«The Anatomy of the Infra-red Sense Organ in the Facial Pit of Pit Vipers By THEODORE H. BULLOCK AND WADE FOX (From the Department of Zoology, ...»
The Anatomy of the Infra-red Sense Organ in the Facial Pit
of Pit Vipers
By THEODORE H. BULLOCK AND WADE FOX
(From the Department of Zoology, University of California, Los Angeles.
Dr. Fox's present address is Department of Anatomy,
Louisiana State University School of Medicine, New Orleans, La.)
With four plates (figs, i, 2, 3, and 5)
1. The histological composition of the sensory membrane in the facial pit of rattlesnakes (Crotalus spp.), the disposition of the nerve-trunks entering it, the fibre-size spectrum of the nerves, and the form of the sensory endings are described.
2. Between the two layers of extremely attenuated epidermis the principal constituent of the membrane is a single layer of specialized parenchyma cells with osmiophil, reticular cytoplasm. These are not regarded as sense cells but they react strongly and locally to degeneration of nerve-endings.
3. The axons enter through numerous trunks from three branches of the trigeminal nerve, from all sides of the membrane, providing a dense innervation. They lose their myelin, taper to about 1 JJ,, then expand into flattened palmate structures which bear many branched processes terminating freely over an average area of about I,SOOJU.3, overlapping only slightly with adjacent units but leaving virtually no area unsupplied.
This means there are from 5 00 to 1,5 00 axons ending per mm 2, an estimate which agrees with the nerve-counts. No other form of ending was found.
4. The mode of the fibre-size spectrum lies in the region 5-7/J, diameter.
5. A transmission spectrum of the fresh membrane shows broad absorption peaks at 3 and 6 /x and about 50% transmitted in other regions out to 16/n. The visible spectrum is at least 50% transmitted and probably much is lost by reflection. Strong absorption takes place at wavelengths shorter than 490 /A.
6. The anatomical adaptations of the sense organ are discussed, especially the concentration of warm receptor fibres, the thinness of the membrane, the extremely superficial position of the nerve-endings—all increasing sensitivity to caloric flux. The overhanging margins of the pit and the richness of supply are believed to permit directionality of reception.
7. It is suggested that the palmate form of the ending has a significance in permitting several independent local sub-threshold activity generators to coexist in the processes and in pooling their coincident, electrotonically spread potentials to influence the initiation of spikes which may take place at the junction of axon and palm.
I N a recent communication the properties of the sense organ in the remarkable facial pit of pit vipers have been analysed physiologically (Bullock and Diecke, 1956). It is shown that this structure is highly sensitive to intermediate and long infra-red radiation to or from the snake. The conclusions are reached that the organ is specialized for the detection of solid objects of slightly different surface temperature from background objects, that the object can be rather small, that there is a certain degree of directional sensitivity, and that the special features are not so much measured by a high temperature sensitivity as a caloric flux sensitivity. The receptors themselves are regarded as warm receptors, a type poorly known heretofore, functionally and anatomically. The three sizeable nerve-branches supplying it are found to be nearly pure populations of warm fibres.
These results, together with the unsatisfactory state of the descriptions in the literature, have prompted us to re-examine the facial pit organ histologically.
The facial pit is peculiar to and characteristic of the crotalid snakes and has hence aroused attention for a long time. Lynn (1931) gives an account of the early studies and lists seven theories of its function that have been proposed.
We may confine our notice here to the five workers who have undertaken microscopical investigation. Desmoulins (1824) was apparently the first to see the rich innervation and concluded that it was sensory, probably olfactory in function. Ley dig (1868) treated it, together with other unfamiliar structures in lower vertebrates, in a memoir on organs of a sixth sense. He provided a rather good description of its histology but described the nerve-fibres as originating in 'terminal ganglion cells' in the sensory membrane in spite of recognizing that they are trigeminal nerve-elements, presumably with cellbodies in the Gasserian ganglion. As we shall see, the strange form of the free nerve-endings does in fact resemble a nerve-cell with several branching processes and it is possible that Leydig saw some of them, only erring in thinking he saw a nucleus. In any case he came closer than later workers for the next 80 years.
