Z. Zelfforseh. 136, 501--510 (1973) © by Springer-Verlag 1973

The Fine Structure of the Pancreatic Nerves of the Domestic Fowl

Erik DaM

Department of Anatomy, Dental Faculty, University of Oslo, Blindern, Norway

Received August 21, 1972

Summary. The fine structure of the pancreatic nerves of the domestic fowl has been studied. Naked axon headings were found in membranous contact with endocrine as well as exocrine cells. From an anatomical point of view it seems reasonable to suggest that the endocrine glands might be subjected to some influence of the autonomic nervous system.

Key word8 : Pancreas - - Fowl - - Innervation - - Electron microscopy.

The inne rva t ion of the pancreas is derived from the celiac plexus main ly through the hepatic, superior mesenteric and splenic plexuses, and enters the gland along the blood vessels (Kunz, 1945).

The presence of unmye l ina ted axons in the islets of Langerhans has been demons t ra ted in u l t ras t ruc tura l studies of pancreas in different species (Fawcett , Long and Jones, 1969) and some invest igators have i l lustrated what they inter- pret to be funct ional nerve endings on the endocrine cells (Beneosme, 1959; Stahl, 1963; Esterhuizen, Spriggs, and Lever, 1968). I n electron mierographs axons are also seen pene t ra t ing the basement lamina to end in contact with the acinar cells (Bloom and Fawcett , 1968). I n a previous invest igat ion (Dahl, 1970) mem- branous contact has been demons t ra ted between the steroid-producing cells of the hen ovary and axon-terminals . The present s tudy was earned out in an a t t e mp t to demonst ra te the fine s t ructure of the pancreat ic nerves of the domestic fowl, and to determine ff membranous contact between hormone-producing cells and the au tonomic nervous system is more regularly encountered t h a n previously conceded.

Materials and Methods

Nine White Leghorn hens 18-24 months old with an average body weight of 1650 g were used for this study. Fixation was performed by intraaortic perfusion with dextran (Intradex, "Nyco"®, Oslo, Norway), followed by 1.7 % glutaraldehyde in either 0.1 M phosphate buffer, 1% Tyrode, or 0.1 M cacodylate buffer, all at pH 7.3. After perfusion for 10 min the pancreas was excised and cut into thin slices under the dissecting microscope. The tissues were then fixed for an additional 2 hr by immersion in the perfusion fixative. Subsequently, the speci- mens were rinsed for 10 rain in 0.15 M phosphate buffer at ptt 7.3 and postfixed in 1% osmium tetroxide at 4°C for 2 hr (Mi]lonig, 1961). After fixation the blocks were rapidly dehydrated in graded series of acetone solution and embedded in Vestopal W. Ultrathin sections were cut on an LKB Ultrotome III. The sections were treated with uranyl acetate for 30 min, followed by lead citrate (Reynolds, 1963) for 5 rain. From the same plastic blocks sections one micron thick were cut for light microscopy.

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Fig. 1. Survey light photomicrograph of an islet of Langerhans (IL) surrounded by aeinar ceils of the exoerine pancreas. Connective tissue cells (Ct) are seen at the periphery of the islet. Note the contact between islets eells and aeinar cells (arrows) without any intervening connec-

t ive tissue (eft. insert in Fig, 2). × 175

Pancreatic Nerves of the Domestic Fowl 503

Observations

Light Microscopy

In toluidine blue sections the islets of Langerhans are seen as richly vascu- larized small cell masses th roughout the exoerine port ion of the pancreas (Fig. 1). They are roughly circular in outline and dispersed almost in a r andom fashion. Each islet is surrounded by a layer of connective tissue cells and septa pass f rom this connective tissue into the islet. I n some areas at the periphery of the islets, no intervening connective tissue is observed between the exocrine and the endo- crine cells. I n these areas there is contact between the islets and the acinar tissue (Fig. 2). Nerves could no t be identified with certainty.

Electron Microscopy

Innervation o/ the Endocrine Cells. The endocrine cells within the islets of Langerhans appear somewhat elongated and crescentic in shape. The most strik- ing feature of these cells is the presence of a large number of closely packed granula embedded in a cytoplasmic matr ix of moderate densi ty (Figs. 2, 3). Asso- ciated with the capillaries small bundles of unmyel inated nerve fibers of different sizes are seen ensheathed by Schwann cell cytoplasm (Figs. 3, 4). Myelinated nerve fibers are not observed. The general distribution of the nerve fibers is perivas- cularly and intercellularly, and in some instances single axons are found invaginat- ed into the surface of the endocrine cells (Fig. 5). As long as the axons are in- vested by Schwann cell cytoplasm they are considered to be fibers. However, round or oval naked processes which are found in a recess of an endocrine cell are regarded as axon terminals (Fig. 5). Nerve processes with a bulbous and broad outline with accumulat ions of vesicles and mitoehondria are presumed to be ex- pansions, or axon beadings of the nerves (Figs. 4, 6, 7). Two types of axon bead- ings are seen (Figs. 4, 5). One type is characterized by containing vesicles with central, dense, osmiophilic granules of an average size of 500 ~ (Fig. 4) and a second type devoid of these granules (Fig. 5), but with vesicles.

