J. Anat. (1970), 107, 2, pp. 215-237 215 With 14 figures Printed in Great Britain An experimental study of the origin and the course of the centrifugal fibres to the in the rat

J. L. PRICE AND T. P. S. POWELL Department of Human , Oxford (Received 25 August 1969)

INTRODUCTION There is now anatomical evidence for the presence of fibres passing from more central to more peripheral levels in all the sensory systems, although the term 'centrifugal' is not always used for such 'returning' fibre pathways. Examples of such fibres at the extreme periphery of the sensory pathways are the y-efferent fibres and the olivo-cochlear bundle, both of which pass out to the receptors, while at the most central level there is now electron microscopic evidence to show that fibres pass back to the relevant sensory relay nuclei of the from the sensory areas of the cerebral cortex (Szentagothai, Hamori & Tombol,- 1966; Jones & Powell, 1969). The elucidation of the functional significance of these 'centrifugal' fibre systems has been a stimulating and fruitful field of electrophysiological work during the past two decades (see Granit, 1955; Matthews, 1964; Whitfield, 1967). In his studies of normal stained with the Golgi method, Ramon y Cajal (1911) found relatively coarse fibres which pass forwards into the olfactory bulb from the lateral . He described these as centrifugal, and clearly distinguished them from the finer, more deeply situated axons of the . Although Cajal was uncertain of the origin of the centrifugal fibres, he suggested that they came from the sphenoidal cortex or the olfactory tract. For many years the presence of these centrifugal fibres was either ignored or denied (see Allison, 1953; Green, Mancia & von Baumgarten, 1962), but experimental anatomical evidence of their presence has now been provided in the rabbit (Cragg, 1962), the rat (Powell & Cowan, 1963; Powell, Cowan & Raisman, 1965; Heimer, 1968), and the coypu (Girgis & Goldby, 1967); and Powell et al. (1965) have shown that these fibres pass in and close to the lateral olfactory tract. The material of Cragg (1962), and of Powell et al. (1965) was not suitable for determining the origin of the centrifugal fibres, but these authors suggested that they arose from the region of the . The recent claim of Heimer (1968) that the centrifugal fibres arise from widespread areas, including the pyriform cortex, cannot be considered substantiated as his lesions may have involved the lateral olfactory tract. Because the centrifugal fibres pass in close relation to the centrally directed axons of the mitral cells of the olfactory bulb, the responses in the olfactory bulb to electri- cal stimulation of the lateral olfactory tract might be due in part to activation of these centrifugal fibres, as well as to antidromic stimulation of the axons (Phillips, Powell & Shepherd, 1963). For an electro-physiological study of the centrifugal fibres, it would be advantageous to know their site of origin, and in the I4-2 216 J. L. PRICE AND T. P. S. POWELL present investigation an attempt has been made to determine this origin by using the Nauta technique to show degenerating axons after lesions have been placed in the areas (Price, 1969).

MATERIALS AND METHODS Approximately 100 rats, none of which were more than 2 months old, were used for this study. For lesions of the pyriform cortex, the temporal approach of Powell et al. (1965) was used, and considerable care was taken to avoid injury to the lateral olfactory tract. Stereotaxic lesions were placed by reference to the atlas of the rat by de Groot (1959). After survival periods of 4-7 d, depending on the staining procedure to be used, the animals were anaesthetized and perfused with normal saline and 10 %0 formalin. After further fixation the brains were cut at 25 ,am on a freezing microtome. The olfactory bulbs and peduncles were removed and sectioned sagittally, while the rest of the brain was cut coronally. A one in ten series was stained in the first instance, using either the method of Nauta & Gygax (1954) or that of Fink & Heimer (1967). Frequently a further series was mounted and stained with thionine. Another series of 13 rats in which one or both olfactory bulbs had been removed in infancy was available for retrograde degeneration studies, and serial coronal sections through several brains prepared by the Golgi-Cox method were also examined.

RESULTS The two silver impregnation procedures which have been used to stain orthograde axonal degeneration, that of Nauta & Gygax (1954), and the more recent method of Fink & Heimer (1967), have given qualitatively similar results. This is clearly shown in several brains which have been stained with both methods; the limits of the degeneration and the relative amounts of degeneration in different regions of the brain have always been the same. The Fink-Heimer method has certain advantages, however, because it gives a lighter background against which degenerating fibres stand out very distinctly, and because it stains terminal degeneration more intensely than the Nauta-Gygax method. This intense staining of terminal degeneration is especially useful when the degenerating terminals are limited to a single layer of cortex, or to a relatively restricted nucleus; a more diffuse termination over a large nucleus or area is often difficult to interpret, however, as isolated granules are sometimes found which do not appear to be related to degeneration. Of particular interest to the present study is the occurrence of a considerable amount of such pseudo-degenera- tion in the olfactory bulb; very commonly the olfactory bulbs of both sides of the brain contain small granules of silver, which are not associated with degenerating fibres, and which bear no relation to the site of the lesion. These granules can be easily distinguished from degenerating fibres, but they closely resemble degenerating terminals; because of this difficulty only well-stained fibre degeneration can be accepted as valid within the olfactory bulb. Centrifugalfibres to olfactory bulb 217 Lesions of the pyriform cortex Several brains with lesions of the pyriform cortex are available from previous in- vestigations, and 15 additional brains have been prepared, both in order to obtain carefully restricted lesions which do not involve the lateral olfactory tract, and for use in other studies (Price & Powell, 1970). Three of the recently prepared brains (R 717, R 718 and R 665) will be described, as they illustrate the results from the whole group. In R 717 the pyriform cortex is damaged in its central part, at the level of the anterior commissure and optic chiasma (Fig. 1). Laterally the lesion involves very slightly the neocortex beyond the rhinal sulcus and although it extends through the entire thickness of the pyriform cortex it does not reach the ; only a

