innervation and nerve terminations of the reptilian lung

INNERVATION AND NERVE TERMINATIONS O F THE REPTILIAN LUNG

ARTHUR C. JONES Anatomical Laboratory, University of Oregon Medical School, Portland

ELEVEN FIGURES

INTRODUCTION

Detailed anatomical knowledge of the nerve supply of most of the visceral organs is meager. This is especially true with reference to submammalian forms. It was with a view to filling some of the gaps in our knowledge of the innervation of one of these organs, namely, the lung, that the present investigation was undertaken. The scope of the present account will be confined to an anatomical description of the nerve trunks and nerve terminations in the lungs of reptiles as represented by the snake and the turtle.

MATERIAL AND METHODS

The common garter snake, Thamnophis, was used for the greater part of the work here reported. Several species of this genus are found in northwestern Oregon, the prevailing ones being T. vagrans (B. and G.) , T. leptocephala (B. and G.), and T. parietalis pickeringii (B. and G.). It was not attempted to determine accurately the species in each of the fifty or more specimens used, and it is likely that repre- sentatives of all three species were utilized. In addition, the turtle, Chrysemys marginata, was employed.

The methylene-blue method described by Larsell ( '21) was easily adapted to the reptilian material. The best results with snake material were obtained by injecting 0.1 per cent methylene blue made up either with Ringer's solution or with

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372 ARTHUR C. J O N E S

0.75 per cent sodium chloride. RIetliylene blue of 0.5 per cent gave the best results with the turtle lung. The material was fixed in 8 per cent cold ammonium molybdate, and after washing, dehydration, and treatment with xylol was either embedded in paraffin f o r sectioning or the lungs were slit open and placed on large slides in balsam as spread preparations. The paraffin material was cut at 50 to 1 0 0 ~ and mounted serially.

A number of preparations were also made by filling the lungs of recently killed animals with 0.25 per cent osmic acid and, after dissecting out the organs, immersing them in a con- siderable quantity of the same solution for thirty-six hours o r longer. They were then washed in water, dehydrated, and either mounted as spread preparations o r sectioned serially, as with the methylene-blue material.. The osmic-acid material was of great advantage in securing general pictures a i d in determining the distribution of myelinated fibers.

DESCRIPTION

Nerve trurzks amil gainglia

Both vagi give fibers to the single lung of Thamiiophis. Connections with the sympathetic trunk are somewhat doubt- ful, according to the experimental results of Carlson and Luckhardt ( '20). There is formed a rather coarse-meshed, simple plexus about the trachea, main bronchus, and pulmonary vessels, composed for the most part of large trunks which are made up chiefly of many myelinated fibers. These lie entirely outside the cartilaginous rings of the trachea and bronchus and correspond to the extrachondrial plexus of the mammalian lung. Groups of ganglion cells (fig. l), to which Schulze ('72) refers, are encountered in the loose areolar tissue about the trachea and bronchus. The connective tissue about these ganglia is thickened, forming capsules, with which the large nerve trunks are in such close connection as to form occasionally part of the wall. From these trunks myelinated nerve fibers penetrate into ganglia and terminate about the

NERVE TERMINATIONS O F REPTILIAX LUNG 373

nerve cells in simple basket-like networks, as shown in the figure. Unmyelinated nerve fibers, many of which are seen to originate from the ganglion cells, are found within the nerve trunks. Isolated nerve cells also are found near the main nerve trunks of the extrachondrial plexus along the bronchi and within the loose tissues just inside the hilum.

ABBREVIATIONS

a&., adventitia alv., alveolus cap., capsule m.bu., muscle bundle n.fi., nerve fiber n.ter., nerve termination

n.tr., nerve trunk po.ji., postganglionic fiber pre.fi., pregangiionic fiber r.b.c., red blood cell sm.in.c., smooth muscle cell

Fig. 1 Large, fusiform ganglion near main bronchus of snake, showing portions of pericellular, basket-like networks and the relation of nerve trunks to capsule. Tliamnopliis sp. hlethylene-blue stain. 100 p. Camera lucida. X 206.

These are of the same cell type and have coiinections similar to those in the larger ganglia.

