Specific olfactory receptor populations projecting to identified glomeruli in the rat olfactory bulb

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1 Proc. NatI. Acad. Sci. USA Vol. 81, pp , August 1984 Neurobiology Specific olfactory receptor populations projecting to identified glomeruli in the rat olfactory bulb (horseradish peroxidase/topogeaphy/nasal cavity/development/modified glomerular complex) P. J. JASTREBOFF*t, P. E. PEDERSEN*I, C. A. GREER*, W. El. STEWART, J. S. KAUER 1I, T. E. BENSON*, AND G. M. SHEPHERD* Sections of *Neuroanatomy, Neurosurgery, and Gross Anatomy, Yale University School of Medicine, New Haven, CT Communicated by Carl Pfaffmann, April 20, 1984 ABSTRACT A critical gap exists in our knowledge of the topographical relationship between the olfactory epithelium and olfactory bulb. The present report describes the application to this problem of a method involving horseradish peroxidase conjugated to wheat germ agglutinin. This material was iontophoretically delivered to circumscribed glomeruli in the olfactory bulb and the characteristics and distribution of retrogradely labeled receptor cells were assessed. After discrete injections into small glomerular groups in the caudomedial bulb, topographically defined populations of receptor cells were labeled. Labeled receptor cell somata appeared at several levels within the epithelium. The receptor cell apical dendrites followed a tight helical course towards the surface of the epithelium. The data thus far demonstrate that functional units within the olfactory system may include not only glomeruli as previously suggested but, in addition, a corresponding matrix of receptor cells possessing functional and topographical specificity. The topographical relationship between receptors and second-order neurons is well known for most sensory systems and is essential information for understanding sensory processing mechanisms in these systems. However, there is limited information concerning this relationship in the olfactory system. The traditional view was that olfactory receptors project in a regional manner to their synaptic targets in the olfactory bulb (1, 2). However, beginning with studies utilizing methods of localized degeneration (3, 4) and amino acid transport (5), evidence was obtained for more localized projections from the receptor sheet to groups of glomeruli in the olfactory bulbs of mammals. The precise organization of this projection at the cellular level has awaited more detailed analysis. An ideal tool for pursuing this problem is the horseradish peroxidase (HRP) method, which has been effective in mapping in detail the topography of other peripheral sensory systems (6, 7). Kauer (8) and DuBois-Dguphin et al. (9) showed in the salamander that, after injections of HRP into the nerve bundles or olfactory bulb, HRP could be transported both retrogradely into receptor neurons and anterogradely into axon terminals in the glomeruli of the olfactory bulb. Thus far there have been no HRP studies of the peripheral olfactory system in the mammal, due in part to the difficulties of adapting the method to include decalcification of the bony structures of the nose. We have modified the HRP method to permit decalcification of the mammalian nose, and we have combined it with microiontophoresis of wheat germ agglutinin-conjugated HRP (WGA-HRP) into the region of a histologically distinct group of glomeruli, the modified glomerulir complex (MGC) in the olfactory bulb. The results reveal specific sets of receptor neurons projecting to The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact identified groups of glomeruli in the mammalian olfactory bulb. This provides essential clues to the principles of organization underlying the neural processing of information about molecular specificity of odor stimuli in the olfactory pathway. METHODS Twelve-day-old Sprague-Dawley rat pups (Charles River Breeding Laboratories) were used. They were anesthetized with Nembutal (20 mg/kg, intraperitoneally) and placed in a stereotaxic device modified for neonatal surgery. A craniotomy was made over the caudal portion of the olfactory bulbs and the frontal pole of the cortex. Glass micropipettes (20-25 Am, inside diameter) were filled with WGA-HRP (Sigma, 4% in Tris HCl buffer, ph 7.5). Microiontophoretic injections were made into the region of the MGC, a histologically distinct group of glomeruli near the surface in the posterior olfactory bulb and along the medial wall of the accessory olfactory bulb (AOB). The MGC is particularly prominent in the first 3 weeks of life (10), and it has been implicated in transmitting information about the odor cue in suckling (10, 11). It is the only known olfactory glomerular group that can be reliably recognized from animal to animal. Placement of the micropipette was determined by stereotaxic coordinates and surface landmarks. Ten minutes after electrode placement, iontophoresis was begun, using 500-msec positive pulses, at 0.5 Hz, for 20 min. Ten minutes later, the electrode was removed, the incision was closed with cyanoacrylate glue, and the animal was placed on nest shavings under a heat lamp for 4-24 hr. The animals were anesthetized and perfused intracardially with buffered 0.9% saline followed by 50 ml of 2.0% (wt/vol) glutaraldehyde in 0.1 M sodium phosphate buffer, ph 7.4, and then 50 ml of 10% sucrose in 0.1 M ph 7.4 phosphate buffer (40C), over a period of 1 hr. The olfactory bulbs were removed and placed in cold 10% sucrose/0. 