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myelin-immunoreactivity n the developing olfactory system
NeuroscienceVol. 67, No. 4, pp. 1009-1019, 1995
Pergamon
0306-4522(95)00095-X
Elsevier Science Ltd Copyright cc, 1995 IBRO Printed in Great Britain. All rights reserved
0306-4522/95 $9.50 + 0.00
OLIGODENDROCYTE/MYELIN-IMMUNOREACTIVITY THE DEVELOPING OLFACTORY SYSTEM B. D. PHILPOT, Department
of Psychology,
A. Y. KLINTSOVA
102 Gilmer
Hall, University
IN
and P. C. BRUNJES*
of Virginia,
Charlottesville,
VA 22903, U.S.A.
Abstract-Immunocytochemistry was used to characterize oligodendrocyte maturation in the developing mammalian olfactory system. Postnatal day 10-16, 20, 30 and adult rats were examined, as well as postnatal day 20, 30, 40 and adult Monodelphis domestica (the grey, short-tailed opossum). In rats, oligodendrocyte/myelin-immunoreactivity first appears in the accessory olfactory bulb by day 11, with labeling rapidly increasing throughout the entire bulb over the next five days. An adult pattern of immunoreactivity, characterized by dense labeling in the granule cell layer, sparse immunoreactivity in the external plexiform layer, and staining along the periphery of glomeruli, is attained by day 30. Staining is apparent in both the lateral olfactory tract and anterior commissure by day 11, and becomes heavy by day 20. While patterns of oligodendrocyte/myelin-immunoreactivity in the adult Monodelphis and rat bulb are similar, staining first appears much later in the opossum (around day 30), and maturation occurs more slowly. For example, rostrakaudal gradients in the development of staining in the anterior commissure were noted which were not seen in the rat. These differences emerge because Monodelphis’ slower growth allows more resolution into developmental sequences. Finally, in rats, unilateral naris closure on the day after birth, which significantly alters normal patterns of bulb development, has no effect on the pattern and level of immunoreactivity even after long (30 day) survival periods. In both normal and naris occluded rats, oligodendrocyte/myelin-immunoreactivity is found in caudal aspects of the rat bulb on day 11 and subsequently progresses throughout the entire bulb over the next five days. Patterns in the Monodelphis bulb mirror those observed in the rat, however, staining appears later and progresses more slowly, suggesting Monodelphis is a useful animal for examining early myelin formation.
During early development, multi-potential 02-A progenitor cells migrate to specific destinations and differentiate into mature oligodendrocytes as well as other cell types. The oligodendrocytes undergo large changes in protein synthesis culminating in the formation of myelin around axons, dendrites, and even somata.29.37 Proper formation of myelin has important consequences for physiological function. Myelination increases membrane resistance and lowers membrane capacitance, thereby increasing signal conduction velocity. Selective augmentation of signal velocity can greatly affect patterns of system processing. Furthermore, the insulative properties of perineuronal oligodendrocytes may serve to isolate signal input on to somata.47 Oligodendrocytes also have a role in morphological development as they release and respond to numerous growth factors.“x3’ For example, myelin-forming oligodendrocytes secrete36 and express4’ factors that can inhibit neurite outgrowth, thus preserving the integrity of established myelinated fibers. Increasing evidence for the important role of oligodendrocytes in cellular dynamics and
*To whom correspondence should be addressed. Abbreuiafions: ABC, avidin-biotin peroxide complex; AC, anterior commissure; AOB, accessory olfactory bulb; LOT, lateral olfactory tract; MOB, main olfactory bulb; P, postnatal day; PBS, phosphate-buffered saline.
the prevalence of devastating demyelinating diseases such as multiple sclerosis have served as an impetus for characterizing the development of oligodendrocytes in mammals. The olfactory bulb is an accessible model for examining the development of myelin/oligodendrocytes for several reasons. First, the bulb is well laminated and thus convenient for tracking myelination patterns. Second, there is sufficient electrophysiological data in the developing rat bulb to correlate the time course of myelination to observed changes in signal conduction velocities. Finally, due to the general posterior-to-anterior gradient of myelin formation and the immaturity of the bulb at birth, the region is one of the last areas where myelination occurs. The bulb therefore provides an easily obtainable source of immature oligodendrocytes and a view of the entire sequence of their expression. Indeed, this feature may prove clinically important, as transplanting olfactory bulb tissue containing undifferentiated oligodendrocytes into demyelinated spinal cord areas has been demonstrated to allow subsequent remyelination.” The present study characterizes myelination and oligodendrocyte maturation in the rat olfactory bulb via immunocytochemical techniques in hope that this information will (I) help elucidate cytoarchitectonic organization of oligodendrocytes and their processes and (2)
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correlate the degree of myelination to known electrophysiological changes in conduction velocity during development. Furthermore, this study examines the possible role of afferent innervation in proper myelination. Blocking airflow through one half of the nasal cavity by unilateral naris closure results in profound changes in metabolic turnover and protein synthesis in the ipsilateral bulb.’ Due to the large requirements of protein synthesis during myelin formation, we hypothesized that global reductions in bulb protein synthesis might reflect alterations in patterns of myelin formation. Finally, myelin/oligodendrocyte development was also traced in Monodelphis domesopossum. Monodelphis has tica, the grey short-tailed become increasingly popular because offspring are born in a very immature state, they develop quite slowly, and, since they are a pouchless species, pups are easily accessible for experimental manipulation. Morphological characteristics of the Monodelphis olfactory bulb have been previously established,” and recent studies suggest the prolonged developmental time course offers a higher degree of resolution into morphological events as compared with studies in rats.38 EXPERIMENTAL
PROCEDURES
et al.
specific binding and increase penetration, the Chemicon antibody and all the avidin-biotin peroxide complex (ABC) solutions were diluted in PBS supplemented with 0.25% lambda carrageenan (Sigma type IV) and 0.25% Triton X-100.‘” Slides were incubated for 24 h at 4°C (Chemicon antibody) or at room temperature (“Rip” antibody), washed four times with PBS, and transferred into a solution containing biotinylated secondary antibodies (Dako, rabbit anti-mouse) for 1 h. Following four rinses in PBS, slides were placed into an ABC solution (Vector; 100~1 each of solutions A and B in 50ml solution, an alteration of the Vector protocol) for 90 min. After four washes in PBS slides were treated with diaminobenzidine for 15.-25 min. Slides were then dehydrated, cleared and coverslipped with DPX (BDH Ltd, Poole U.K.). In some instances sections were lightly counterstained with Methylene Blue. Control experiments, consisting of the same protocol but omitting exposure to the primary antibody, consistently resulted in the absence of staining. The specificity of the antibodies used in this study have been previously examined.“,” The Chemicon antibody detects a unique myelin/oligodendrocyte-specific protein associated with microtubules.” The “Riu” antibodv co-localizes with myelin basic protein and is absent from processes staining for glial fibrillary acidic protein.15 Furthermore, electron microscopic studies show oligodendrocytes to be immunoreactive for “Rip”.j Results outlined below indicate that patterns of immunoreactivity are similar for the two antibodies, although the staining with the “Rip” antibody was more pronounced and labeled somata in addition to their processes. Photomicrographs are of immunoreactivity using the Chemicon antibody unless otherwise noted.