R717 R718 ABBREVIATIONS AC Anterior commissure LOT Lateral olfactory tract AOB Accessory olfactory bulb M Medial nucleus of AON Anterior olfactory nucleus MD Medio-dorsal nucleus of thalamus C Central nucleus of the amygdala MFB Co Cortical nucleus of the amygdala NOT Nucleus of the lateral olfactory tract EPL External plexiform layer ofolfactory bulb OT Olfactory tubercle GCL layer of olfactory bulb PC Pyriform cortex GL Glomerular layer of olfactory bulb S Corpus HDB Nucleus of the horizontal limb of the SM Stria medullaris diagonal band VDB Nucleus of the vertical limb of the LH Lateral habenular nucleus diagonal band Fig. 1. The site and extent of the lesions and the resulting degeneration in experiments R717 and R718. The surface extent of the two lesions is shown in the lower middle figure, and the degeneration in the anterior olfactory nucleus, which was the same in both experiments, is shown in a tracing of a sagittal section in the upper middle figure. In this and subsequent figures the lesion is shown in solid black, the fibre degeneration in dashes, and the terminal degeneration by stipple. The coronal sections are close to the anterior and posterior ends of each lesion. 218 J. L. PRICE AND T. P. S. POWELL few degenerating fibres are present in the . In no place does the lesion encroach upon the lateral olfactory tract, and this is confirmed by the absence of degeneration in the tract behind the lesion. The lesion in R718 is similar to that in R717, but it is farther anterior in the pyriform cortex, and it does not extend beyond the rhinal sulcus (Fig. 1). The lateral olfactory tract is immediately adjacent to the lesion, but is not damaged, and again there are no degenerating fibres in the tract caudal to the lesion. In both brains, which are stained with the Fink-Heimer method, well-stained degeneration is present in the sites already described in detail by Powell et al. (1965), including the rest of the pyriform cortex, the anterior commissure, the medial forebrain bundle, the , and the thalamus. Terminal degenera- tion is especially clear in the medio-dorsal and lateral in the

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R 665 Fig. 2. The lesion and the fibre and terminal degeneration in experiment R 665. The lesion is shown on a surface reconstruction and on outlines of coronal section of the cerebral hemi- sphere; the fibre and terminal degeneration in the anterior olfactory nucleus and the olfactory bulb are shown on a sagittal section. The surface extent of three lesions which did not cause degeneration of centrifugal fibres to the olfactory bulb (R681, R685 and R699) and of one lesion which encroached upon the lateral olfactory tract and which did cause such degeneration (R668). thalamus. Rostral to the lesion, degenerating fibres of medium size are present in the medial forebrain bundle, and in the deep layers of the pyriform cortex; these fibres are found, in the sagittal sections of the frontal pole of the hemisphere, to pass into the anterior olfactory nucleus, where terminal degeneration is very prominent in the deep half of the plexiform layer along the dorso-lateral margin of the nucleus. This terminal degeneration forms a distinct band, extending rostrally to the margin of the accessory olfactory bulb, but leaving relatively clear the super- ficial half of the plexiform layer, and the deeper areas of the nucleus (Fig. 9). A little degeneration may also be present in the ventral part of the nucleus, but this is very much less than in the dorsal part. There is no degeneration in, or associated with, the lateral olfactory tract, and all degenerating fibres within the anterior olfactory Centrifugalfibres to olfactory bulb 219 nucleus terminate caudal to the posterior end of the olfactory bulb. No degeneration could be found within the olfactory bulb of either side. In R665 the lesion is in the anterior part of the pyriform cortex, but, in contrast to those in R 717 and R 718, it does slightly involve the lateral olfactory tract (Fig. 2). In this brain, which is stained by the Nauta-Gygax method, the pattern of degenera- tion is precisely the same as in the previous brains, except that there are also a few degenerating fibres in the lateral olfactory tract, both caudal and rostral to the lesion; those which are rostral to the lesion lie deep to, as well as within, the tract, and follow the course of the centrifugal fibres as described by Ramon y Cajal (191 1), Cragg (1962) and Powell et al. (1965): they enter the posterior end of the olfactory bulb from the deep aspect of the lateral olfactory tract and run through all its layers, finally reaching the level of the glomeruli. From these experiments it may be concluded that the centrifugal fibres to the olfactory bulb itself (as opposed to those which terminate in the anterior olfactory nucleus) do not degenerate after a lesion confined to the pyriform cortex. Degenera- tion of these centrifugal fibres has been found with lesions of the pyriform cortex only when the lateral olfactory tract had also been damaged. The pyriform cortex does send fibres rostrally into the olfactory peduncle, but they terminate behind the olfactory bulb in the anterior olfactory nucleus, and for the most part in the deep half of the plexiform layer over its dorso-lateral portion. These results are con- firmed by all the other brains which have been used, four of which are shown in Fig. 2. The lesions in R681 and R685 are well clear of the lateral olfactory tract, and the one in R699 is caudal to its posterior limit; in all three brains degenerating fibres are seen running medially into the medial forebrain bundle and then rostrally to the anterior olfactory nucleus, but none of them enter the olfactory bulb. In R668, however, the tract has been encroached upon very slightly, and in this experiment, in addition to the degeneration in the medial forebrain bundle and the anterior olfactory nucleus, there are degenerating fibres in the lateral olfactory tract and the olfactory bulb. Lesions of the amygdala In five of the brains studied, the amygdala has been directly involved by the lesion. Three of the lesions were placed stereotaxically, while the other two are lesions of the pyriform cortex which involve the amygdala on their deep aspect. One of the stereotaxically placed lesions (R736) is shown in Fig. 3. the baso-lateral nuclei have been completely destroyed, and the cortical nucleus damaged on its lateral part. In all of these brains there is fibre degeneration in the , the medial forebrain bundle, and the anterior commissure, and terminal degeneration in the postero-medial part of the olfactory tubercle, the hypothalamus, and the medio- dorsal, medio-ventral, and lateral habenular nuclei in the thalamus. The degeneration rostral to the olfactory tubercle corresponds precisely with that found in experiments with lesions restricted to the pyriform cortex; fragmenting fibres pass forwards into the anterior olfactory nucleus, where they terminate in the deep half of the plexi- form layer, but none enter the olfactory bulb. Whether the fibres to the anterior olfactory nucleus arise in the amygdala as well as the pyriform cortex, cannot be determined from these experiments, as all of the lesions may have interrupted fibres from the cortex. 220 J. L. PRICE AND T. P. S. POWELL Lesions of the olfactory tubercle Lesions were made in the olfactory tubercle, by the direct temporal approach, in five experiments. As degeneration is present in the ipsilateral olfactory bulb in three of these experiments (R664, R675, and R679) but not in the other two (R661 and R677), they will be described in some detail. In R661 (Fig. 4) the lesion involves all layers of the central and lateral parts of the tubercle and the ventral portion of the medial forebrain bundle. The damage does not extend caudal to the level of the vertical limb of the diagonal band, and is well medial to the lateral olfactory tract. In R677 (Fig. 4) the superficial lesion of the tubercle extends farther medial and caudal than the one in R661, and again there is slight damage to the ventro-lateral