West (1900) provided a more complete account and may also have seen some actual endings. But he also believed they were cells and identified as nerve-terminations in sections of embryos what must have been epidermal cells and in sections of adults what we call the parenchymatous cells of the membrane. It is remarkable that these workers made out as much as they did.
when we recall that they had only a few, poorly fixed, mostly spirit specimens of these snakes. Not until Lynn (1931) took up this object did a worker with Bullock and Fox—Infra-red Sense Organ in Pit Vipers 221 access to fresh material exploit this advantage. With respect to the microscopic structure of the membrane, however, little could be added and the same mistake was perpetuated concerning fifth cranial nerve-fibres with cellbodies in the periphery. This mistake was corrected by Noble (1934) and Noble and Schmidt (1937), who identified the cells of West and Lynn as epidermal cells preparing for shedding and with silver impregnation described free nerve-endings. As we now see, they still did not see the endings. Their impregnations, like many of ours, were incomplete. There is also some reason to doubt their identification of parenchyma as outer epidermis.
MATERIALS AND METHODSSeveral species of rattlesnake were used, Crotalus atrox, C. cerastes, C. ruber, C. horridus, C. adamanteus, but mostly C. viridis. No consistent differences between the species were noted in respect to the characters under study; it is probable, from incidental observations, that there are differences in the degree of pigmentation of the pit and especially the sensory membrane. This is likely to have little functional significance.
The membrane at the bottom of the facial pit was studied microscopically in its natural position by reflected and by transmitted light. It was removed and observed while fresh. Intravitam methylene blue staining was attempted but without significant success. Overfixing in 1% buffered osmium tetroxide (Palade, 1952) was found to be useful for visualizing the disposition of the nerve-branches in the membrane and following individual fibres to the point where myelin stops abruptly. Alcohol/formaldehyde/acetic, Carnoy, Bouin, and the fixatives required by special silver methods were used to reveal fibreendings in both whole mounts and sections. Sections were cut in low viscosity nitrocellulose or in paraffin or double embedded and stained with Masson's trichrome stain or silver-on-the-slide methods (Holmes, 1943; Romanes, 1950). A large number of whole mounts of the membrane were treated according to these as well as the silver procedures of Palmgren (1951), Weddell and Zander (1950), and Bodian (1936), and variations of these. A small number of these showed nerve-fibres in various degrees of completeness and a still smaller number showed the palmate expansions beyond the end of the myelin sheath, with their rich aborization of branching processes. We believe a reasonable argument can be made that these preparations show the endings virtually completely, but of course we have no assurance of this.
Snakes in which a nerve or two nerves had been cut were kept for various periods and then the pit membrane was removed and over-fixed in osmium tetroxide to show the pattern of degeneration from that nerve.
One very large specimen yielded a membrane so large as to permit mounting, fresh, in specially made adapters. The transmission spectrum between 2 and 16/j, wavelength was recorded with a Baird Associates automatic infrared spectrophotometer, and transmission through the visible into the near ultra-violet was recorded with a Beckman DU and a Beckman IR2.
In order to estimate the fibre-size spectrum in the nerves supplying the pit, 222 Bullock and Fox—Infra-red Sense Organ in Pit Vipers these were fixed in Flemming's fluid (Lillie, 1948), embedded in paraffin, cut, and mounted without staining.
RESULTS Histological composition of the sensory membrane A cutaway drawing of the pit showing the sensory membrane and its relation to the rest of the head is given in the physiological paper (Bullock and Diecke, 1956). Further details of the gross anatomy are provided by the earlier authors mentioned above. The portion of concern here is the thin, richly vascularized and innervated, slackly suspended, dry membrane which forms the floor of the pit. It separates the pit or outer chamber from an air-filled inner chamber which has communication with the outside through a special duct opening into the adnexa of the eye. This means that the membrane is in contact with air on both sides. Its thickness in recently-shed adult rattlesnakes is IOJU. or slightly less except for local thickenings, especially where nerve-bundles lie.