Within the trachea between the cartilage plates and the muscle bundles lies a much finer plexus of nerves, many of which are myeliiiated, but 'the majority are devoid of a sheath. These fibers form a finer network than that outside the trachea, the meshes lying for the most part transversely to the long axis of the air tube; only a few of the larger

374 ARTHUB C. JONES

myelinated fibers parallel the lumen. This may be called the subchondrial plexus. It is derived from branches of the outer plexus which penetrate the wall. The fin& fibers are for the most part postganglionic. Many of them are seen to send out fine collaterals to the smooth-muscle layer beneath which the plexus lies.

As the single main bronchus approaches the lung sac, the cartilage plates become more irregular, anastomosing with each other, and forming unevenly shaped plates which become more sparse caudally and are lost entirely at or near the point where the air tube enters the atrium of the lung. The disappearance of the cartilage at this level leads to a fusion of the two formerly separated plexuses, and from this point distally their fibers proceed as a single network. I have not encountered ganglia beyond this point. The nerve strands and blood vessels take their course within the larger smooth- muscle trabeculae which form the boundaries of the so-called alveoli, or chambers, which line the cephalic one-third of the snake’s lung. As the trabeculae become thinner and fewer in the more caudal portions of the lung, the nerve fibers can often be seen crossing the open floors of the atria just beneath the epithelium. In those regions where the lung wall contains no muscle at all, only a few myelinated trunks can be dis- cerned. Here they usually parallel the larger blood vessels, although they are sometimes isolated. They ramify through the submucons connective tissue and send fine rami into the basement membrane.

The arrangement of nerve fibers about the trachea and bronchi of the turtle is almost identical with that in the snake. The trunks of the extrachondrial plexus are large and heavy, while those of the subchondrial plexus are fewer in number, small and fine, with a larger number of unmyelinated fibers. Ganglia (fig. 2) of a much greater size than those of the snake are connected with the nerve trunks which parallel the trachea and main-stem bronchi.

The main bronchi in the turtle lung subdivide at the hilurn, and cartilage plates of irregular shape are found well down

NERVE TERMINATIONS O F REPTILIAN LUNG 375

within the upper portion of the lung, as far as the infundibula which empty into the upper atria. The arrangement of the plates is quite loose in this region, so there is not a definite point of fusion of the extra- and subchondrial plexuses, but they gradually merge and are continued into the substance

Fig. 2 Ganglioii in liilum of tur t le lung, showing portion of nerve t runk in relation t o capsule. Chrpemys niaryinata. Methyleue-blue stain. 100 p.

Camera lucida. X 227.

of the lung as one plexus. Ganglia do not appear beyond the first branching of the main-stem bronchus.

The larger nerve trunks lie within the smooth-muscle bands. Frequently smaller branches depart from the main trabeculae and traverse the alveoli for long distances, passing over the intervening muscle bands beneath the basement membrane in

376 ARTHUR C . JONES

a direct course, oiily to become lost again within the substance of another muscle fasciculus.

Sensory terminations

A few sensory nerve endings have been seen in the epithelium of the trachea and main-stem bronchus. These are of a rather simple type, showing less arborization than those described in the mammalian lung (Larsell, '2 l ) , but they appear to liavc the same general structure. Three or four principal branches proceed from the medullated nerve fiber, and from these secondary and tertiary divisions cxtend into the epithe-

Fig. 3 Sensory nerve trrmiiiations in epithelium of a n alveolus in the ccplialic third of snake's lung. Thamiiopliis sp. Metliyleiie-blue stain. Spread preparation. Cninera 1ucid:L. x 280.

lium. The main nerve fiber and all the branches are studded with varicosities which vary in size and shape, and upon all the fine terminal endings are small, rounded knobs (fig. 3). I n the bronchial epithelium thesc terminal knobs lie at varying depths between the epithelial cells, some of the finer arborizations crossing others above and below.