R736 Fig. 3. The lesion and the resulting fibre and terminal degeneration in R 736.

portion of the medial forebrain bundle. In the latter brain, in addition to the super- ficial damage, the involvement of blood vessels which traverse the tubercle has caused extensive necrosis in the corpus striatum, although the between the olfactory tubercle and the striatum has not been damaged. In neither of these brains is there any evidence of degeneration in the lateral olfactory tract or the olfactory bulb. Terminal degeneration is present in the medio-dorsal and lateral habenular nuclei in the thalamus, and there is fibre degeneration in the medial forebrain bundle as far caudal as the posterior hypothalamus. Only a few de- generating fibres are found in the anterior olfactory nucleus; as they are more numerous in R661, where the involvement of the medial forebrain bundle is greater, they may again be due to the interruption of fibres from the pyriform cortex. Centrifugalfibres to olfactory bulb 221 In R679 the lesion is very similar to those in the two previous experiments except that this lesion encroaches slightly upon the medial side of the lateral olfactory tract (Fig. 5). In addition to the degeneration present in the other brains there are few degenerating fibres running from the lateral olfactory tract into the granule cell layer of the olfactory bulb, and degenerating fragments are found in the external plexiform layer and among the periglomerular cells. As with experiments with lesions of the pyriform cortex, therefore, superficial lesions of olfactory tubercle do not cause the centrifugal fibres to the olfactory bulb to degenerate unless the lateral olfactory tract is also damaged.

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R 661 R 677 Fig. 4. The lesions and the degeneration in R661 and R677. It may be noted that in neither of these experiments was there degeneration in the olfactory bulb, and that the degeneration in the anterior olfactory nucleus was the same in both. The superficial part of the damage in the remaining two experiments of this group (R664 and R675) differs little from those in the first two, but injury to penetrating vessels has again caused necrosis deep to the olfactory tubercle, and in these brains the substantia innominata as well as the corpus striatum has been involved (Fig. 5). In R664 the necrosis extends as a band from the tubercle to the central part of the striatum, beginning at the level of the vertical limb of the diagonal band and con- 222 J. L. PRICE AND T. P. S. POWELL tinuing to the posterior limit of the olfactory tubercle. In R675 the superficial part of the lesion in the tubercle is relatively small, but there is extensive damage caudal to the lesion in the substantia innominata and the corpus striatum. In neither experi- ment is the lateral olfactory tract involved in the lesion. In both of these brains, as well as the degeneration due to the damage of the olfactory tubercle, medial fore- brain bundle, and corpus striatum, degenerating fibres are found in the lateral olfactory tract. These fibres pass fowards into the olfactory bulb to ramify in all its

R679 R675 R664 Fig. 5. The lesions and the degeneration in R664, R675 and R679. Following each of these lesions, there was degeneration in the anterior olfactory nucleus and olfactory bulb.

