The Pattern of Olfactory Innervation by W

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J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from

THE PATTERN OF OLFACTORY INNERVATION BY W. E. LE GROS CLARK and R. T. TURNER WARWICK From the Department of Anatomy, University of Oxford (RECEIVED 31ST JULY, 1946)

IT is desirable that, from time to time, commonly Methods accepted statements regarding anatomical pathways Most of the observations recorded in this paper were and connexions in the peripheral and central nervous made on rabbit material. The fixation of the olfactory systems should be carefully reviewed in the light of mucosa presented considerable difficulty. The method modern technical methods of investigation, for it finally selected, because it gave the best results with must be admitted that not a few of these statements protargol and was also adequate for the other stains are based on old methods which are now recognized employed, was perfusion of 70 per cent. alcohol through to be too crude to permit of really accurate con- the aorta, after preliminary washing through with normal clusions. In recent years, indeed, a number of saline, as recommended by Bodian (1936). Another facts have been shown unexpected difficulty arose from the fact that a large apparently well-established number of laboratory rabbits suffer from a chronic by critical studies to be erroneous. For example, rhinitis which leads to gross pathological changes in the the so-called ventral nucleus of the lateral geniculate olfactory mucosa. Consequently, a considerable pro- Protected by copyright. body and the pulvinar are no longer accepted as portion of our material, experimental and otherwise, terminal stations of the optic tract, and the strie had to be discarded as useless. In the latter part of our acoustice have been shown to be unconnected with investigation, rabbits a few weeks old were used in the auditory system. Of all the systems in the brain order to avoid this difficulty, but experimental lesions which call for a careful revision, that related to in them were found to be more susceptible to infection. olfaction seems most urgently in need of treatment. In the histological study of the olfactory epithelium in of the rhinencephalon, as they are rabbits the septal mucosa was always used, since in these The complexities animals the greater part of the nasal septum is carti- expounded in most text-books, are largely based on laginous and thus does not require decalcification. How- the accounts of early investigators who had to rely ever, certain of our observations were confirmed on mice for their conclusions on the examination of Weigert in which the whole of the nasal cavity was sectioned for sections of normal material or of Marchi prepara- histological study after decalcification with 5 per cent. tions-methods which, as is now well recognized, nitric acid and staining with a modification of Bielschow- are both highly unreliable by themselves even for sky's method. From the rabbit material, paraffin fibre connexions of an apparently simple and direct sections were prepared and stained by Bodian's protargol kind. There is reason to think, indeed, that some method, hkmatoxylin and eosin, or methylene blue. http://jnnp.bmj.com/ components of the so-called rhinencephalon should The experimental lesions in the olfactory bulb were from this system. Thus, made by the exposure (under nembutal anasthesia and be excluded altogether with strict aseptic technique) of the olfactory bulb and there is now grave doubt whether the hippocampus the frontal pole of the cerebral hemisphere. Total or (together with its direct and indirect connexions, partial removal of the bulb was then effected by cutting such as the mamillary body and the anterior nucleus through the bulb or its peduncle with a fine-bladed of the thalamus) can in any way be said to be scalpel (care being taken not to injure the underlying primarily concerned with smell. dura mater which covers the cribriform plate of the In view of these uncertainties, it was decided to ethmoid), and gently scooping out the detached portion. on September 24, 2021 by guest. investigate anew the olfactory connexions of the Hxmorrhage was usually very slight, and was easily con- brain, and in this paper we are concerned with the trolled by pressure with a pledget of cotton wool. In pattern of innervation of the olfactory mucosa, and most cases, the animals made a rapid and uneventful with the projection of the latter on the olfactory recovery from the operation. bulb. Our main intention was to approach these problems by studying the retrograde degeneration The Olfactory Receptors in the Rabbit in the olfactory epithelium which follows partial or The olfactory sensory cells of mammals have been total destruction of the olfactory bulb. In the described by a number of histologists (e.g. Exner, course of the investigation, material was also ob- 1872; von Brunn, 1875, 1892; Cajal, 1889; Grassi tained which permitted certain observations to be and Castronuovo, 1889; van Gehuchten, 1890; made on the olfactory receptors, on the cytological Kallius, 1905; Read, 1908; van der Stricht, 1909; details of their retrograde degeneration, and on the Alcock, 1910; Kolmer, 1927), and it is now well intranasal course of the olfactory nerves. recognized that each consists of a small cell-body I* 101 102 W. E. LE GROS CLARK AND R. T. TURNER WARWICK J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from with a round nucleus embedded in a minimal amount may even appear to be a little thickened (Figs. 1 of cytoplasm, an exceedingly fine proximal