Innervation o/ the Exocrine Cells. As in the islets of Langerhans the nerve fibers are located in the vicinity of the capillaries also in the exocrine par t of the pancreas (Figs. 2, 6). The nerves to the exoerine cells seem to be organized in the similar manner as to the endocrine cells. Unmyel ina ted nerve fibers containing vesicles with a dense central osmiophilic granule of an average size of 500 ~ are found, bu t the second type, devoid of granules is only seldom encountered. Naked processes are found in recesses of exocrine cells (Fig. 7) with membranous contact (Fig. 8) and regarded as axon terminals. - - Membranous contact between axon terminals and endothelial cells were never observed.

Fig. 2. Survey electron micrograph with acinar cells (A C) (upper part) and islet of Langerhans (115) (lower part) surrounding a capillary (Ca) deep in the pancreas. Arrows indicate nerve fibers adjacent to the capillary, and also between aeinar cells. D Lumen of an aeinus, N nu- cleus of acinar cells. Inset upper right corner illustrates the membranous contact between

endocrine and exocrine ceils (arrow) (efr. Fig. 1). × 4060

504 E. Dahl: Pancreatic Nerves of the Domestic Fowl

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Fig. 3. Electron micrograph at low magnification of a capillary (Ca) with a distinct basement membrane (arrows) surrounded by endocrine cells in an islet of Langerhans. Note the axons

(A) in the lower right corner. Ed Endothelial cells. × 16800

Fig. 4. Electron micrograph at high magnification of pericapillary axons (A) ensheathed by Sohwann cell cytoplasm (Sw). Note the granular vesicles with the dense cores (arrows) in the

large axons. The small axons contain only agranular vesicles. × 56000

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Fig. 5. This electron micrograph illustrates axons adjacent to a capillary (Ca) in an islet of Langerhans (IL). To the r ight is a naked axon terminal (A 1) in membranous apposition with an endocrine cell. The axons to the left are still surrounded by Schwann cell cytoplasm. Note the axon (A 2) which is relatively large, probably representing a beading witb vesicles bu t

devoid of granules. × 56000

Fig. 6. Electron micrograph at low magnification of a capillary (Ca) from the exocrine par t of the pancreas (AC). Axons (A) are seen adjacent to the capillary. Note the axon beading (Ab), with the dense granules which are also located in the ordinary par t of the fiber. × 11200

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Fig. 7. This electron micrograph at low magnification illustrates two axon terminals (A) found in recesses of acinar cells (A C). A possibly small beading (arrow) is seen close to the basement

membrane of the capillary (Ca). × 24000

Fig. 8. Electron micrograph at high magnification of the axon (A) seen to the r ight in Fig. 7. Note the membranous contact between the acinar cells and the naked axon (arrows), and also

the granular vesicles in the axon. x 120000

P~ncreatic Nerves of the Domestic Fowl 507

Discussion

The classical silver impregnation technique of light microscopy demonstrated numerous nerves in pancreas, thyroid and certain other ductless glands. Although many of these nerves were associated with the blood vessels, some were reported to ramify in the parcnchyma and to end upon the endocrine ceils. Silver stains were notoriously capricious, however, and the possibility of confusing reticular fibers and small axons was ever present (Fawcett et al., 1969).

Many authors have described beadings of autonomic nerves in their terminal distribution in a wide variety of methylene blue-stained preparations (e. g. Wollard, 1926; Millen, 1948). Electron microscopy has confirmed that the autonomic ter- minal nerves show successive narrowings and expansions where accumulations of mitochondria and microvesiclcs may be found (Thaemert, 1966). Adrenergic (Lever, Spriggs, and Graham, 1968) and eholinergic (Esterhuizen et al., 1968) beadings have been described, and it is accepted, that in adrenergic nerves, t ransmit ter release may occur at the expansions (Hertting and Axelrod, 1961; Lever et al., 1965, 1968; Spriggs et al., 1966). Terminal axon varicosities have been demonstrated in which both large (600-1000 A) and small (300-500 ~) ves- icles are located, and some of the smaller vesicles possess a dense central granule surrounded by a ring of less dense material (Ruskell, 1967). Chemical analysis of tissue fractions (Michaelson et al., 1964), and electron microscopic autoradio- graphy (Wolfe et al., 1962) have revealed tha t norepinephrine is stored in granular vesicles measuring about 500 ~ in diameter, demonstrable electron microscopi- cally in adrenergie nerves (I~ichardson, 1964; Bondareff, 1965). Such granular vesicles in adrenergic nerve fibers can be depleted by administration of metar- amino] (Bondareff and Gordon, 1966; Dahl, 1970), Ruskell (1967), in his s tudy of axon terminals of the lacrimal gland in monkeys, found that axon terminals populated with small granular vesicles (300-500 ~) are sympathetic, while large granular vesicles (650-1000 ~) are present in both sympathetic and parasympa- thetic terminals.

