureter is in good agreement with the observations of Disselhorst (1894) who has reported on only few nerve fibers in this organ. The sparse


innervation, even around the time of spermiation, markedly contrast with the situation seen in the seminal excretory duct system. Particularly in the vas deferens of mammals and birds, fluorescence microscopic studies have revealed a rich supply of adrenergic nerves (Baumgarten et al., 1968 ; Baumgarten, Holstein and Rosengren, 1971; Falck, Owman, and Sjhstrand, 1965; Ljunggren, 1969; Norberg, 1967; Norberg, Risley and Ungerstedt, 1967; Owman and Sjhstrand, 1965). This is in accordance with the large amounts of axons and axon terminals observed in the electron microscope (Baumgarten, Holstein, and Rosengren, 1971 ; H6kfelt, 1966; Merrillees, Burnstock, and Holman, 1963; Taxi, 1969; and others). Close neuromuscular contacts of the types described by Lane and Rhodin (1964), Merrillees, Burnstock, and Holman (1963), Richardson (1962), Yamauchi and Burnstock (1969) in the ductus deferens of guinea-pig, mouse, and rat are never seen in the amphibian ureter. The density of innervation in the mesonephric amphibian ducts is also considerably lower than in the ureter of mammals (Dixon and Gosling, 1971; Gosling and Dixon, 1972; Lange, 1974; Notley, 1969). The scarce adrenergic nerve supply in the amphibian ureter, as well as the occurrence of contractile cells both in intra- and extratesticular ducts and ureter (Unsicker, 1975b), resemble the situation in the upper portions of seminal excretory ducts in man. Baumgarten, Holstein and Rosengren (1971) have suggested tha t a high level of differentiation of smooth muscle and a dense direct adrenergic innervation may suppress intrinsic contractility, and that the reverse conditions may rather be related to spontaneous motility.

Administration of 5- and 6-OHDA induces a complete labeling of all terminals in the amphibian ureter and there is thus no evidence for a separate cholinergic nerve supply as shown for the guinea-pig vas deferens (Bell, 1967; Burnstock and Robinson, 1967; Robinson, 1969). Further, there is no evidence for the existence of short adrenergic neurons (Falck, Owman and Sjhstrand, 1965 ; Owman and Sj6strand, 1965; Sjhstrand, 1965): no peripheral adrenergic ganglion formations are found near or within the ureter, and the adrenergic terminals in the ureter do not show a lower in vivo sensitivity to 6-OHDA (cf. Malmfors and Sachs, 1968). In Rana temporaria there is a more pronounced adrenergic inner- ra t ion in the seminal vesicle than in the ureter, suggesting a nervous involvement in the control of sperm ejaculation. Whether the few terminals contacting epithelial cells play a role in secretory functions remains to be elucidated.

Kidney. The pat tern of innervation of the amphibian kidney shares many features characteristic for that of the mammalian kidney; thus, the renal arteries and afferent arterioles are supplied by a dense plexus of adrenergic nerves (Dole~el, 1970; McKenna and Angelakos, 1968; Munkacsi, 1969; Nilsson, 1965; Norvell, 1969; Ws Ungerstedt, and Ljungqvist, 1968). The adrenergic innervation of the amphibian renal veins proper is comparably scarce, but cells containing high monoamine levels are distributed along the veins and may contri- bute to the control of their muscular walls. The nerve fibers which innervate the renal vessels have been reported to be vasoconstrictor in the toad (Yamagishi and Azuma, 1963). The rich adrenergic innervation of the portal veins suggests an important role of these vessels in the regulation of renal blood flow. Vessels in the amphibian kidney lack a cholinergic nerve supply contrary to what has been described for the kidney of mammals (Dole~el, 1958; Gosling, 1969; McKenna


and Angelakos, 1968 ; Norvell 1969 ; Norvell, Weitsen and Sheppek, 1970; Weitsen and Norvell, 1969; Williams, 1961). This conclusion is drawn from the observation that all the terminals display characteristic label after administration of 5- or 6- OHDA. I t must be stressed tha t the occurrence of small " syaap t i c " , agranular vesicles in terminals on vessels and tubules repeatedly seen in the course of this s tudy cannot be taken as a criterion for cholinergic nerves. The complete lack of cholinergic fibers in amphibian renal vessels is notable in view of the finding that atropine blocks the excitatory response to stimulation of intramural nerves in isolated toad arteries, indicating tha t a small cholinergic excitatory component in the nervous control may be present (Burnstock, 1969, p. 298). The differences in the amount of arteriolar nerve fibers between Xenopus on one hand, and Rana and Bu/o on the other, may possibly be related to different environmental conditions: in aquatic life there is little need for elaborate adaptive mechanisms in contrast to terrestrial life with its rapidly changing conditions (Burnstock, 1969). The origin of neither the vascular nor the tubular nerve fibers in the amphibian kidney could be determined in the present study. I t is probable that the few fibers which could be traced from the adrenal gland into the kidney belong to chromaffin cells. The vast majori ty of renal nerves is likely to be derived from extrarenal sympathetic ganglia. The observation of pronounced adrenergic nerve supply to the juxtaglomerular granular epitheloid cells located in the media of afferent arterioles gives strong support to the view that the renin-secreting cells in amphibia are under influence of the sympathetic nervous system. Investigations on the innervation of the juxtaglomerular apparatus in various mammals has provided evidence for the involvement of adrenergic nerves in renin production and release (Barajas, 1964; Barajas and Miiller, 1973; Miiller and Barajas, 1972; Ganong, 1972; Nilsson, 1965; Silverman and Barajas, 1974; Ws Ungerstedt, and Ljungqvist, 1968). I t is notable that, so far, typical granulated epitheloid cells have not been found in Xenopus (Unsicker, 1975b). The presence of a dense adrenergic innervation of both granulated and non- granulated (i.e., "ordinary" smooth muscle cells) lying close together raises the question whether a single axon can activate both types of cells leading to a synergistic effect to increase renin secretion, as has been suggested by Barajas and Miiller (1973). The macula densa-like differentiation of the adjacent distal tubules occasionally observed in Bu]o (Unsicker, 1975b) and the occurrence of adrenergic varicosities in close juxtaposition to this structure, indicates a complex nature of the adrenergic mechanism involved in renin secretion.

The morphology of the renal tubular innervation has been extensively dealt with in mammals (Barajas and Miiller, 1973; Miiller and Barajas, 1972; Ohgushi et al., 1970; Zimmermann, 1972), and there is some physiological evidence that autonomic nerves promote sodium reabsorption (Gill, 1969). The topographical relation between adrenergic nerve terminals and various tubular portions suggests that a similar function operates in amphibia, too. The presence of a "contractile appara tus" in the bases of the proximal and distal tubule cells (Newstead, 1971; Pease, 1969; Ross and Reith, 1970; Rostgaard et al., 1972; Unsicker and Krisch, 1974 and of filamentous interstitial cells (Unsicker, 1975b) indicates that also a mechanical activity, influenced by adrenergic nerves, may be involved in the tubular function.


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Documents

Innervation of the male genital tract and kidney in the amphibia, Xenopus laevis daudin, Rana temporaria L., and Bufo bufo L