- Email: [email protected]
The shape of the olfactory bulb influences axon targeting
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The shape of the olfactory bulb influences axon targeting Fatemeh Chehrehasaa,c , Brian Keya,b , James A. St John a,⁎ a
Brain Growth and Regeneration Lab, Discipline of Anatomy and Developmental Biology, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Australia b Centre for Functional and Applied Genomics, The University of Queensland, Brisbane 4072, Australia c The Zahedan University of Medical Sciences of Iran, Zahedan, Iran
A R T I C LE I N FO
AB S T R A C T
Article history:
Each primary olfactory neuron in the mouse expresses a single type of odorant receptor. All
Accepted 26 June 2007
neurons expressing the same odorant receptor gene typically project to two topographically
Available online 19 July 2007
fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb. While topographic gradients of guidance receptors and their ligands help to establish the
Keywords:
retinotectal projection, similar orthogonal distributions of cues have not yet been
Bulbectomy
detected within the olfactory system. While odorant receptors are crucial for the final
Development
targeting of axons to glomeruli, it is unclear whether the olfactory bulb itself provides
Glomerulus
instructive cues for the establishment of the topographic map. To begin to understand the
Guidance
role of the olfactory bulb in the formation of the olfactory nerve pathway, we developed a
Neuron
model whereby the gross shape of the bulb in the P2-IRES-tau-LacZ line of mice was radically
Topography
altered during postnatal development. We have shown here that the topography of axons expressing the P2 odorant receptor is dependent on the shape of the olfactory bulb. When the dorsoventral axis of the olfactory bulb was compressed during the early postnatal period, newly developing P2 axons projected to multiple inappropriate glomeruli surrounding their normal target site. These results suggest that the distribution of local guidance cues within the olfactory bulb is influenced by the shape of the olfactory bulb and that these cues contribute to the topographic positioning of glomeruli. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved.
1.
Introduction
Primary olfactory neurons in the mouse express one of ~1000 odorant receptors. Those neurons expressing the same receptor type are mosaically distributed throughout zones in the nasal cavity (Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993) and typically project axons to two topographically fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb (Ressler et al., 1993; Vassar et al., 1994). What are the mechanisms that enable these axons to navigate to their target sites and form
glomeruli in the appropriate topographic location in the olfactory bulb during development? The odorant receptors themselves are known to play a role in topographic targeting (Mombaerts et al., 1996) and several adhesion molecules and guidance receptors such as galectin-1 (Puche et al., 1996), NCAM (Treloar et al., 1997), neuropilin-1 (Pasterkamp et al., 1998) and its ligand Semaphorin 3A (Crandall et al., 2000) as well as neuropilin-2 (Cloutier et al., 2002; Walz et al., 2002) are involved in olfactory axon guidance. However, the specific cues responsible for generating the topographic map remain elusive.
⁎ Corresponding author. Fax: +61 7 3365 1299. E-mail address: [email protected] (J.A. St John). 0006-8993/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.073
18
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
While the expression of spatially restricted guidance cues in the olfactory bulb has been proposed to determine the location of glomeruli (Key and St John, 2002; St John et al., 2002), others have suggested that intrinsic self-organizational properties of olfactory axons are sufficient for emergence of the map (Feinstein and Mombaerts, 2004). In this latter model, the olfactory bulb is believed to merely provide a permissive substrate for axon growth. Rather than olfactory axons recognizing specific topographic cues, as in the retinotectal system, they are instead hypothesized to sort out autonomously in a highly stereotyped spatiotemporal order. Following genetic or physical ablation of the olfactory bulb, axons arising from neurons expressing the P2 odorant receptor are able to form glomerular-like loci which suggests that these axons have a cell autonomous ability to, at least, converge (Bulfone et al., 1998; St John et al., 2003). Furthermore, we have previously shown that regenerating olfactory axons are able to form the olfactory nerve and glomerular layers in the presence of an artificial biological scaffold, similar in size and shape to the olfactory bulb, although they are not able to form glomeruli in appropriate positions (Chehrehasa et al., 2006). However, it remains to be determined what role the olfactory bulb itself plays in the establishment of the topographic map. One means of assessing the role of the olfactory bulb in the formation of the olfactory nerve pathway is to experimentally
alter the normal shape of this tissue during development. We developed a model whereby the gross shape of the olfactory bulb in P2-IRES-tau-LacZ mice was disrupted without perturbing existing olfactory nerve connections. We have demonstrated here that this altered shape caused growing P2 axons to project to multiple inappropriate glomeruli rather than to a single glomerulus. These results demonstrate for the first time that the shape of the olfactory bulb is an important determinant in the targeting of olfactory axons to specific glomeruli.
2.
Results
2.1. Unilateral bulbectomy alters the gross morphology of the remaining olfactory bulb We hypothesized that the gross shape of the bulb contributes to the topographic organization of glomeruli. It is envisaged that the shape either influences the spatial and temporal ordering of axons as they converge to form glomeruli or that it determines the positioning of intrinsic axon guidance cues. We developed a model whereby the gross shape of the olfactory bulb was disrupted during development, without perturbing the existing projections of olfactory axons to their glomeruli. This was achieved by unilaterally removing an olfactory bulb and the accompanying rostral forebrain from
Fig. 1 – Unilateral bulbectomy perturbs the gross morphology of the remaining olfactory bulb. Panels are coronal sections through the olfactory bulb, with dorsal to the top. (A, B) In control animals at 4 weeks, the width of the olfactory bulb was approximately one-half of its height with a longer vertical than horizontal axis (dashed line). (C) However, at 4 weeks post-surgery in animals which underwent unilateral bulbectomy at P3, the rostral portion of the remaining olfactory bulb appeared flattened with a horizontal axis almost twice the width of its height (dashed line). This collapsed morphology was most prominent in the rostral half of the bulb. Asterisk indicates the position of tissue that has filled the bulbectomized cavity. (D) In the caudal region, the collapsed morphology was less prominent and the olfactory bulb had a longer vertical than horizontal axis (dashed line). Asterisk indicates tissue in the bulbectomized cavity. Scale bar = 400 μm in panel A; 600 μm in panel B; 550 μm in panels C, D.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
neonatal P2-IRES-tau-lacZ transgenic mice (Mombaerts et al., 1996). These mice express the β-galactosidase transgene specifically in olfactory sensory neurons expressing the P2 odorant receptor gene. The unilateral bulbectomy caused a change in the gross shape of the contralateral olfactory bulb as it spread to fill the resultant space in the bulbar cavity. The removal of the rostral forebrain in these unilateral bulbectomies prevented the forebrain pushing forward into the
19
bulbar cavity as usually happens during bulbectomy (Graziadei and Monti Graziadei, 1986; St John et al., 2003). The bulbectomized mice were then allowed to survive for four weeks at which time they were examined both for changes in the bulbar shape and for defects in the topography of the P2 axons. In control animals at 4 weeks of age, coronal sections revealed that the dorsoventral axis of the olfactory bulb was near vertical, both at rostral and caudal levels (dashed lines,
Fig. 2 – The topography of axons is affected in the remaining olfactory bulb after unilateral bulbectomy. Panels are coronal sections through the olfactory bulb of animals 4 weeks post-surgery, with medial to the right. (A) In a control animal, X-gal staining revealed that P2 axons projected to one glomerulus on the lateral surface of the olfactory bulb. (B) However, in the bulbectomized animals at 4 weeks post-surgery, P2 axons projected to multiple glomeruli on the lateral surface of the collapsed olfactory bulb. In some animals, P2 axons projected to three glomeruli, which were located either at the same (C, arrowheads) or different levels along the rostrocaudal axis (D–F). (G–I) Projection to multiple glomeruli (arrowheads) was observed on the medial surface of the collapsed olfactory bulbs. Scale bar = 400 μm in panels A, B, 200 μm in panels C–F and 300 μm in panels G–I.