Subjects Offspring of Long-Evans hooded rats purchased from the Charles River Breeding Laboratories (Wilmington, MD) were used. Rats were housed in polypropylene cages (48 x 25 x 16 cm) and given food and water ad libitum. The colony room was maintained on a 16/8 h light/dark cycle. Litters were culled to 10 on the day after birth postnatal day 1 (Pl). PlO-P16, P20, P30 and adult rats were examined. For studies of unilaterally naris occluded pups, PI subjects were cold anesthetized and received either unilateral occlusion of the right external naris via cautery or a sham manipulation consisting of a cautery on the dorsal surface of the nose.32 Pups were then warmed and returned to their mothers. Naris occluded and sham operated rat pups were examined at PlO, P20 and P30. Breeding stock for the opossum colony was purchased from the Southwest Foundation for Biomedical Research (San Antonio, TX) and maintained according to the recommendations of Fadem et al.‘* Opossum litters were not culled due to small litter sizes (n = 3-9). P20, P30, P40 and adults were examined. In all experiments, two to six animals from different litters were used at each age (with the exception of only using animals from one litter in P40 opossums) to ensure a representative sample. Tissue preparation Subjects were given an overdose of barbiturates and perfused intracardially with 0.1 M phosphate-buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde in PBS with 10% sucrose. Brains were then carefully dissected and cryoprotected overnight with 20-30% sucrose in 0.1 M PBS. The following day, 20pm sections were cut on a cryostat in either the coronal or horizontal plane. Sections were thaw-mounted onto gelatin-coated slides, placed in an evacuated chamber, and stored overnight at 4°C. Slides were then rinsed three to four times with PBS and incubated with a mouse monoclonal antibody for myelin/oligodendrocytes. Primary antibodies used were either from Chemicon (antimyelin/oligodendrocyte specific protein), used in a 1: 1000 dilution, or “Rip”, (generously supplied by Dr Joel Black, Yale University) used in a 1: 10 dilution. To reduce non-
Levels of oligodendrocyte-immunoreactivity were determined in four sections from each of two animals that had undergone unilateral naris closure on Pl and were reared until P30. Both left and right bulbs (i.e. contralateral and ipsilateral to the occluded naris, respectively) were analysed. Each bulb sections was divided into rostral, middle and caudal portions, and from each of these subdivisions, 300 pm long segments from the medial wall were selected. Within these zones, the entire width of the granule cell, external plexiform, and glomerular layers were examined. Images were thresholded such that areas containing diaminobenzidine reaction products were black and the background white. An image analysis system (Olympus Cue-2) then calculated the ratio of the area stained to the total test area. Care was taken to ensure that thresholding was identical in both left and right bulbs. RESULTS
Oligodendrocyte-immunoreactivity in the olfactory bulb proceeded in a caudal-to-rostra1 gradient. Accordingly, staining was present in the accessory olfactory bulb (AOB) before it was observable in rostra1 areas of the main olfactory bulb (MOB). Rat accessory
olfactory
bulb
Oligodendrocyte-immunoreactivity was first present in the AOB of the rat by PlO or Pll (Fig. IA), with most staining concentrated in the internal plexiform layer. Occasionally, immunoreactive fibers were present in lateral aspects of the granule cell layer. By PI6 immunoreactivity had dramatically increased in both regions (Fig. lB), although mature patterns were not seen until P20 (Fig. 1C). Dense labeling in the internal plexiform layer and numerous
Oligodendrocytes
and the olfactory
system
Fig. I. Horizontal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat accessory olfactory bulb (the dashed lines in panel B delineate the accessory olfactory bulb). (A) PI 1. Staining is apparent primarily in the internal plexiform layer (ipl). (B) P16. Labeling becomes apparent along the periphery of the granule cell layer (gel), marked by arrows. (C) P20. Immunoreactivity is dense in the internal plexiform layer. (D) P30. An adult-like distribution is reached, characterized by heavy labeling in the internal plexiform layer and minimal staining in the granule cell layer. epl, external plexiform layer. Scale bar = 200 pm.
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Philpot et al.
immunoreactive fibers extending from the periphery of the granule cell layer were also observed. Staining intensity continued to increase until P30, when adultlike levels were observed (Fig. 1D). Unlike patterns seen in the MOB (see below), oligodendrocyteimmunoreactivity was notably absent in all ages along the periphery of glomeruli in the AOB. Rat main olfactory bulb
Staining in the MOB was first observed about P15. At this time, immunoreactivity was confined to submitral areas. By P16, however, oligodendrocyteimmunoreactivity was discerned along the periphery of the glomeruli (Fig. 2A). Staining was very sparse in the external plexiform layer. Staining density increased in the MOB over the subsequent days, with the most noticeable changes occurring along the deep border of the glomeruli by P20 (Fig. 2B). Ten days later, at P30, fibers in the granule cell layer were densely immunoreactive (Fig. 2C). Furthermore, although oligodendrocyte-immunoreactive processes remained sparse in the external plexiform layer, this layer now contained an increased level of positively stained processes. Remarkably, glomeruli were now delicately delineated by oligodendrocyte-immunoreactivity along their entire peripheral extent. Quantitative analysis did confirm visual assessments of a clear laminar difference in oligodendrocyteimmunoreactivity levels: staining densities in the granule cell layer were approximately three times that of the external plexiform or glomerular layers (Table 1). In the mature animal, staining densities had increased in’all laminae of the MOB (Fig. 2D). Rat retrobulbar regions
Diffuse staining was observed uniformly across the entire extent of the lateral olfactory tract (LOT) on Pll (Fig. 3A). At this time, fibers running in the medial forebrain bundle also exhibited oligodendrocyte-immunoreactivity (Fig. 3A). By P14, the LOT of the rat showed moderate oligodendrocyte-immunoreactivity. By P20, the LOT was stained darkly and, once again, the entire tract appeared evenly labeled (Fig. 3B). The medial forebrain bundle also exhibited increased immunoreactivity (Fig. 3B). Oligodendrocyte-immunoreactivity in the anterior commissure was evident by Pll (Fig. 4A). Interestingly, immunoreactive fibers were first present in the mid-portion (rostro-caudally) of the commissure. Near the start of the third postnatal week, P20, the full breadth of the commissure was darkly labeled (Fig. 4B). EfSects of naris occlusion
Unilaterally naris occluded pups were observed at PlO, P20 and P30 (although oligodendrocyte-immunoreactivity in the bulb of PlO pups was minimal). As in normal rats, oligodendrocyte-immunoreactivity first appeared in caudal aspects of the bulb ipsilateral to occlusion around PI 1 and rapidly progressed
throughout the entire bulb over the next five days. Qualitatively, immunoreactive patterns and density of naris occluded rats appeared similar in both left and right bulbs at each age. Because past research has indicated that the severest effects of the procedure emerge after the longest survival times, quantitative analysis was performed on the P30 rats. Staining densities appeared similar in left and right bulbs in all three laminae examined (Table 1). Developmental changes in Monodelphis
domestica
Oligodendrocyte-immunoreactivity appeared much later in the opossum bulb than in the rat. In fact, although staining was heavy in hindbrain regions, by P20 the opossum bulb was consistently devoid of immunoreactivity. By P30, oligodendrocyteimmunoreactivity was rarely seen and was constrained to caudal aspects (Fig. 5A, B). It was not until P40 that staining was pronounced in the MOB (Fig. 5C). At this age, immunoreactive fibers were distinct in submitral regions and were just becoming apparent along the periphery of the glomeruli. Patterns and density of immunoreactivity in adult Monodelphis were similar to that observed in the adult rat (Fig. 5D). Characteristically, oligodendrocyte-immunoreactivity extended along the periphery of glomeruli. Also, submitral regions were densely labeled whereas the external plexiform layer contained only moderate staining. Oligodendrocyte-immunoreactivity in the P40 animals provided an interesting suggestion of topographical variations in the ontogeny of the anterior commissure (AC) as at this age, staining was seen only along its rostra1 extent (Fig. 6). These immunoreactive fibers of the commissure appeared to arise from the olfactory bulb. DISCUSSION
Rat
Oligodendrocyte-immunoreactivity is first present by Pl 1 in the region of the AOB. Immunoreactivity rapidly increases throughout the entire bulb over the next five days, with more gradual changes observable until P30. The pattern of labeling indicates that dramatic myelination occurs in the bulb beginning about Pll and continues through P30. These results are corroborated by the appearance and rapid increase in expression of myelin-specific mRNAs (e.g. myelin basic protein mRNA transcripts) in the rat bulb from P10-P15,2’ and the continued accrual of myelin basic protein in mice until P30.20 Although oligodendrocyte-immunoreactivity is initially present in the internal plexiform layer of the AOB, this may not imply that these immunoreactive fibers arise from neurons intrinsic to the AOB. Axons from both the MOB and AOB coalesce in the internal plexiform layer of the AOB as they exit the bulb to form the lateral olfactory tract,** and there are also substantial afferent projections to the bulb
Oligodendrocytes
and the olfactory
system
of oligodendrocyte/myelin-immunoreactivity in the Fig. 2. Horizontal sections depicting the developmenl rat main olfactory bulb (layers are delineated in panel B; glm, glomerular layer; epl, external plexiform layer; gel, granule cell layer). (A) Pl6. Staining is apparent in the granule cell layer, and labeling is just evident along the border of some glomeruli (curved arrow). (B) P20. Immunoreactivity extends along the deep border of the glomerular layer. (C) P30. Labeling increases in the granule cell layer, external plexiform layer and the glomerular layer. An adult-like distribution is achieved. (D) Adult. The entire extent of the glomerular border is encapsulated by immunoreactivity. Scale bar = 100 pm.