1 M phosphate buffer for 24 hr. The intact nose was separated from the overlying muscle and fascia. Decalcification was carried out by immersion in 0.1 M ethylenediaminetetraacetic acid (EDTA, Fisher) and 10% sucrose in 0.1 M phosphate buffer, ph 7.4, at 40C for 24 hr (6). Both the olfactory bulbs and nasal tissue were then embedded in gelatin and placed in 10% sucrose/0.1 M phosphate buffer (40C) for an additional 24 hr. Sections were cut at 50 pum (olfactory bulbs) and 70 gum (nose). The sections were allowed to react according to the TMB method of Mesulam (12) and then were mounted on Abbreviations: HRP, horseradish peroxidase; WGA, wheat germ agglutinin; MGC, modified glomerular complex; AOB, accessory olfactory bulb. ton leave from: Nencki Institute of Experimental Biology, Warsaw, Poland. tto whom reprint requests should be addressed. IPresent address: Department of Neurosurgery, Tufts New England Medical Center, 171 Harrison Avenue, Boston, MA

2 Neurobiology: Jastreboff et al gelatin/chrome alum-coated slides, dried, stained with neutral red, and examined by conventional and dark-field light microscopy. RESULTS In five animals the injections were restricted to small dorsomedial regions of the caudal olfactory bulb. An example of an injection within the region of the MGC is illustrated in Fig. 1. The injection site is approximately indicated by an Proc. NatL. Acad. Sci. USA 81 (1984) 5251 asterisk. In this section, labeling was restricted to three glomeruli within the region of the MGC and five glomeruli at the nearby border of the main olfactory bulb. Extending a short distance from the injection site can be seen labeled fibers of the olfactory nerve, a series of mitral cells with their dendritic processes in the deeper part of the external plexiform layer, and axons in the bundle coursing through the AOB. In addition, there is a small amount of labeling at the margin of the AOB, within the AOB glomerular layer. This section rep- FIG. 1. Representative injection site. (A) Dark-field photomicrograph of a coronal section through the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) as well as the MGC of a 12-day-old rat pup. An asterisk indicates the probable injection site of WGA-HRP. The bright areas are regions that are labeled with WGA-HRP as a result of iontophoretic injection into this part of the bulb. Higher magnification (B) demonstrates more clearly that label is confined to a sector of the bulb composed of MGC glomeruli, and portions of the olfactory nerve layer (onl), main olfactory glomerular layer (glm), external plexiform layer (epl), and mitral body layer (mbl). Label is also apparent in axons coursing through the AOB. Note the small amount of labeling that is present within the AOB at its border with the MGC.

3 5252 Neurobiology: Jastreboff et al. resents the maximal extent of the injection site. In sections rostral and caudal to this, glomerular labeling was less; the total rostral-caudal extent of the labeled region was approximately 1200,um. The olfactory glomeruli contain the terminals of afferent axons arising from the receptor cells in the olfactory epithelium. HRP taken up by axon terminals in the glomeruli was retrogradely transported to their receptor cells of origin. Examples of these cells are illustrated in Fig. 2A, a photomicrograph of a coronal section through the nasal cavity at the midpoint of the extent of the olfactory epithelium. The only olfactory region containing labeled receptor cells lies between the arrows on the septal wall. This population of labeled cells is seen in higher magnification in Fig. 2 B and C. The labeled cell bodies and slender apical dendrites ending at the epithelial surface are typical of olfactory receptor cells. The dentrites have a tortuous configuration. This may be to some extent an artefact of the HRP method. It is of interest that Rafols and Getchell (13) observed a similar configuration of these dendrites when they utilized the Golgi method. Also, as they described, the cell bodies vary in shape, becoming more spherical as they approach the surface. In some cases a terminal knob was observed on the dendrite, but we have not yet seen clear evidence of labeled cilia arising from the knob, as reported by Kauer for the salamander (8). Occasionally labeled receptor cells were observed with dendrites that appeared not yet to have reached the surface; this population probably included immature cells, as well as cells with incompletely labeled dendrites and cells with dendrites sheared off during sectioning. The distribution of labeled receptor cells was of considerable interest. It was clear that only a portion of the receptor cell population within a given region was labeled. The labeled cells were often distributed relatively evenly throughout a region. In Fig. 2, the ventral border of the labeled region corresponded to the border between olfactory and respiratory epithelium; the dorsal border, however, lies within the olfactory epithelium of the septum. Note the distinct border between labeled and unlabeled regions within the olfactory epithelium. This appeared to be a general finding in rostral portions of the nasal cavity in those pups that had HRP injection sites mostly confined within the MGC borders. However, in more caudal sections of the nasal cavities of these same pups, the population of labeled neurons extended into the dorsal recess, and the border between labeled and unlabeled