20
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
Figs. 1A, B). This alignment was also clearly depicted by the predominantly vertical orientation of the granule cell laminae in the nuclear stained sections. The width of the olfactory bulb at this age was approximately one-half of its height. In contrast, in unilateral bulbectomized animals, the rostral portion of the remaining intact olfactory bulb had collapsed at 4 weeks postsurgery (Fig. 1C). In these bulbs, the granule cell laminae were now aligned along the horizontal plane and the width of the bulb was about twice its height. Despite this dramatic change in shape, the bulb still maintained its normal cytoarchitectural layers. In addition, immunostaining for glial fibrillary acidic protein indicated that the change in the morphology of the remaining olfactory bulb did not cause any abnormal proliferation of astrocytes or olfactory ensheathing cells within the olfactory bulb that could interfere with axon growth (not shown). The collapsed morphology was predominantly restricted to the rostral half of the bulb. Caudally, the olfactory bulb was less affected and retained a longer vertical than horizontal axis (Fig. 1D), although the shape was obviously distorted and clearly not the same as in control animals (Fig. 1B). This change in morphology of the rostral half of the contralateral olfactory bulb, which we refer to as a collapsed phenotype, only occurred when the bulbectomy was performed concurrently with removal of the frontal pole of the telencephalon in young neonatal mice, between 2 and 3 days of postnatal age. No significant collapse of the bulb was observed when the surgery was performed in animals at 4 days or older; and no axon targeting defects were observed in these animals.
2.2.
of animals exhibiting this phenotype on the lateral surface of the bulb, and was not significantly different from controls. This indicates that the collapsed phenotype affects the targeting of the laterally projecting axons to a greater extent than that of the medially projecting axons. It should be noted that we did not observe any axons passing from the bulbectomized side of the embryo into the collapsed bulb which could account for the targeting errors. Interestingly, P2 axons innervating the medial glomerulus must first pass through the collapsed portion of the olfactory bulb before reaching their glomeruli, as on the lateral surface of the bulb. Thus, despite the gross shape changes in the rostral bulb, axons exhibited fewer targeting errors on the medial side. These results suggest that targeting errors are not a result of inappropriate sorting of axons in the rostral bulb but are more likely a result of changes occurring at the level of each glomerulus. Taken together, the increased targeting defects observed on both surfaces of the collapsed olfactory bulb indicate that the gross bulbar shape was regulating the final targeting of primary olfactory axons.
2.3. Primary olfactory axons remain intact in the collapsed bulb following unilateral bulbectomy In order to assess whether the existing primary olfactory axons innervating the contralateral olfactory bulb were perturbed by the bulbectomy, we examined the appearance of P2 neurons and their axons at various post-surgery intervals. Four days
Olfactory bulb shape affects the topography of axons
In order to assess whether the topography of the olfactory pathway was affected by the collapsed nature of the rostral olfactory bulb in animals at 4 weeks post-surgery, we X-gal stained coronal sections to selectively reveal the trajectory of the P2 subpopulation of axons. In control animals, P2 axons project to either one or two glomeruli on the lateral surface of the olfactory bulb (Fig. 2A) as previously described (Royal and Key, 1999; Schaefer et al., 2001). In contrast, in the collapsed olfactory bulb following bulbectomy, 50% of the animals now had P2 axons that projected to three or more glomeruli on the lateral surface (Figs. 2B–F) which was significantly more than in control animals (p b 0.05, Mann–Whitney U test). The P2 axons often appeared to form a broad band across the surface of numerous glomeruli in the nerve fiber layer (Fig. 2B). In 20% of the operated animals, P2 axons projected to three glomeruli, that were located either at the same rostrocaudal level (Fig. 2C, arrowheads) or at different levels along the rostrocaudal axis (Figs. 2D–F). Only about 20% of the bulbectomized animals had P2 axons that innervated a single lateral glomerulus which was considerably less than the 60% of control animals that had a single P2 glomerulus on the lateral surface of the bulb. Next we assessed whether the topography of P2 glomeruli on the medial surface of the olfactory bulb was also affected by the bulbectomy at 4 weeks post-surgery. Since the medial P2 glomeruli are located more caudally than those on the lateral bulbar surface (Royal and Key, 1999), they occur in the region with a more normal shape of the olfactory bulb (Fig. 1D). In ∼20% of animals, the medial P2 axons projected to three or more glomeruli (Figs. 2G–I) which was considerably less than the 50%
Fig. 3 – Primary olfactory axons remain intact in the remaining bulb following unilateral bulbectomy. Panels are coronal sections through the olfactory bulb and neuroepithelium, with medial to the right for panels B–D. (A) Four days after surgery, P2 neurons in the nasal cavity were unaffected on the control contralateral (collapsed) side while there was an obvious loss of these neurons in the neuroepithelium on the operated side. (B–D) The P2 glomeruli also persisted in the contralateral OB at 4 days, 7 days and 2 weeks post-surgery. Scale bar = 200 μm in panel A and 400 μm in panels B–D.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
after surgery, P2 neurons in the nasal cavity were unaffected on the unoperated contralateral (collapsed) side while there was an obvious loss of these neurons in the neuroepithelium on the bulbectomized side (Fig. 3A). The P2 glomeruli also persisted in the collapsed olfactory bulb at all survival times examined (4 days, 7 days and 2 weeks post-surgery) (Figs. 3B–D). It should be noted that the olfactory bulb rapidly collapsed within 4 days of bulbectomy. Therefore the newly growing axons entering into the collapsed olfactory bulb post-surgery were immediately exposed to an altered morphology. Interestingly, it was at 2 weeks post-surgery when the first clear signs of abnormal axon targeting were observed in the glomerular layer of the olfactory bulb (arrow, Fig. 3D). In summary, the persistence of the P2 olfactory neurons, axons and glomeruli in the collapsed olfactory bulb indicated that the existing olfactory nerve pathway between the peripheral neuroepithelium and the olfactory bulb was undamaged and remained intact following the dramatic change in shape induced by unilateral bulbectomy.