The close spatial relationship between nerve fibers and islet cells has been demonstrated by Stahl (1963), Legg (1967) and Esterhuizen et al. (1968). In the present s tudy unmyelinated axons were found lodged between islets cells or in recesses in their base, beneath the basal lamina and without any Schwann cell investment. The membranous contact between the axons and the endocrine cells, and the presence of different vesicles in the axons similar to tha t described by Bencosme (1959), and Legg (1967) indicate that the nerve fibers represent axon terminals of autonomic origin. I t seems therefore reasonable to suggest tha t this membranous contact constitutes a site of innervation of the endocrine cells.

Nerve terminals containing numerous sympathetic vesicles have also been found in intimate contact with the base of acinar cells in the bat (Bloom and Fawcett, 1968). In the present study bundles of unmyelinated nerve fibers were found in close proximity to the blood vessels and exoerine cells. The nerve fibers adjacent to the capillaries were found ensheathed by Sehwann cell cytoplasm. However, naked axons were found lodged in recesses of the exocrine cells, and in membranous contact in the same manner as for the endocrine cells, probably representing a site of innervation of the exoerine cells.

508 E. Dahh

In some of the axon terminals granules measuring about 500 ~ were regularly encountered. I t is highly probable that these nerve fibers are adrenergic as they contain the type of granular vesicles commonly associated with adrenergie nerves (Richardson, 1964; Thaemert, 1966). Cegrell et al. (1964) using fluorescence micro- scopy, reported the presence of a network of adrenergic nerves surrounding small lobu]es of islets cells, but they were uncertain whether the axons represented a vascular or true parenchymatous innervation. On the other hand, many of the nerve fibers contain agranular vesicles but it is not known whether these agranular vesicles contain either acetyleho]ine or an amine (Burnstock and Merfllees, 1964). However, it is possible that some of these nerves correspond to the cholinergic fibers of the islet of Langerhans, described by Libman and Suthcrland (1965) and Coupland (1958). Compared with previous investigations of the hen ovary (Dahl, 1970), one may therefore conclude that the exocrine as well as the endocrine tissue of the pancreas seem to be furnished with an anatomic innervation rich in autonomic fibers, and that their vat]cos]ties most likely represent axon terminals.

I t is generally assumed that regulation of secretion of both the exocrine and endocrine pancreas depend largely upon humoral factors (Bloom and Fawcett, 1968). However, stimulation of the vagi causes secretion of a small amount of pancreatic juice rich in enzymes. This secretion is blocked by atropin and de- nervation of the pancreas (e.g. Ganong, 1967). I t therefore seems reasonable to indicate that the terminals which have been demonstrated in membranous contact with the exocrine cells in the present study represent nerves which may stimulate the exocrine cells.

Regarding the influence on insulin release by vagal stimulation, the results of physiological experiments are somewhat contradictory (Fawcett et al., 1969). However, both the present study and previous investigations have demonstrated nerves to the endocrine pancreas. In a previous study is has also been demon- strated that hormone producing cells of the ovary may be subjected to nervous control (Dahl, 1970). These demonstrations of nerves to hormone producing cells raises anew questions as to the exact role of nerves in the normal physiology of the endocrine cells. So far, it has not been possible to reveal the function of the nerves to the endocrine cells. On the other hand, the present investigation sup- ports the view (Fawcett et al., 1969) that it seems reasonable to suggest that the nervous system generally must have some influence on the function of the endo- crine glands, directly or inderectly, perhaps by modulating the sensivity of the cells to humoral factors.

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Pancreatic Nerves of the Domestic Fowl 509

Burnstock, G., Merrillees, N. C. R.: Structural and experimental studies on autonomic nerve endings in smooth muscle. Proceedings of the 2nd International Pharmacological Meeting, Prague, 1963, vol. 6, p. 1-17, London: Pergamon Press and Czechoslovak Medical Press 1964.

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Wolfe, D. E., Potter, L. T., Richardson, K. C., Axelrod, J.: Localizing of tritiated norepine- phrine in sympathetic axons by electron microscopic autoradiography. Science 188, 440-442 (1962).

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Erik Dahl Department of Anatomy Dental Faculty University of Oslo P. O. Box 1052 Blindem, Norway