studies on the avian gizzard: morphology and innervation of the smooth muscle

Z. Zellforsch. 96, 173--185 (1969)

Studies on the Avian Gizzard: Morphology and Innervation of the Smooth Muscle*

TERENCE BENNETT and J . L. S. COBB

Zoology Department, Melbourne University, Australia

Received January 14, 1969

Summary. Light and electron-microscope studies of the smooth muscle of the gizzard and its innervation in the chick and the pigeon have been made. The muscle is organised into extensive interlocking bundles, with frequent anastomoses between adjacent bundles. Nexuses are common between apposing smooth muscle cell membranes. The irmervation of the smooth muscle is comparable to that in other gastrointestinal circular muscle. These results are discussed in regard to the contractility and electrophysiology of the tissue.

Introduction

Many early studies described the ana tomy and extrinsic innerva t ion of the

av ian gizzard (BAYER, 1901; MANGOLD, 1906; NOLF, 1934a, b; and others). This

work has been summarised by FARNER (1960). There are, however, only few recent

accounts of the l ight microscopical a n a t o m y or u l t ras t ruc ture of the smooth

muscle of this organ (BRADLEY and GRAHAME, 1951; CALHOUN, 1954; CItOI, 1962;

PANNER and HoNm, 1968; KELLY and RICE, 1968).

The present s tudy deals with the morphology of the smooth muscle and its

innervat ion, and forms the basis for his tochemical and electrophysiological studies

of the gizzard of the chick and pigeon (BENNETT, 1969a, b).

Materials and Methods White leghorn chicks [Gallus domesticus (L.)] between 1 day and 12 weeks old, and adult

pigeons [Columba domestica (L.)] of unknown age were used in this study.

Light Microscopy Masson-Trichrome Stain. Small pieces of tissue taken from the lateral muscles and from

the outer circular and inner longitudinal layers of the intermediate muscles of the gizzard (see MA•GOLD, I906) were fixed for one week in a 10% formol-saline solution, neutralised with calcium carbonate. The tissue was then dehydrated and cleared and finally vacuum embedded in paraffin wax (m.p. 63 ~ C). Sections were cut 10 ~z thick and mounted on albu- minised slides. They were then dewaxed, hydrated and stained with a Masson-Trichrome solution (Paragon Ltd.) for 20 minutes. The sections were differentiated in 1% acetic acid prior to dehydration, clearing and mounting in canada balsam.

Champy-MaiUet Stain. The osmium tetroxide technique of CHAMPY (1913) as modified by MAILLET (1968) was used to stain the intramural nerve fibres. Just prior to use, one part of 2% osmium tetroxide solution (buffered with veronal acetate to pH 7.4) was mixed with 3 parts of zinc iodide solution (prepared by dissolving 3 g zinc iodide in 100 mls distilled

* We would like to thank Prof. G. BURNSTOCK for his constructive criticism of the manuscript.

** Supported by grants from Australian Research Grants Committee, the National Heart Foundation of Australia and J.L.S.C. was supported by a Queen Elizabeth Fellowship.

174 T. BENNETT and J. L. S. COBB:

water and filtering). Small pieces of fresh tissue were dropped into this mixture and left in the dark for periods between 5 and 24 hrs. The best results were obtained after 8--10 hrs staining. The tissue was then washed, dehydrated, cleared, and vacuum embedded in paraffin wax. Serial sections 10--20 ~z thick were mounted in paraffin oil and viewed through a microscope equipped with a heated stage. This last step gave a very much clearer picture than was seen after mounting in the usual media.

Nitric Acid Maceration. Small pieces of tissue from the various regions of the gizzard, fixed under isometric conditions in a formol-saline solution, were left in 10% nitric acid for periods up to 7 days in order to separate the cells. The single cells were examined using a phase-contrast microscope.

