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Keywords:

  • pax6;
  • dendritogenesis;
  • olfactory bulb;
  • cultures;
  • mice;
  • telencephalon

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

We have established previously that, although the olfactory epithelium is absent in the homozygous Pax-6 mutant mouse, an olfactory bulb-like structure (OBLS) does develop. Moreover, this OBLS contains cells that correspond to mitral cells, the primary projection neurons in the olfactory bulb. The current study aimed to address whether the dendrites of mitral cells in the olfactory bulb or in the OBLS mitral-like cells, exhibit a change in orientation in the presence of the olfactory epithelium. The underlying hypothesis is that the olfactory epithelium imparts a trophic signal on mitral and mitral-like cell that influences the growth of their primary dendrites, orientating them toward the surface of the olfactory bulb. Hence, we cultured hemibrains from wild-type and Pax 6 mutant mice from two different embryonic stages (embryonic days 14 and 15) either alone or in coculture with normal olfactory epithelial explants or control tissue (cerebellum). Our results indicate that the final dendritic orientation of mitral and mitral-like cells is directly influenced both by age and indeed by the presence of the olfactory epithelium. Developmental Dynamics 232:325–335, 2005. © 2004 Wiley-Liss, Inc.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

One fundamental issue in developmental neuroscience is the extent to which peripheral structures influence the formation of brain maps. In this context, the olfactory system is a valuable model to understand neuronal development and plasticity. The formation of the olfactory bulb (OB) involves the generation and differentiation of a wide variety of cells, whose axons contribute to the formation of the central olfactory projections at a time when the specific temporal and spatial patterns of gene expression are also being established (for review, see López-Mascaraque and De Castro, 2002).

The development of mitral neurons, the main output cells from the OB, is complex. Although these cells do not mature until well into adulthood, their neurogenesis begins on embryonic day (E) 10 and continues until E14 in mice (Hinds, 1968a; Jiménez et al., 2000). Dendrites of mitral cells are radially oriented in the mature OB, although during development exhibit a tangential orientation with a wide variety of morphologies (Hinds, 1968a, b; Hinds and Ruffett, 1973; Matsunami and Yamamoto, 2000). Some controversy remains regarding the factors that influence the differentiation and development of the mitral cells. Indeed, the degree to which the olfactory epithelium (OE) and afferent inputs influence the dendritic orientation of mitral cells has still to be defined.

In homozygous Pax-6 mutant mice, no OE develops, although an early olfactory bulb-like structure (OBLS) can be detected, which develops a lateral tract (LT) similar to the lateral olfactory tract (LOT; López-Mascaraque et al., 1998; Jiménez et al., 2000). Recently, Nomura and Osumi (2004) indicated that the mislocation of the olfactory bulb in this mutant is not caused by loss of olfactory nerve innervation but the abnormal migration of mitral cell progenitors provoked by the absence of Pax6. However, the failure of the Pax 6 mutant mouse to laminate correctly, rather is due to the absence of the OE, as occurs in different mutants (Dlx5, Long et al., 2003; Emx-2, Yoshida et al., 1997; Mash-1, Guillemot et al., 1993; for review, see López-Mascaraque and De Castro, 2002), where the contact between the OE and OB is lost, but all of them present alterations in the lamination of the OB (as occurs in the OBLS).

Nevertheless, it seems probable that signals released from the primary olfactory axons in the OE stimulate the reorientation of mitral cells in the OB. To gain insight into the role of peripheral structures on the formation of the OB, we analyzed the influence exerted by OE. Specifically, we have explored the orientation of dendrites in projection neurons of the OB and OBLS at both E14 and E15 in wild-type and Pax6 mutant mice in the presence or absence of the OE. Combined with previous results, our current findings suggest that the OE is not necessary for the initial development of mitral cells but rather for their later orientation.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