process and 3). Moreover, the hairs radiate out in a single (which is continued as an olfactory nerve fibre), and series from the peripheral part of the terminal a coarser peripheral process. The latter terminates swelling, their attached ends forming a single ring; at the surface in a small thickening which gives rise they do not project as spinous processes from all to very fine olfactory hairs. In the course of our parts of the swelling as sometimes portrayed in experimental studies, we have had occasion to diagrams. This latter appearance is probably examine many protargol preparations of the normal based on the examination of abnormal sensory cells. olfactory mucosa of the rabbit, in which the peri- In some of our sections, the appearance and dis- pheral processes of the sensory cells are displayed position of the olfactory hairs show rather marked with unusual clarity. Their appearance in these variations. Thus there may be a single stout sections suggests that current descriptions of the process projecting straight up into the overlying olfactory receptors of mammals may require some layer of mucus, sometimes terminating in a sharp revision, and it is for this reason that the following point or in a minute vesicle (Fig. 5). In other cases, brief description is given. one or more of the hairs may end in darkly staining For convenience of terminology, the main part of end-bulbs of irregular size (Fig. 4), or the hairs may the peripheral process is here called the "olfactory be clumped together (Fig. 2). Sometimes, again, rod", and the thickening of its free end is called the the hairs appear to be prolonged into long and "terminal swelling ". The olfactory rods vary in exceedingly fine filaments which eventually pass length from 20,uk to 90,. In the greater part of beyond the range of visibility as they are traced in their length, their diameter is slightly uneven and the film of mucus covering the epithelium. In this measures 06-1 5,u. They stain deeply with pro- connexion it may be noted that, in 1901, Jagodowski targol' and can be seen to have a neurofibrillar described such filaments (or " flagella " as he termed structure. Throughout most of their length the them) in the olfactory cells of the pike, and sug- neurofibrillae are disposed peripherally so that the gested that the so-called olfactory hairs are really rods (particularly if viewed in cross-section) have a only their thickened proximal ends. But it is diffi- " tubular " appearance. Proximally the neuro- cult to exclude the possibility (at least in our own Protected by copyright. fibrilbe' fan out into the meagre cytoplasm of the preparations) that the "filaments " are nothing cell body. Distally they become more compactly more than mucinous threads of adventitious origin arranged as they pass into the terminal swelling, which have attached themselves to the tips of the and here the olfactory rod is constricted to a thick- olfactory hairs. This, indeed, seems to be the most ness of 0 3-0-6,t. probable explanation of the appearance sometimes Some doubt has been expressed (for example, by seen in protargol sections. The other variations von Brunn, 1892) whether the terminal swellings of mentioned above may be (at least in part) abnormal the olfactory rods are normal structures or whether phenomena of degenerative origin. Our reason for they are post-mortem artifacts. But, as Kallius such a conclusion is based on sections (tangential to (1905) observes, if they are not present in life they the surface) through the olfactory mucosa of a must be formed extraordinarily quickly after death normal rabbit (R286) in which the impregnation is since they are always to be seen even in freshly fixed usually successful and uniform. In these sections animal material (though, according to the same every rod which is traced to the surface ends in the author, they appear smaller in osmic acid prepara- same way, i.e. in a small terminal swelling from the tions than in preparations fixed in Muller's solu- upper circular margin of which the olfactory hairs http://jnnp.bmj.com/ tion). In successful impregnations in our own diverge in stellate fashion (Figs. 7 and 9). The material, the terminal swellings are regularly de- hairs usually do not stain as deeply as the olfactory veloped at the distal end of all the olfactory rods. rods. Counts made on the tangential sections show They show a variation in size, but in general appear that. the number of hairs at the distal end of each relatively' somewhat larger than they are commonly sensory cell is usually 10-12, and in some instances depicted to be in text-book figures. Van der Stricht there may be as many as 13 or 14. In other stellate (1909) has given the name " olfactory vesicle " to formations there seem to be fewer, but in these cases the terminal svwelling, but it does not appear to be there are often obvious gaps in the radial series on September 24, 2021 by guest. vesicular in the usually accepted sense of the term. which are apparently due to the displacement of Rather, the swelling forms a cup-shaped hollow some of the fine hairs so that they cannot be easily which narrows down to a circular margin from defined. It thus appears that in the rabbit the which the olfactory hairs splay out in a stellate number ofhairs considerably exceeds that commonly arrangement, the whole formation bearing a close given for mammals, e.g. 5-6 in man (von Brunn, 1892; resemblance to the gamopetalous corolla of a cam- Kallius, 1905) or 5-8 in the pig (Alcock, 1910). panulate or urceolate flower with the free portions The only difference to be noted in these stellate of the