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 olfactory bulb in the rat J. L. PRICE AND T. P. S. POWELL Department of Human Anatomy, 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 thalamus 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 brains stained with the Golgi method, Ramon y Cajal (1911) found relatively coarse fibres which pass forwards into the olfactory bulb from the lateral olfactory tract. He described these axons as centrifugal, and clearly distinguished them from the finer, more deeply situated axons of the anterior commissure. 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 olfactory tubercle. 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 mitral cell 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 basal forebrain 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 brain 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 white matter; only a R717 R718 ABBREVIATIONS AC Anterior commissure LOT Lateral olfactory tract AOB Accessory olfactory bulb M Medial nucleus of amygdala AON Anterior olfactory nucleus MD Medio-dorsal nucleus of thalamus C Central nucleus of the amygdala MFB Medial forebrain bundle Co Cortical nucleus of the amygdala NOT Nucleus of the lateral olfactory tract EPL External plexiform layer ofolfactory bulb OT Olfactory tubercle GCL Granule cell layer of olfactory bulb PC Pyriform cortex GL Glomerular layer of olfactory bulb S Corpus striatum 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 external capsule. 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 hypothalamus, and the thalamus. Terminal degenera- tion is especially clear in the medio-dorsal and lateral habenular nuclei in the GL EPL C_ AON ';t:&~' AC GCL R LO LOT R 681- ~~~R685 LOILOT ~~~~R699 AC 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).