Electron Microscopy Animals were killed with ether and dissected. Small pieces of tissue from the gizzard were

removed and dropped into a 1% solution of osmium tetroxide buffered with veronal acetate to a pH of 7.3. The tissue was left in the fixative 15--30 minutes at 4 ~ C; it was then chopped into pieces about 1 mm 2 and fixed for another hour. After fixation the tissue was rinsed in 20 % acetone and dehydrated over a period of not more than 30 minutes. It was then left in a mixture of 50% Araldite in absolute acetone for 4 hours, and finally embedded in Araldite using a vacuum embedding chamber.

Small blocks of the embedded material were trimmed and pale gold sections were cut using a Huxley ultra-microtome. The sections were placed on uncoated 300 mesh copper grids and stained in a solution of lead citrate (prepared by adding 0.5 ml I0 N NaOH to 0.04 g lead citrate in l0 ml distilled water) for 2 minutes, followed by staining in a 2 % solution of uranyl acetate for 3 minutes. Sections were examined using a Hitachi HU-11 b electron microscope at 50 kV.

Results

Light Microscopy

The gizzard of graminivorous birds is different iated into lateral muscles, which

form the bulk of the organ, and which are separated f rom each other by the

smaller in te rmedia te muscles (MANGOLD, 1906; Pm{NKOeF, 1930). The present

s tudy showed tha t one of the most characteris t ic features of the muscula ture was

its extensive and orderly a r rangement into inter locking bundles, or sheets, se-

pa ra ted by connect ive tissue. The a r rangement of these bundles differed in the

lateral and in termedia te muscles.

The lateral muscles were character ized by their overall thickness and the t ight-

packing of the const i tuent muscle bundles. In the in termedia te muscles, which

were thinner, the muscle bundles were loosely packed into circularly orien-

t a ted columns separated by connect ive tissue. The size of muscle bundles was

much greater in the lateral than in the in te rmedia te muscles. In the central region

of the lateral muscles the bundles were f requent ly 50--150 ~ deep and 0 . 6 - - 4 . 0 m m

wide (Fig. 1). Towards the serosal surface, and in the basal region, the muscle

bundles were less extensive, measuring 40 ~ deep and 90 ~z wide, on average

(Fig. 2). I n longi tudinal section the muscle bundles were identif ied for distances some-

t imes as great as 2 cms. Along their length they f requent ly anastomosed, the

branches running to join adjacent bundles. The in te rmedia te muscles possessed an inner longi tudinal smooth muscle layer

which was absent from the lateral regions. In both the chick and the pigeon the lateral muscles were composed ent i rely of circularly or ienta ted smooth muscle cells. Contract ion bands were prominent th roughout the tissue.

Gizzard Smooth Muscle 175

Fig. 1. Transverse section through centre region of the lateral muscle of the pigeon gizzard. Axon bundles (ab) are cut in transverse section, whilst blood vessels (bv) running in the muscle bundles are cut in longitudinal section in places. The muscle bundles are separated by connective tissue (ct). The extent of one small muscle bundle (centre field) is seen. Champy- Maillet. 200 x . In Figs. 1 and 2 the material has been overstained to show connective tissue

between muscle bundles

Fig. 2. Transverse section through the lateral muscle of the pigeon gizzard towards the serosal surface showing small muscle bundles. Axon bundles are seen either accompanying

blood capillaries (arrow) or separate from them. Champy-Maillet. 400 x

The s m o o t h musc l e cells, m e a s u r e d f r o m t issue which h a d b e e n f ixed a n d m a c e r a t e d , were 3 0 0 - - 4 5 0 {z long a n d f r o m 4 8 ~ in d i a m e t e r , a n d h a d a t yp i ca l

fu s i fo rm appea rance . T h e m u s c u l a t u r e was s e p a r a t e d f r o m t h e u n d e r l y i n g se-

c r e t o r y r eg ion b y a t h i ck c o n n e c t i v e t i ssue l aye r - - t h e s t r a t u m c o m p a c t u m (OePEL, 1896).