3.
Discussion
The point-to-point topography between the retina and the tectum in the visual system is dependent on retinal axons responding to the graded expression pattern of guidance cues along orthogonal axes in the tectum (Inatani, 2005). The existence of these topographically distributed guidance cues was elegantly demonstrated by tectum rotation experiments in chicks (Nakamura et al., 1994). The question of whether topographically distributed guidance cues are also distributed across the surface of the olfactory bulb in the olfactory system remains unresolved. While it has not been possible to rotate the olfactory bulb, we postulated that if the shape of the olfactory bulb could be distorted during development of the olfactory nerve pathway then the existence of topographic cues could be revealed. We have developed an experimental model whereby the gross shape of the mouse olfactory bulb was disrupted without perturbing the projections of existing axons to their glomeruli. By performing a unilateral bulbectomy in neonatal mice, we induced the remaining olfactory bulb to collapse and fill the vacant cranial cavity. The dorsoventral axis of the remaining olfactory bulb became severely flattened and the granule cell laminae were radically reoriented in the horizontal plane by this procedure. Despite these gross changes, the development of the cytoarchitectonic layers was unaffected by the collapsed nature of the bulb. Importantly, the existing olfactory axon connections between the olfactory neuroepithelium and the glomerular layer remained intact. We examined the topography of the olfactory nerve projection by using the P2-IRES-tau-LacZ line of mice that expresses the β-galactosidase reporter in the cell bodies and axons of the subpopulation of primary olfactory neurons expressing the P2 odorant receptor. P2 axons typically project to two glomeruli, one each on the medial and lateral surfaces of the bulb (Royal and Key, 1999). While these topographically fixed glomeruli are already present at birth, the majority of P2 neurons are born in the postnatal period, with the number of P2 neurons increasing from around 600 at birth to over 4000 by the end of the second
21
postnatal week (Royal and Key, 1999). Thus, by examining the P2 subpopulation of axons 4 weeks after bulbectomy, we were able to clearly observe whether the perturbed morphology of the bulb had affected the postnatal development of this pathway. We demonstrated that the newly growing P2 olfactory axons, which projected into the bulb after the induced change in bulb shape, inappropriately projected to multiple glomeruli surrounding the normal target site. Our results clearly indicate that the shape of the olfactory bulb is important for the normal glomerular targeting of olfactory axons.
3.1.
Olfactory bulb shape alters axon guidance
It appears that olfactory axons are able to project from the periphery and reach the vicinity of their normal target but then fail to terminate in their correct glomerulus in the experimentally manipulated bulb. While axons are still able to converge, they appear to do so with increasing error in the experimental animals. Thus, the altered bulb shape has affected the positioning of the converged axons rather than the convergence per se. We consider that it is likely that the altered morphology of the olfactory bulb has caused a change in topographically localized axon guidance cues in this tissue. An altered local distribution of guidance cues would clearly confuse the targeting of growing axons and cause them to terminate in aberrant sites. Similar targeting defects have been observed in retinal ganglionic axons in the visual system when topographic cues have been perturbed locally in the optic tectum (Udin, 1977). While there were fewer errors in the targeting of the medially projecting P2 axons compared to the laterally projecting axons, this is likely due to the rostral–caudal positions of these different glomerular targets. The medial P2 glomeruli are located in the region of the olfactory bulb where there was reduced collapse, whereas the lateral P2 axons project to the rostral bulb which underwent the greatest collapse. Thus it is not surprising that targeting of the medial P2 axons was less affected. Rather this result is fortuitous since it clearly demonstrates that perturbations in the olfactory nerve fiber layer that could alter axon sorting were not responsible for changes in axon guidance. The projection of P2 olfactory axons to a broad area of the olfactory bulb has been reported during regeneration after olfactory nerve lesion; however, this aberrant growth has been attributed to both widespread degeneration and the possibility of scar formation (Costanzo, 2000). However, similar mistargeting has been reported following chemical ablation of the olfactory neuroepithelium with dichlobenil in adult mice (St John and Key, 2003). While this technique does not physically perturb the olfactory bulb, the widespread degeneration may cause reactive changes in the bulb that contribute to the aberrant regeneration. The dichlobenil-induced regeneration caused P2 axons to terminate in numerous inappropriate glomeruli which were widely dispersed over a large area in the rostrocaudal and ventrodorsal regions of the olfactory bulb. These regenerated P2 axons terminated in up to seven glomeruli on the lateral side and to about four glomeruli on the medial surface of the bulb (St John and Key, 2003). However, this dichlobenil regeneration model was performed in adult mice and thus developmentally regulated guidance cues may no longer be expressed in the adult which may account for the numerous axon targeting defects. Thus these regeneration
22
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
models may not be appropriate for finding evidence of bulbar axon guidance cues in the developing animal since there is considerable nerve damage or altered spatial relations. This is highlighted by the fact that when P2 neurons are selectively ablated by genetic techniques, the regenerating P2 axons were able to converge without error to approximately the same site as wild type P2 glomeruli (Gogos et al., 2000).
3.2.
Local guidance cues in the olfactory bulb affect targeting
It has been proposed that the self-organizational property of olfactory axons is sufficient for the establishment of olfactory map and as a consequence there is no need for guidance cues in the olfactory bulb (Feinstein and Mombaerts, 2004). However, we have previously proposed that the shape of the olfactory bulb would influence the positioning of glomeruli (Key and St John, 2002) and indeed Feinstein and Mombaerts (2004) also suggested that the shape of the bulb could alter the ability of axons to selforganize. We have now determined that the shape of the olfactory bulb does influence axon targeting, but we believe that the collapse of the olfactory bulb has altered the location of positional cues within the bulb. In the vast majority of animals where there were multiple glomeruli on the lateral surface, it was quite clear that the numerous P2 axons were closely positioned to each other with opportunity for selffasciculation and correct targeting, and yet they instead projected to multiple dispersed glomeruli. We suggest that local guidance cues are distorted which then cause axons to converge inappropriately. In mutant animals lacking an olfactory bulb, and presumably any intrinsic topographic guidance cues, axons are still able to sort out and converge to form glomerular-like structures (St John et al., 2003). In these animals the spatiotemporal ordering of axons is distorted and yet the axons are still able to converge at one or two loci, as in normal animals. Interestingly, the mere passage of P2 axons through a collapsed nerve fiber layer in the present study could not account for aberrant targeting. Together, these observations do not support the idea that precise spatiotemporal ordering of axons is essential for convergence and formation of the topographic map. Our results instead support the hypothesis that local guidance cues in the bulb contribute to the final targeting of olfactory axons. The nature of these cues remains to be determined.