Pax 6 Does Not Directly Influence Outgrowth of Neurites From Mitral Cells

During embryogenesis, the lateral olfactory tract becomes organized into a compact axonal bundle that runs rostrocaudally through the telencephalic vesicle (Fig. 1A,C). Despite the absence of the OE, olfactory-like components do appear to form in Pax-6 mutant mice, where an OBLS can be detected in the rostral pole of the telencephalon (compare Fig. 1A,B). The overall shape of the tract and the pathway followed by the axons that originate in the OB/OBLS appears to be remarkably similar throughout the developmental period analyzed (Fig. 1C,D). The effect of the OE on neurite outgrowth from the OB already has been established (De Castro et al., 1999). Hence, to examine whether the OE exerts the same influence on axonal outgrowth from the OBLS, we compared outgrowth in explants from OB/OBLS, which were cultured alone or cocultured with OE and/or control tissue explants. When the OB (Fig. 1E) or OBLS (Fig. 1F) explants were grown alone in collagen gel matrices, the axonal processes of mitral cells grew symmetrically and haphazardly into the matrigel, without demonstrating any preference in terms of direction (Fig. 1E,F). Of interest, the growth of these processes was poorer in the OBLS (Fig. 1F) than in the OB explant (Fig. 1E). As previously described in vitro (De Castro et al., 1999), the OE exerted a strong to moderate repulsive effect on the outgrowth of axons from the OB. Thus, it was no surprise that,, when an OBLS explant was cocultured next to an OE, the neurite outgrowth was inhibited in the proximity of the OE tissue (Fig. 1G). This finding provides further evidence that the OBLS really can be considered as a prospective OB. Moreover, in the presence of both OE or control tissue, the behavior of the OB and OBLS explants was only influenced by the OE. Thus, it appeared that axogenesis of mitral cells is a process that is independent of the arrival of olfactory sensory axons and that the Pax-6 is apparently not needed for the formation of this tract.

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Figure 1. A,B: Olfactory bulb (OB) and olfactory bulb-like structure (OBLS) projections in whole brain hemispheres of embryonic day (E) 15 mice. A: Brain hemisphere of a wild-type mouse (+/+) with a small 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) injection made into the OB to label the lateral olfactory tract (LOT) by anterograde transport (arrow). B: Brain hemisphere of Pax-6 mutant mouse (−/−) with a DiI injection into the OBLS to label the lateral tract (LT, arrow). C,D: Growth of the LOT (C) and LT (D) throughout different embryonic stages (P, postnatal day), respectively, in both wild-type and mutant mice. E–G: Explants of OB and OBLS from E14 cultured for 2 days in a collagen gel and stained with the TuJ1 antibody. E: Olfactory bulb explants cultured alone. Axons from OB cells grow symmetrically in the collagen gel without demonstrating any preferred orientation. F: OBLS explant cultured alone in which axons grow symmetrically with no preferred orientation, although to a lesser degree than in E. G: OBLS explant cocultured with an olfactory epithelium (OE) tissue explant from a wild-type mouse. Note the OBLS axons growing away from the OE explant. In A–D, rostral is right and dorsal is up. Scale bars = 100 μm in A (applies to A,B), in C (applies to C,D), in E, in F, in G.

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Presence of the OE Influences Mitral Cell Processes Orientation

To better understand the influence of the OE on the OB primordium, we explored the effect of the OE on the dendritic reorientation of projection cells in the OB/OBLS. We concentrated our efforts on studying the effects of age and the presence or absence of the OE on dendritic morphological development of mitral cells. To investigate these interactions and to analyze whether the direction in which the dendritic processes grew was affected by the presence of the OE, we carried out a variety of different tissue culture assays. Hemibrains from E14 and E15 wild-type and mutant mice were cultured either alone, with control tissue (cerebellum), or with an OE tissue explant (Table 1). Mitral/mitral-like cells were visualized by retrograde labeling with lipophilic dyes or biotinylated dextran amine (BDA) injected into the LOT area, such that the only cells labeled are the efferent (projection) neurons of the OB/OBLS. Furthermore, the filling of the processes of these labeled cells with the dye enabled us to study the disposition of their dendrites. Consequently, after sectioning the cultured tissue, the number of projecting cells labeled in the different sections were counted and classified according to their dendrite disposition. Based on the orientation of the dendrites, the cultures were assigned to one of the three following categories (see Experimental Procedures section): (1) tangential, (2) radial, or (3) radial + tangential.

Table 1. Summary of Collagen Gel Experimentsa
Culture assayDendrite orientation
AgeTotal no. of hemibrainsTangentialRadialRad + Tang
  • a

    Percentage of hemibrain cultures with dendritic processes in a given orientation. Overall dendritic orientation for a culture dish was categorized as follows: radial, if more than 60% of labeled cells had dendritic processes oriented between 30° and 150° with respect to a line drawn tangential to the OB surface; tangential, if more than 60% of labeled cells had dendritic processes oriented between 210° and 330° and Rad + Tang, if the fewer than 60% of cells were oriented in either direction. OE, olfactory epithelium; OB, olfactory bulb; OBLS, olfactory bulb-like structure; E, embryonic day.