176 T. BENNETT and J. L. S. CO~B:

Fig. 3. Longitudinal section through the lateral muscle of the chick gizzard. Muscle bundles and branching axon bundles are seen. Champy-Maillet. 90 x

Af ter Champy-Mai l le t s taining, the axon bundles running wi thin the muscle bundles were clear ly seen (Fig. 1). Most axon bundles were be tween 2 - - 8 ~ in diameter . Throughou t bo th the la te ra l and in t e rmed ia te muscles the dens i ty of innerva t ion was r e m a r k a b l y cons tan t when viewed in t ransverse section, being abou t 1 axon bundle per 670 ~t 2 of tissue. At high magnif ica t ions (Fig. 2) axon bundles were often seen to be accompanying capillaries, a l though nerves and blood vessels occurred separa te ly on occasion. The dens i ty of vascular isa t ion, judged f rom examina t ion of high magnif ica t ion micrographs , appea red to be much grea ter in the pigeon t han in the chick gizzard.

I n longi tud ina l sect ion (Fig. 3) the axon bundles ran paral le l to one ano ther and ex tended th rough the muscle bundles. The m a j o r i t y of axon bundles ran in the body of the muscle bundles ra the r t han in the connect ive t issue bands separ- a t ing them. There were f requent side branches joining ad j acen t axon bundles bo th in the same and separa te muscle bundles.


Fig. 4. Large axon bundles in a longitudinal section of the lateral muscle of the chick gizzard. Side branches occasionally give rise to single varicose fibres (arrow). Chamoy-Maillet. 350 •

Fig. 5. Electron micrograph of chick gizzard showing portions of three cells cut in transverse section. Dense areas (da) along the unit membrane alternate with regions in which caveolac intracellulares (ci) arc numerous. The cell cytoplasm contains mitochondria (mr) and irregularly distributed dense bodies (db). Myofilaments are present, but show no obvious orientation. A small plaque-like nexus (n) occurs between two adjacent smooth muscle cells.

32,000 •

A t h igh m a g n i f i c a t i o n s t h e large a x o n bund les were seen to be c o m p o s e d of n u m b e r s of s e p a r a t e b u t i n t e r w e a v i n g smal l e r n e r v e fascic les ; occas iona l ly single

var icose f ibres b r a n c h e d o u t f r o m the m a i n bund le a n d e x t e n d e d b e t w e e n t h e musc le cells (Fig. 4).


Fig. 6. Adjacent muscle cells exhibiting interlocking unit membranes and nexal fusion at two points (arrows). Caveolae intracellulares are particularly prominent in the region of the

nexuses. 34,000 •

Fig. 7. Small plaque-like nexus between two adjacent smooth muscle cells. 84,000 •

Fig. 8. Plaque-like nexus showing some interdigitation of the apposing smooth muscle cells. 66,00 •

Fig. 9. Nexus showing marked interdigitation of one cell into the other. The quintuple-layered nature of the structure is clearly seen. 84,000 •

Electron Microscopy The smooth muscle cells of both the chick and pigeon gizzard were 3 - -8 ~ in

diameter. I n non-contrac ted tissue, as judged by the smooth profiles of the muscle cells, the intercellular distance was usual ly between 500--700/i~ (Fig. 5).

The cells showed features now considered to be typical of smooth muscle (see review by DEWEY and BA]~R, 1968). The un i t membrane of the cells, where i twas


Fig. 10. Connective tissue band separating muscle cells in adjacent bundles. 8,000 •

resolvable, was 70--75 A in width. Along the cytoplasmic side of the unit membrane dark areas alternated with regions where caveolae intracellulares were numerous (Fig. 5). In some cases, such caveolae could be seen to be invaginated from the unit membrane. Dense bodies were irregularly distributed throughout the cytoplasm (Fig. 5). In the present study, it appeared that both thick and thin myofilaments were present in the smooth muscle cells. A feature of the smooth muscle cells was the size of the mitochondria. In vertebrate intestinal smooth muscle the mitochondria are normally not longer than 2 ~ or wider than 0.2--0.5 (DEWEY and BARR, 1968). In both the chick and the pigeon, the mitochondria were up to 1--2 ~ wide and frequently 6 ~ long. Apart from their size they ex- hibited no unique features.