B. D. Philpot
1014 Table 1. Oligodendrocyte/myelin-immunoreactivity ous bulb layers of normal and experimental staining density (+S.E.M.) Control Glomerular layer External plexiform layer Granule cell layer
bulbs:
in varimean
Experimental
0.0532 (0.0288)
0.0572 (0.0341)
0.0400 (0.0332) 0.1848 (0.0435)
0.0351 (0.0164) 0.1637 (0.0454)
that may follow this course. Using the techniques employed in this study, it is difficult to assess the origin of immunoreactive fibers. The early appearance of staining in the AOB may be due to the general caudal to rostra1 gradient seen in myelination.‘4 Perhaps, however, cells in the AOB may become myelinated prior to those in the MOB. Hinds” has demonstrated that neuroglial precursors in the mouse bulb undergo their final division in the AOB before they do in the MOB, and Schwab and Price42 have suggested AOB fibers develop just prior to MOB fibers. In the adult rat MOB, the granule cell layer is approximately three times more heavily labeled than the external plexiform and glomerular layers. The dense labeling probably results from the large number of myelinated processes found in the region, including mitral/tufted cell axons exiting the bulb and afferent fibers traversing through the area to more superficial bulb regions. On the other hand, oligodendrocyte-immunoreactivity in the external plexiform layer Mayo represent myelinated mitral/tufted dendrites rather than axons. Previous electron microscopic studies in the mouse,9 rat,39 cat,48 monkey,40,46 and human4 have shown lamellar wrappings of dendrites in the external plexiform layer and/or peri-
et al.
glomerular region. The oligodendrocyte sheaths around dendrites have been hypothesized to play a role in the transmission of mitral cell dendritic action studies have potentials.33 Electron microscopic suggested there may not be myelinated axons present in the external plexiform layer.48 Adult glomeruli are conspicuously enveloped in immunoreactive processes, although there is a lack of staining within the glomerular neuropil. Similar patterns have been observed in other animals (see, for example Ref. 48, results above for Monodelphis). In the hedgehog, for example, electron microscopic observations coupled with “Rip” immunocytochemistry have demonstrated that oligodendrocytes encompass glomeruli and are found in the interstices separating them.47 As previously mentioned, the oligodendrocyte-immunoreactivity around glomeruli may reflect myelinated dendritic segments as well as perineuronal oligodendrocytes. These oligodendrocytes have been hypothesized to play a role in both glomerular structural support and in isolation of glomerular cells from extra-glomerular influences.47 Interestingly, perineuronal oligodendrocytes have been shown to have re-myelinating abilities.26,27 The juxtaposition of perineuronal oligodendrocytes against glomeruli is notable due to the unique resculpting occurring at the glomerular level. Because turnover of olfactory receptor neurons occurs regularly, new synapses are constantly being formed at the glomerular level. I3 Perineuronal oligodendrocytes may participate in the remyelination of the distal portions of mitral/tufted cell dendrites during glomerular remodeling. While the dendrites of mitral and tufted cells form synapses in glomeruli, their axons form the lateral olfactory tract. By Pl 1, oligodendrocyte-labeling in
Fig. 3. Coronal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat lateral olfactory tract and medial forebrain bundle. (A) Pl 1. The lateral olfactory tract (arrow) exhibits moderate levels of staining, and the medial forebrain bundle (mfb) is diffusely labeled. (B) P20. The lateral olfactory tract is heavily labeled, and a large extent of the medial forebrain bundle is immunoreactive. Scale bar = 500 pm.
Oligodendrocytes
and the olfactory system
1015
Fig. 4. Horizontal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat AC. (A) Pl 1. The midline of the commissure exhibits sparse labeling. (B) P20. The entire extent of the commissure is heavily stained. 3V, third ventricle. Scale bar = 100 pm.
the LOT is observed across the entire course of the tract. Moderate levels of immunoreactivity are seen by P14, and heavy labeling is demonstrated by P20. Studies using transgenic mice with a myelin basic protein-Lac Z transgene suggest similar findings: myelination of the LOT is initiated along its course starting around P9.14 Adult conduction velocities in the rat LOT are not reached until after P15,43 also paralleling our immunocytochemical observations. Many fibers enter the bulb via the medial forebrain bundle.j4 These centrifugal projections arise from various areas including the raphe, the ventral tegmental area, substantia innominata, and preoptic area.5*34 The data presented here shown that oligodendrocyteimmunoreactivity in the medial forebrain bundle is present by P14 and is densely distributed by P20. Other histological studies for myelin have shown that most of the rostra1 extent of the medial forebrain bundle fibers is myelinated, mirroring our immunocytochemical observations.34 However, Azmitia and Gannon’ have shown that both myelinated and unmyelinated serotonergic projections run in the medial forebrain bundle, although most serotonergic fibers are unmyelinated. In order to elucidate which of the centrifugal projections to the bulb are myelinated, further work, including electron microscopic immunocytochemistry, may be necessary. Initial oligodendrocyte-immunoreactivity is seen in the rat anterior commissure by Pl 1, concomitant to the appearance of labeling in the LOT. We observed only sparse immunoreactivity along the mid-portion of the commissure, but oligodendrocytes-immunoreactivity increases steadily until heavy labeling is seen by P20. Our immunocytochemical evidence is supported by the appearance and rapid increase in immunoreactivity for myelin-associated glycoprotein in the commissure from Pl 1 to P29” and by electron microscopic observations suggesting myelination starts around P12.” There are few electrophysiologi-
cal studies of AC development (but see Math and Davrainville3”), but interestingly, the appearance of oligodendrocyte-immunoreactivity coincides with the onset of function in the commissure’s olfactory component.23,24 Effects of naris closure in rats
The role of possible external influences on the development of myelination in the olfactory bulb was examined by surgically sealing one external naris on Pl. This procedure produces rapid changes in the bulb, including flattened EEG responses,” decreases in metabolic markers and reductions of protein synthesis.‘,** The possibility that it might also affect the development of bulb oligodendrocytes was examined for several reasons. First, there is a large requirement for protein synthesis during myelination,6 and protein formation is seriously affected by the treatment. Second, manipulations of other sensory modalities have been shown to affect patterns of myelination. For example, treating the optic nerve with tetrodotoxin, effectively blocking voltage-gated sodium channels, decreases oligodendrocyte precursor development during myelin formative periods,* while preaccelerates myelination.45 cocious eye opening However, naris closure did not affect patterns of immunoreactivity. Perhaps (1) the low level of metabolic and physiological activity remaining in deprived bulbs7,49 is sufficient to induce and maintain myelination and/or (2) myelination may potentially be conserved at a high metabolic cost to other events within the bulb. Monodephis
domestica
Patterns of oligodendrocyte-immunoreactivity are similar in both the adult rat and opossum (e.g. Figs 2, 5). Furthermore, in both species, the bulb is the last area in the brain where oligodendrocyte maturation occurs (e.g. Fig. 5). Indeed, as mentioned,
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et al.