+/+ hemisphere (OB) aloneE141010000
E1511282745
+/+ hemisphere (OB) + cerebellumE141090010
E1510503020
+/+ hemisphere (OB) + OEE142826371
E152413879
−/− hemisphere (OBLS) aloneE141010000
E15119604
−/− hemisphere (OBLS) + cerebellumE14109406
E151090010
−/− hemisphere (OBLS) + OEE143080416
E1528431443

We first analyzed the orientation of olfactory bulb projecting cells by culturing wild-type hemibrains for 4 days alone from the two different ages, E14 and E15 (Fig. 2A–G). At E14, after tracer injection into the LOT, we observed that, in all cultures (n = 10), the retrograde labeled cells were oriented tangential to the OB surface (Fig. 2A,B,H). However, when E15 telencephalon hemispheres were analyzed, only in 28% of cultures were the projection neurons tangentially oriented (Fig. 2C, black arrows and F; n = 11, Table 1), whereas in 27% of the cultures, they were oriented radially to the OB surface (Fig. 2E,G). Of interest, 45% of the cultured had the same amount of radial and tangential mitral cell dendrites. A remarkable finding was that the radial dendrite orientation was higher in E15 than in E14. Thus, at E15 (but not at E14), some mitral cells themselves were already oriented radial to the OB surface.

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Figure 2. Orientation of olfactory bulb (OB) projection cell dendritic processes in brain hemispheres from both embryonic day (E) 14 (A,B) and E15 (C–G) mouse embryos after 4 days in culture. A,B: Mitral cells are tangentially oriented with respect to the olfactory bulb pial surface in brain hemispheres from E14 embryos (inset) after a biotinylated dextran amine (BDA) injection into the lateral olfactory tract (LOT). C: Wild-type E15 brain hemisphere shows tangentially (black arrows) and radially (open arrows) oriented mitral cells with respect to the olfactory bulb pial surface labeled after a BDA injection into the LOT. D: E15 brain hemisphere cultured for 4 days with the 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) injected into the LOT area. E–G: Retrograde labeled mitral cells in the OB (dash line corresponds to the OB boundary) oriented either tangentially (E and F) and radially (E, arrow, and G). H: Histograms correspond to E14 (n = 10) and E15 (n = 11) hemibrains, comparing the percentage of cultures where more than 60% of cells were oriented either tangentially, radially, or with the same amount of cells radially or tangentially (Rad + Tang) oriented. DIV, days in vitro. Scale bars = 20 μm in A–C,E,G, 200 μm in D, 100 μm in F.

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To determine whether the OE influenced the direction in which the dendrites grew, wild-type mouse hemibrains (E14 and E15) were cultured either alone or cocultured with OE or control (cerebellum) tissue explants during 4 days (Fig. 3). When wild-type E14 brain hemispheres were cocultured facing an OE explant (Fig. 3A), only in 3% of the cultures were the mitral cell dendritic processes oriented toward the direction of the OE explant (n = 28). In contrast, 71% of the cultures had the same amount of cells oriented tangentially and radially with respect to the OE explant (Fig. 3B). Thus, 26% of cultures had their cell processes oriented in a tangential manner (Fig. 3J). This finding was distinct when E15 hemispheres were cultured with OE explants (Fig. 3C–I). At this age, only 13% of E15 hemisphere cultures had most of the mitral cell processes in the OB oriented tangential to its surface (n = 24), whereas in 8%, the cell processes were primarily oriented radially toward the control tissue. Indeed, in 79%, an equal amount of cell processes were directed either tangential to the surface of the explant or radially toward the OE explant (Fig. 3K; Table 1). The pattern of mitral dendrite reorientation from E14 hemibrains cocultured in proximity to cerebellum explants (n = 10), was similar to the growth of hemibrains cultured alone (Fig. 2H). Thus, there was a significant difference in the orientation of projection neurons in OB at E14 cultured alone vs. hemibrains cocultured with the OE (P < 0.001; Kruskal–Wallis and Dunn's test). There was also a significant difference in mitral dendritic orientation when E14 hemibrains are cultured with the OE vs. hemibrains cocultured with cerebellum (P < 0.01; Kruskal–Wallis test and Dunn's test). However, no statistical differences were observed at E15 by comparing the different assays cultures combinations (P > 0.05; Kruskal–Wallis and Dunn's test for all comparisons). Those data reflect the influence of the OE on the orientation of the mitral cell processes at E14, suggesting the existence of a critical period for the mitral cells to receive the OE influences between E14 and E15. Moreover, in more than 95% of cultures, projecting neurites from the OE explants did not penetrate the OB/OBLS explants (Fig. 3H,I). This finding suggests that the effect of the OE on the OB is exercised by proximity, and it is likely to be due to one or several diffusible molecules.