petals slender and pointed (P1. I, Fig. 1). formations is one ofsize, their total diameter varying The olfactory hairs are short, straight, or slightly from 4,u to 7,u. In the large formations the olfactory curved processes, l-2,u in length and about 01,u in hairs are coarser and stain more heavily. It hNs width, and in each sensory element they are remark- not been possible to establish that there is any ably even both in length and width. They end definite pattern in the distribution of stellate fbrma- abruptly in blunt tips, and in some cases the latter tions of different sizes. In general -the olfactory OLFACTORY INNERVATION 103 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from hairs of adjacent receptors do not overlap with one the case in text-book illustrations. On the con- another, though they may be close enough to come trary, they run in parallel formation, following a into contact. They project up definitely beyond straight course upwards and backwards to the the distal margins of the supporting cells and thus cribriform plate, and any interchange of fibres lie free within the mucinous film which covers the between adjacent fasciculi appears to be quite olfactory epithelium during life. exceptional (Fig. 11). Thus it may be said that It had been noted above that in different prepara- from each localized area of the olfactory mucosa tions (even though these had been fixed and stained the olfactory nerve fibres proceed by the shortest by the same technique) the terminal swellings show and most direct route to reach the intracranial some variation in size. It now seems clear that this cavity. On the other hand, as soon as they have is due to the varying extent to which the olfactory pierced the cribriform plate they immediately enter hairs become clumped together from their base into the formation of a most complicated and upwards. Thus, to continue our floral simile, they intricate plexus on the surface of the olfactory bulb, may be widely splayed like the petals of an ox-eye and it is clear from the study of silver sections that daisy (in which case they lie almost flat on the the fibres of all the fasciculi here undergo a com- surface of the free extremities of the supporting plete rearrangement before the latter are sorted out cells), or they may diverge only in their distal portion into the terminal fasciculi which enter the glomeruli. like the petals of a bluebell, or in some cases (Fig. 2) The possible significance of this " sorting out " is they may be grouped together like the closed petals discussed later in reference to the experimental of a tulip. In the last case, the clumped hairs give studies. the appearance of an unusually large terminal swelling. Whether these are normal variations Retrograde Degeneration of the Olfactory Epithelium which occur in life, it is not yet possible to state. Previous studies of the effect on the olfactory At the distal extremities, the olfactory rods are epithelium of destruction of the olfactory bulb, or evenly disposed over the surface of the olfactory of section of the olfactory nerves, have led to equi- epithelium (except at the points where the ducts of vocal and sometimes conflicting results so far as the olfactory glands of Bowman break through). mammals are concerned. Baginsky (1894) reported Protected by copyright. At a deeper level they are disposed in a hexagonal that section of the olfactory nerves in young rabbits pattern between the supporting cells, each of the led to atrophy of the entire olfactory mucosa (in- latter having 6 to 10 rods applied to its surface cluding both the epithelial and the subepithelial (Fig. 10). On the basis of counts made on tan- tissues), the degree of atrophy being more severe in gential sections, it is estimated that, in the rabbit's the long-term experiments (up to 68 days). How- olfactory epithelium, the density of olfactory rods ever, he considered the possibility that these results is of the order of 150,000 per sq. mm. may have been, at least in part, due to interference Because of their fineness, the proximal processes with the vascular supply. Lustig (1884) destroyed of the olfactory sensory cells, extending from the one or both olfactory bulbs in 12 rabbits and cell bodies to the basement membrane, are exceed- examined the olfactory mucosa after intervals of ingly difficult to demonstrate, and when they are 45-65 days. He found that all the cell elements demonstrable to the eye with the use of an oil- of the epithelium underwent degeneration (that is, immersion lens, they are very difficult to reproduce supporting and basal cells as well as sensory cells). convincingly in a photograph. They are illustrated It seems not improbable that the results reported in the microphotograph shown in Fig. 8, which has by Baginsky and Lustig may both be vitiated by http://jnnp.bmj.com/ been partly retouched. The processes pass verti- the fact that some of the changes observed in the cally down towards the basement membrane and, olfactory mucosa were due to the rhinitis which not as they approach the latter, a number converge to uncommonly affects laboratory animals. Takata form a small fasciculus of olfactory nerve fibres. (1929) expressed the opinion that destruction of the These fasciculi, which are formed at fairly regular olfactory bulb in rats is not followed by changes in intervals immediately beneath the epithelium, join the olfactory region so long as the vascular supply the main longitudinal fasciculi which are directed to the nasal cavity is not interfered with. On the upwards and backwards towards the cribriform other hand, he found that trauma of