Fig. 5. Horizontal sections depicting the development of oligodendrocyte/myehn-immunoreactivity in Monodelphis domestica. (A) P20. Staining is confined to hindbrain regions (arrow) and is absent from the olfactory bulb (ob). (B) P30. Immunoreactivity is most apparent in caudal portions of the brain (arrow), and is rarely apparent in the olfactory bulb. (C) P40 main olfactory bulb. Labeling is apparent in the granule cell layer, and staining is just apparent along the periphery of glomeruli, marked by a curved arrow (“Rip” antibody). (D) Adult main olfactory bulb. Immunoreactivity is dense in the granule cell layer, moderate in the external plexiform layer, and present along the border of glomeruli. In C and D, the deep glomerular border lies across from the top arrowhead, and the mitral cell layer lies across from the bottom arrowhead. glm, glomerular layer; epl, external plexiform layer; gel, granule cell layer. Scale bar A, B = 1000 pm; scale bar C, D = 100 pm.
Ohgodendrocytes
and the olfactory system
1017
Fig. 6. Horizontal section (Rostra1 at top) depicting “Rip” immunoreactivity in the P40 Monodelphis AC. Staining is primarily present in rostra1 position, likely corresponding to the olfactory component of the commissure. 3V, third ventricle. Scale bar = 250 pm.
remyelination studies using tissue grafts have already taken advantage of the late maturation of oligodendrocytes in the mouse olfactory bulb.16 However, in the rodent, there is only a brief postnatal window when oligodendrocytes remain immature enough to succeed bulb presents a in remyelination. I6 The opossum structure from which immature oligodendrocytes are easily accessible over an extended postnatal period. For example, initial staining in the rat bulb occurs by PI 1 whereas staining in the opossum is not apparent until P30. Furthermore, only five days separate the appearance of oligodendrocyte-immunoreactivity in the rat bulb and the subsequent expression at the glomerular level; however, this same series of events occurs over 10 days in the opossum bulb. The slow maturation of the opossum bulb allows increased resolution into the time course of ontogenetic changes in oligodendrocyte-immunoreactivity, providing unique glimpses into developmental changes. For example, we examined patterned changes of oligodendrocyte-immunoreactivity in the anterior commissure because studies in the mouse suggested myelination occurs first in the anterior limb (Sturrock, 1976, cited in Ref. 25) whereas myelination in the hamster bulb is initiated in caudal portions.25 The opossum anterior commissure first exhibits oligodendrocyte-immunoreactivity in rostra1 aspects (e.g. Fig. 6) likely corresponding to the olfactory component of this commissure. Whereas a gradient of oligodendrocyte-immunoreactivity is easily discerned over the development of the opossum commissure, a similar pattern in the rat was not observed, perhaps due to its rapid maturation.
The relatively late onset and slow maturation of the Monodelphis bulb allow examination of the sequential expression of oligodendrocyte-specific proteins at a high resolution, Biochemical changes in myelin composition during rat brain development have given insights into the process of myelination,3s but, as more myelin-specific proteins are identified. it will become increasingly important to understand sequential events in myelinogenesis. This work, in turn, will lead to an increased understanding of the transformation of oligodendrocytes that permit them to act as a permissive substrate for neurite outgrowth at immature stages of development to acting as a nonpermissive substrate in more mature forms4’ CONCLUSIONS
In sum, oligodendrocyte-immunoreactivity first appears in the rat AOB by Pll, and levels rapidly increase in the entire bulb over the next five days. Also, oligodendrocyte-immunoreactivity in the LOT and AC are first present by PI 1 and are heavily stained by P20. Furthermore, oligodendrocyteimmunoreactivity in the bulb is not altered by unilateral naris closure, despite the large changes in protein synthesis associated with this manipulation. Finally, oligodendrocyte changes in the Monodelphis bulb mirror changes in the rat bulb, although initial staining appears much later and the time course of development occurs over an extended period in the opossum. The late onset and slow development of oligodendrocyte-immunoreactivity in the opossum bulb suggest it is a promising laboratory animal for the study of myelinogenesis.
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Acknowledgements-Thanks to M. Paternostro, C. Byrd, D. Cummings and D. Malun for thoughtful advice. Special thanks to E. Geisert and C. Dyer for suggestions on antibody use, and to J. Black for a gener-
et al.
ous supply of the “Rip” antibody. This work was supported by grants from the National Institutes of Health (DC-00338) and National Sciences Foundation (BNS-8919751)
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(1994) Unilateral naris closure and olfactory system development. Brain Res. Rev. 19, 146-160. 8. Brunjes P. C., Jazaeri A. A. and Sutherland M. J. (1992) Olfactory bulb organization and development in Monodelphis domestica (grey short-tailed opossum). J. camp. Neural. 320, 5444554. 9. Burd G. D. (1980) Myelinated dendrites and neuronal perikarya in the olfactory bulb of the mouse. Brain Res. 181, 450-454. 10. Culhnan W. E. and Zaborsky L. (1991) Organization of ascending hypothalamic projections to the rostra1 forebrain with special reference to the innervation of cholinergic projection neurons. J. camp. Neural. 306, 631-667. 11. Dyer C. A., Hickey W. F. and Geisert E. E. (1991) Myelin/oligodendrocyte-specific protein: a novel surface membrane protein that associates with microtubules. J. Neurosci. Res. 28, 607-613. 12. Fadem B. H., Trupin G. L., Maliniak E., VandeBerg J. L. and Hayssen V. (1982) Care and breeding of the gray, short-tailed opossum (Monodelphis domestica). Lab. Anim. Sci. 32, 405-409. 13. Farbman A. I. (1992) Cell BiologJt of Olfaction. Cambridge University Press, New York. 14. Foran D. R. and Peterson A. C. (1992) Myelin acquisition in the central nervous system of the mouse revealed by and MBP-Lac Z transgene. J. Neurosci. 12, 4890-4897. IS. Friedman B., Hockfield S., Black J. A., Woodruff K. A. and Waxman S. G. (1989) In situ demonstration of mature oligodendrocytes and their processes: an immunocytochemical study with a new monoclonal antibody, Rip. Glia 2, 380-390. 16. Gout O., Gansmuller A., Baumann N. and Gumpel M. (1988) Remyehnation by transplanted oligodendrocytes of a demyelinated lesion in the spinal cord of the adult shiverer mouse. Neurosci. Lett. 87, 195-199. 17. Gray C. M. and Skinner J. E. (1988) Centrifugal regulation of neuronal activity in the olfactory bulb of the waking rabbit as revealed by reversible cryogenic blockade. Exp. Brain Res. 69, 378-386. 18. Guadano-Ferraz A., Berbel P., Balboa R. and Innocenti G. M. (1992) The development of the anterior commissure in normal and hypothyroid rats. Eur. J. Neurosci. Suppl. 4, 225. 19. Hinds J. W. (1968) Autoradiographic study of histogenesis in the mouse olfactory bulbI. Time of origin of neurons and neuroglia. J. camp. Neural. 134, 2877304. of the oligodendrocytes 20. Jacque S. M., Collet A., Raoul M., Monge M. and Gumpel M. (1985) Functional maturation and myelin basic protein expression in the olfactory bulb of the mouse. Devl Brain Res. 21, 277-282. 21. Kanfer J., Parenty M., Gouejet-Zalc C., Monge M., Bernier L., Campagnoni A. T., Dautigny A. and Zalc B. (1989) Developmental expression of myelin proteolipid, basic protein, and 2’,3’-cyclic nucleotide 3’-phosphoesterase transcripts in different rat brain regions. J. molec. Neurosci. 1, 39946. 22. Korol D. L. and Brunjes P. C. (1990) Rapid changes in 2-DG uptake and amino acid incorporation following unilateral odor deprivation: a laminar analysis. Deul Bruin Res. 52, 75585. D. and Hall W. G. (1987) New routes to early memories. Science 238, 7866788. 23. Kucharski 24. Kucharski D., Burka N. and Hall W. G. (1990) The anterior limb of the anterior commissure is an access route to contralaterally stored olfactory preference memories. Psychobiology 18, 195-204. of the cerebral commissures of the hamster, as revealed by a monoclonal 25. Lent R. and Jhaveri S. (1991) Myelination antibody specific for oligodendrocytes. Deal Brain Res. 66, 193-201. satellite oligodendrocyte: a role in remyelination. Acfa Neuropath. 47, 49-53. 26. Ludwin S. K. (1979) The perineuronal S. K. (1984) The function of perineuronal satellite ohgodendrocytes: an immunohistochemical study. 27. Ludwin Neuropath. Appl Neurobiol. 10, 143-149. 28. Macrides F. and Davis B. J. (1983) The olfactory bulb. In Chemical Neuroanatomy (ed. Emson P. C.), pp. 391-426. Raven Presq New York. R. E. (1992) Myelin: Biology and Chemistry. CRC Press, West Palm Beach, FL. 29. Martenson J. L. (1980) Electrophvsiological study on the postnatal development of mitral cell activity 30. Math F. and Davrainville in the rat olfactory bulb. Brain Res. .190, 2431247. _ F. A.. Mozell R. L.. Carson M. J.. Shinar Y.. Mever M. D. and Marchetti N. (1993) Reeulation of 31. McMorris oligodendrocyte development and central nervous system myelination by insulin-like growth factors. km. N. Y. Acad. Sci. 692, 321-334. 32. Meisami E. (1976) Effects of olfactory deprivation on postnatal growth of the rat olfactory bulb utilizing a new method for the production of neonatal unilateral anosmia. Brain Res. 107, 437-444. 33. Mori K. and Takagi S. F. (1975) Spike generation in the mitral cell dendrite of the rabbit olfactory bulb. Brain Res. 100, 685-689. R., Geeraedts L. M. G. and Veening J. G. (1982) The medial forebrain bundle of the rat--I. General 34. Nieuwenhuys introduction. J. camp. Neural. 206, 49-81.