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Figure 3. Influence of olfactory epithelium (OE) explants on the orientation of the mitral cells of the olfactory bulb (OB) after whole embryonic day (E) 14 and E15 brain hemispheres are cultured for 4 days. A,B: Wild-type brain hemisphere from an E14 embryo was cocultured for 4 days next to an OE explant. The hemisphere was stained with bisbenzimide (blue), and DiA was injected into the lateral olfactory tract (LOT, green). B: Retrograde labeled cells (magnification from inset in A) oriented toward the OE. C–I: Hemibrains from E15 embryos of wild-type mice cocultured with OE. C: Hemisphere cocultured with an OE explant. The inset corresponds to D. D,E: A biotinylated dextran amine injection into the LOT labels tangential (D, black arrows) and radially (D, open arrow) oriented cells, facing the OE explant. E: Magnification of the box in D shows a detail of two radially oriented mitral cells. F: Wild-type brain hemispheres cocultured with an OE explant. G: Magnification of inset in F. The 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) injection into the LOT labels radially oriented cells facing the OE explant. H: Hemibrain cocultured with an OE explant. In this case, DiI has been injected into the LOT and 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA) into the OE. I: Magnification of inset in H. Radially oriented mitral cell in the OB facing the OE, but there are no yellow (DiA) fibers entering into the hemisphere. J,K: Histograms show the percentage of E14 and E15 cultures that had more than 60% of dendritic processes oriented tangentially or radially and those that had a similar amount of dendrite processes oriented tangentially or toward the collagen gel (Rad + Tang), in the different culture conditions. Cb, cerebellum; DIV, days in vitro. Scale bars = 200 μm in A,C,F, 20 μm in B,D,E,G,I, 100 μm in H.

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Analyses of Dendrite Orientation in OBLS Projection Cells

When brain hemispheres from E14 and E15 mutant mice were cultured alone, the OBLS projection cells in most cultures showed a tangential orientation (Fig. 4A,B and histogram in 4C). However, when the hemisphere faced an OE explant at E14 (Fig. 5A,B), the dendritic processes of mitral-like cells of the OBLS became oriented tangential to the surface of this structure in 80% of cases (n = 30; Fig. 5E). At E15 (Fig. 5A–D), this percentage fell to 43% of the cultures (n = 28; Fig. 5F). However, an equal amount of dendritic processes were oriented either tangentially or radially in 16% (E14, n = 30) or 43% (E15, n = 28) of cultures. Finally, the majority of dendrites were oriented toward the OE explant in 4% (E14) and 14% (E15; Fig. 5C,F). When cultured with control tissue, none of the OBLS hemispheres had predominantly radially oriented mitral-like cell dendrites (histograms 5E and F). Indeed, there was no clear evidence of preferential growth toward the control explant. Statistical quantification of the results in the mutants, show a significant difference in the orientation of projection neurons in OBLS at E15 cultured alone vs. hemibrains cocultured with the OE (P < 0.01; Kruskal–Wallis and Dunn's test). There was also a significant difference in mitral dendritic orientation when E15 hemibrains are cultured with the OE vs. hemibrains cocultured with cerebellum (P < 0.05; Kruskal–Wallis test and Dunn's test). However, no statistical differences were observed at E14 by comparing the different assays cultures combinations (P > 0.05; Kruskal–Wallis and Dunn's test for all comparisons). Thus, the presence of the OE explant at E15 (and not at E14) provoked an important increase in the number of radial oriented mitral-like cells in the OBLS, providing further evidence that the direction of dendritic growth is strongly influenced by the presence or absence of an input from the OE (see Table 1). Hence, these results provide evidence that the OE exerts a directional and positional effect on the outgrowth of the dendrites in OB/OBLS explants and that this effect could be also related to the embryonic age.

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Figure 4. Dendritic orientation of olfactory bulb-like structure (OBLS) projection cells after culturing embryonic day (E) 14 (A) and E15 (B) mutant hemibrains for 4 days. A: Mitral-like cells tangentially oriented after biotinylated dextran amine injection into the lateral tract. B: Mitral-like cells tangentially oriented after 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) injection into the lateral olfactory tract. C: Histogram corresponds to E14 (n = 10) and E15 (n = 11), showing no differences among between the two ages. DIV, days in vitro; Rad + Tang, radially and tangentially. Scale bars = 25 μm in A,B.