4.
Experimental procedures
4.1.
Surgical ablation of olfactory bulbs
Unilateral bulbectomies were performed on 25 P2-IRES-tauLacZ transgenic mice (Mombaerts et al., 1996) at postnatal day (P) 3.5 as described in Chehrehasa et al. (2006). Animals were allowed to recover for 4 days (n = 5), 7 days (n = 5), 14 days (n = 5) and 4 weeks (n = 10). All procedures were carried out with the approval of, and in accordance with, the University of Queensland Animal Ethics Experimentation Committee.
4.2.
Animal preparation
Following decalcification, heads were vacuum embedded with OCT (Optimal Cutting Temperature, Sakura Fintek Japan) and
snap frozen in isopentane that had been cooled by liquid nitrogen and serial 30 μm coronal sections were cut on a cryostat.
4.3. Analysis of P2 glomeruli in the control and collapsed olfactory bulb X-gal staining for the LacZ reporter was performed as previously described (Royal and Key, 1999). For each animal, every section of the olfactory cavity was analyzed for the presence of P2-LacZpositive axons within glomeruli. A P2 positive glomerulus was defined as any glomerulus containing three or more P2-LacZ axons. Glomeruli on the lateral and medial surfaces of the collapsed and control bulbs for each animal were examined. Only glomeruli that were clearly separated between serial sections were counted.
Acknowledgments This work was supported by grants from the National Health and Medical Research Council to J.St.J and B.K and the government of Iran to F.C. We thank Prof. Mombaerts for the P2-IRES-tau-LacZ mice.
REFERENCES
Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S., Meneses, J., Pedersen, R., Axel, R., Rubenstein, J.L., 1998. An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons. Neuron 21, 1273–1282. Chehrehasa, F., St John, J.A., Key, B., 2006. Implantation of a scaffold following bulbectomy induces laminar organization of regenerating olfactory axons. Brain Res. Cloutier, J.F., Giger, R.J., Koentges, G., Dulac, C., Kolodkin, A.L., Ginty, D.D., 2002. Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877–892. Costanzo, R.M., 2000. Rewiring the olfactory bulb: changes in odor maps following recovery from nerve transection. Chem. Senses 25, 199–205. Crandall, J.E., Dibble, C., Butler, D., Pays, L., Ahmad, N., Kostek, C., Puschel, A.W., Schwarting, G.A., 2000. Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb. J. Neurobiol. 45, 195–206. Feinstein, P., Mombaerts, P., 2004. A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Cell 117, 817–831. Gogos, J.A., Osborne, J., Nemes, A., Mendelsohn, M., Axel, R., 2000. Genetic ablation and restoration of the olfactory topographic map. Cell 103, 609–620. Graziadei, P.P., Monti Graziadei, G.A., 1986. Neuronal changes in the forebrain of mice following penetration by regenerating olfactory axons. J. Comp. Neurol. 247, 344–356. Inatani, M., 2005. Molecular mechanisms of optic axon guidance. Naturwissenschaften 92, 549–561. Key, B., St John, J., 2002. Axon navigation in the mammalian primary olfactory pathway: where to next? Chem. Senses 27, 245–260. Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Nemes, A., Mendelsohn, M., Edmondson, J., Axel, R., 1996. Visualizing an olfactory sensory map. Cell 87, 675–686. Nakamura, H., Itasaki, N., Matsuno, T., 1994. Rostrocaudal polarity formation of chick optic tectum. Int. J. Dev. Biol. 38, 281–286.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
Pasterkamp, R.J., De Winter, F., Holtmaat, A.J., Verhaagen, J., 1998. Evidence for a role of the chemorepellent semaphorin III and its receptor neuropilin-1 in the regeneration of primary olfactory axons. J. Neurosci. 18, 9962–9976. Puche, A.C., Poirier, F., Hair, M., Bartlett, P.F., Key, B., 1996. Role of galectin-1 in the developing mouse olfactory system. Dev. Biol. 179, 274–287. Ressler, K.J., Sullivan, S.L., Buck, L.B., 1993. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73, 597–609. Royal, S.J., Key, B., 1999. Development of P2 olfactory glomeruli in P2-internal ribosome entry site-tau-LacZ transgenic mice. J. Neurosci. 19, 9856–9864. Schaefer, M.L., Finger, T.E., Restrepo, D., 2001. Variability of position of the P2 glomerulus within a map of the mouse olfactory bulb. J. Comp. Neurol. 436, 351–362. St John, J.A., Key, B., 2003. Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium. Chem. Senses 28, 773–779. St John, J.A., Clarris, H.J., Key, B., 2002. Multiple axon guidance cues establish the olfactory topographic map: how do these cues interact? Int. J. Dev. Biol. 46, 639–647.
23
St John, J.A., Clarris, H.J., McKeown, S., Royal, S., Key, B., 2003. Sorting and convergence of primary olfactory axons are independent of the olfactory bulb. J. Comp. Neurol. 464, 131–140. Strotmann, J., Wanner, I., Helfrich, T., Beck, A., Meinken, C., Kubick, S., Breer, H., 1994. Olfactory neurones expressing distinct odorant receptor subtypes are spatially segregated in the nasal neuroepithelium. Cell Tissue Res. 276, 429–438. Treloar, H., Tomasiewicz, H., Magnuson, T., Key, B., 1997. The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform. J. Neurobiol. 32, 643–658. Udin, S.B., 1977. Rearrangements of the retinotectal projection in Rana pipiens after unilateral caudal half-tectum ablation. J. Comp. Neurol. 173, 561–582. Vassar, R., Ngai, J., Axel, R., 1993. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74, 309–318. Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B., Axel, R., 1994. Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981–991. Walz, A., Rodriguez, I., Mombaerts, P., 2002. Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice. J. Neurosci. 22, 4025–4035.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
The shape of the olfactory bulb influences axon targeting Fatemeh Chehrehasaa,c , Brian Keya,b , James A. St John a,⁎ a
Brain Growth and Regeneration Lab, Discipline of Anatomy and Developmental Biology, School of Biomedical Sciences, The University of Queensland, Brisbane 4072, Australia b Centre for Functional and Applied Genomics, The University of Queensland, Brisbane 4072, Australia c The Zahedan University of Medical Sciences of Iran, Zahedan, Iran
A R T I C LE I N FO
AB S T R A C T
Article history:
Each primary olfactory neuron in the mouse expresses a single type of odorant receptor. All
Accepted 26 June 2007
neurons expressing the same odorant receptor gene typically project to two topographically
Available online 19 July 2007
fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb. While topographic gradients of guidance receptors and their ligands help to establish the
Keywords:
retinotectal projection, similar orthogonal distributions of cues have not yet been
Bulbectomy
detected within the olfactory system. While odorant receptors are crucial for the final
Development
targeting of axons to glomeruli, it is unclear whether the olfactory bulb itself provides
Glomerulus
instructive cues for the establishment of the topographic map. To begin to understand the
Guidance
role of the olfactory bulb in the formation of the olfactory nerve pathway, we developed a
Neuron
model whereby the gross shape of the bulb in the P2-IRES-tau-LacZ line of mice was radically
Topography
altered during postnatal development. We have shown here that the topography of axons expressing the P2 odorant receptor is dependent on the shape of the olfactory bulb. When the dorsoventral axis of the olfactory bulb was compressed during the early postnatal period, newly developing P2 axons projected to multiple inappropriate glomeruli surrounding their normal target site. These results suggest that the distribution of local guidance cues within the olfactory bulb is influenced by the shape of the olfactory bulb and that these cues contribute to the topographic positioning of glomeruli. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved.