Nexuses, i.e. regions of fusion between apposing smooth muscle cell membranes (DEWEY and BA~R, 1962) were frequently observed (Figs. 5--9). These nexuses appeared to be of three main types, namely, small planar contacts showing no interdigitation, small interdigitations, and large bulbous protrusions of one cell into another. Nexuses of all types appeared to occur more frequently at the ends of cells than at other points along their length. A fuller description of these struc- tures has been given elsewhere (COBB and BENNETT, 1969).

The connective tissue separating the muscle bundles (Fig. 10) was 10 tz or more wide, and frequently contained numerous fibroblasts. The amount of connective tissue between adjacent smooth muscle cells in the same bundle was small.

Nerve trunks passed from Auerbach's plexus into the muscle. They were often 50 ~ in diameter, and contained several hundred axons (Fig. 11). As they pene- trated into the muscle the nerve trunks branched, became progressively smaller, and contained fewer axons. However, the density of innervation did not decrease with increasing distance from Auerbach's plexus.

Fig. 11. Small portion of large axon bundle cut in semi-transverse section. The neurotubules in the individual axons are particularly marked. Collagen fibrils occur throughout the bundle.

6,000 •

Fig. 12

Fig. 13. Adjacent axon profiles one of which contains large granular vesicles (gv). The other contains typical agranular vesicles (agv). 45,000 x

Fig. 14. Small axon bundle coursing between smooth muscle cells. Two axon profiles (a) containing vesicles and free of Schwann cell sheath approach within 400 A of a smooth muscle

cell (mc). 27,000 •

Fig. 12. A blood capillary (c) accompanied by a small axon bundle (ab). Large granular and small agranular vesicles can be seen in some of the axon profiles. The nucleus (sn) of the Schwann cell surrounding the axon bundle can be seen. There is a large gap (about 2,000 A)

between the axon bundle and the nearest smooth muscle cell (mc). 24,000 x

182 T. BEN~CE~r and J. L. S. COBB:

Blood capillaries were frequently accompanied by axon bundles (Fig. 12), which were normally enclosed within a Schwann cell sheath. Even where axon profiles were surrounded by Schwann cells they often contained vesicles. At high magnifications it was possible to identify the vesicles as either agranular vesicles 300 450 A in diameter or large granular vesicles 700--1000/~ in diameter (Fig. 13). These would seem to correspond to the small agranular and large granular vesicles described in other autonomically innervated tissues (GRILLO, 1966; BURNSTOCK and ROBINSON, 1967).

Usually, axon bundles enclosed by Schwann cells were separated by 800 A or more from the nearest muscle cells. In some cases, however, the neuromuscular contact was closer, axons free of a Schwann cell sheath approaching to within 400 A of a muscle cell (Fig. 14). These axon profiles frequently contained large numbers of vesicles. In no instance was there seen to be any pre- or postsynaptic membrane specialisation at these presumed neuromuscular junctions.

Discussion

I t has been suggested tha t the effector in visceral smooth muscle is a muscle bundle, rather than a single cell (BvRNSTOCK and PROSSER, 1960; BENNETT and ROGERS, 1967; BENNETT and BURNSTOCK, 1968). The association between ef- fectors is thought to be through the branches joining adjacent muscle bundles, whilst the association between adjacent smooth muscle cells in an effector appears to be through nexuses (BENNETT and ROGERS, 1967; BENNETT and BURNSTOCK, 1968). The present study has shown tha t the arrangement of smooth muscle cells into muscle bundles delineated by connective tissue is more extensive in the avian gizzard than has been seen in other visceral smooth muscle tissues. Moreover, nexuses between adjacent smooth muscle cells appear to be more common in the gizzard than has been described in other tissues, although no quantitat ive studies with serial sections have yet been made (DEWEY and BARR, 1964). However, it seems tha t the anatomical basis of conducted electrical activity is more clearly defined in the gizzard than in other visceral smooth muscle. I t is possible that this organisation provides, in part, the basis for the massive contractility of this organ (KATO, 1914).