Figs. 6 and 7. Photomicrographs to show degeneration of centrifugal fibres in the olfactory bulb after a lesion of the nucleus of the horizontal limb of the diagonal band (Fig. 7) as com- pared with the appearance of the bulb of the unoperated side (Fig. 6). The degeneration is concentrated around the glomeruli superficially, and immediately deep to the mitral cell layer, and there are only a few degenerating fibres present in the intervening external plexiform layer. Fink-Heimer method. x 140. Fig. 8. To show degenerating centrifugal fibres passing rostrally within and immediately deep to the lateral olfactory tract after a lesion of the nucleus of the horizontal limb of the diagonal band. Fink-Heimer method. x 170. Fig. 9. Photomicrograph to show terminal degeneration in the deep half of the plexiform layer of the anterior olfactory nucleus after a lesion of the ipsilateral pyriform cortex. Fink- Heimer method. x 370. Centrifugalfibres to olfactory bulb 223

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R 754 Fig. 10. The lesions and the degeneration in R754 and R776. The distribution of the degenera- tion in the anterior olfactory nucleus and the olfactory bulb, shown in a sagittal section, was the same in both experiments. Lesions deep and caudal to the olfactory tubercle In order to confirm this hypothesis and to rule out the possibility that the fibres might arise in the hypothalamus and run through this area within the medial fore- brain bundle, stereotaxic techniques have been used to produce lesions deep to the olfactory tubercle and in the rostral hypothalamus in 14 animals. Three of these experiments (R776, R754 and R715) will be described, the results of which are Centrifugalfibres to olfactory bulb 225 entirely consistent with those of the whole group. The lesion in R 776 destroys a large area in the medial and ventral part of the corpus striatum, and extends into the substantia innominata to damage the dorsal edge of the medial forebrain bundle and a large part of the nucleus of the horizontal part of the diagonal band (Fig. 10). All layers of the olfactory tubercle are unharmed, as is the ventral portion of the medial forebrain bundle. The posterior limb of the anterior commissure is severed, but the anterior limb is not directly injured and is almost clear of degeneration. The lesion extends from the anterior edge of the vertical limb of the diagonal band to the anterior hypothalamus. In addition to the degenerating fibres passing to the thalamus and hypothalamus, and through the diagonal band to the septum, there are two groups of degenerating fibres which pass rostral to the olfactory tubercle. The first

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R71 5 R753 Fig. 11. The lesions and the resulting degeneration in R715 (A) and R753 (B). It should be noted that the nucleus of the horizontal limb of the diagonal band was not injured in either of these experiments, anld that the degeneration in the olfactory bulb of R715 was due to in- volvement of the anterior commissure. of these is made up of fibres which run in the medial forebrain bundle to the anterior olfactory nucleus; these fibres are seen in all parts of the nucleus, but terminal de- generation is present only in the plexiform layer. Most, and possibly all, of this degeneration is likely to be due to the interruption of fibres from the pyriform cortex. The second group is composed of the large diameter centrifugal fibres to the olfactory bulb itself. From the lesion they pass posteriorly and laterally around the posterior edge of the olfactory tubercle to reach the caudal end of the lateral olfactory tract under the medial edge of the pyriform cortex; few, if any, centrifugal fibres pass through the pyramidal cell layer of the olfactory tubercle or the pyriform cortex. Upon reaching the tract the fibres turn rostrally and run along the deep edge of the tract, some within it, and some in the plexiform layer immediately deep to it (Fig. 8). 226 J. L. PRICE AND T. P. S. POWELL In the sagittal sections of the olfactory bulb and peduncle the coarse fibre fragments can be seen to fan out in a cone and enter the granule cell layer through all parts of the posterior border of the olfactory bulb; they do not appear to change appreciably in size or number caudal to the bulb. From the granule cell layer they continue into the external plexiform layer and eventually ramify among the periglomerular cells (Fig. 7). These fragmenting fibres are best seen in the granule cell layer and in the glomerular layer where they virtually outline the glomeruli; within the external plexiform layer there is a lower concentration of degenerating fibres, although there is some evidence of terminal degeneration here. The granule cell layer and glomeruli of the accessory olfactory bulb are completely clear of degeneration, and this absence of degeneration is especially striking when compared with the intense fragmentation in the immediately adjoining parts of the main bulb in the same sections. There is, however, fibre degeneration in that part of the olfactory tract lying between the granule and external plexiform layers of the accessory bulb, and some of this de- generation extends into the deep half of the external plexiform layer. These differences between the main and accessory bulbs have been a constant feature of all the brains studied. R754 will be presented as an experiment in which the damage to the horizontal part of the diagonal band is minimal; for technical reasons no current was passed, and the lesion is due to the passage of the electrode only. This needle track passes behind the posterior limb of the anterior commissure, disrupts the medial forebrain bundle, and damages the central part of the nucleus of the horizontal limb of the diagonal band; it reaches the central surface of the brain between the caudal edge of the olfactory tubercle and the nucleus of the lateral olfactory tract (Fig. 10). As in the experiments with larger lesions in this region, degenerating fibres pass rostrally from the lesion in both the medial forebrain bundle and the lateral olfactory tract and end in the anterior olfactory nucleus and the olfactory bulb respectively; the number of degenerating fibres passing to the olfactory bulb is about the same as in R776, but there are many fewer which terminate in the anterior olfactory nucleus. R 715 provides a very useful control experiment with which to compare the previous two brains. The lesion is similar to that in R 754, but it is considerably more anterior, and disrupts the anterior limb of the anterior commissure as well as a large part of the medial forebrain bundle immediately anterior and lateral to the horizontal limb of the diagonal band before passing through the central part of the olfactory tubercle (Fig. 11). There is degeneration in the medial forebrain bundle and the anterior olfac- tory nucleus, but no degenerating fibres are present in or deep to the lateral olfactory tract. The only degeneration which is present in the olfactory bulb can be attributed to the involvement of the anterior commissure; it can easily be distinguished from de- generating centrifugal fibres because it is not associated with the lateral olfactory tract, it is almost completely restricted to the granule cell layer, and comparable frag- mentation is found in the opposite olfactory bulb. The degenerating fibres of the anterior commissure are also very much finer than the thick centrifugal fibres (Cajal, 1911). It is of interest that the granule cell layer of the accessory olfactory bulb, as compared with the corresponding layer of the main bulb, is relatively free of de- generation, containing only an occasional fragmented fibre. R753 will be described as representative of five brains with lesions of the anterior Centrifugalfibres to olfactory bulb 227 hypothalamus. The damage in this experiment is immediately caudal to the nucleus of the horizontal limb of the diagonal band and destroys the dorsal two-thirds of the medial forebrain bundle and the initial part of the stria medullaris (Fig. 11). There is intense degeneration in the thalamus and hypothalamus, and in the vertical limb of the diagonal band. More rostrally in the medial forebrain bundle, there are de- generating fibres which pass to the nucleus of the horizontal limb of the diagonal band, and a smaller number which continue as far as the rostral tip of the olfactory tubercle and possibly into the anterior olfactory nucleus, but there is no degenera- tion in or associated with the lateral olfactory tract or in the olfactory bulb. The experiments described above indicate that the site of origin of the centrifugal fibres to the olfactory bulb is in the substantia innominata immediately deep to the caudal half of the olfactory tubercle in the vicinity of the diagonal band. Lesions which damage this area or which interrupt the centrifugal fibres in the lateral olfactory tract produce degeneration in the olfactory bulb, while lesions outside but surrounding this area are ineffective in this regard. In particular, the lesions in R715 and R753 are important negative evidence in that they limit the antero- posterior extent of the origin to the region of the horizontal limb of the diagonal band. This conclusion is confirmed by the following experiments in which the method of retrograde cell degeneration has been used. Retrograde degeneration experiments Rat A2 (Fig. 12) shows most clearly the degenerative changes which have been found in these brains. In this animal the right olfactory bulb was completely removed and there is some damage to the rostral portion of the anterior olfactory nucleus; the survival time was 8 months. A comparison of the two sides of the brain shows that there is no difference between the ipsilateral and contralateral pyriform cortex, amygdala, or olfactory tubercle, either in the form of cell loss or cell shrinkage. However, within the area deep to the caudal part of the olfactory tubercle, in the nucleus of the horizontal limb of the diagonal band, there is a marked difference between the two sides (Fig. 13). On the normal side this nucleus is conspicuous as a group of large, densely staining cells contiguous anteriorly with the nucleus of the vertical limb of the diagonal band, and disappearing posteriorly at the level of the nucleus of the lateral olfactory tract. Laterally it spreads deep to the olfactory tubercle towards the anterior amygdala and , through and around the medial fore- brain bundle. At the lateral edge of the nucleus a small tongue of cells extends forward, so that the anterior limit of the nucleus is formed by a few cells both medial and lateral to the medial forebrain bundle. On the experimental side the entire extent of the nucleus appears depleted of cells at low magnification, and is difficult to identify. With a higher magnification the are found to be still present, but they are markedly shrunken and stain palely; cell shrinkage rather than complete cell loss is therefore the characteristic feature of this degenerative change. In other experiments with smaller lesions the same effect is present, but it is more localized. This is well illustrated in B 6 and B 8, in which the olfactory bulb has been only partially destroyed, leaving the postero-medial region of the bulb and almost all of the anterior olfactory nucleus intact; in these brains marked cell shrinkage is limited to the caudal parts of the nucleus. Cellular degeneration is also 228 J. L. PRICE AND T. P. S. POWELL present in experiments with shorter survival periods, although the effect is less striking than after a longer survival period. It is important to note that in all of these experiments the degeneration stops abruptly at the junction between the vertical and horizontal limbs of the diagonal band. In spite of the term 'nucleus of the horizontal limb of the diagonal band' which is usually applied to this nucleus, and the direct contiguity between it and the nucleus of the vertical limb of the diagonal band, the latter is quite obviously a septal structure and can be distinguished from the more lateral nucleus on architectonic grounds: the cells of the nucleus of the vertical limb are arranged in parallel rows along the