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35. Norton W. T. and Poduslo S. E. (1973) Myelination in rat brain: changes in myelin composition during brain maturation. J. Neurochem. 21, 759-773. 36. Pesheva P., Spiess E. and Schachner M. (1989) 51-160 and 51-180 are oligodendrocyte-secreted nonpermissive substrates for cell adhesion. J. Cell Biol. 109, 176551778. 37. Pfeiffer S. E., Warrington A. E. and Bansal R. (1993) The oligodendrocyte and its many cellular processes. Trends Cell Biol. 3, 191-197. 38. Philpot B. D., Jazaeri A. A. and Brunjes P. C. (1994) The development of serotonergic projections to the olfactory opossum). Devl Brain Res. 77, 265-270. bulb of Monodelphis domestica (the grey, short-tailed 39. Pinching A. J. (1970) Synaptic connections in the glomerular layer of the olfactory bulb. J. Physiol., Land. 210, 14-15. 40. Pinching A. J. (1971) Myelinated dendritic segments in the monkey olfactory bulb. Brain Res. 29, 133-138. 41. Schwab M. E. and Caroni P. (1988) Oligodendrocytes and CNS myelin are nonpermissive substrates for neurite growth and fibroblast spreading in vitro. J. Neurosci. 8, 2381-2393. in fetal and neonatal 42. Schwab J. E. and Price J. L. (1978) The cortical projection of the olfactory bulb: development rats correlated with quantitative variations in adult rats. Brain Res. 151, 369-374. 43. Schwab J. E., Haberly L. B. and Price J. L. (1984) The development of physiological responses of the piriform cortex in rats to stimulation of the lateral olfactory tract. J. camp. Neural. 223, 223-237. 44. Sternberger N. H., Quarles R. H., Itoyama Y. and Webster H. DeF. (1979) Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myehn-forming cells of developing rat. Neurobiology 76, 1510-1514. 45. Tauber H., Waehneldt T. V. and Neuhoff V. (1980) Myelination in rabbit optic nerves is accelerated by artificial eye opening. Neurosci. Left. 16, 235-238. 46. Tigges M. and Tigges J. (1980) Distribution of morphology of myelinated perikarya and dendrites in the olfactory bulb of primates. J. Neurocytol. 9, 825-834. 47. Valverde F. and Lopez-Mascaraque L. (1991) Neuroglial arrangements in the olfactory glomeruli of the hedgehog. J. camp. Neural. 308, 658-674. 48. Willey J. T. (1973) The ultrastructure of the cat olfactory bulb. J. camp. Neural. 152, 21 I-232. 49. Wilson D. A. and Wood J. G. (1992) Functional consequences of unilateral olfactory deprivation: time course and age sensitivity. Neuroscience 49, 183-192. (Accepted 30 January 1995)
Pergamon
0306-4522(95)00095-X
Elsevier Science Ltd Copyright cc, 1995 IBRO Printed in Great Britain. All rights reserved
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OLIGODENDROCYTE/MYELIN-IMMUNOREACTIVITY THE DEVELOPING OLFACTORY SYSTEM B. D. PHILPOT, Department
of Psychology,
A. Y. KLINTSOVA
102 Gilmer
Hall, University
IN
and P. C. BRUNJES*
of Virginia,
Charlottesville,
VA 22903, U.S.A.
Abstract-Immunocytochemistry was used to characterize oligodendrocyte maturation in the developing mammalian olfactory system. Postnatal day 10-16, 20, 30 and adult rats were examined, as well as postnatal day 20, 30, 40 and adult Monodelphis domestica (the grey, short-tailed opossum). In rats, oligodendrocyte/myelin-immunoreactivity first appears in the accessory olfactory bulb by day 11, with labeling rapidly increasing throughout the entire bulb over the next five days. An adult pattern of immunoreactivity, characterized by dense labeling in the granule cell layer, sparse immunoreactivity in the external plexiform layer, and staining along the periphery of glomeruli, is attained by day 30. Staining is apparent in both the lateral olfactory tract and anterior commissure by day 11, and becomes heavy by day 20. While patterns of oligodendrocyte/myelin-immunoreactivity in the adult Monodelphis and rat bulb are similar, staining first appears much later in the opossum (around day 30), and maturation occurs more slowly. For example, rostrakaudal gradients in the development of staining in the anterior commissure were noted which were not seen in the rat. These differences emerge because Monodelphis’ slower growth allows more resolution into developmental sequences. Finally, in rats, unilateral naris closure on the day after birth, which significantly alters normal patterns of bulb development, has no effect on the pattern and level of immunoreactivity even after long (30 day) survival periods. In both normal and naris occluded rats, oligodendrocyte/myelin-immunoreactivity is found in caudal aspects of the rat bulb on day 11 and subsequently progresses throughout the entire bulb over the next five days. Patterns in the Monodelphis bulb mirror those observed in the rat, however, staining appears later and progresses more slowly, suggesting Monodelphis is a useful animal for examining early myelin formation.
During early development, multi-potential 02-A progenitor cells migrate to specific destinations and differentiate into mature oligodendrocytes as well as other cell types. The oligodendrocytes undergo large changes in protein synthesis culminating in the formation of myelin around axons, dendrites, and even somata.29.37 Proper formation of myelin has important consequences for physiological function. Myelination increases membrane resistance and lowers membrane capacitance, thereby increasing signal conduction velocity. Selective augmentation of signal velocity can greatly affect patterns of system processing. Furthermore, the insulative properties of perineuronal oligodendrocytes may serve to isolate signal input on to somata.47 Oligodendrocytes also have a role in morphological development as they release and respond to numerous growth factors.“x3’ For example, myelin-forming oligodendrocytes secrete36 and express4’ factors that can inhibit neurite outgrowth, thus preserving the integrity of established myelinated fibers. Increasing evidence for the important role of oligodendrocytes in cellular dynamics and
*To whom correspondence should be addressed. Abbreuiafions: ABC, avidin-biotin peroxide complex; AC, anterior commissure; AOB, accessory olfactory bulb; LOT, lateral olfactory tract; MOB, main olfactory bulb; P, postnatal day; PBS, phosphate-buffered saline.