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Figure 5. Embryonic day (E) 14 and E15 mutant embryo hemibrains cocultured with an olfactory epithelium (OE) explant during 4 days. A,B: Hemibrain was injected with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI) in the lateral tract (mutant, LT, asterisk in B marks the tract counterstained with bisbenzimide). C,D: Retrograde labeled cells in the olfactory bulb-like structure (OBLS) oriented toward the OE explant. E,F: Histograms show the percentage of E14 and E15 cultures in different conditions that had more than 60% of dendritic processes oriented tangentially or radially and those that had similar amount of dendritic processes oriented toward the collagen gel or tangentially (Rad + Tang), in different culture conditions. Cb, cerebellum. Scale bars = 25 μ m in B (applies to A,B), in C, 200 μm in D.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

The present study set out to explore the dendritic development of the main projection neurons in the OB, in relation to the age and the presence of the olfactory epithelium. Moreover, the presence of an OBLS in Pax-6 mutant mice has enabled us to examine the effects that peripheral neural elements located in the OE have on OB development in vitro. Thus, we have analyzed the influence that the OE has on the fate of mitral cell and mitral-like cell orientation.

Over recent years, much evidence has accumulated to support the idea that in the OB, neurogenesis and axogenesis can occur in the absence of OE innervation (López-Mascaraque et al., 1996; Brunjes and Greer, 2003; Tolbert et al., 2004). Indeed, a nonevaginated olfactory bulb-like structure develops in the Pax-6 mutant mice that completely lack an OE (López-Mascaraque et al., 1998; Jiménez et al., 2000; López-Mascaraque and De Castro, 2002). Moreover, in Dlx5−/− mutant mice, where OSNs form in the OE yet their axons fail to innervate the OB, induction and growth of the OB occurs albeit to a lesser extent (Long et al., 2003; Levi et al., 2003). Nevertheless, although contact between the projections from the OE and the OB may not be necessary to induce OB formation (López-Mascaraque et al., 1998; Jiménez et al., 2000; López-Mascaraque and De Castro, 2002; Long et al., 2003), several reports have proposed that the OE and its sensory afferent inputs have a key role in initiating neurogenesis and the development of the OB (Graziadei and Monti-Graziadei, 1992; LaMantia et al., 1993, 2000; Gong and Shipley, 1995).

In the absence of an OE, an OB-like structure does develop, and mitral and mitral-like cells project their axons independently of the arrival of the OSN axons (López-Mascaraque et al., 1998; Jimenez et al., 2000). Indeed, we have identified these central projections of the main OB/OBLS neurons, running in a rostrocaudal direction along the ventrolateral part of the telencephalic vesicle and giving rise to the LOT/LT. We have seen that the OE exerts a moderate repulsive effect on the outgrowth of axons from both the OB, as described De Castro et al. (1999), and OBLS. However, we do not think that this effect is indispensable for the formation of the LOT/LT. Nevertheless, the signals provided by the arrival of OSN axons at later stages, and eventually other tissue-derived cues, certainly do contribute to confer the mature appearance on the OB (for a review, see López-Mascaraque and De Castro, 2002). Furthermore, in Dlx5 mutant mice the OB lamination is disrupted (Long et al., 2003), in Emx-2 mutants the mitral cell layer is disorganized and most of the OSN are missing (Yoshida et al., 1997), and in Mash-1 mutants embryos there is a slight perturbance of some OB layers (Guillemot et al., 1993). Indeed, studies of other mutant mouse strains have also provided evidence of the uncoupling of OB development from the OE (Long et al., 2003; Hebert et al., 2003).