a large number on September 24, 2021 by guest. plate (Fig. 6). So far as can be determined from of the olfactory fibres in the immediate neighbour- our material, the olfactory fibres maintain their hood of the cribriform plate led to degeneration of individuality from their origin in the cell bodies of the olfactory sensory cells. He also stated that, in the sensory cells to their termination in the olfactory regions of severe degeneration, the olfactory epithe- bulb; in other words, each nerve fibre serves only lium becomes replaced by the type of ciliated one receptor. Further, unlike sensory surfaces such epithelium characteristic of the respiratory region as the skin, there is practically no overlap in the of the nasal cavity. Here again, however, the histo- epithelial distribution of different fasciculi and logical picture may have been obscured by inflam- there is no subepithelial plexus. Indeed, our matory changes, for, according to Nagahara (1940), material also confirms the statement made by Read such metaplasia may occur in chronic rhinitis. An (1908) that the main olfactory fasciculi do not examination of the microphotographs reproduced anastomose to form an interweaving plexus in the in Takata's paper makes it quite evident that his olfactory cavity as is commonly represented to be histological material was of very poor quality and 104 W. E. LE GROS CLARK ANMO R. T. TURNER WARWICK J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from that some of his experimental animals actually were basal cell. It is clear from the histological picture suffering from quite severe rhinitis. Nagahara that the supporting cells of the olfactory epithelium studied the effect of. olfactory bulb ablation and ,play a significant part in the phagocytosis of the olfactory nerve section in mice, and he noted that products of degeneration of the olfactory sensory such lesions were followed by a rapid degeneration cells. Indeed, it is doubtful whether the removal of of the olfactory epithelium involving naost of the the debris involves the activity of any mobile olfactory cells. But he states that, as soon as three elements of extraneous origin such as macrophages. days after the operation, the basal cells start to A careful search has failed to show any indubitable proliferate by mitotic division, and in approximately macrophages in the olfactory epithelium at the 90 days the regeneration of the olfactory epithelium height of retrograde degeneration (provided no is complete. From the regenerating cells, the central infection is present), though occasionally a cell is processes first appear, and later the peripheral seen in the basal layers which may have such an processes. The centripetal growth of the re- origin. However, it has not been found possible to generating axons finally leads to the establishment decide with certainty whether such cells are not of new endings in glomerular formations in the simply basal cells of the epithelium which have -olfactory bulb. Nagahara also affirms that the taken up some of the cellular debris. In view of presence of the olfactory bulb is not necessary for Nagahara's statement that mitotic activity is very the regeneration of the olfactory cells and their pronounced in the basal cells a few days after processes, for in this case regenerating olfactory destruction of the olfactory bulb in mice, special nerve fibres still grow out from the olfactory mucosa. attention has. been given to this in the study of our In anticipation of the discussion of our own experi- histological material. Mitoses are very occasion- mental studies, it may be said that the latter pro- ally seen, but they are so rare that they can hardly vided no evidence of such a regenerative process, be regarded as significant for a true regenerative and it is possible that Nagahara may have been process. misled partly by the incompleteness of his lesions In an experiment in which the olfactory bulb had and partly by the fact that pathological conditions been removed three days before death, the degree

affecting both the olfactory bulb and mucosa (which of atrophy of the olfactory epithelium was found to Protected by copyright. might simulate repair phenomena) are by no means be very similar to that just described in the forty- uncommon in laboratory mice. eight hours' material (Fig. 17). The effect of total ablation of the olfactory bulb. After one week, the great majority of the olfactory In a number of rabbits the right olfactory bulb was cells have completely degenerated, and much of the removed, and the animals killed at intervals ranging cellular debris resulting from this degeneration has from twelve hours to eight weeks after the operation. now disappeared. The few olfactory rods which The changes observed in the olfactory epithelium remain are beaded and broken up into fusiform are as follows. segments (Fig. 18). No fibrillary structure is After twenty-four hours degeneration is very evident in these remnants. distinct (Figs. 12 to 15). The total thickness of the In the eight weeks' specimen, retrograde degenera- epithelium already shows a reduction, and the nuclei tion of the sensory elements is already complete of the sensory cells are relatively fewer. Many of (from other material we have reason to suppose that the nuclei which remain show various stages of it is actually completed earlier-between two and pyknosis and karyorrhexis, and, in hiematoxylin three weeks after ablation of the olfactory bulb). sections, deeply staining granules of nuclear debris The olfactory epithelium is reduced to practically http://jnnp.bmj.com/ are seen scattered in the