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Fig. 12. Experiment A2. To show the extent of the damage to the olfactory bulb and anterior olfactory nucleus, and the distribution of the retrograde cell degeneration in the nucleus of the horizontal limb of the diagonal band. fibres of the diagonal band as it sweeps dorsally into the septum, while those of the nucleus of the horizontal limb are evenly dispersed throughout the nucleus, which therefore appears to be of quite uniform composition. Furthermore, at all levels the nucleus of the horizontal limb lies lateral to the 'diagonal band' fibres passing from the hypothalamus to the septum (Guillery, 1957); this can be readily determined in horizontal sections where the medial forebrain bundle is seen to divide into three parts immediately caudal to the nucleus: the medial division runs along the medial side of the nucleus and into the septum, the lateral one turns laterally towards the amygdala along the postero-lateral border of the nucleus, and the central division continues rostrally through the nucleus and into the olfactory peduncle. The nucleus of the horizontal part of the diagonal band can also be distinguished in Golgi-Cox material (Fig. 14); the large cells are even more obviously clustered in a seemingly random manner into a clearly defined nucleus lateral to the vertical limb of the diagonal band. In contrast to the vertical limb where the dendrites tend to be arranged either parallel or at right angles to the fibres of the diagonal band, in the nucleus of the Centrifugalfibres to olfactory bulb 229