1.
Introduction
Primary olfactory neurons in the mouse express one of ~1000 odorant receptors. Those neurons expressing the same receptor type are mosaically distributed throughout zones in the nasal cavity (Ressler et al., 1993; Strotmann et al., 1994; Vassar et al., 1993) and typically project axons to two topographically fixed glomeruli, one each on the medial and lateral surfaces of the olfactory bulb (Ressler et al., 1993; Vassar et al., 1994). What are the mechanisms that enable these axons to navigate to their target sites and form
glomeruli in the appropriate topographic location in the olfactory bulb during development? The odorant receptors themselves are known to play a role in topographic targeting (Mombaerts et al., 1996) and several adhesion molecules and guidance receptors such as galectin-1 (Puche et al., 1996), NCAM (Treloar et al., 1997), neuropilin-1 (Pasterkamp et al., 1998) and its ligand Semaphorin 3A (Crandall et al., 2000) as well as neuropilin-2 (Cloutier et al., 2002; Walz et al., 2002) are involved in olfactory axon guidance. However, the specific cues responsible for generating the topographic map remain elusive.
⁎ Corresponding author. Fax: +61 7 3365 1299. E-mail address: [email protected] (J.A. St John). 0006-8993/$ – see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2007.06.073
18
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
While the expression of spatially restricted guidance cues in the olfactory bulb has been proposed to determine the location of glomeruli (Key and St John, 2002; St John et al., 2002), others have suggested that intrinsic self-organizational properties of olfactory axons are sufficient for emergence of the map (Feinstein and Mombaerts, 2004). In this latter model, the olfactory bulb is believed to merely provide a permissive substrate for axon growth. Rather than olfactory axons recognizing specific topographic cues, as in the retinotectal system, they are instead hypothesized to sort out autonomously in a highly stereotyped spatiotemporal order. Following genetic or physical ablation of the olfactory bulb, axons arising from neurons expressing the P2 odorant receptor are able to form glomerular-like loci which suggests that these axons have a cell autonomous ability to, at least, converge (Bulfone et al., 1998; St John et al., 2003). Furthermore, we have previously shown that regenerating olfactory axons are able to form the olfactory nerve and glomerular layers in the presence of an artificial biological scaffold, similar in size and shape to the olfactory bulb, although they are not able to form glomeruli in appropriate positions (Chehrehasa et al., 2006). However, it remains to be determined what role the olfactory bulb itself plays in the establishment of the topographic map. One means of assessing the role of the olfactory bulb in the formation of the olfactory nerve pathway is to experimentally
alter the normal shape of this tissue during development. We developed a model whereby the gross shape of the olfactory bulb in P2-IRES-tau-LacZ mice was disrupted without perturbing existing olfactory nerve connections. We have demonstrated here that this altered shape caused growing P2 axons to project to multiple inappropriate glomeruli rather than to a single glomerulus. These results demonstrate for the first time that the shape of the olfactory bulb is an important determinant in the targeting of olfactory axons to specific glomeruli.
2.
Results
2.1. Unilateral bulbectomy alters the gross morphology of the remaining olfactory bulb We hypothesized that the gross shape of the bulb contributes to the topographic organization of glomeruli. It is envisaged that the shape either influences the spatial and temporal ordering of axons as they converge to form glomeruli or that it determines the positioning of intrinsic axon guidance cues. We developed a model whereby the gross shape of the olfactory bulb was disrupted during development, without perturbing the existing projections of olfactory axons to their glomeruli. This was achieved by unilaterally removing an olfactory bulb and the accompanying rostral forebrain from
Fig. 1 – Unilateral bulbectomy perturbs the gross morphology of the remaining olfactory bulb. Panels are coronal sections through the olfactory bulb, with dorsal to the top. (A, B) In control animals at 4 weeks, the width of the olfactory bulb was approximately one-half of its height with a longer vertical than horizontal axis (dashed line). (C) However, at 4 weeks post-surgery in animals which underwent unilateral bulbectomy at P3, the rostral portion of the remaining olfactory bulb appeared flattened with a horizontal axis almost twice the width of its height (dashed line). This collapsed morphology was most prominent in the rostral half of the bulb. Asterisk indicates the position of tissue that has filled the bulbectomized cavity. (D) In the caudal region, the collapsed morphology was less prominent and the olfactory bulb had a longer vertical than horizontal axis (dashed line). Asterisk indicates tissue in the bulbectomized cavity. Scale bar = 400 μm in panel A; 600 μm in panel B; 550 μm in panels C, D.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
neonatal P2-IRES-tau-lacZ transgenic mice (Mombaerts et al., 1996). These mice express the β-galactosidase transgene specifically in olfactory sensory neurons expressing the P2 odorant receptor gene. The unilateral bulbectomy caused a change in the gross shape of the contralateral olfactory bulb as it spread to fill the resultant space in the bulbar cavity. The removal of the rostral forebrain in these unilateral bulbectomies prevented the forebrain pushing forward into the
19
bulbar cavity as usually happens during bulbectomy (Graziadei and Monti Graziadei, 1986; St John et al., 2003). The bulbectomized mice were then allowed to survive for four weeks at which time they were examined both for changes in the bulbar shape and for defects in the topography of the P2 axons. In control animals at 4 weeks of age, coronal sections revealed that the dorsoventral axis of the olfactory bulb was near vertical, both at rostral and caudal levels (dashed lines,
Fig. 2 – The topography of axons is affected in the remaining olfactory bulb after unilateral bulbectomy. Panels are coronal sections through the olfactory bulb of animals 4 weeks post-surgery, with medial to the right. (A) In a control animal, X-gal staining revealed that P2 axons projected to one glomerulus on the lateral surface of the olfactory bulb. (B) However, in the bulbectomized animals at 4 weeks post-surgery, P2 axons projected to multiple glomeruli on the lateral surface of the collapsed olfactory bulb. In some animals, P2 axons projected to three glomeruli, which were located either at the same (C, arrowheads) or different levels along the rostrocaudal axis (D–F). (G–I) Projection to multiple glomeruli (arrowheads) was observed on the medial surface of the collapsed olfactory bulbs. Scale bar = 400 μm in panels A, B, 200 μm in panels C–F and 300 μm in panels G–I.