the prevalence of devastating demyelinating diseases such as multiple sclerosis have served as an impetus for characterizing the development of oligodendrocytes in mammals. The olfactory bulb is an accessible model for examining the development of myelin/oligodendrocytes for several reasons. First, the bulb is well laminated and thus convenient for tracking myelination patterns. Second, there is sufficient electrophysiological data in the developing rat bulb to correlate the time course of myelination to observed changes in signal conduction velocities. Finally, due to the general posterior-to-anterior gradient of myelin formation and the immaturity of the bulb at birth, the region is one of the last areas where myelination occurs. The bulb therefore provides an easily obtainable source of immature oligodendrocytes and a view of the entire sequence of their expression. Indeed, this feature may prove clinically important, as transplanting olfactory bulb tissue containing undifferentiated oligodendrocytes into demyelinated spinal cord areas has been demonstrated to allow subsequent remyelination.” The present study characterizes myelination and oligodendrocyte maturation in the rat olfactory bulb via immunocytochemical techniques in hope that this information will (I) help elucidate cytoarchitectonic organization of oligodendrocytes and their processes and (2)
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correlate the degree of myelination to known electrophysiological changes in conduction velocity during development. Furthermore, this study examines the possible role of afferent innervation in proper myelination. Blocking airflow through one half of the nasal cavity by unilateral naris closure results in profound changes in metabolic turnover and protein synthesis in the ipsilateral bulb.’ Due to the large requirements of protein synthesis during myelin formation, we hypothesized that global reductions in bulb protein synthesis might reflect alterations in patterns of myelin formation. Finally, myelin/oligodendrocyte development was also traced in Monodelphis domesopossum. Monodelphis has tica, the grey short-tailed become increasingly popular because offspring are born in a very immature state, they develop quite slowly, and, since they are a pouchless species, pups are easily accessible for experimental manipulation. Morphological characteristics of the Monodelphis olfactory bulb have been previously established,” and recent studies suggest the prolonged developmental time course offers a higher degree of resolution into morphological events as compared with studies in rats.38 EXPERIMENTAL
PROCEDURES
et al.
specific binding and increase penetration, the Chemicon antibody and all the avidin-biotin peroxide complex (ABC) solutions were diluted in PBS supplemented with 0.25% lambda carrageenan (Sigma type IV) and 0.25% Triton X-100.‘” Slides were incubated for 24 h at 4°C (Chemicon antibody) or at room temperature (“Rip” antibody), washed four times with PBS, and transferred into a solution containing biotinylated secondary antibodies (Dako, rabbit anti-mouse) for 1 h. Following four rinses in PBS, slides were placed into an ABC solution (Vector; 100~1 each of solutions A and B in 50ml solution, an alteration of the Vector protocol) for 90 min. After four washes in PBS slides were treated with diaminobenzidine for 15.-25 min. Slides were then dehydrated, cleared and coverslipped with DPX (BDH Ltd, Poole U.K.). In some instances sections were lightly counterstained with Methylene Blue. Control experiments, consisting of the same protocol but omitting exposure to the primary antibody, consistently resulted in the absence of staining. The specificity of the antibodies used in this study have been previously examined.“,” The Chemicon antibody detects a unique myelin/oligodendrocyte-specific protein associated with microtubules.” The “Riu” antibodv co-localizes with myelin basic protein and is absent from processes staining for glial fibrillary acidic protein.15 Furthermore, electron microscopic studies show oligodendrocytes to be immunoreactive for “Rip”.j Results outlined below indicate that patterns of immunoreactivity are similar for the two antibodies, although the staining with the “Rip” antibody was more pronounced and labeled somata in addition to their processes. Photomicrographs are of immunoreactivity using the Chemicon antibody unless otherwise noted.
Subjects Offspring of Long-Evans hooded rats purchased from the Charles River Breeding Laboratories (Wilmington, MD) were used. Rats were housed in polypropylene cages (48 x 25 x 16 cm) and given food and water ad libitum. The colony room was maintained on a 16/8 h light/dark cycle. Litters were culled to 10 on the day after birth postnatal day 1 (Pl). PlO-P16, P20, P30 and adult rats were examined. For studies of unilaterally naris occluded pups, PI subjects were cold anesthetized and received either unilateral occlusion of the right external naris via cautery or a sham manipulation consisting of a cautery on the dorsal surface of the nose.32 Pups were then warmed and returned to their mothers. Naris occluded and sham operated rat pups were examined at PlO, P20 and P30. Breeding stock for the opossum colony was purchased from the Southwest Foundation for Biomedical Research (San Antonio, TX) and maintained according to the recommendations of Fadem et al.‘* Opossum litters were not culled due to small litter sizes (n = 3-9). P20, P30, P40 and adults were examined. In all experiments, two to six animals from different litters were used at each age (with the exception of only using animals from one litter in P40 opossums) to ensure a representative sample. Tissue preparation Subjects were given an overdose of barbiturates and perfused intracardially with 0.1 M phosphate-buffered saline (PBS), pH 7.4, followed by 4% paraformaldehyde in PBS with 10% sucrose. Brains were then carefully dissected and cryoprotected overnight with 20-30% sucrose in 0.1 M PBS. The following day, 20pm sections were cut on a cryostat in either the coronal or horizontal plane. Sections were thaw-mounted onto gelatin-coated slides, placed in an evacuated chamber, and stored overnight at 4°C. Slides were then rinsed three to four times with PBS and incubated with a mouse monoclonal antibody for myelin/oligodendrocytes. Primary antibodies used were either from Chemicon (antimyelin/oligodendrocyte specific protein), used in a 1: 1000 dilution, or “Rip”, (generously supplied by Dr Joel Black, Yale University) used in a 1: 10 dilution. To reduce non-
Levels of oligodendrocyte-immunoreactivity were determined in four sections from each of two animals that had undergone unilateral naris closure on Pl and were reared until P30. Both left and right bulbs (i.e. contralateral and ipsilateral to the occluded naris, respectively) were analysed. Each bulb sections was divided into rostral, middle and caudal portions, and from each of these subdivisions, 300 pm long segments from the medial wall were selected. Within these zones, the entire width of the granule cell, external plexiform, and glomerular layers were examined. Images were thresholded such that areas containing diaminobenzidine reaction products were black and the background white. An image analysis system (Olympus Cue-2) then calculated the ratio of the area stained to the total test area. Care was taken to ensure that thresholding was identical in both left and right bulbs. RESULTS
Oligodendrocyte-immunoreactivity in the olfactory bulb proceeded in a caudal-to-rostra1 gradient. Accordingly, staining was present in the accessory olfactory bulb (AOB) before it was observable in rostra1 areas of the main olfactory bulb (MOB). Rat accessory
olfactory
bulb
Oligodendrocyte-immunoreactivity was first present in the AOB of the rat by PlO or Pll (Fig. IA), with most staining concentrated in the internal plexiform layer. Occasionally, immunoreactive fibers were present in lateral aspects of the granule cell layer. By PI6 immunoreactivity had dramatically increased in both regions (Fig. lB), although mature patterns were not seen until P20 (Fig. 1C). Dense labeling in the internal plexiform layer and numerous
Oligodendrocytes
and the olfactory
system
Fig. I. Horizontal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat accessory olfactory bulb (the dashed lines in panel B delineate the accessory olfactory bulb). (A) PI 1. Staining is apparent primarily in the internal plexiform layer (ipl). (B) P16. Labeling becomes apparent along the periphery of the granule cell layer (gel), marked by arrows. (C) P20. Immunoreactivity is dense in the internal plexiform layer. (D) P30. An adult-like distribution is reached, characterized by heavy labeling in the internal plexiform layer and minimal staining in the granule cell layer. epl, external plexiform layer. Scale bar = 200 pm.