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DISCUSSION The results obtained in the present study show that the centrifugal fibres to the olfactory bulb in the rat arise from a discrete nucleus in the basal forebrain, the ' nucleus of the horizontal limb of the diagonal band' (Price, 1969). Here, and through- out this discussion, the term 'centrifugal fibres to the olfactory bulb' is used in the sense that it was originally used by Cajal (1911) and refers only to those fibres which arise from the ipsilateral side of the brain posterior to the olfactory peduncle, pass forwards in or close to the lateral olfactory tract, and terminate within the olfactory bulb. This definition excludes the fibres of the anterior commissure from the contra- lateral anterior olfactory nucleus, as well as any fibres which terminate within the anterior olfactory nucleus posterior to the bulb. As has been mentioned, pseudo-degeneration in the form of grains of silver of varying sizes is often found in the olfactory bulbs of brains stained with the Nauta- Gygax or Fink-Heimer methods (Fink & Heimer, 1967; Heimer, 1968); this bears no relation to any lesion and is commonly found on the non-experimental side, or in brains in which the lesion is completely unrelated to the olfactory pathways. Well- stained fragmenting fibres can easily be distinguished from this pseudo-degeneration, and in these experiments only fibre degeneration together with a clear difference be- tween the bulbs of the two sides has been accepted as valid. As the pseudo- degeneration masks any terminal degeneration which may be present, it is probable that terminal degeneration in the olfactory bulb can be satisfactorily studied only with the electron microscope (Price, 1968). In all experimental investigations of this type on the basal forebrain areas the possible involvement of fibres passing through the area of damage must be taken into account, and in the present study three fibre tracts in particular must be con- sidered: the lateral olfactory tract, the anterior commissure, and the medial forebrain bundle. The centrifugal fibres to the olfactory bulb from the ipsilateral forebrain have been known to pass rostrally in and close to the lateral olfactory tract since Cajal (1911) first described them, and this has been confirmed experimentally by Cragg (1962), Powell & Cowan (1963) and Powell et al. (1965). The present experiments demonstrate that even the slightest encroachment upon the medial or lateral side of the tract will produce degeneration within the bulb, and extreme care has therefore been taken to obtain several lesions of the pyriform cortex and olfactory tubercle which unequivocally do not damage the lateral olfactory tract. From the anterior commissure fibres pass into the bulbs of both sides, but there is no difficulty in identifying these fibres as they are much finer than the centrifugal fibres, they are confined to the anterior commissure, are not related to the lateral olfactory tract, and only the occasional fibre passes superficial to the mitral cell layer. Furthermore, because degeneration within the anterior commissure is always bilateral, it can be checked by comparison with the non-experimental side. In order to ensure that the I5-2 232 J. L. PRICE AND T. P. S. POWELL positive results are not due to the inevitable involvement of the medial forebrain bundle by lesions of the nucleus of the horizontal limb of the diagonal band, lesions have been placed in the hypothalamus which destroy the medial forebrain bundle immediately caudal to the nucleus. As possible sites of origin of the olfactory centrifugal fibres, those structures which are known to receive direct connexions from the olfactory bulb were investi- gated first. However, lesions of all parts of the pyriform cortex and amygdala, pro- vided they were carefully placed to avoid damage to the lateral olfactory tract, have always failed to cause degeneration within the olfactory bulb, and the fact that these same brains contained well-stained fibre and terminal degeneration in the anterior olfactory nucleus immediately behind the bulb indicates that this negative finding is not due to incomplete impregnation. Superficial lesions of the olfactory tubercle also gave negative results, but any lesion in the tubercle which extended into the nucleus of the horizontal limb of the diagonal band did cause the centrifugal fibres to degenerate. Only lesions which involve this diagonal band nucleus, or which interrupt the centrifugal fibres themselves in the lateral olfactory tract, have given positive results in this respect. Thus, in several brains, damage to the striatum, the medial forebrain bundle either immediately rostral or caudal to the nucleus of the diagonal band, or the hypothalamus, has been ineffective in producing degeneration in the lateral olfactory tract or the olfactory bulb,- whereas this degeneration has been found after lesions which selectively damage various parts of the nucleus of the horizontal limb of the diagonal band. Although it is impossible to place a lesion in this nucleus without injury to the medial forebrain bundle, the lesions in the rostral hypothalamus immediately posterior to the nucleus indicate that no fibres to the olfactory bulb arise caudal to the nucleus of the diagonal band. As there appears to be some doubt as to whether the centrifugal fibres found in experimental material in recent years corresponds precisely to the centrifugal fibres described by Cajal (1911) (see Dennis & Kerr, 1968), it is important to consider the course of the centrifugal fibres in fuller detail. They pass laterally and caudally from their nucleus of origin around the posterior edge of the olfactory tubercle and under the medial margin of the pyriform cortex to enter the caudal end of the lateral olfactory tract; in this part of their course they are in close relation to the anterior, dorsal, and ventral sides of the nucleus of the lateral olfactory tract. They run for- wards in the deepest part of the tract, and also in the most superficial part of the plexiform layer deep to the tract. As they approach the olfactory bulb the fibres begin to fan out in a cone to enter the entire breadth of the posterior end of the bulb; they pass through the granule cell layer into the external plexiform layer and eventually ramify extensively among the periglomerular cells which outline the glomeruli (Fig. 7). These fibres correspond, therefore, in all respects to the thick centrifugal fibres described in normal material by Cajal (1911) except that he concluded that they did not pass superficial to the mitral cell layer; it seems probable either that he was unable to follow the finer preterminal axons into the external plexiform layer, or that in the neonatal animals which he usually used these fibres had not yet fully developed. The