intercellular spaces of the half its normal thickness and now consists entirely epithelium. The olfactory rods are distorted and of supporting and basal cells (Fig. 19). No sensory frequently show varicose swellings, and, for the cells remain. The supporting cells retain their most part, the terminal swellings and olfactory hairs original morphological characters rfiore or less un- have undergone disintegration. The proximal pro- changed (except that their infra-nuclear portion has cesses of the sensory cells which remain tend to take shrunk to less than half the normal length), and on a darker stain with protargol, and the basement they show no tendency to become transformed into membrane of the whole epithelium has become other types of cell; nor do they develop cilia at on September 24, 2021 by guest. more sharply defined than in the normal mucosa. their -free extremities as has been reported by After 48 hours (Fig. 16) the degenerative changes, Takata (1929). All the olfactory glands of Bowman are much more marked. The whole epithelium has have undergone distinct atrophy; their cells are shrunk to approximately two-thirds of its normal shrunken and irregular in outline, and they stain depth and the majority of the nuclei of the sensory feebly with eosin. The fasciculi of the olfactory celjs have now undergone disintegration. The pro- nerves in the submucosa show some degree of ducts of the disintegration, in the form of nuclear fibrosis. On the other hand, at no time up to eight deibris and eosinophil granules, are partly to be seen weeks do these fasciculi show any proliferation of in intercellular spaces, but a great deal has now the Schwann nuclei (as might perhaps be anticipated been taken up into the cytoplasm of the supporting to occur if there is any attempt at regeneration). cells, and in many cases is contained there within The effect ofpartial lesions of the olfactory bulb.- large vacuoles. Sometimes also (but much less In a series of experiments a portion only of the frequently) particles of debris may be found in a olfactory bulb of one side was excised in order to OLFACTORY INNER VATION 105 note the effect of such a partial lesion on the spread over the whole extent of the olfactory J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from olfactory epithelium. The bulb was exposed in the mucosa. It may thus be inferred that the normal usual way, and a varying amount removed. The glomeruli which remain on the medial surface of the proportional extent of the ablation was estimated bulb receive some olfactory fibres from the entire approximately at the time of operation by direct area of the olfactory epithelium. This specimen is inspection, and by naked-eye examination after of particular interest since it confirms the results of death. However, the remains of the bulb were not the experimental studies in rabbits and justifies the serially sectioned for microscopic study, and thus in assumption that, in these studies, the septal mucosa no case could the possibility be excluded that the may be taken as a representative sample of the whole actual lesion was rather more extensive than had olfactory area. been intended, as the result of local trauma and hemorrhage. In all the experiments, the animals Discussion were killed seven days after the operation. Our experiments have demonstrated that partial In three rabbits approximately half the bulb was or complete ablation of the olfactory bulb gives rise removed, in two the rostral half and in one animal to a rapid degeneration of the olfactory epithelium. the upper or dorsal half. In all cases the ensuing If care is taken to exclude all material in which atrophy of the olfactory epithelium was found to there is any sign of rhinitis, the evidence is quite be spread over all the septal mucosa, and a rough clear that the immediate degeneration involves only count of the nuclei of the sensory cells shows that the sensory elements of the epithelium. In twenty- approximately half of these cells had disappeared. four hours many of these are already in process of There is a corresponding thinning out of the olfac- disintegration, and by the end of a week most have tory rods (Fig. 21), and this thinning out also disappeared altogether. The process of atrophy appears to be evenly spread over the septal epithe- appears at first to be a simple autolysis, for we have lium. In another experiment in which approxi- found no clear evidence of phagocytic agents mately the rostral two-thirds of the bulb were invading the epithelium. The fragments of the removed, the ensuing atrophy of the olfactory disintegrated nuclei of the sensory cells at the epithelium was found to be more severe in degree end of 24 hours are seen lying free in the inter- Protected by copyright. (see Fig. 22). But in this case rather more than cellular spaces of the epithelium. By 48 hours, two-thirds of the sensory elements in the olfactory however, much of the debris has been collected epithelium have undergone total atrophy, which in large vacuoles formed within the cytoplasm may well be due to the fact that the effects of the of the supporting cells. The latter thus appear lesion were somewhat more severe than had been to play a significant part in the removal of intended. It should again be noted, however, that the products of degeneration. In some experi- the atrophy is spread evenly over the septal mucosa. ments, after removal of the olfactory bulb a few Lastly, in one experiment, approximately the rostral sensory cells may persist throughout the epithelium. third of the bulb was removed at operation, and in This is certainly due to the