As other authors have shown, it is thicker in developmental stages and we believe it may be thicker in some of the other genera of the family. At 10^, it seems to have reached a kind of limit dictated by the size of the erythrocytes.
Added to this thickness there will normally be a few microns of the multilayered cornified epidermis preparing to be shed.
On both outer and inner surfaces there is a cornified epidermal layer (fig. 1).
This doubtless varies in thickness and number of layers with the stage in the moult cycle; in our sections it is from 0-5 to i-5/x, whereas in a figure given by Noble and Schmidt (1937) it is several times thicker. According to Lange (1931) the layer with which this is continuous in the ordinary epidermis should not be called a cuticle, for he believes it is a proper cellular layer and he applies the term Oberhdutchen. We can confirm that the shed skin of the pit consists of two cellular layers but there is also a third layer without cell outlines or nuclei which appears to be a cuticle. For all its thinness, the outer epidermis must have remarkable physical properties since there is virtually nothing else standing between the soft tissues and the dry air. It is not essentially different on the two sides of the membrane except that the inner layers are less cornified.
Under the cornified layer, since it is periodically shed, should be a germinative epidermal layer. This is easily seen in the embryo (Noble and Schmidt, 1937; Lynn, 1931), and in occasional thicker places in the adult membrane.
But in the typical adult structure it is so thin that it cannot be traced continuously in our sections. Much flattened- nuclei, 0-5 ^ thick, are encountered at long intervals applied to the underside of the superficial cuticular layer and these we believe represent the basal germinative cell-layer. This layer seems to be even more reduced on the inner surface than on the outer.
FIG. 1 (plate). Cross-sections of sensory membrane.
A, the junction of the membrane and the side wall of the facial pit; anterior chamber above, posterior chamber below; Masson stain. C. atrox.
B, reduced silver method; region of a bundle of myelinated fibres some distance from their endings; outer side of membrane above. C viridis. Note sections through fine unmyelinated endings (arrows).
"1OJL B Fie. i T. H. BULLOCK «»«/ WADK FOX Bullock and Fox—Infra-red Sense Organ in Pit Vipers 223 Under the epidermal layers on both surfaces is a distinct stratum staining green with the Masson trichrome in contrast to the layers above and below it.
This is generally 0-5 to i-$fi thick or even vanishingly thin on the outer side but 1 to 2-5 fj. or even much thicker in local regions on the inner side of the membrane. We believe these layers to represent connective tissue of the dermis or corium, contrary to Noble and Schmidt (1937), who identify everything from the outer surface through the next and deepest layer as outer epidermis and thus recognize but a single connective tissue-layer, just beneath the inner epidermis. To be sure, the outer layer of connective tissue is so thin in most of the membrane that our only reason for so identifying it is its staining affinity. But the inner has discernible collagenous connective tissue fibres.
Both can be followed into thicker regions where they become continuous with typical connective tissue. Fibres which appear to be collagenous connective tissue-fibres are yisible in some silver impregnations of whole mounts where they reveal a sparse array of thin, straight fibres running in all directions for long distances. Both outer and inner layers of connective tissue show nuclei though in the thin parts of the membrane these are far apart and very much flattened (fig. 1).
This brings us to the middle and thickest layer which may be called the parenchyma. This is a single-cell layer, generally between the limits of 4 and 12 jj. thick. The cells are quite specialized in having, unlike any others in the membrane, an abundant cytoplasm which is coarsely reticular, almost granular, and stains darkly. In Masson's stain it takes a dull red, distinct from the brighter red of the epidermis in the side walls of the pit. In osmium preparations and in some silver impregnations this cytoplasm takes up considerable colour. The cell-type is not easily identified with any in ordinary skin. It is here regarded as part of the dermis since it lies between connective tissuelayers and is cut up into lobules by the capillary bed which occupies the same level of the cross-section. The nuclei are rich in chromatin and often show two nucleoli, but are mainly distinguished from the epidermal nuclei by being commonly a little irregular in outline rather than smoothly oval as are those of the epidermis of the sensory membrane.