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Philpot et al.
immunoreactive fibers extending from the periphery of the granule cell layer were also observed. Staining intensity continued to increase until P30, when adultlike levels were observed (Fig. 1D). Unlike patterns seen in the MOB (see below), oligodendrocyteimmunoreactivity was notably absent in all ages along the periphery of glomeruli in the AOB. Rat main olfactory bulb
Staining in the MOB was first observed about P15. At this time, immunoreactivity was confined to submitral areas. By P16, however, oligodendrocyteimmunoreactivity was discerned along the periphery of the glomeruli (Fig. 2A). Staining was very sparse in the external plexiform layer. Staining density increased in the MOB over the subsequent days, with the most noticeable changes occurring along the deep border of the glomeruli by P20 (Fig. 2B). Ten days later, at P30, fibers in the granule cell layer were densely immunoreactive (Fig. 2C). Furthermore, although oligodendrocyte-immunoreactive processes remained sparse in the external plexiform layer, this layer now contained an increased level of positively stained processes. Remarkably, glomeruli were now delicately delineated by oligodendrocyte-immunoreactivity along their entire peripheral extent. Quantitative analysis did confirm visual assessments of a clear laminar difference in oligodendrocyteimmunoreactivity levels: staining densities in the granule cell layer were approximately three times that of the external plexiform or glomerular layers (Table 1). In the mature animal, staining densities had increased in’all laminae of the MOB (Fig. 2D). Rat retrobulbar regions
Diffuse staining was observed uniformly across the entire extent of the lateral olfactory tract (LOT) on Pll (Fig. 3A). At this time, fibers running in the medial forebrain bundle also exhibited oligodendrocyte-immunoreactivity (Fig. 3A). By P14, the LOT of the rat showed moderate oligodendrocyte-immunoreactivity. By P20, the LOT was stained darkly and, once again, the entire tract appeared evenly labeled (Fig. 3B). The medial forebrain bundle also exhibited increased immunoreactivity (Fig. 3B). Oligodendrocyte-immunoreactivity in the anterior commissure was evident by Pll (Fig. 4A). Interestingly, immunoreactive fibers were first present in the mid-portion (rostro-caudally) of the commissure. Near the start of the third postnatal week, P20, the full breadth of the commissure was darkly labeled (Fig. 4B). EfSects of naris occlusion
Unilaterally naris occluded pups were observed at PlO, P20 and P30 (although oligodendrocyte-immunoreactivity in the bulb of PlO pups was minimal). As in normal rats, oligodendrocyte-immunoreactivity first appeared in caudal aspects of the bulb ipsilateral to occlusion around PI 1 and rapidly progressed
throughout the entire bulb over the next five days. Qualitatively, immunoreactive patterns and density of naris occluded rats appeared similar in both left and right bulbs at each age. Because past research has indicated that the severest effects of the procedure emerge after the longest survival times, quantitative analysis was performed on the P30 rats. Staining densities appeared similar in left and right bulbs in all three laminae examined (Table 1). Developmental changes in Monodelphis
domestica
Oligodendrocyte-immunoreactivity appeared much later in the opossum bulb than in the rat. In fact, although staining was heavy in hindbrain regions, by P20 the opossum bulb was consistently devoid of immunoreactivity. By P30, oligodendrocyteimmunoreactivity was rarely seen and was constrained to caudal aspects (Fig. 5A, B). It was not until P40 that staining was pronounced in the MOB (Fig. 5C). At this age, immunoreactive fibers were distinct in submitral regions and were just becoming apparent along the periphery of the glomeruli. Patterns and density of immunoreactivity in adult Monodelphis were similar to that observed in the adult rat (Fig. 5D). Characteristically, oligodendrocyte-immunoreactivity extended along the periphery of glomeruli. Also, submitral regions were densely labeled whereas the external plexiform layer contained only moderate staining. Oligodendrocyte-immunoreactivity in the P40 animals provided an interesting suggestion of topographical variations in the ontogeny of the anterior commissure (AC) as at this age, staining was seen only along its rostra1 extent (Fig. 6). These immunoreactive fibers of the commissure appeared to arise from the olfactory bulb. DISCUSSION
Rat
Oligodendrocyte-immunoreactivity is first present by Pl 1 in the region of the AOB. Immunoreactivity rapidly increases throughout the entire bulb over the next five days, with more gradual changes observable until P30. The pattern of labeling indicates that dramatic myelination occurs in the bulb beginning about Pll and continues through P30. These results are corroborated by the appearance and rapid increase in expression of myelin-specific mRNAs (e.g. myelin basic protein mRNA transcripts) in the rat bulb from P10-P15,2’ and the continued accrual of myelin basic protein in mice until P30.20 Although oligodendrocyte-immunoreactivity is initially present in the internal plexiform layer of the AOB, this may not imply that these immunoreactive fibers arise from neurons intrinsic to the AOB. Axons from both the MOB and AOB coalesce in the internal plexiform layer of the AOB as they exit the bulb to form the lateral olfactory tract,** and there are also substantial afferent projections to the bulb
Oligodendrocytes
and the olfactory
system
of oligodendrocyte/myelin-immunoreactivity in the Fig. 2. Horizontal sections depicting the developmenl rat main olfactory bulb (layers are delineated in panel B; glm, glomerular layer; epl, external plexiform layer; gel, granule cell layer). (A) Pl6. Staining is apparent in the granule cell layer, and labeling is just evident along the border of some glomeruli (curved arrow). (B) P20. Immunoreactivity extends along the deep border of the glomerular layer. (C) P30. Labeling increases in the granule cell layer, external plexiform layer and the glomerular layer. An adult-like distribution is achieved. (D) Adult. The entire extent of the glomerular border is encapsulated by immunoreactivity. Scale bar = 100 pm.
B. D. Philpot
1014 Table 1. Oligodendrocyte/myelin-immunoreactivity ous bulb layers of normal and experimental staining density (+S.E.M.) Control Glomerular layer External plexiform layer Granule cell layer
bulbs:
in varimean
Experimental
0.0532 (0.0288)
0.0572 (0.0341)
0.0400 (0.0332) 0.1848 (0.0435)
0.0351 (0.0164) 0.1637 (0.0454)
that may follow this course. Using the techniques employed in this study, it is difficult to assess the origin of immunoreactive fibers. The early appearance of staining in the AOB may be due to the general caudal to rostra1 gradient seen in myelination.‘4 Perhaps, however, cells in the AOB may become myelinated prior to those in the MOB. Hinds” has demonstrated that neuroglial precursors in the mouse bulb undergo their final division in the AOB before they do in the MOB, and Schwab and Price42 have suggested AOB fibers develop just prior to MOB fibers. In the adult rat MOB, the granule cell layer is approximately three times more heavily labeled than the external plexiform and glomerular layers. The dense labeling probably results from the large number of myelinated processes found in the region, including mitral/tufted cell axons exiting the bulb and afferent fibers traversing through the area to more superficial bulb regions. On the other hand, oligodendrocyte-immunoreactivity in the external plexiform layer Mayo represent myelinated mitral/tufted dendrites rather than axons. Previous electron microscopic studies in the mouse,9 rat,39 cat,48 monkey,40,46 and human4 have shown lamellar wrappings of dendrites in the external plexiform layer and/or peri-
et al.
glomerular region. The oligodendrocyte sheaths around dendrites have been hypothesized to play a role in the transmission of mitral cell dendritic action studies have potentials.33 Electron microscopic suggested there may not be myelinated axons present in the external plexiform layer.48 Adult glomeruli are conspicuously enveloped in immunoreactive processes, although there is a lack of staining within the glomerular neuropil. Similar patterns have been observed in other animals (see, for example Ref. 48, results above for Monodelphis). In the hedgehog, for example, electron microscopic observations coupled with “Rip” immunocytochemistry have demonstrated that oligodendrocytes encompass glomeruli and are found in the interstices separating them.47 As previously mentioned, the oligodendrocyte-immunoreactivity around glomeruli may reflect myelinated dendritic segments as well as perineuronal oligodendrocytes. These oligodendrocytes have been hypothesized to play a role in both glomerular structural support and in isolation of glomerular cells from extra-glomerular influences.47 Interestingly, perineuronal oligodendrocytes have been shown to have re-myelinating abilities.26,27 The juxtaposition of perineuronal oligodendrocytes against glomeruli is notable due to the unique resculpting occurring at the glomerular level. Because turnover of olfactory receptor neurons occurs regularly, new synapses are constantly being formed at the glomerular level. I3 Perineuronal oligodendrocytes may participate in the remyelination of the distal portions of mitral/tufted cell dendrites during glomerular remodeling. While the dendrites of mitral and tufted cells form synapses in glomeruli, their axons form the lateral olfactory tract. By Pl 1, oligodendrocyte-labeling in
Fig. 3. Coronal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat lateral olfactory tract and medial forebrain bundle. (A) Pl 1. The lateral olfactory tract (arrow) exhibits moderate levels of staining, and the medial forebrain bundle (mfb) is diffusely labeled. (B) P20. The lateral olfactory tract is heavily labeled, and a large extent of the medial forebrain bundle is immunoreactive. Scale bar = 500 pm.