fact that in these cases this case again, although the thinning out of the the ablation of the bulb was not total, for the subse- sensory elements is rather greater than had been quent histological examination of stumps left after anticipated (presumably because of the fact that the apparently total ablations showed that small por- effects of the lesion were more severe than had been tions of the bulb may not infrequently be left behind intended), it corresponds roughly to the extent of in the region where it meets the olfactory peduncle. the lesion. This is of some importance in the consideration of http://jnnp.bmj.com/ In the course of the study of retrograde degenera- the statement by Nagahara that rapid regeneration tion in the olfactory mucosa of mice, complete of the olfactory epithelium occurs in the mouse, for serial sections were prepared through the nasal it seems possible that his " regenerated " elements cavity and olfactory bulbs of a supposedly normal may have been original sensory cells left behind as animal, after decalcification and staining with a the result of an incomplete destruction of the bulb. modified Bielschowsky's method. In this specimen, However, he also reported that active mitotic pro- however, it was found that a localized part of the liferation of the basal cells of the olfactory epithe- bulb had undergone atrophy (presumably as the lium was to be observed a few days after the opera- on September 24, 2021 by guest. result either of a development defect or of some tion. In our own experiments a careful search has infective process), thus providing further material failed to show any significant evidence of such for the study of the effects on the olfactory epithe- mitotic activity; nor, indeed, have we found any lium of a partial lesion of the bulb. The atrophy evidence of a regenerative process up to eight weeks had involved the glomeruli and external plexiform after removal of the bulb. After such an interval, layer of the bulb mainly over its dorsal, lateral, and the olfactory epithelium consists only of supporting ventral surfaces. The olfactory epithelium on the cells and basal cells, and the olfactory fasciculi in affected side is shrunk to a little less than two-thirds the submucosa have undergone some fibrosis with of the thickness of the epithelium on the opposite no indication of any Schwann cell activity. It normal side, and the olfactory sensory cells are remains possible, of course, that the epithelium of reduced to approximately one-third of their normal the mouse possesses a regenerative capacity not number. This partial atrophy of the olfactory found in the rabbit. receptor cells, it should again be noted, is evenly The rapidity of the epithelial degeneration in 1061W. E. LE GROS CLARK AND RP T. TURNER WARWICK these experiments suggests the possibility that it stimulation. If olfactory discrimination is ulti- J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from may be due to an interference with the vascular mately dependent on peripheral analysers in the supply of the olfactory mucosa and, as already form of different olfactory receptors responsive to noted, this has been put forward in explanation of different types of stimulus (analogous to the peri- the results recorded by previous workers. The pheral analysing mechanisms in the retina or vessels involved would presumably be the ethmoidal cochlea), it must be assumed that the olfactory arteries, though their extradural position should fibres derived from each type of sensory cell require safeguard them from injury in a careful ablation of to be segregated so as to permit of some form of the olfactory bulb. However, so far as our experi- localization 'in their projection on the olfactory ments are concerned, the possibility can be excluded centres of the brain. We have noted that in our for the following reasons. In the first place, the preparations the terminal swellings of the olfactory immediate degenerative process in the olfactory rods, together with the olfactory hairs, show certain mucosa only affects the sensory cells; the supporting morphological variations in form and size, but these and basal cells of the epithelium and the subepi- variations show no regularity in their distribution thelial tissues remain unchanged. Secondly, partial which might suggest a relationship to functional lesions of the olfactory bulb are followed by an even types of receptor. On the other hAnd, the sensory thinning-out of sensory cells throughout the septal cells all over the olfactory mucosa vary greatly in epithelium, and not by circumscribed changes in the length (20,u-90,u) of their distal processes (the local vascular territories. Thirdly, removal of the olfactory rods), and the idea suggests itself that such dorsal half of the bulb (which involves no inter- a spectrum of rod lengths might provide a possible ference with the immediate neighbourhood of the basis for the analysis of olfactory stimuli of different cribriform plate) is also followed by a partial atrophy kinds. However, we have not been able to deter- of olfactory cells. Lastly, in order to study directly mine that partial destruction of the olfactory bulb the effect of unilateral ablation of the bulb on the leads to a selective atrophy of olfactory rods of any vascularity of the olfactory mucosa, in one rabbit particular length, though it remains possible that 6 c.cm. of a 4 per cent. solution of bromophenol much more localized lesions in the bulb might