A more obvious correlation would be expected between contractility and the contractile protein of the smooth muscle of the gizzard, although the role of such protein in smooth muscle contraction is still not clear. There is disagreement as to whether or not both thick and thin myofilaments are present in the smooth muscle cells of the avian gizzard. PANNER and HONIG (1968) have recently claimed that myosin does not occur as longitudinally orientated thick filaments, whereas KELLY and RICE (1968) have identified such filaments in the smooth muscle cells of the chicken gizzard. The present study apparently supports the claim tha t both thick and thin filaments are present in the smooth muscle cells of the chicken gizzard (CHoI, 1962 ; KELLY and RICE, 1968), although it is feasible tha t the thick filaments seen represent aggregated aetin filaments. The presence of large mitochondria and prominent myofilaments in the muscle cells possibly indicates a more active contractile mechanism in this tissue compared to other visceral smooth muscle tissues.


BRADLEY and GRAttAME (1951) claimed that slow striated muscle fibres were present in the region of the keel in the chicken gizzard. CALHOUN (1954) could not confirm this observation, but pointed out tha t contraction banding of the smooth muscle of the gizzard might account for the observations of BRADLEY and GRAHAME (1951). In the present study, the whole musculature of the gizzard of the chick and the pigeon was found to be composed of smooth muscle cells, although contraction bands were frequently observed throughout the tissue, giving the appearance of coarse striations.

Unlike the taenia coli of the guinea pig (BENNETT and ROGERS, 1967) the density of innervation of the muscle did not decrease with increasing distance from Auerbach's plexus. This indicates that there may have been another source of innervation for the muscle cells. Such an innervation may have been provided by the ganglionic masses which lie embedded in the body of the lateral and intermediate muscles (IwANOW, 1930; BENNETT and COBB, 1969). Electron microscope studies of gut have usually shown the innervation of the circular muscle (TttAEMERT, 1963, 1966; ROGERS and BURNSTOCK, 1966; NAGASAWA and MITO, 1967) to be more extensive and intimate than that seen in the longitudinal muscle (RICHARDSON, 1958; YAMAMOTO, 1960; YAMAUCHI, 1964; TAXI, 1965; ROGERS and BUR~STOCK, 1966; THAEMERT, 1966; BENNETT and ROGERS, 1967; and others). The gizzard musculature derives from the primordial circular muscle layer and thus the extent of its innervation is not unusual.

There have been few correlated electrophysiological and electron-microscopical studies on vertebrate smooth muscle. BURNSTOCK and HOLMAN (1962) observed spontaneous miniature excitatory junction potentials (M.E.J.P.s) in smooth muscle cells of the guinea pig vas deferens and subsequently MERRILLEES, BURI~- STOCK and HOLMAN (1963) showed close associations between single axons and some smooth muscle cells in this tissue. BENI~ETT and ROGERS (1967) described the innervation of the guinea pig taenia coli using both electrophysiological and electron-microscopical techniques. They never observed axons closer than 1000 A to smooth muscle cells or recorded M.E.J.P.s comparable to those seen in the guinea pig vas deferens. EIectrophysiological studies (BENNETT, 1969b) have shown that spontaneous miniature excitatory junction potentials (M.E.J.P.s) can be recorded from some smooth muscle cells of the avian gizzard. In the present study of the gizzard of both the chick and the pigeon, naked axons were seen, on occasions, to approach within 400 A of smooth muscle cells. The axon profile at this point frequently contained large numbers of vesicles. The evidence thus suggests that M.E.J.P.s may be recorded from smooth muscle cells which are closely associated with naked axons.

In the gizzard of the pigeon and the chick, axons containing agranular, large granular or both types of vesicle have been seen. Histochemical and electrophysiological studies (BENNETT, 1969a, b) have shown that the smooth muscle cells may be innervated by excitatory cholinergic and non-adrenergic inhibitory fibres (BENNETT, BURNSTOCK and HOLMAN, 1966) and also affected by overflow from noradrenergic vasomotor fibres. However, it was not possible in the present study to correlate the types of axonal vesicles seen with the identity of the trans- mit ter released by these nerves.

13 Z. Zellforsch., Bd. 96


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TERENCE BENNETT Zoology Department , Universi ty of Melbourne Parkville, 3052, Victoria, Australia

13"

studies on the avian gizzard: morphology and innervation of the smooth muscle

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