A similar type of ending has been observed in both snake and turtle preparations lying within the respiratory epithelium of the alveoli. They resemble in both form and position the endings described by Wolff ('02) in the interalveolar septa of the frog's lung. Rather large, varicosed, medullatecl fibers follow the muscle bands and branch upon entering the

NERVE TERMINATIONS O F ItEPTILIAN LUNG 377

alveoli ; and f rom these primary rami secondary branches may be given off. At the ends of the latter lie the terminations, which a re composed of principal branches, from which secondary a i d tertiary arborizations extend, all ending in fine, rounded knobs (figs. 3 and 4). Fine rugosities are seen upon all the terminal fibers of the nerve endings, often more marked and larger at points of branching, thus appearing as nodal points. Cross-sections t.hrough these endings and

Fig. 4 Group of sensory nerve terminations in epitlielium of alveolar floor. Tlianinophis sp. Metliylene-blue stain. Spread prepra t ion . Camera lucida. x 170.

oblique views of the same make it quite certain that they lie within the respiratory epithelium. The manner of branching of the main nerve fiber gives rise to groups of sensory terminations. These a re scattered over the floor of an alveolus, most of them entirely isolated from the smooth-muscle fasciculi which form the walls, but others a re in close contact with the fasciculi.

Like endings hare been noted in the caudal portion of the snake’s lung, well beyond the limit of smooth muscle, though here they are much less frequently encountered and are of

378 ARTHUR C. JONES

simpler structure. Some myelinated fibers fail to branch to form a group, as is the usual condition, but terminate in single endings of the type described. The larger groups of sensory endings of this type are most numerous in the upper, alveolated portion of the snake’s lung.

The terminatioiis of this type in the turtle’s lung are so similar that separate description of them is unnecessary.

Fig. 5 A. Eiicapsulatcd sensory nerve endings situated in subepithelial eon- nectire tissue near a muscle bundle. Thamnophis sp. Metliyleiie-blue stain. Spread preparation. Caniera lucida. X 265. B. Terminal encapsulated ending of sensory type overlying a junction of smooth-muscle bauds. Thamnophis sp. Nethylene-blue stain. Spread preparation. Camera lucida. X 265.

A second type of sensory termination (fig. 5, a and b) is also encountered. These endings are found in the walls of the alveoli, but are distinctly seen to lie below the epithelium within the connective tissue. Usually they are situated at the ends of very short side branches of a large nerve fiber. Some of the large fibers, however, terminate in endings of this type (fig. 5, b). They are peculiar in the fact that they are surrounded by a capsule, quite distinctly seen even in

NEIiVF, TERBIINATlONS O P REPTILIAN L U N G 379

preparations in which no counterstain has been used. As the figures show, the capsule appears as a series of concentric lamellae about the rather complex ending. The latter is composed of two to four primary branchings from which finer secondary and tertiary fibers arise, all terminating in rounded knobs. The latter are usually quite short and often bent as though confined within the limits of the capsule. The larger of these encapsulated sensory nerve endings are rather complex, with nuinerous tertiary branchings, and such large endings are, as a rule, the terminations of myelinated fibers. Those which lie along the course of the fibers are smaller, exhibit less arborization, and ha\-e fewer tertiary rami. Thp medullary substance of the main nerve fiber terminates im- mediately proximal to the capsule.

The position of these endings is of interest, for they always are found near a point of junction of two or more smooth- muscle trabecnlae and where the nerve trunk leaves its usual course within a trabecula to traverse the floor of an alveolus. I have failed to find them in the epithelium of the main bronchus. This encapsulated ending is not found in large groups, as is the simpler type first described, though two are often found together.

Encapsulated sensory terminations in the turtle lung are larger and more complex than those seen in the snake’s lung. The secondary and tertiary branches are characteristically short, and the entire ending is quite rounded, giving the impression of a flattened disc. They less frequently lie a t the ends of short collaterals from the medullated trunks in the turtle, but usually are placed singly a t their terminations. 1 have never found these endings in groups in the turtle.

