the map. Although transient ectopic glomeruli
may be generated during normal postnatal development, they are pruned in adult animals (30, 31).
The pruning process may reflect a refinement
process at a later postnatal stage.
Our study puts the olfactory system in line
with other sensory systems in that it undergoes a
change in circuit plasticity during the critical period of development (32). Although, traditionally,
the discussions of critical period have focused on
how sensory deprivation affects the development
of neural circuits, our study reveals an intrinsic
developmental program that unfolds whether or
not neural activity is perturbed. Even though the
olfactory system is regulated by a critical period,
late-generated neurons adopt a different strategy
for axon projection. Thus, a developmental critical period may function to restrict the reorganization of the neural circuit and to maintain an
References and Notes
1. G. L. Ming, H. Song, Annu. Rev. Neurosci. 28, 223–250
2. A. Mackay-Sim, P. Kittel, J. Neurosci. 11, 979–984 (1991).
3. J. A. Gogos, J. Osborne, A. Nemes, M. Mendelsohn,
R. Axel, Cell 103, 609–620 (2000).
4. R. M. Costanzo, Ciba Found. Symp. 160, 233–242,
discussion 243–248 (1991).
5. R. M. Costanzo, Chem. Senses 25, 199–205 (2000).
6. R. M. Costanzo, Chem. Senses 30 (suppl. 1), i133–i134
7. P. P. Graziadei, G. A. Monti Graziadei, Ann. N. Y. Acad. Sci.
457, 127–142 (1985).
8. P. Mombaerts et al., Cell 87, 675–686 (1996).
9. S. J. Royal, B. Key, J. Neurosci. 19, 9856–9864 (1999).
10. M. S. Bailey, A. C. Puche, M. T. Shipley, J. Comp. Neurol.
415, 423–448 (1999).
11. S. M. Potter et al., J. Neurosci. 21, 9713–9723 (2001).
12. A. Bulfone et al., Neuron 21, 1273–1282 (1998).
13. A. Mackay-Sim, P. W. Kittel, Eur. J. Neurosci. 3, 209–215
14. P. Mombaerts, Annu. Rev. Cell Dev. Biol. 22, 713–737 (2006).
15. J. A. John, B. Key, Chem. Senses 28, 773–779 (2003).
16. C. R. Yu et al., Neuron 42, 553–566 (2004).
17. T. Bozza, J. P. McGann, P. Mombaerts, M. Wachowiak,
Neuron 42, 9–21 (2004).
18. L. Ma et al., Proc. Natl. Acad. Sci. U.S.A. 109, 5481–5486
19. P. Feinstein, P. Mombaerts, Cell 117, 817–831 (2004).
20. G. Barnea et al., Science 304, 1468 (2004).
21. K. T. Nguyen-Ba-Charvet, T. Di Meglio, C. Fouquet,
A. Chédotal, J. Neurosci. 28, 4244–4249 (2008).
22. H. Takeuchi et al., Cell 141, 1056–1067 (2010).
23. D. M. Lin et al., Neuron 26, 69–80 (2000).
24. E. J. Clowney et al., Cell 151, 724–737 (2012).
25. P. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 95, 11371–11376
26. T. Imai et al., Science 325, 585–590 (2009).
27. S. Serizawa et al., Cell 127, 1057–1069 (2006).
28. A. Fleischmann et al., Neuron 60, 1068–1081 (2008).
29. T. Imai, H. Sakano, Eur. J. Neurosci. 34, 1647–1654 (2011).
30. D. J. Zou et al., Science 304, 1976–1979 (2004).
31. H. B. Treloar, P. Feinstein, P. Mombaerts, C. A. Greer,
J. Neurosci. 22, 2469–2477 (2002).
32. T. K. Hensch, Nat. Rev. Neurosci. 6, 877–888 (2005).
Acknowledgments: We thank L. Nay, E. S. Gillespie, and
the Laboratory Animal Services at the Stowers Institute for
technical assistance and S. Lomvardas, R. Axel, N. Ryba,
and P. Mombaerts for sharing transgenic mice. We appreciate
helpful discussions with H. Taniguchi, H. Sakano, Y. Zou,
and members of the Yu lab. This work fulfills, in part,
requirements for Y. W.’s Ph.D. thesis with the Open University,
United Kingdom. This work is supported by funding
from Stowers Institute.