regions was less distinct. Separate small populations of labeled cells were also seen on the most caudal dorsolateral turbinates. No labeled neurons were seen in any other parts of the olfactory epithelium. In the experiment of Fig. 1, epithelium containing the labeled cells extended approximately 4.5 Am along the rostralcaudal axis of the septal wall ipsilateral to the injection site. In addition, a few labeled cells resembling receptor cells were seen in the ipsilateral vomeronasal organ. Presumably, this indicates transport from the AOB, although we cannot rule out the possibility of some transport from the MGC. Within the labeled regions, the receptor cell distribution was of particular interest. Labeled cells appeared to be arranged in several distinct levels that extended laterally to different extents. Most labeled cells were at relatively superficial levels. Further, there was often a close proximity in a radial direction of two cells within different tiers. All of the labeled cells appeared to be receptor cells. We have not seen any clear examples of labeled cells corresponding to supporting or basal cells at these survival times. The discrete clusters of retrogradely labeled receptor cells were all obtained with WGA-HRP iontophoresis as described in Methods. In contrast, pressure injections of small volumes ( ,ul) of 10% HRP (type VI, Sigma) into the olfactory bulb MGC region resulted in spread of label Proc. NatL Aca'd Sci. USA 81 (1984) throughout the olfactory bulb and into the forebrain and was associated with dense labeling of receptor cells throughout the ipsilateral olfactory epithelium. DISCUSSION The present results support and extend previous studies of the topographical organization of the olfactory periphery (1-5, 8, 9) by showing that there is a histologically distinct group of glomeruli that receives input from a topographically defined group of olfactory receptors. The results suggest that olfactory receptor cells projecting to specific glomeruli are organized into relatively clearly demarcated populations in the olfactory epithelium. Within a given region, the labeled cells constitute only part of the total population of receptor cells. This confirms the results of Kauer (8) for the salamander, with the important implication that other receptor cells within that region project to other glomeruli, which was also suggested by Land and Shepherd (5). These results thus provide evidence supporting the hypothesis that, in terms of their connections to different glomeruli, the olfactory receptors form an overlapping mosaic in the epithelial sheet. The distribution of labeled cells within a region appears to be highly significant. It is known from previous studies that receptor cells differentiate from basal cells deep in the epithelium and mature as they move outward toward the surface, where they eventually degenerate and are phagocytized (14). The radial alignments of two or three cells is consistent with the cell columns previously noted by Graziadei and Monti Graziadei (14). It suggests the radial migration of clones of cells that differentiate successively from a common precursor. The change in shape of the cells with proximity to the epithelial surface is consistent with previous descriptions of the receptor cells as they mature and age (14). The arrangement of labeled neurons in several layers has not been observed previously, to our knowledge. We hypothesize that the several labeled cells in a given layer may reflect synchronized differentiation and migration of cells in different columns projecting to a common glomerular target. The restriction of injection sites in the olfactory bulb with iontophoresis of WGA-HRP contrasted greatly with the diffuse spread of label throughout the bulb with pressure injections of HRP (cf. ref. 15). Thus, the method described here should be useful in mapping neural pathways in small neonatal brains at early stages of development. The main target of these experiments has been the MGC, a recently described histological entity in the olfactory bulb that is prominent very early in development (10). It has been implicated in the transmission of information about the odor cue that is essential for suckling by the rat pup (10, 11). The present results suggest that at least part of the input to the MGC comes from a specific population of receptor cells that, in its maximal extent, is distributed along the septal wall, the caudodorsal recess, and small portions of the dorsolateral turbinates. Since our injections usually included adjacent glomeruli of the main olfactory bulb, the input to the MGC most likely comes from a correspondingly smaller population of receptor cells. Our injections included various numbers of these adjacent glomeruli high on the medial wall of the main olfactory bulb, and it is interesting to note that these may also be involved with processing the suckling cue (10). Further work will be necessary to define the precise distribution of cells projecting only to the MGC, including the possibility of input from the vomeronasal organ. Since the injections were aimed at the glomerular level, we assume that most of the HRP uptake occurred in the axon terminals in the glomeruli. We cannot rule out some degree of uptake by olfactory axons in the overlapping olfactory nerve layer. However, this was minimized by the depth of the injection below the surface and by the location of the MGC at the caudal border of the olfactory bulb.