During development, after the formation of the protoglomeruli, the dendrites of mitral/tufted cells rearrange their dendritic arbor dramatically, reducing their multiple apical branches to generate a unique dendritic tuft restricted to one glomerulus (Malun and Brunjes, 1996). Axodendritic synapses are formed between OSN axons and mitral cell dendrites from E19–E20 (Bailey et al, 1999; Treolar et al., 1999), long after mitral cell reorientation takes place. Thus, synapses cannot influence the reorientation process, although, synaptic activity might play different roles, such as arbor maintenance, promotion of primary dendrite differentiation, and/or dendritic maturation (Matsutani and Yamamoto, 2000). On the other hand, although contact between projections from the OE and the OB may not be necessary to induce OB formation (López-Mascaraque et al., 1998; Jiménez et al., 2000; López-Mascaraque and De Castro, 2002; Long et al., 2003), it is possible that signals derived from the olfactory epithelium (Graziadei and Monti-Graziadei, 1992; LaMantia et al., 1993, 2000; Gong and Shipley, 1995) stimulate the reorientation of the dendrites from tangential to radial. Assuming that both diffusible and cell adhesion molecules are involved, a large number of molecules have been proposed that could play a critical role on these dendritic movements. Semaphorin 3A (Sema3A) can act as a diffusible chemoattractant for cortical apical dendrites and as a chemorepellent for the axons due to the asymmetric distribution of one component of the signal transduction machinery (Polleux et al., 2000). This ligand is expressed either by olfactory sensory neurons (Williams-Hogarth et al., 2000) or by both mitral and ensheathing cells (Pasterkamp et al., 1998) where olfactory axons are known to sort out (Schwarting et al., 2000). Moreover, the analysis of Sema3A homozygous mutant mice revealed that the sorting of axons within the nerve fiber layer was disrupted and axons terminated in topographically inappropriate glomeruli (Schwarting et al., 2000). Similarly, Taniguchi et al. (2003) described a critical role for Sema3A in the spatial arrangement of glomeruli in the OB. Thus, a Sema3A gradient could exist in the superficial layers of the olfactory bulb that would allow both dendritic growth and axonal guidance during the formation of glomeruli.

Here, we present evidence that, in the absence of the OE, mitral cell dendrites do not show any radial orientation in the OB at E14 hemibrains cultured alone and that the exposure to an OE explant in a collagen gel matrix provokes the reorientation of their dendritic processes (P < 0.001). However, in isolated E15 brain hemispheres, many dendrites of mitral cells are already oriented radially to the OB surface, and the presence of an OE explant increases the number of radially oriented dendritic processes, although with no statistical significance (P > 0.05). In contrast, when OB explants were cocultured facing a piece of cerebellum, there was no influence in the growth or orientation of the dendritic processes. These data reflect either the existence of a critical period for the mitral cells to receive the OE influences and/or that, at E15, some mitral cells already have reoriented the dendrites due to the arrival of OSN axons. On the other hand, in the mutant, there was no significant difference in mitral-like dendritic orientation when E14 hemibrains were cultured alone vs. OBLS facing an OE (P > 0.05), although significant differences were found when compared E15 mutant hemibrains alone vs. cocultured with OE (P < 0.01). Taken together, the above findings indicate that (1) the OE influences the orientation of mitral/mitral-like cell dendrites; (2) there is a critical period for the OB (E14/E15) to receive the OE influences; and (3) there is a 1-day delay in the development of the olfactory system of the Pax-6 mutant mice when compared with the wild-type, as occurs during the development of the cerebral cortex in this mutant (Jiménez et al., 2002).

In relation to the mutant, we also observed that, at very early developmental stages, the mitral-like cells in the OBLS were positioned in a tangential manner with respect to the boundary of this structure. As development proceeded, these cells become radially oriented, but directed their dendritic processes toward the core of the OBLS, instead of toward the surface as occurs in the OB. In contrast, when the OBLS was cocultured next to an OE explant, these mitral-like cells reoriented their dendritic processes toward the OE explant rather than the core of the OBLS. Thus, the percentage of tangential oriented cells that we observed in these cultures might be a transitory orientation, from dendrites facing the core of the OBLS to dendrites finally oriented toward the outside of the structure.

Thus, it is likely that diffusible molecules liberated by OE are implicated in the process of dendritic reorientation. From our cultures, we realized that OE axons do not need to enter into the OB/OBLS to achieve this effect; thus, the dendrite reorientation does not depend on synaptic contact. In a similar manner, the dendrites of projection neurons in the Drosophila antennal lobe have undergone significant growth and branching before the OSN axons mediate any influence, so the axons only refine and consolidate this prototypic map (Jefferis et al., 2003). Furthermore, mitral-like neurons in Manduca are not essential for the formation of olfactory glomeruli (Oland and Tolbert, 1998; Tolbert et al., 2004). Thus, in different species, at least certain aspects of connectivity in the olfactory pathway appear to be “hardwired” during development.