Materials and Methods
Figs. S1 to S3
21 November 2013; accepted 28 February 2014
A Critical Period Defined
by Axon-Targeting Mechanisms
in the Murine Olfactory Bulb
Lulu Tsai* and Gilad Barnea†
The olfactory system remains plastic throughout life because of continuous neurogenesis of sensory
neurons in the nose and inhibitory interneurons in the olfactory bulb. Here, we reveal that
transgenic expression of an odorant receptor has non–cell autonomous effects on axons expressing
this receptor from the endogenous gene. Perinatal expression of transgenic odorant receptor
causes rerouting of like axons to new glomeruli, whereas expression after the sensory map is
established does not lead to rerouting. Further, chemical ablation of the map after rerouting does
not restore the normal map, even when the transgenic receptor is no longer expressed. Our
results reveal that glomeruli are designated as targets for sensory neurons expressing specific
odorant receptors during a critical period in the formation of the olfactory sensory map.
Critical periods are epochs of increased brain plasticity when neural circuits are specially sensitive to shaping by stimu-
li. In the olfactory system, enhanced plasticity is
not confined to early development; rather, it is
maintained throughout adult life (1). This pro-
longed plasticity is achieved by the continuous
generation of the inhibitory granule cells that
migrate into the olfactory bulb and integrate
into the circuits and by the generation of olfactory
sensory neurons (OSNs) that incorporate into
the circuits throughout life (2, 3). Although we
know that plasticity is retained in the mature
olfactory system, does a critical period exist in
the formation of the sensory map in the olfac-
In mice, each OSN expresses only one of the
~1300 odorant receptor (OR) genes (4–7) from
only one allele (8). The OSNs that express the
same OR are randomly dispersed within a broad
zone in the main olfactory epithelium in the nose
(9, 10). In the olfactory bulb, the first olfactory
center in the brain, the axons of OSNs expressing
the same OR converge on spatially fixed neuropil
structures called glomeruli (9–11). Further, ORs
actively participate in the axon guidance of OSNs
to particular glomeruli (12, 13). In the glomeruli,
the axons synapse with the dendrites of mitral
and tufted cells, the projection neurons in the
bulb. Each projection neuron receives input from
a single glomerulus and sends its axon to the ol-
factory cortex. Thus, an olfactory sensory map
is formed in the bulb. In this map, the identity
of each odor is encoded by the combination of
glomeruli that it activates (3). In contrast to the
somatosensory, auditory, and visual maps, neigh-
boring relations between peripheral sensory neu-
rons are not maintained in the olfactory sensory
map. Because OSNs continue to integrate into
the circuits throughout life, the challenge of axon
guidance persists in adulthood (3).
We devised a strategy for ectopic expression
of a specific OR, MOR28, in a temporally controlled manner using the tetracycline response
element (tetO) to drive its expression. The tetO
promoter is activated by the tetracycline-controlled
transcription activator t TA, which is inhibited by
the antibiotic doxycycline. When doxycycline
is removed, expression from the tetO promoter
is induced within days (14–16). A similar approach for inducing ectopic expression of ORs
was previously used (17–19). Our strategy involved the use of three alleles (fig. S1A). In
the first, designated OMP-IRES-tTA, the olfactory marker protein (OMP) drives expression of
t TA in all OSNs (16). In the second, designated
tetO::MOR28-IRES-tau-LacZ (TO28), tetO
drives the expression of MOR28 and the fusion
protein tau–b-galactosidase (b-gal). To distinguish between the OSNs that express MOR28
from its endogenous genomic locus (
endogenous MOR28 OSNs) versus OSNs that express
MOR28 from the transgene (transgenic MOR28
OSNs), we introduced a third allele, designated
MOR28-IRES-GFP. OSNs that express MOR28
from this allele also express green fluorescent
protein (GFP) (20). Thus, GFP expression marks
OSNs expressing MOR28 from its endogenous
locus. Because b-gal and GFP are exogenous to
mice, staining for each identifies transgenic or
endogenous MOR28 OSNs, respectively (fig. S1B).
Department of Neuroscience, Brown University, Providence, RI
*Present address: Department of Biology, Drexel University,
Philadelphia, PA 19104, USA.
†Corresponding author. E-mail: firstname.lastname@example.org