A third and entirely different type of ending is found within the smooth-muscle bundles themselves (fig. 6). This differs widely from those previously described. The nerve fibers leading to such endings are large and mednllated, their trunks studded with heavy varicosities. As they near their terminations they branch much after the manner of those which end in the alveolar epithelium. The nerve trunks

THE J O r R N A L OF COXP.\RATIVF, NEVROLOGY, VOL. 40, NO. 2

380 ARTHUR. C. JONES

always lie within the smooth-muscle bundles. Their branches follow the smaller subdivisions of the trabeculae, in which the endings are found lying among the smooth-muscle cells. Each secondary branch terminates in a single nerve ending. Careful focusing reveals a series of fine fibers which proceed usually from nodal varicosities of the primary branches of the ending. These fibers penetrate between the smooth- muscle cells or embrace the muscle bundle. Cross-sections show clearly that the fibers of these endings lie between the smooth-muscle cells. Their rounded terminations are not in direct relation to the cells as are the simple motor knobs. Some of the terminal twigs cross each other or may

Pig. 6 Rmootli-muscle nerve spindle erribracing a small muscle bundle. Tham- nophis sp. Metliylene-blue stain. Spread preparation. Camera lueida. X 280.

anastomose, and in larger endings of this type the primary and secondary branches cross and wind about the muscle band in an intricate manner. I n the proximal portion of the snake’s lung groups of these endings are quite frequently encountered, as many as eight or ten arising from the branchings of a single, large myelinated fiber (fig. 7 ) . As the smooth-muscle bands become fewer in proceeding caudad, these endings are less often seen. Also they are not so frequently grouped, but more usually lie singly as terminal endings. It is interesting to note that these endings also are almost invariably found near points of bifurcation of the smooth-muscle bands which line the alveoli.

The name of ‘smooth muscle nerve spindles’ has been ap- plied to similar endings by Larsell ( ’22), who described them


first in the mammalian lung. The structure in the snake, while simpler, is so nearly identical as to justify a similar interpretation and nomenclature. The position and structure of these spindles would indicate that they are stimulated by contraction and relaxation or stretching of the smooth-muscle bands in which they lie. The similarity to the neuromuscular spindles of skeletal muscle and the sensory spindles in tendon is quite striking.

Fig. 7 Aggregation of smooth-muscle nerve spindles in relation t o fascicnli of wide muscle hand in cephalic portion of snake’s lung. Thanmophis sp. 90 P.

Metliglene-blue stain. Camera lucida. X 280.

The muscle spindles are large in the turtle’s lung. They are usually embedded among the smooth-muscle cells of a trabecula, but may lie superficially, their rami surrounding the muscle bundle. Numerous anastomoses between the see- ondary and tertiary branches in this type of ending are the rule. The varicosities are heavy and often appear as plate- like structures rather than as small knobs. The latter are

382 ARTHUR C. J O N E S

numerous, however, and a11 terminals lie between the muscle cells.

Ploschko ( '97) describes iiitermuscular 'Endbaumchen ' in the clog's trachea which are somewhat similar to the muscle spindles, save that they lie between, rather than in, the muscle bands. Carpenter ( '24) has found tufted or arborescent endings in the longitudinal smooth-muscle coat of the dog's small intestine, to which he ascribes a sensory function. These lie free between the muscle bands and do not embrace the muscle strands as do those above described, but are made up of exceedingly delicate, heavily varicosed fibers, compactly ar- ranged. These fibers are more numerous and are set more closely together than are those of the smooth-muscle nerve spindles, hence the latter are probably distinct forms, although related. More recently Cole ( ' 2 5 ) has described intramuscular receptive endings within the muscle of the frog's cloaca, ~vhich are also apparently of the same general type.

Xotor terminations Motor endings of a simple type are found in the snake's

lung, in the musculature of trachea and bronchus, and also in the tuiiica media of the pulmonary arteries and veins.

Several writers have described motor endings in the smooth muscle of the trachea in the mammal, and Ploschko ('97) described small ganglion cells with fine processes which pass to the smooth-muscle bundles, where they divide and terminate in small rounded knobs upon the individual muscle cells. Larsell ('21) also has described these ganglion cells, which he found both singly and in groups. These cells are surrounded by networks of finely varicosed fibers which seem to originate from the vagus, splitting to form basket-like pericellular synapses abont the small ganglion cells. This arrangement provides the typical pre- and postganglionic mechanism which is found throughout the visceral efferent spst em.