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

The main findings of this study are that both mitral and mitral-like cells become reoriented toward OE explants when cocultured in vitro. Indeed, this only occurs in the presence of the OE but not with a control tissue such as cerebellum. The penetration of the OSN axons into the OBLS explant is not necessary to influence the reorientation of mitral/tufted cell dendritic processes; thus, it is highly probable that one or several diffusible molecules are implicated. This conclusion is compatible with previous findings indicating that the inputs provided by OSN axons and other tissue-derived cues are essential for later stages of OB development (for a review, see Tolbert et al., 2004). In fact, whereas mitral cell generation and axonal projections are processes that are independent of the OE, the orientation of mitral cell dendrites does depend upon the presence of olfactory epithelium.

EXPERIMENTAL PROCEDURES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

Animals

Wild-type (+/+), heterozygous (Pax-6Sey-Neu/+), and homozygous (Pax-6Sey-Neu/Pax-6Sey-Neu) mouse embryos were used in this study (n = 192). The homozygous Pax-6 mutants will be referred to as “mutant” and will be compared with their morphologically normal littermates, pooling both +/+ and Pax-6Sey-Neu/+ embryos together as “wild-type.” The embryos were obtained from a colony established at the Instituto Cajal that has been described elsewhere (from four heterozygous females generously donated by Jack Favor, GSF-Institut, Neuherber, Germany; Jiménez et al., 2000). The day of detection of the vaginal plug was counted as E0, and the first 24 hr after birth were considered as postnatal day 0 (P0). We have studied animals from E12 to P0, combining tracer injections and coculture experiments in three-dimensional collagen gel matrices, with axonal labeling. All the procedures for handling and killing animals used in this study were in accordance with the European Commission guidelines (86/609/CEE) approved by the animal care and use committee of the Cajal Institute.

Pregnant heterozygous dams were anesthetized with Equithesin (3 ml/kg body weight) before surgery and killed with an overdose of anesthetic. Embryos were removed by midline laparotomy, anesthetized by hypothermia and decapitated when cultured or transcardially perfused with 4% paraformaldehyde (PFA) when used in other experiments.

Carbocyanine Injections

To analyze the temporal growth pattern of mitral axons (LOT/LT formation), wild-type and mutant embryos (aged from E12 to P0) were perfused with 4% PFA in phosphate buffer. Crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate (DiI, Molecular Probes) and of 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA, Molecular Probes) were placed into the OB/OBLS area with a fine tungsten wire. After maintaining for 1 week at 37°C, Vibratome sections (100 μm) were obtained from the brains and they were counterstained with 0.002% bisbenzimide (Hoechst 33258, Sigma) in phophate buffered saline (PBS). Carbocyanine labeling was also used to visualize mitral and mitral-like cell dendritic processes in the culture assays (see below).

Culture Assays

We performed two types of culture assays in three-dimensional collagen gel matrices: slice and hemibrain cultures. Brains from E14–E15 wild-type and mutant mouse embryos, were dissected in ice-cold L-15 medium (GIBCO) with 10% horse serum. In both types of cultures, OE and cerebellum, for the same age wild-type mice were cut into 250- to 300-μm coronal slices by using a McIlwain tissue chopper (Mickle Laboratory Engineering Co., Ltd., Gomshall, UK). The cultures were established by placing a small drop of collagen (4.11 mg/ml, rat tail collagen Type 1, Biosciences), supplemented with 0.35% sodium bicarbonate and 0.95% 10× MEM, into a four-well Nunc tissue culture plate and positioning the different explants at a distance of 0.1–0.5 mm. The collagen was allowed to polymerize at room temperature for 15 min, and culture wells were then filled with DMEM (Dulbecco's minimal essential medium) supplemented with L-glutamine 2 mM, peniciline/streptomicin 100 IU/ml, and 10% horse serum. Finally, cultures were incubated in 95% O2 and 5% CO2 at 37°C.

Slice cultures.

To compare the axonal outgrowth from the OB and OBLS, explants were cultured either alone or combination with OE. The OB and OBLS from E14 embryos was dissected out in a single piece, and the explants were sliced coronally (300–350 μm) with a tissue chopper in a collagen gel or matrigel as described above. In all cases, only the rostral two thirds of the OB were used to avoid including tissue from the accessory olfactory bulb. After two days in culture, the explants were labeled with the neuronal anti-class III β-tubulin monoclonal antibody (1:1,000, TuJ1 antibody; gift from Dr. Frankfurter), which was visualized with a goat anti-mouse biotinylated IgG (1:200; Jackson ImmunoResearch) using the ABC method, diaminobenzidine (DAB) as the substrate, and H2O2 (Immuno Pure Ultra-Sensitive ABC Peroxidase Staining Kit; Pierce). After immunostaining, 50- to 100-μm Vibratome sections were obtained for analysis.