blue was injected into the ear vein a few minutes demonstrate such a relationship. If there is anyProtected by copyright. after the operation and the animal was killed ten form of localization within the olfactory bulb in minutes later. By this time most of the tissues the sense that olfactory receptors related to different normally supplied with blood are stained an intense kinds of olfactory stimuli are projected on to dif- blue, and in this particular experiment the mucous ferent glomeruli within the bulb, it might be expected membrane in both sides of the nasal cavity was that partial lesions of the bulbs (bilaterally sym- stained with equal intensity. Thus it seems clear metrical in extent) would lead to a partial anosmia that the degeneration of the sensory elements of the of a selective type. Hitherto, however, there is no olfactory epithelium is a true retrograde degenera- evidence for such a supposition as far as we are tion. aware, possibly because such circumscribed lesions If the septal mucosa may be regarded as repre- only very rarely become available for study. sentative of the olfactory mucosa as a whole, it can be stated that fractional removal of the olfactory Summary bulb is followed, not by a complete atrophy of the 1. In the rabbit, the olfactory receptors form sensory elements limited to a corresponding area of distally small terminal swellings from each of which the mucosa, but to a general thinning-out of the 10-12 olfactory hairs commonly arise. The varia- http://jnnp.bmj.com/ sensory cells over the entire mucosa. This general- tions in the appearance of the terminal swellings ization was confirmed in the study of silver sections and the hairs are discussed. of the whole of the olfactory area in a mouse in 2. The density of olfactory receptors in the rabbit which a partial atrophy of unknown origin was is estimated to be of the order of 150,000 per sq. mm. found incidentally in one olfactory bulb. While our 3. There is no overlapping in the distribution data do not permit of an accurate quantitative within the olfactory epithelium of fasciculi of olfac- assessment, the results of six experiments permit tory nerve fibres, nor is there a subepithelial plexus. the statement that the degree of thinning-out of the 4. The main fasciculi of the olfactory nerves pro- on September 24, 2021 by guest. olfactory sensory cells is approximately proportional ceed in parallel bundles by the shortest and most to the amount of the olfactory bulb which is de- direct route to the cribriform plate of the ethmoid, stroyed. The immediate significance of the intricate and there is no plexiform arrangement within the plexus formed by the olfactory nerve fasciculi after nasal cavity. piercing the cribriform plate is now made apparent, 5. As soon as they have pierced the cribriform for it is clear that it provides for a sorting out of plate, the olfactory nerve fasciculi enter into the the filres in the fasciculi in such a way that each formation of a most intricate plexus. local area of the olfactory epithelium is projected on 6. Total ablation of the olfactory bulb is followed to all parts of the olfactory bulb. The further by a very rapid retrograde degeneration in the significance of the sorting out remains to be deter- olfactory epithelium which is already well advanced mined. It seems difficult to explain, however, twenty-four hours later, and within one week most except on the basis of the segregation of fibres con- of the sensory elements have disappeared. veying impulses related to different types ofolfactory 7. The supporting cells of the olfactory epithelium 107 OLFACTORY INNERVATION J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from play a significant part in the phagocytosis of the Bodian, D. (1936). Anat. Rec., 65, 89. products of degeneration of the sensory cells. Brunn, A. von (1875). Arch. mikr. Anat. 11, 468. 8. No evidence of regeneration is apparent up to (1892). Ibid., 39, 632. Cajal, Ramon y. (1889). Gac. med. Catalana, 12, 6 eight weeks after ablation of the olfactory bulb. (quoted by the same author in "Histologie du 9. Partial ablation ofthe olfactory bulb is followed Systeme Nevreux ", vol. 2. Paris, 191 1). by a general thinning-out of the olfactory sensory Exner, S. (1872). Sitzung. K. Akad. Wiss., 65, 7. cells over all the olfactory area of the corresponding Gehuchten, A. van (1890). La Cellule, 6, 393. side. The degree of this thinning-out is approxi- Grassi, B. and Castronuovo, A. (1889). Arch. mikr. mately proportional to the size of the lesion. Anat., 34, 385. Jagodowski, K. P. (1901). Aaiat. Anz., 19, 257. 10. It is inferred that olfactory fibres from each Kallius, E. (1905). " Handbuch der Anatomie des local area of the olfactory mucosa are distributed to Menschen." Edited by Bardeleben. Vol. 5, 115, all parts of the olfactory bulb. The significance of Fischer, Jena. this type of projection is discussed. Kolmer, W. 1927). " Handbuch mikr. Anat." Edited by von Mollendorf. Vol. 3, teil 1, p. 192. We wish to express our gratitude to Mr. E. Thompson Lustig, A. (1884). Sitzung. Math-Naturwiss. Classe for his expert assistance in the preparation of the histo- Kais. Akad. Wissen., 89, 119. logical material, and to Mr. W. Chesterman for his Nagahara, Y. (1940). Jap. J. med. Sci. (Sect. 5: Pathol.), microphotographic skill. 5, 165. Read, E. A. (1908). Amer. J. Anat., 8, 17. Stricht, 0. van der (1909). Mem. Cour. Acad. Roy. REFERENCES Med. Belg., 2nd Ser., 2, 20. Alcock, N. (1910). Anat. Rec., 4, 123. Takata, N. (1929). Arch. Ohren Nasen Kehlkopfh., 121, Baginsky, B. (1894). Virch. Arch., 137, 389. 31. Protected by copyright.

EXPLANATION OF PLATES Except where otherwise mentioned, all the microphotographs are of sections stained with Bodian's protargol method.