20
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
Figs. 1A, B). This alignment was also clearly depicted by the predominantly vertical orientation of the granule cell laminae in the nuclear stained sections. The width of the olfactory bulb at this age was approximately one-half of its height. In contrast, in unilateral bulbectomized animals, the rostral portion of the remaining intact olfactory bulb had collapsed at 4 weeks postsurgery (Fig. 1C). In these bulbs, the granule cell laminae were now aligned along the horizontal plane and the width of the bulb was about twice its height. Despite this dramatic change in shape, the bulb still maintained its normal cytoarchitectural layers. In addition, immunostaining for glial fibrillary acidic protein indicated that the change in the morphology of the remaining olfactory bulb did not cause any abnormal proliferation of astrocytes or olfactory ensheathing cells within the olfactory bulb that could interfere with axon growth (not shown). The collapsed morphology was predominantly restricted to the rostral half of the bulb. Caudally, the olfactory bulb was less affected and retained a longer vertical than horizontal axis (Fig. 1D), although the shape was obviously distorted and clearly not the same as in control animals (Fig. 1B). This change in morphology of the rostral half of the contralateral olfactory bulb, which we refer to as a collapsed phenotype, only occurred when the bulbectomy was performed concurrently with removal of the frontal pole of the telencephalon in young neonatal mice, between 2 and 3 days of postnatal age. No significant collapse of the bulb was observed when the surgery was performed in animals at 4 days or older; and no axon targeting defects were observed in these animals.
2.2.
of animals exhibiting this phenotype on the lateral surface of the bulb, and was not significantly different from controls. This indicates that the collapsed phenotype affects the targeting of the laterally projecting axons to a greater extent than that of the medially projecting axons. It should be noted that we did not observe any axons passing from the bulbectomized side of the embryo into the collapsed bulb which could account for the targeting errors. Interestingly, P2 axons innervating the medial glomerulus must first pass through the collapsed portion of the olfactory bulb before reaching their glomeruli, as on the lateral surface of the bulb. Thus, despite the gross shape changes in the rostral bulb, axons exhibited fewer targeting errors on the medial side. These results suggest that targeting errors are not a result of inappropriate sorting of axons in the rostral bulb but are more likely a result of changes occurring at the level of each glomerulus. Taken together, the increased targeting defects observed on both surfaces of the collapsed olfactory bulb indicate that the gross bulbar shape was regulating the final targeting of primary olfactory axons.
2.3. Primary olfactory axons remain intact in the collapsed bulb following unilateral bulbectomy In order to assess whether the existing primary olfactory axons innervating the contralateral olfactory bulb were perturbed by the bulbectomy, we examined the appearance of P2 neurons and their axons at various post-surgery intervals. Four days
Olfactory bulb shape affects the topography of axons
In order to assess whether the topography of the olfactory pathway was affected by the collapsed nature of the rostral olfactory bulb in animals at 4 weeks post-surgery, we X-gal stained coronal sections to selectively reveal the trajectory of the P2 subpopulation of axons. In control animals, P2 axons project to either one or two glomeruli on the lateral surface of the olfactory bulb (Fig. 2A) as previously described (Royal and Key, 1999; Schaefer et al., 2001). In contrast, in the collapsed olfactory bulb following bulbectomy, 50% of the animals now had P2 axons that projected to three or more glomeruli on the lateral surface (Figs. 2B–F) which was significantly more than in control animals (p b 0.05, Mann–Whitney U test). The P2 axons often appeared to form a broad band across the surface of numerous glomeruli in the nerve fiber layer (Fig. 2B). In 20% of the operated animals, P2 axons projected to three glomeruli, that were located either at the same rostrocaudal level (Fig. 2C, arrowheads) or at different levels along the rostrocaudal axis (Figs. 2D–F). Only about 20% of the bulbectomized animals had P2 axons that innervated a single lateral glomerulus which was considerably less than the 60% of control animals that had a single P2 glomerulus on the lateral surface of the bulb. Next we assessed whether the topography of P2 glomeruli on the medial surface of the olfactory bulb was also affected by the bulbectomy at 4 weeks post-surgery. Since the medial P2 glomeruli are located more caudally than those on the lateral bulbar surface (Royal and Key, 1999), they occur in the region with a more normal shape of the olfactory bulb (Fig. 1D). In ∼20% of animals, the medial P2 axons projected to three or more glomeruli (Figs. 2G–I) which was considerably less than the 50%
Fig. 3 – Primary olfactory axons remain intact in the remaining bulb following unilateral bulbectomy. Panels are coronal sections through the olfactory bulb and neuroepithelium, with medial to the right for panels B–D. (A) Four days after surgery, P2 neurons in the nasal cavity were unaffected on the control contralateral (collapsed) side while there was an obvious loss of these neurons in the neuroepithelium on the operated side. (B–D) The P2 glomeruli also persisted in the contralateral OB at 4 days, 7 days and 2 weeks post-surgery. Scale bar = 200 μm in panel A and 400 μm in panels B–D.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
after surgery, P2 neurons in the nasal cavity were unaffected on the unoperated contralateral (collapsed) side while there was an obvious loss of these neurons in the neuroepithelium on the bulbectomized side (Fig. 3A). The P2 glomeruli also persisted in the collapsed olfactory bulb at all survival times examined (4 days, 7 days and 2 weeks post-surgery) (Figs. 3B–D). It should be noted that the olfactory bulb rapidly collapsed within 4 days of bulbectomy. Therefore the newly growing axons entering into the collapsed olfactory bulb post-surgery were immediately exposed to an altered morphology. Interestingly, it was at 2 weeks post-surgery when the first clear signs of abnormal axon targeting were observed in the glomerular layer of the olfactory bulb (arrow, Fig. 3D). In summary, the persistence of the P2 olfactory neurons, axons and glomeruli in the collapsed olfactory bulb indicated that the existing olfactory nerve pathway between the peripheral neuroepithelium and the olfactory bulb was undamaged and remained intact following the dramatic change in shape induced by unilateral bulbectomy.