4 Neurobiology: Jastreboff et al Proc. Natl. Acad. Sci. USA 81 (1984) t '44< - / I' 'r V -0 It a IF. i 1. a.104, T 0.,. A t;i.4- afi 0~~~~~. I, *,i 11X ^. = at ' ' P. "Nba k A 200pm a ~ ~ 9 a ~~~~~~i S O~~~um1 ' k i * or a. A 4 ' ",4, tq V a. 49 "Is _.~~~~~~~~~~~~~~f' is* N 50.._m i j 10pm &1" It C. FIG. 2. Retrogradely labeled olfactory receptor cells. Iontophoretic injection of WGA-HRP into the MGC and surrounding regions depicted in Fig. 1 resulted in retrograde transport of WGA-HRP to ipsilateral peripheral receptors in the olfactory epithelium that line the nasal cavity. (A) Representative frontal section through the rostral part of the nose, in which the olfactory epithelium extends between the asterisks, and the region of the septal epithelium containing the population of retrogradely labeled receptor cells extends between the arrows. Higher magnification of this region (B and C) emphasizes more clearly the pear-shaped cell bodies, elongated dendrites, and terminal knobs of receptor cells filled with HRP. These cells had a granular accumulation of HRP, characteristic of retrograde transport, although this is not apparent because of the contrast in the photomicrograph. Labeled cells in B appear to be arranged in several distinct levels. Note (arrow) the radial alignment of two cell bodies.

5 5254 Neurobiology: Jastreboff et al. Further studies employing injections into other parts of the olfactory bulb will be required to obtain a complete picture of topographical organization. However, within the main bulb one cannot identify and inject the same glomerulus from one animal to the next, as in the case of the MGC. Even smaller injections will be needed in the smaller and more tightly packed main bulb glomeruli. With regard to the MGC, the present results provide some intriguing functional clues. The 2-deoxyglucose data indicate that individual olfactory glomeruli may be considered as functional units in the processing of specific odor information (16). This concept may now be extended to include within a functional unit all of the receptors projecting to a glomerulus or functionally related group of glomeruli. Since, as noted above, the receptors that project to the MGC are intermingled with many that project to other glomeruli, it implies that receptors form a functional as well as anatomical mosaic in the epithelium. As already noted, the 2-deoxyglucose mapping data have further indicated that the MGC and nearby glomeruli are active in the neonatal rat pup during suckling (10, 11). This suggests that the receptors projecting to the MGC are responsive to the suckling odor cue. This hypothesis is supported by the recent finding that an analogous distinct glomerular complex in insects receives its input from olfactory receptors that are preferentially responsive to an important insect pheromone (17, 18). To the extent that the receptors projecting to the MGC may be responsive to a highly specific olfactory stimulus, they present an attractive target for electrophysiological and biochemical analysis of olfactory receptor mechanisms. We are grateful to the National Institute of Neurological and Communicative Disorders and Stroke for support by Grants NS-07609, Proc. NatL Acad ScL USA 81 (1984) NS-10174, NS-16993, NS-06978, NS-06990, and NS and to the March of Dimes Birth Defects Foundation for Basil O'Connor Starter Research Grant to C.A.G. 1. LeGros Clark, W. E. (1951) J. Neurol. Neurosurg. Psychiatry 14, LeGros Clark, W. E. (1957) Proc. R. Soc. London Ser. B 146, Land, L. J., Eager, R. P. & Shepherd, G. M. (1970) Brain Res. 23, Land, L. J. (1973) Brain Res. 63, Land, L. J. & Shepherd, G. M. (1974) Brain Res. 70, Kiang, N. Y. S., Rho, J. M., Northrop, C. C., Liberman, M. C. & Ryugo, D. K. (1982) Science 217, LaVail, J. H. & LaVail, M. D. (1974) J. Comp. Neurol. 157, Kauer, J. S. (1981) Anat. Rec. 200, Dubois-Dauphin, M., Tribollet, E. & Dreifuss, J. (1981) Brain Res. 219, Greer, C. A., Stewart, W. B., Teicher, T. H. & Shepherd, G. M. (1982) J. Neurosci. 2, Teicher, M. H., Stewart, W. B., Kauer, J. S. & Shepherd, G. M. (1980) Brain Res. 194, Mesulam, M. M. (1976) J. Histochem. Cytochem. 24, Rafols, J. A. & Getchell, T. V. (1983) Anat. Rec. 206, Graziedei, P. P. C. & Monti Graziadei, G. A. (1978) in Handbook of Sensory Physiology, ed. Jacobson, M. (Springer, Berlin), Vol. 9, pp Grafe, M. & Leonard, C. M. (1982) Dev. Brain Res. 3, Lancet, D., Greer, C. A., Kauer, J. S. & Shepherd, G. M. (1982) Proc. Natl. Acad. Sci. USA 79, Matsumotu, S. G. & Hildebrand, J. G. (1981) Proc. R. Soc. London Ser. B 213, Tolbert, L. P. & Hildebrand, J. G. (1981) Proc. R. Soc. London. Ser. B 213,