The reptilian lung forms no exception to this rule. Gang- lion cells are found along the trachea and bronchus as already

NERVE TERniIINATIONS OF REPTILIAN LUNG 383

described, and within the cellular tissue of the hilum of the lung. They appear in small groups or singly in the snake's lung, the larger ganglia being surrounded by capsules of connective tissue. The nerve trunks lie in close relation to the capsules and sometimes form part of them. Bundles of fibers from the trunks enter the ganglia to terminate in relation to the cells of the latter (fig. 1). The cells give off axones which are sometimes distributed directly to the smooth-muscle bundles. Here their collateral branchings may be traced to fine, knob-like terminations upon the smooth-muscle cells (fig. 8). In other instances the axone fibers join the larger trunks and become lost, as it is impossible to trace individual fibers of so small a size. It is apparent, however, that similar small, unmyelinated fibers are given off from these nerve trunks within the lung parenchyma. Their branches can be seen everywhere in favorable preparations as numerous filaments lying parallel between the smaller smooth-muscle bundles of the trabeculae, these in turn giving off collateral twigs at right angles which end in the familiar knob-like motor endings described.

Motor fibers in the smooth muscle of the trachea and bronchi are much more numerous than are those in the alveolated portion of the lung itself, where they become propor- tionately less in number as the smooth-muscle fasciculi become fewer and farther apart.

Efferent visceral fibers are seen in all the turtle preparations as fine, non-medullated filaments dotted with minute enlargements, lying between the smooth-muscle cells, upon which they terminate. The source of these fibers in the mammal has been found by Larsell and Mason ('21) to be the vagus nerve, through which preganglionic fibers pass to the terminal ganglia. These lie on the main-stem bronchi or within the hilum of the lung. The ganglia of the turtle lung (fig. 2) are larger, and include a greater number of nerve cells than do those in the snake. The pericellnlar networks (fig. 9 ) about the cells are also somewhat more complex, but the essential arrangement of afferent and efferent nerve

384 ARTHUR C. JONES

fibers, nerve trunks, and capsule are identical in these two reptiles.

Innervation of the pulmonary vessels

Sections of the snake's lung stained with the methylene- blue technique are very favorable for observations on the innervation of the pulmonary blood vessels. The nerve supply is quite rich in these vessels and particularly

Fig. 8 Motor nerve terminations in smooth muscle of small trabecula of snake's lung. Thamnophis sp. Methylme-blue stain. Spread preparation. Camera lucida. X 280.

Cluster of ganglion cells near broiiclius of turtle lung, showing typical basket-like, pericellular networks about the sympathetic ganglion cells. Chrys- emys marginata. Meth j -he-b lue stain. 100 EL. Camera lucida. x 475.

Fig. 9

in the larger branches of the pulmonary artery a very thickly set fibrillar network is present. The nerve bundles lie in the adventitia, winding about the lumen in an irregular manner roughly parallel to the lumen and anastomosing with or crossing other bundles at frequent iiiter- vals. They give off secondary collateral fibers to the smooth muscle of the tunica media, ia much the same manner as do the motor fibers in the smooth muscle of the lung (fig. 10).


These secondary branches usually run in the same direction as the nerve trunks in the adventitia, and from them proceed ex- tremely fine, beaded fibers which parallel the circumference of the blood vessel. They pass between the muscle cells, and their terminal twigs bear motor knobs similar to those described in the lung itself.

The origin of these fibers to the blood vessels in the snake is problematical. I<arsner ( '11) demonstrated a fine network of nerve fibers with small end knobs in the tunica media of

Fig. 10 Distribution of unmyelinated nerve fibers in tunica incvlia of a small pulmonary artery. Tli~niiiopliis sp. Methylene-blue stain. 50 p. Camera lucida. x 280.

the pulmonary artery of the dog, but merely suggested an origin from the anterior and posterior pulmonary plexuses. Berkeley ('93) describes fibers to the pulmonary vessels in the mouse. Larsell ('21 and '22) describes them in the dog and rabbit, and believes that the periarterial plexus from which they are derived in these animals is formed by a segregation of the sympathetic fibers from the pulmonary plexuses. The experimental results of Carlson and Luckhardt ( '20) indicate that these vasomotor nerves are derived chiefly from the vagus in the reptile and amphibian.

386 ABTHUIt C. JONES

The networks of minute, unmyelinated fibers in the walls of the pulmonary vessels are exceedingly rich in the turtle. They appear to be more numerous than in the arteries of the snake’s lung. The arrangement, however, is almost identical, and does not necessitate a second detailed description.