PLATE I PLATE II FIG. 1.-Distal processes of olfactory cells showing the FIG. 9.-Tangential section through the surface of the olfactory rods, terminal swellings, and olfactory olfactory epithelium showing the olfactory hairs hairs. Note that the " double" appearance of the radiating in stellate fashion from the terminal rods is due to the fact that the neurofibrillk are swellings of the olfactory rods. Note the variation situated peripherally so that the rods here appear to in size of these stellate formations. x 2000. have a " tubular " structure. x 1800. FIG. 10.-Tangential section through the olfactory FIG. 2.-Distal processes of olfactory cells showing the epithelium near the surface of the latter. The appearance produced by the clumping of the olfactory rods are seen disposed in a hexagonal olfactory hairs. The terminal swellings of the pattern between the adjacent sides of the supporting http://jnnp.bmj.com/ olfactory rods are thereby made to appear unusually cells. Towards the upper part of the microphoto- large. x 1800. graph the level of the free extremities of the sup- FIG. 3.-Distal processes showing the terminal swellings porting cells is reached, and here the olfactory rods and olfactory hairs. x 1500. are more evenly dispersed. At the uppermost part FIG. 4.-Distal processes showing darkly staining end- of the microphotograph the actual surface of the bulbs at the end of olfactory hairs-probably de- mucosa is shown, and at this level are the stellate generative formations. x 1500. formations of olfactory hairs (not clearly visible at FIG. 5.-Distal processes of olfactory cells in which the this magnification). x 600. terminal swellings and olfactory hairs show certain FIG. 11.-Tangential section through the submucosa in variations from their usual appearance, which may the olfactory area of the nasal septum, showing the on September 24, 2021 by guest. possibly be abnormal. x 1500. main fasciculi of the olfactory nerves passing in FIG. 6.-Section of the olfactory mucosa showing one parallel formation and demonstrating the absence of the main fasciculi of the olfactory nerves receiving of the plexiform arrangement which is commonly accessions of fibres from the olfactory epithelium. depicted in diagrams. Between the fasciculi can be x 84. seen alveoli of the olfactory glands of Bowman. FIG. 7.-Tangential section through the surface of the x 46. olfactory epithelium showing the olfactory hairs radiating from the terminal swellings of the olfactory rods. x 2300. FIG. 8.-Section of normal olfactory mucosa showing the proximal processes of the sensory cells passing through the basement membrane to form a fasciculus of olfactory nerve fibres. Since it proved impossible to bring all the proximal processes into focus simul- to taneously, this microphotograph has been slightly retouched in order to demonstrate them clearly. x 770. 108 W. E. LE GROS CLARK AND R. T. TURNER WARWICK J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from

PLATE III FIG. 12.-Section ofnormal olfactory epithelium, stained with hematoxylin and eosin. x 900. FIG. 13.-Section of the olfactory epithelium (stained with h2ematoxylin and eosin). 24 hours after total ablation of the olfactory bulb of the same side. Note, in comparison with the preceding figure (which is taken from the normal side of the same animal), the reduction in the total thickness of the epithelium and in the number of nuclei of sensory cells. The disintegration of some of these nuclei is also demonstrated. x 900. FIG. 14.-Section of normal olfactory epithelium, for comparison with succeeding figures. x 600. FIG. 15.-Section of the olfactory epithejium 24 hours after total ablation of the olfactory bulb of the same side. Note, in comparison with the preceding figure (which is taken from the unoperated side of the same animal), the reduction in the total thickness of the epithelium and in the number of nuclei of sensory cells, the distortion of the olfactory rods, the virtual disappearance of the terminal swellings, and the accentuation of the basement membrane. x 600. FIG. 16.-Section of the olfactory epithelium 48 hours Protected by copyright. after total ablation of the olfactory bulb of the same side. There is a further reduction in the total thickness of the epithelium, and many of the olfactory rods have disappeared. x 600. FIG. 17.-Section of the olfactory epithelium three days after total ablation of the olfactory bulb of the same side. Only a few olfactory rods now remain. x 600. FIG. 18.-Section of the olfactory epithelium one week after total ablation of the olfactory bulb. Most of the sensory cells have disappeared, and the few remaining remnants of the rods are fragmented. This section is taken from material fixed in formol- saline, and is thus not directly comparable with the other microphotographs of this series, which are taken from material fixed in alcohol. x 600. FIG. 19.-Section of the olfactory epithelium eight weeks after total ablation of the olfactory bulb, stained http://jnnp.bmj.com/ with hxematoxylin and eosin. All the sensory cells have disappeared and only the supporting and basal cells remain. x 600. FIG. 20.-Section of normal olfactory epithelium for comparison with the succeeding microphotographs. x 600. FIG. 21.-Section of the olfactory epithelium one week after ablation of half the olfactory bulb of one side.

Note, in comparison with the preceding figure on September 24, 2021 by guest. (which is taken from the unoperated side of the same animal), the reduction of the nuclei of the sensory cells to approximately half their normal number, and a corresponding thinning-out of the olfactory tods. x 600. FIG. 22.-Section of the olfactory epithelium one week after ablation of two-thirds of the olfactory bulb of the same side. There is a very marked reduction of the nuclei of the sensory cells and of the olfactory rods. x 600. OLFACTORY INNER VATION 109 J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.9.3.101 on 1 July 1946. Downloaded from PLATE I

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