3.
Discussion
The point-to-point topography between the retina and the tectum in the visual system is dependent on retinal axons responding to the graded expression pattern of guidance cues along orthogonal axes in the tectum (Inatani, 2005). The existence of these topographically distributed guidance cues was elegantly demonstrated by tectum rotation experiments in chicks (Nakamura et al., 1994). The question of whether topographically distributed guidance cues are also distributed across the surface of the olfactory bulb in the olfactory system remains unresolved. While it has not been possible to rotate the olfactory bulb, we postulated that if the shape of the olfactory bulb could be distorted during development of the olfactory nerve pathway then the existence of topographic cues could be revealed. We have developed an experimental model whereby the gross shape of the mouse olfactory bulb was disrupted without perturbing the projections of existing axons to their glomeruli. By performing a unilateral bulbectomy in neonatal mice, we induced the remaining olfactory bulb to collapse and fill the vacant cranial cavity. The dorsoventral axis of the remaining olfactory bulb became severely flattened and the granule cell laminae were radically reoriented in the horizontal plane by this procedure. Despite these gross changes, the development of the cytoarchitectonic layers was unaffected by the collapsed nature of the bulb. Importantly, the existing olfactory axon connections between the olfactory neuroepithelium and the glomerular layer remained intact. We examined the topography of the olfactory nerve projection by using the P2-IRES-tau-LacZ line of mice that expresses the β-galactosidase reporter in the cell bodies and axons of the subpopulation of primary olfactory neurons expressing the P2 odorant receptor. P2 axons typically project to two glomeruli, one each on the medial and lateral surfaces of the bulb (Royal and Key, 1999). While these topographically fixed glomeruli are already present at birth, the majority of P2 neurons are born in the postnatal period, with the number of P2 neurons increasing from around 600 at birth to over 4000 by the end of the second
21
postnatal week (Royal and Key, 1999). Thus, by examining the P2 subpopulation of axons 4 weeks after bulbectomy, we were able to clearly observe whether the perturbed morphology of the bulb had affected the postnatal development of this pathway. We demonstrated that the newly growing P2 olfactory axons, which projected into the bulb after the induced change in bulb shape, inappropriately projected to multiple glomeruli surrounding the normal target site. Our results clearly indicate that the shape of the olfactory bulb is important for the normal glomerular targeting of olfactory axons.
3.1.
Olfactory bulb shape alters axon guidance
It appears that olfactory axons are able to project from the periphery and reach the vicinity of their normal target but then fail to terminate in their correct glomerulus in the experimentally manipulated bulb. While axons are still able to converge, they appear to do so with increasing error in the experimental animals. Thus, the altered bulb shape has affected the positioning of the converged axons rather than the convergence per se. We consider that it is likely that the altered morphology of the olfactory bulb has caused a change in topographically localized axon guidance cues in this tissue. An altered local distribution of guidance cues would clearly confuse the targeting of growing axons and cause them to terminate in aberrant sites. Similar targeting defects have been observed in retinal ganglionic axons in the visual system when topographic cues have been perturbed locally in the optic tectum (Udin, 1977). While there were fewer errors in the targeting of the medially projecting P2 axons compared to the laterally projecting axons, this is likely due to the rostral–caudal positions of these different glomerular targets. The medial P2 glomeruli are located in the region of the olfactory bulb where there was reduced collapse, whereas the lateral P2 axons project to the rostral bulb which underwent the greatest collapse. Thus it is not surprising that targeting of the medial P2 axons was less affected. Rather this result is fortuitous since it clearly demonstrates that perturbations in the olfactory nerve fiber layer that could alter axon sorting were not responsible for changes in axon guidance. The projection of P2 olfactory axons to a broad area of the olfactory bulb has been reported during regeneration after olfactory nerve lesion; however, this aberrant growth has been attributed to both widespread degeneration and the possibility of scar formation (Costanzo, 2000). However, similar mistargeting has been reported following chemical ablation of the olfactory neuroepithelium with dichlobenil in adult mice (St John and Key, 2003). While this technique does not physically perturb the olfactory bulb, the widespread degeneration may cause reactive changes in the bulb that contribute to the aberrant regeneration. The dichlobenil-induced regeneration caused P2 axons to terminate in numerous inappropriate glomeruli which were widely dispersed over a large area in the rostrocaudal and ventrodorsal regions of the olfactory bulb. These regenerated P2 axons terminated in up to seven glomeruli on the lateral side and to about four glomeruli on the medial surface of the bulb (St John and Key, 2003). However, this dichlobenil regeneration model was performed in adult mice and thus developmentally regulated guidance cues may no longer be expressed in the adult which may account for the numerous axon targeting defects. Thus these regeneration
22
BR A IN RE S EA RCH 1 1 69 ( 20 0 7 ) 1 7 –23
models may not be appropriate for finding evidence of bulbar axon guidance cues in the developing animal since there is considerable nerve damage or altered spatial relations. This is highlighted by the fact that when P2 neurons are selectively ablated by genetic techniques, the regenerating P2 axons were able to converge without error to approximately the same site as wild type P2 glomeruli (Gogos et al., 2000).
3.2.
Local guidance cues in the olfactory bulb affect targeting
It has been proposed that the self-organizational property of olfactory axons is sufficient for the establishment of olfactory map and as a consequence there is no need for guidance cues in the olfactory bulb (Feinstein and Mombaerts, 2004). However, we have previously proposed that the shape of the olfactory bulb would influence the positioning of glomeruli (Key and St John, 2002) and indeed Feinstein and Mombaerts (2004) also suggested that the shape of the bulb could alter the ability of axons to selforganize. We have now determined that the shape of the olfactory bulb does influence axon targeting, but we believe that the collapse of the olfactory bulb has altered the location of positional cues within the bulb. In the vast majority of animals where there were multiple glomeruli on the lateral surface, it was quite clear that the numerous P2 axons were closely positioned to each other with opportunity for selffasciculation and correct targeting, and yet they instead projected to multiple dispersed glomeruli. We suggest that local guidance cues are distorted which then cause axons to converge inappropriately. In mutant animals lacking an olfactory bulb, and presumably any intrinsic topographic guidance cues, axons are still able to sort out and converge to form glomerular-like structures (St John et al., 2003). In these animals the spatiotemporal ordering of axons is distorted and yet the axons are still able to converge at one or two loci, as in normal animals. Interestingly, the mere passage of P2 axons through a collapsed nerve fiber layer in the present study could not account for aberrant targeting. Together, these observations do not support the idea that precise spatiotemporal ordering of axons is essential for convergence and formation of the topographic map. Our results instead support the hypothesis that local guidance cues in the bulb contribute to the final targeting of olfactory axons. The nature of these cues remains to be determined.