Fine nerve fibers to the capillary walls (fig. 11) are present in several of my preparations of the snake’s lung, but not in the turtle, probably because of variations of technique. Such fibers have been repeatedly seen and described in mam-

Fig. 11 Ketwork of capillaries with accompanying plexus of fine nerve fibers, seen in wall of a n alveolus. Thamnophis sp. Metliylene-blue stain. Spread preparation. Camera lucidn. x 373.

mals, reptiles, and amphibians and require no further description, save to call attention to their presence in the pnlmonary capillaries, as well as in other parts of the body.

SUMMARY

1. Sensory terminations are present in the epithelium of the main bronchi and the alveoli of the lungs of both snake and turtle. Two structural types are present, the first being non- eneap~u1atc.d~ finely branched, with endings lying between the

NERVE TERMINATIONS OF REPTILIAN LUNG 387

epithelial cells. The second type is encapsulated and is located in the connective tissue just beneath the epithelium, usually near the wall of an alveolus or at a bifurcation of smooth-muscle bands.

2. A specialized intramuscular nerve ending is found in the lungs of both snake and turtle, the fibers of which lie between the smooth-muscle cells or twine about the muscle fasciculi. The fibers with which they are connected are myelinated and are derived from the trunks of the plexuses about the bronchi.

3. Nerve endings of motor type are present in the smooth- muscle trabeculae of the trachea, bronchi, and lung parenchyma of both forms investigated. 4. Intrapulmonary ganglia of varying size are located in

the hilum of the lungs, near the bronchi, in these reptiles. The ganglion cells are surrounded by basket-like, pericellular networks, which probably represent the terminations of preganglionic myelinated nerve fibers from the pulmonary plexuses. Postganglionic axones are distributed to the smooth musculature of the bronchi and lungs.

5. The pulmonary vessels are supplied by a rich plexus of very fine nerve fibers, which terminate in relation to the smooth-muscle cells of the tunica media.

The capillaries are seen to be supplied by fine networks of nerve fibers which parallel them o r wind spirally about the lumina.

LITERATURE CITED

BERKELEY, H. J. 1893 The intrinsic pulmonary nerve in mammals. Jour. Comp. Neur., vol. 3, pp. 107-111.

CARLSON, A. J., AND LUCKHARDT, A. B. Studies on the visceral sensory nervous system. 111. Lung automatism and lung reflexes in Reptilim. (Turtles : Chrysemps elrgans and Malacocleinmys lesueurii. Snake : Eutenia elegans). Amer. Jour. Physiol., vol. 54, pp. 261-306.

CARPENTER, F. W. 1924 Intramuscular nerve endings of sensory type in the small intestine, with a consideration of their probable function. Jour. Comp. Neur., vol. 37, pp. 439-454.

Intramuscular nerve endings of a receptive type in the cloaca of the frog. Preliminary note. Jour. Comp. Xeur., vol. 38, pp.

KARSNER, H.T. 1911 Nerve fihrillae in the pulmonary artery of the dog. Jour.

1920

COLE, E. C. 1925

3 69-3 74.

Exper. Med., vol. 14, pp. 322-325.

388 ARTHUR C. JONES

LARRELL, 0. 1921 Nerve terminations in tlic lung of the rabbit. Jour. Comp.

The ganglia, plexuses, and nerve terminations of the mam- Jour. Comp. Neur., vol. 35, pp.

Experimental degeneration of the vagus nerve and its relation to the nerve terminations in the lung of the rabbit. Jour. Comp. Neur., vol. 33, pp. 509-516.

PLOWHO, A. 1897 Nervenendigungen und den Ganglien der Respirations- organe. Anat. Anz., Bd. 13, S. 12-22.

SCHULZE, 1". E. 1872 I n Stricker's Manual of Histology. English transla- tion. New York.

WOLFF, MAS 1902 Ueber Elirlich 'sclie Mrtliylenbl~u-Farbung und uber Lage und Bau einiger peripherer Nervenendigungen. Arch. f u r Anat. u. Entwick., S. 155-183.

Neur., vol. 33, pp. 105-131.

malian lung and pleura pulnionalis. 1922

97-132. LARRELL, o., AND %,[ASON, &f. L. 1921

innervation and nerve terminations of the reptilian lung

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