accessory olfactory bulb has been known for some time to receive a specific input from the vomero-nasal organ, and it is therefore of interest that in the present Centrifugalfibres to olfactory bulb 233 material no degenerating centrifugal fibres have been found in relation to the glomeruli of the accessory bulb and only an occasional degenerating commissural fibre in the granule cell layer. Because of the difficulty of identifying terminal de- generation in the olfactory bulb with the light microscope it is not possible to state whether any fibres from these two systems terminate in the accessory bulb. These differences in the pattern of degeneration in the accessory bulb as compared with the main bulb may be further evidence to support the suggestion that the accessory bulb has a specific function different from that of the main olfactory bulb. The conclusion that the nucleus of the horizontal limb of the diagonal band is the nucleus of origin of the olfactory centrifugal fibres is extended and confirmed by the retrograde cell degeneration experiments. After damage to the olfactory bulbs of very young animals and survival periods of several months, marked cell shrinkage is present in this nucleus, but no change is found in the pyriform cortex, the olfactory tubercle, the septum (including the nucleus of the vertical limb of the diagonal band), or the hypothalamus. The fact that cell shrinkage rather than cell loss occurs may be due to the presence of collaterals of the centrifugal axons which pass in the stria medullaris and the habenular commissure to the nucleus of the opposite side (Price & Powell, 1970). Because the lesions used in these retrograde cell degeneration experiments also damage a small portion of the anterior olfactory nucleus, one possible criticism is that the cell shrinkage might be due to axons which terminate in the anterior olfactory nucleus and not in the olfactory bulb, but taken together with the previous experiments which indicate that orthograde degenerating fibres can be followed into the bulb only after lesions which damage this nucleus or which interrupt the fibres from it, the retrograde degeneration experiments provide very good evidence that the large cells of the nucleus of the horizontal limb of the diagonal band are the cells of origin of the centrifugal fibres to the olfactory bulb. These results confirm and extend the earlier tentative conclusions of Cragg (1962), Powell & Cowan (1963) and of Powell et al. (1965) that the centrifugal fibres arise from the region of the olfactory tubercle, but they are in disagreement with the more recent work of Heimer (1968), who reported that the centrifugal fibres to the olfactory bulb arise from the pyriform cortex, as well as from the olfactory tubercle or regions caudal or deep to it. He therefore considered the centrifugal fibres to be part of a rostrally directed association system which includes at least all parts of the pyriform cortex, the olfactory tubercle and the olfactory peduncle. It may be significant, how- ever, that the only photomicrograph of degenerating fibres within the olfactory bulb which he included in his paper was from a brain with a lesion of the lateral olfactory tract, and that he was unable to find degeneration in the bulb after lesions behind the anterior amygdaloid area, i.e. caudal to the lateral olfactory tract. It is probable that the degeneration which he found in the olfactory bulb was due to damage to the lateral olfactory tract and not to the pyriform cortex. Similarly, as all of his lesions of the olfactory tubercle destroyed 'centrifugal fibres originating in basal forebrain regions caudal to the tubercle', the degeneration of centrifugal fibres in these experiments was probably due to injury to the nucleus of the diagonal band, or of the fibres leaving it. The term 'nucleus of the horizontal limb of the diagonal band' has been used in order to distinguish it from the nucleus of the vertical limb, which is closely related in 234 J. L. PRICE AND T. P. S. POWELL structure and connexions to the . The nucleus of the horizontal limb extends laterally and caudally from itsjunction with the vertical limb, occupying the area deep and caudal to the postero-medial part of the olfactory tubercle, and disappearing at the level of the nucleus of the lateral olfactory tract. In both Golgi and Nissl material the boundary between the horizontal and vertical limbs of the diagonal band is very sharp, the cells of the horizontal limb being grouped together in a seemingly random fashion, with their dendrites radiating in all directions, while the cells of the vertical limb are arranged in parallel rows along the fibres of the vertical limb, and their dendrites tend to lie along or across these fibres. There is al- ready good evidence that these two portions of what is commonly termed simply the 'nucleus of the diagonal band', e.g. Fox (1940) in the cat, and Lohman (1968) in the guinea-pig, have different connexions; the cells of the nucleus of the vertical limb, together with those of the medial septal nucleus, undergo retrograde degenera- tion after section of the fimbria, while the cells of the nucleus of the horizontal limb are unaffected (Daitz & Powell, 1954). The present observations of retrograde de- generation in the nucleus of the horizontal limb without change in the nucleus of the vertical limb after removal of the olfactory bulb are precisely complementary to the earlier findings. The nucleus of the horizontal limb of the diagonal band also corre- sponds to at least the lateral part of Gurdjian's 'interstitial nucleus of the septal portion of the medial forebrain bundle' (1927). Although the nucleus is at all times lateral to the region described by Gurdjian (1927) as the lateral pre-optic area, in other accounts, e.g. de Groot (1959), the lateral pre-optic area is enlarged and made to lie more laterally so that the nucleus would be within it. It may also be noted that the diagonal band as indicated by de Groot (1959) corresponds only to the vertical limb, and that he does not illustrate the horizontal limb of the diagonal band. The area within which the nucleus lies has also been referred to as the substantia innominata (of Reichert) (Cragg, 1961; Miodonsky, 1967). The finding that the nucleus of the horizontal limb of the diagonal band is directly connected with the olfactory bulb, taken in conjunction with the clear limits of the nucleus in Nissl and Golgi material, suggest that it is a discrete nucleus, separate from and probably independent of the septum; the reference to the diagonal band in the name is there- fore inappropriate, and the term has been used here only because there is no commonly recognized alternative. The projection from the pyriform cortex to the anterior olfactory nucleus was noted by Sanders-Woudstra (1961) and by