Hemibrain cultures.

Brains were cut into two halves along the midline, and the telencephalic hemispheres were placed on collagen with the ventricular side down. Hemibrains from wild-type and mutants were cultured individually or cocultured with either OE or cerebellum from wild-type litter mates and positioned at a distance of 0.1–0.5 mm (see Table 1). After 4 days in culture, the mitral/mitral-like cells and their dendritic processes were visualized by injecting a 1% solution of DiI/DiA in dimethylformamide or inserting small crystals of the dye into the lateral tract of the OB/OBLS hemispheres. The labeled cultures were placed in the dark for 7 days to allow the tracer to diffuse and were viewed and photographed under a fluorescence microscope. In other cultures, BDA (3,000 molecular weight, Molecular Probes D7135) was used. The day before the cultures were fixed with 4% paraformaldehyde, a 5% solution of BDA was microinjected into the LOT/LT through the collagen matrix using a glass pipette. The brain was Vibratome sectioned at 75 μm, and the tracer was developed using an avidin–biotin–peroxidase complex solution, with DAB as the peroxidase substrate (ABC Elite Kit, Vector Laboratories). The sections were mounted on gelatinized slides and counterstained with thionin (0.25%). We carefully compared labeled neurons from hemibrains obtained by the two different methods and did not observe any morphological differences. Labeled cells were clearly identifiable as mitral/mitral-like on the basis of their morphology, revealed by the retrograde labeling from the axons.

The retrograde labeling allowed the orientation of dendritic processes of OB/OBLS neurons to be assessed and assigned to one of three categories (Table 1). Dendritic orientation was defined in relation to a line drawn tangential to the OB surface, and angles were measured in the clockwise direction. For each culture dish, at least 15 neurons were scored in each of 6 to 10 OB/OBLS slices. Overall dendritic orientation for a culture dish was categorized as: “radial,” if more than 60% of labeled cells had dendritic processes oriented between 30 degrees and 150 degrees with respect to the tangential line; “tangential,” if more than 60% of labeled cells had dendritic processes oriented between 210 degrees and 330 degrees; and “radial + tangential,” if the fewer than 60% of cells were oriented in either direction. In the coculture assays, the analysis only included the cells on the side of the OB/OBLS facing the explant.

Data Analysis

Brightfield images (Nikon, Eclipse E600) were captured by using a digital camera (Nikon DXM 1200F). Fluorescent sections and explants were analyzed and studied using the same microscope with appropriate rhodamine (560–610 nm) or fluorescein (450–490 nm) filter cubes to visualize DiI and DiA, respectively. In some experiments, the sections were counterstained with bisbenzimide (0.002% in PBS; Sigma) and photographed under ultraviolet illumination. Selected areas were also studied using a Leica TCS 4D confocal microscope. Statistical analysis was performed using GraphPad Prisma V.4 statistical software. The variables of the different culture assays were compared using nonparametric Kruskal–Wallis ANOVA test and multiple comparisons were made using Dunn's multiple test. A probability value of less than 0.05 was regarded as significant (Table 2).

Table 2. Statistical Significance for Different Culture Assay Comparisonsa
AgeDunn's multiple comparison test
Culture assayP value
  • a

    The variables of the different culture assays have been compared by using the nonparametric Kruskal–Wallis analysis of variance test and multiple comparisons have been made using Dunn's multiple test. A probability value of less than 0.05 was regarded as significant. Cb, cerebellum; OE, olfactory epithelium; OB, olfactory bulb; OBLS, olfactory bulb-like structure.

E14OB vs OB + Cb> 0.05
OB vs OB + OE< 0.001***
OB + Cb vs OB + OE< 0.01**
E15OB vs OB + Cb> 0.05
OB vs OB + OE> 0.05
OB + Cb vs OB + OE> 0.05
E14OBLS vs OBLS + Cb> 0.05
OBLS vs OBLS + OE> 0.05
OBLS + Cb vs OBLS + OE> 0.05
E15OBLS vs OBLS + Cb> 0.05
OBLS vs OBLS + OE< 0.01**
OBLS + Cb vs OBLS + OE< 0.05*

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES

We thank Dr. Jose Borrell and Fernando García for helpful comments and M.L. Poves for her technical assistance.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. RESULTS
  5. DISCUSSION
  6. CONCLUSION
  7. EXPERIMENTAL PROCEDURES
  8. Acknowledgements
  9. REFERENCES