4.
Experimental procedures
4.1.
Surgical ablation of olfactory bulbs
Unilateral bulbectomies were performed on 25 P2-IRES-tauLacZ transgenic mice (Mombaerts et al., 1996) at postnatal day (P) 3.5 as described in Chehrehasa et al. (2006). Animals were allowed to recover for 4 days (n = 5), 7 days (n = 5), 14 days (n = 5) and 4 weeks (n = 10). All procedures were carried out with the approval of, and in accordance with, the University of Queensland Animal Ethics Experimentation Committee.
4.2.
Animal preparation
Following decalcification, heads were vacuum embedded with OCT (Optimal Cutting Temperature, Sakura Fintek Japan) and
snap frozen in isopentane that had been cooled by liquid nitrogen and serial 30 μm coronal sections were cut on a cryostat.
4.3. Analysis of P2 glomeruli in the control and collapsed olfactory bulb X-gal staining for the LacZ reporter was performed as previously described (Royal and Key, 1999). For each animal, every section of the olfactory cavity was analyzed for the presence of P2-LacZpositive axons within glomeruli. A P2 positive glomerulus was defined as any glomerulus containing three or more P2-LacZ axons. Glomeruli on the lateral and medial surfaces of the collapsed and control bulbs for each animal were examined. Only glomeruli that were clearly separated between serial sections were counted.
Acknowledgments This work was supported by grants from the National Health and Medical Research Council to J.St.J and B.K and the government of Iran to F.C. We thank Prof. Mombaerts for the P2-IRES-tau-LacZ mice.
REFERENCES
Bulfone, A., Wang, F., Hevner, R., Anderson, S., Cutforth, T., Chen, S., Meneses, J., Pedersen, R., Axel, R., Rubenstein, J.L., 1998. An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons. Neuron 21, 1273–1282. Chehrehasa, F., St John, J.A., Key, B., 2006. Implantation of a scaffold following bulbectomy induces laminar organization of regenerating olfactory axons. Brain Res. Cloutier, J.F., Giger, R.J., Koentges, G., Dulac, C., Kolodkin, A.L., Ginty, D.D., 2002. Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons. Neuron 33, 877–892. Costanzo, R.M., 2000. Rewiring the olfactory bulb: changes in odor maps following recovery from nerve transection. Chem. Senses 25, 199–205. Crandall, J.E., Dibble, C., Butler, D., Pays, L., Ahmad, N., Kostek, C., Puschel, A.W., Schwarting, G.A., 2000. Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb. J. Neurobiol. 45, 195–206. Feinstein, P., Mombaerts, P., 2004. A contextual model for axonal sorting into glomeruli in the mouse olfactory system. Cell 117, 817–831. Gogos, J.A., Osborne, J., Nemes, A., Mendelsohn, M., Axel, R., 2000. Genetic ablation and restoration of the olfactory topographic map. Cell 103, 609–620. Graziadei, P.P., Monti Graziadei, G.A., 1986. Neuronal changes in the forebrain of mice following penetration by regenerating olfactory axons. J. Comp. Neurol. 247, 344–356. Inatani, M., 2005. Molecular mechanisms of optic axon guidance. Naturwissenschaften 92, 549–561. Key, B., St John, J., 2002. Axon navigation in the mammalian primary olfactory pathway: where to next? Chem. Senses 27, 245–260. Mombaerts, P., Wang, F., Dulac, C., Chao, S.K., Nemes, A., Mendelsohn, M., Edmondson, J., Axel, R., 1996. Visualizing an olfactory sensory map. Cell 87, 675–686. Nakamura, H., Itasaki, N., Matsuno, T., 1994. Rostrocaudal polarity formation of chick optic tectum. Int. J. Dev. Biol. 38, 281–286.
BR A IN RE S E A RCH 1 1 69 ( 20 0 7 ) 1 7 –2 3
Pasterkamp, R.J., De Winter, F., Holtmaat, A.J., Verhaagen, J., 1998. Evidence for a role of the chemorepellent semaphorin III and its receptor neuropilin-1 in the regeneration of primary olfactory axons. J. Neurosci. 18, 9962–9976. Puche, A.C., Poirier, F., Hair, M., Bartlett, P.F., Key, B., 1996. Role of galectin-1 in the developing mouse olfactory system. Dev. Biol. 179, 274–287. Ressler, K.J., Sullivan, S.L., Buck, L.B., 1993. A zonal organization of odorant receptor gene expression in the olfactory epithelium. Cell 73, 597–609. Royal, S.J., Key, B., 1999. Development of P2 olfactory glomeruli in P2-internal ribosome entry site-tau-LacZ transgenic mice. J. Neurosci. 19, 9856–9864. Schaefer, M.L., Finger, T.E., Restrepo, D., 2001. Variability of position of the P2 glomerulus within a map of the mouse olfactory bulb. J. Comp. Neurol. 436, 351–362. St John, J.A., Key, B., 2003. Axon mis-targeting in the olfactory bulb during regeneration of olfactory neuroepithelium. Chem. Senses 28, 773–779. St John, J.A., Clarris, H.J., Key, B., 2002. Multiple axon guidance cues establish the olfactory topographic map: how do these cues interact? Int. J. Dev. Biol. 46, 639–647.
23
St John, J.A., Clarris, H.J., McKeown, S., Royal, S., Key, B., 2003. Sorting and convergence of primary olfactory axons are independent of the olfactory bulb. J. Comp. Neurol. 464, 131–140. Strotmann, J., Wanner, I., Helfrich, T., Beck, A., Meinken, C., Kubick, S., Breer, H., 1994. Olfactory neurones expressing distinct odorant receptor subtypes are spatially segregated in the nasal neuroepithelium. Cell Tissue Res. 276, 429–438. Treloar, H., Tomasiewicz, H., Magnuson, T., Key, B., 1997. The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform. J. Neurobiol. 32, 643–658. Udin, S.B., 1977. Rearrangements of the retinotectal projection in Rana pipiens after unilateral caudal half-tectum ablation. J. Comp. Neurol. 173, 561–582. Vassar, R., Ngai, J., Axel, R., 1993. Spatial segregation of odorant receptor expression in the mammalian olfactory epithelium. Cell 74, 309–318. Vassar, R., Chao, S.K., Sitcheran, R., Nunez, J.M., Vosshall, L.B., Axel, R., 1994. Topographic organization of sensory projections to the olfactory bulb. Cell 79, 981–991. Walz, A., Rodriguez, I., Mombaerts, P., 2002. Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice. J. Neurosci. 22, 4025–4035.