Powell et al. (1965), and the laminar termination of these fibres in the plexiform layer was emphasized by Heimer (1968). The last author also recognized the important relation between fibres from the olfactory bulb which terminate in the superficial half of the plexiform layer, and those from the pyri- form cortex, which end in the deep half. These observations on this projection are largely confirmed in the present study, and it may be noted that the fibres from the pyriform cortex to the anterior olfactory nucleus pass rostrally in the medial fore- brain bundle as well as in the longitudinal association system and that they are not related to the lateral olfactory tract. The olfactory terminals therefore appear to act on the distal parts of the apical dendrites of the pyrimidal cells of the anterior olfactory nucleus, and those from the pyriform cortex on the more proximal seg- ments. No definite statement can be made on a possible projection to the anterior Centrifugal fibres to olfactory bulb 235 olfactory nucleus from the olfactory tubercle or the amygdala, but the degeneration which is found in the olfactory peduncle after lesions of these two structures could be due to the involvement of the pyriform cortex itself or to damage to fibres from it in the medial forebrain bundle. Valverde (1965) has reported from studies of Golgi-stained material that collaterals of the axons from the anterior olfactory nucleus axons turn rostrally and enter the ipsilateral olfactory bulb along with the anterior commissure fibres from the opposite side, and this has been confirmed by our own unpublished electron micro- scopic studies. There are, therefore, at least two fibre systems by which the olfactory bulb could be influenced by more central structures: the centrifugal fibres from the nucleus of the horizontal limb of the diagonal band which pass forwards in and deep to the lateral olfactory tract, and the fibres from the pyriform cortex which run in the medial forebrain bundle and in the anterior olfactory nucleus. These fibre systems are quite separate, both in the origin and course of the fibres, and also probably in the influences which play upon them; the major afferent supply to the pyriform cortex and anterior olfactory nucleus is from the olfactory bulb, whereas the major afferent connexions to the nucleus of the horizontal limb of the diagonal band is from the hypothalamus and midbrain (Price & Powell, 1970). The recent report of Dennis & Kerr (1968) of responses in the olfactory bulb after stimu- lation of the pyriform cortex and other parts of the basal forebrain, as well as many of the earlier results of Kerr & Hagbarth (1955), may be at least partially explained in terms of the pyriform cortex-anterior olfactory nucleus-olfactory bulb fibre system: although these authors prefer to regard the bulbopetal system which they are stimulating as a direct, monosynaptic system, they admit that there is no real evidence that it is not polysynaptic. As Dennis & Kerr (1968) suggest, it is very probable that with the recording techniques used any response in the olfactory bulb due to stimula- tion of the centrifugal fibres in the lateral olfactory tract would be masked by the re- sponses due to antidromic stimulation of mitral cell axons and their collaterals. It is of interest to compare our results with the cholinergic 'olfactory radiation' reported by Shute & Lewis (1967). They describe a projection of acetyl cholinesterase- containing fibres from the lateral preoptic area which passes laterally in a compact bundle and enters the deep aspect of the lateral olfactory tract; the fibres then continue to the olfactory bulb where terminal staining is located mainly in the glomeruli and the mitral cell layer. Apart from a few discrepancies, (Shute & Lewis consider that a few cells from the olfactory tubercle also contribute to the projection) these observations made with histochemical techniques agree remarkably well with the present de- scription of the origin and course of the centrifugal fibres to the olfactory bulb. The termination of the centrifugal fibres has been studied with the electron micro- scope (Price, 1968) and they have been found to end upon the peripheral processes of the granule cells in the external plexiform layer, in close relation to the unusual 'reciprocal synapse' between mitral cell dendrites and the granule cell processes. Because the centrifugal fibres arise from a fairly large, discrete nucleus, which is situated apart from the rest of the , and because their course and termination are now known, it would appear that the influence of these fibres upon the olfactory bulb could be studied with electrophysiological techniques. It should now be possible to stimulate the centrifugal fibres selectively without interference 236 J. L. PRICE AND T. P. S. POWELL due to antidromic stimulation of the mitral cells and their collaterals and this might provide a method for investigating the action of 'reciprocal ' upon mitral cells. However, it must be pointed out that it would be difficult to stimulate the nucleus of the horizontal limb of the diagonal band without exciting fibres to the anterior olfactory nucleus in the medial forebrain bundle; as collaterals from this nucleus also run into the olfactory bulb and end upon the granule cells, the resultant effect would be due to the activation of both of these systems.

SUMMARY The site of origin of the centrifugal fibres to the olfactory bulb has been investigated in the rat using the axonal degeneration techniques of Nauta & Gygax (1954) and of Fink & Heimer (1967), and by the method of retrograde cell degeneration. The centrifugal fibres have been found to arise from the cells of the nucleus of the horizontal limb of the diagonal band. They pass laterally from this nucleus to join the caudal end of the lateral olfactory tract, within and deep to which they then run forwards to the olfactory bulb. The cells of the nucleus of the horizontal limb of the diagonal band undergo marked shrinkage and pallor after removal of the olfactory bulb in infant rats. No centrifugal fibres to the olfactory bulb have been found to arise from the pyri- form cortex, amygdala or olfactory tubercle; degeneration of centrifugal fibres after damage of these structures occurs only if the lateral olfactory tract is concomitantly involved. From the pyriform cortex (and possibly also from the amygdala and olfactory tubercle) fibres do pass forwards in the medial forebrain bundle and longi- tudinal association system to the anterior olfactory nucleus where they end in the deep half of the plexiform layer.

We wish to acknowledge grants from the Medical and Science Research Councils. J. L. P. was supported by a U.S.P.H.S. Fellowship and a personal grant from the Wellcome Trust.

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