Oligodendrocytes
and the olfactory system
1015
Fig. 4. Horizontal sections depicting the development of oligodendrocyte/myelin-immunoreactivity in the rat AC. (A) Pl 1. The midline of the commissure exhibits sparse labeling. (B) P20. The entire extent of the commissure is heavily stained. 3V, third ventricle. Scale bar = 100 pm.
the LOT is observed across the entire course of the tract. Moderate levels of immunoreactivity are seen by P14, and heavy labeling is demonstrated by P20. Studies using transgenic mice with a myelin basic protein-Lac Z transgene suggest similar findings: myelination of the LOT is initiated along its course starting around P9.14 Adult conduction velocities in the rat LOT are not reached until after P15,43 also paralleling our immunocytochemical observations. Many fibers enter the bulb via the medial forebrain bundle.j4 These centrifugal projections arise from various areas including the raphe, the ventral tegmental area, substantia innominata, and preoptic area.5*34 The data presented here shown that oligodendrocyteimmunoreactivity in the medial forebrain bundle is present by P14 and is densely distributed by P20. Other histological studies for myelin have shown that most of the rostra1 extent of the medial forebrain bundle fibers is myelinated, mirroring our immunocytochemical observations.34 However, Azmitia and Gannon’ have shown that both myelinated and unmyelinated serotonergic projections run in the medial forebrain bundle, although most serotonergic fibers are unmyelinated. In order to elucidate which of the centrifugal projections to the bulb are myelinated, further work, including electron microscopic immunocytochemistry, may be necessary. Initial oligodendrocyte-immunoreactivity is seen in the rat anterior commissure by Pl 1, concomitant to the appearance of labeling in the LOT. We observed only sparse immunoreactivity along the mid-portion of the commissure, but oligodendrocytes-immunoreactivity increases steadily until heavy labeling is seen by P20. Our immunocytochemical evidence is supported by the appearance and rapid increase in immunoreactivity for myelin-associated glycoprotein in the commissure from Pl 1 to P29” and by electron microscopic observations suggesting myelination starts around P12.” There are few electrophysiologi-
cal studies of AC development (but see Math and Davrainville3”), but interestingly, the appearance of oligodendrocyte-immunoreactivity coincides with the onset of function in the commissure’s olfactory component.23,24 Effects of naris closure in rats
The role of possible external influences on the development of myelination in the olfactory bulb was examined by surgically sealing one external naris on Pl. This procedure produces rapid changes in the bulb, including flattened EEG responses,” decreases in metabolic markers and reductions of protein synthesis.‘,** The possibility that it might also affect the development of bulb oligodendrocytes was examined for several reasons. First, there is a large requirement for protein synthesis during myelination,6 and protein formation is seriously affected by the treatment. Second, manipulations of other sensory modalities have been shown to affect patterns of myelination. For example, treating the optic nerve with tetrodotoxin, effectively blocking voltage-gated sodium channels, decreases oligodendrocyte precursor development during myelin formative periods,* while preaccelerates myelination.45 cocious eye opening However, naris closure did not affect patterns of immunoreactivity. Perhaps (1) the low level of metabolic and physiological activity remaining in deprived bulbs7,49 is sufficient to induce and maintain myelination and/or (2) myelination may potentially be conserved at a high metabolic cost to other events within the bulb. Monodephis
domestica
Patterns of oligodendrocyte-immunoreactivity are similar in both the adult rat and opossum (e.g. Figs 2, 5). Furthermore, in both species, the bulb is the last area in the brain where oligodendrocyte maturation occurs (e.g. Fig. 5). Indeed, as mentioned,
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et al.
Fig. 5. Horizontal sections depicting the development of oligodendrocyte/myehn-immunoreactivity in Monodelphis domestica. (A) P20. Staining is confined to hindbrain regions (arrow) and is absent from the olfactory bulb (ob). (B) P30. Immunoreactivity is most apparent in caudal portions of the brain (arrow), and is rarely apparent in the olfactory bulb. (C) P40 main olfactory bulb. Labeling is apparent in the granule cell layer, and staining is just apparent along the periphery of glomeruli, marked by a curved arrow (“Rip” antibody). (D) Adult main olfactory bulb. Immunoreactivity is dense in the granule cell layer, moderate in the external plexiform layer, and present along the border of glomeruli. In C and D, the deep glomerular border lies across from the top arrowhead, and the mitral cell layer lies across from the bottom arrowhead. glm, glomerular layer; epl, external plexiform layer; gel, granule cell layer. Scale bar A, B = 1000 pm; scale bar C, D = 100 pm.
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Fig. 6. Horizontal section (Rostra1 at top) depicting “Rip” immunoreactivity in the P40 Monodelphis AC. Staining is primarily present in rostra1 position, likely corresponding to the olfactory component of the commissure. 3V, third ventricle. Scale bar = 250 pm.
remyelination studies using tissue grafts have already taken advantage of the late maturation of oligodendrocytes in the mouse olfactory bulb.16 However, in the rodent, there is only a brief postnatal window when oligodendrocytes remain immature enough to succeed bulb presents a in remyelination. I6 The opossum structure from which immature oligodendrocytes are easily accessible over an extended postnatal period. For example, initial staining in the rat bulb occurs by PI 1 whereas staining in the opossum is not apparent until P30. Furthermore, only five days separate the appearance of oligodendrocyte-immunoreactivity in the rat bulb and the subsequent expression at the glomerular level; however, this same series of events occurs over 10 days in the opossum bulb. The slow maturation of the opossum bulb allows increased resolution into the time course of ontogenetic changes in oligodendrocyte-immunoreactivity, providing unique glimpses into developmental changes. For example, we examined patterned changes of oligodendrocyte-immunoreactivity in the anterior commissure because studies in the mouse suggested myelination occurs first in the anterior limb (Sturrock, 1976, cited in Ref. 25) whereas myelination in the hamster bulb is initiated in caudal portions.25 The opossum anterior commissure first exhibits oligodendrocyte-immunoreactivity in rostra1 aspects (e.g. Fig. 6) likely corresponding to the olfactory component of this commissure. Whereas a gradient of oligodendrocyte-immunoreactivity is easily discerned over the development of the opossum commissure, a similar pattern in the rat was not observed, perhaps due to its rapid maturation.
The relatively late onset and slow maturation of the Monodelphis bulb allow examination of the sequential expression of oligodendrocyte-specific proteins at a high resolution, Biochemical changes in myelin composition during rat brain development have given insights into the process of myelination,3s but, as more myelin-specific proteins are identified. it will become increasingly important to understand sequential events in myelinogenesis. This work, in turn, will lead to an increased understanding of the transformation of oligodendrocytes that permit them to act as a permissive substrate for neurite outgrowth at immature stages of development to acting as a nonpermissive substrate in more mature forms4’ CONCLUSIONS
In sum, oligodendrocyte-immunoreactivity first appears in the rat AOB by Pll, and levels rapidly increase in the entire bulb over the next five days. Also, oligodendrocyte-immunoreactivity in the LOT and AC are first present by PI 1 and are heavily stained by P20. Furthermore, oligodendrocyteimmunoreactivity in the bulb is not altered by unilateral naris closure, despite the large changes in protein synthesis associated with this manipulation. Finally, oligodendrocyte changes in the Monodelphis bulb mirror changes in the rat bulb, although initial staining appears much later and the time course of development occurs over an extended period in the opossum. The late onset and slow development of oligodendrocyte-immunoreactivity in the opossum bulb suggest it is a promising laboratory animal for the study of myelinogenesis.
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Acknowledgements-Thanks to M. Paternostro, C. Byrd, D. Cummings and D. Malun for thoughtful advice. Special thanks to E. Geisert and C. Dyer for suggestions on antibody use, and to J. Black for a gener-
et al.
ous supply of the “Rip” antibody. This work was supported by grants from the National Institutes of Health (DC-00338) and National Sciences Foundation (BNS-8919751)
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