Hypomorphic CEP290/NPHP6 mutations result
in anosmia caused by the selective loss of G
proteins in cilia of olfactory sensory neurons
Dyke P. McEwen*, Robert K. Koenekoop†, Hemant Khanna‡, Paul M. Jenkins*, Irma Lopez†, Anand Swaroop‡§¶,
and Jeffrey R. Martens*?
Departments of *Pharmacology,‡Ophthalmology, and§Human Genetics, University of Michigan, Ann Arbor, MI 48105; and†McGill Ocular Genetics
Laboratory, Montreal Children’s Hospital Research Institute, McGill University Health Centre, Montreal, QC, Canada H3H 1P3
Edited by Randall R. Reed, The Johns Hopkins University School of Medicine, Baltimore, MD, and accepted by the Editorial Board August 11, 2007
(received for review May 3, 2007)
Cilia regulate diverse functions such as motility, fluid balance, and
sensory perception. The cilia of olfactory sensory neurons (OSNs)
compartmentalize the signaling proteins necessary for odor detec-
tion; however, little is known regarding the mechanisms of protein
sorting/entry into olfactory cilia. Nephrocystins are a family of
ciliary proteins likely involved in cargo sorting during transport
from the basal body to the ciliary axoneme. In humans, loss-of-
function of the cilia–centrosomal protein CEP290/NPHP6 is associ-
ated with Joubert and Meckel syndromes, whereas hypomorphic
mutations result in Leber congenital amaurosis (LCA), a form of
early-onset retinal dystrophy. Here, we report that CEP290–LCA
patients exhibit severely abnormal olfactory function. In a mouse
model with hypomorphic mutations in CEP290 [retinal dystro-
phy-16 mice (rd16)], electro-olfactogram recordings revealed an
anosmic phenotype analogous to that of CEP290–LCA patients.
Despite the loss of olfactory function, cilia of OSNs remained intact
in the rd16 mice. As in wild type, CEP290 localized to dendritic
knobs of rd16 OSNs, where it was in complex with ciliary transport
proteins and the olfactory G proteins Golfand G?13. Interestingly,
we observed defective ciliary localization of Golfand G?13but not
odorant signaling pathway in the rd16 OSNs. Our data implicate
distinct mechanisms for ciliary transport of olfactory signaling
proteins, with CEP290 being a key mediator involved in G protein
trafficking. The assessment of olfactory function can, therefore,
serve as a useful diagnostic tool for genetic screening of certain
syndromic ciliary diseases.
nephrocystin ? olfaction
numerous biological functions, including motility, sensory per-
from the plasma membrane at a basal body, originating from the
mother centriole of the centrosome complex (3). In multiciliated
cells, such as in olfactory sensory neurons (OSNs), ciliogenesis
requires the synthesis and assembly of multiple basal bodies
formed en masse from centrioles (4). OSNs terminate in a
dendritic knob containing multiple basal bodies from which
sensory cilia project into the nasal mucosa. These cilia compart-
mentalize the signaling molecules necessary for odorant detec-
tion. Odorant signal transduction is initiated by odorant binding
to G protein-coupled receptors, leading to activation of olfactory
G proteins (5, 6). The G protein heterotrimer consists of the
adenylyl cyclase-stimulating G?olf as well as the ??-subunit,
including G?13. Odor transduction through this complex effi-
ciently couples external stimuli to action potential generation (5,
6). The loss of olfactory cilia or deletion of selected components
of the olfactory signaling cascade leads to anosmia (7–11).
Although each component of this signaling cascade is enriched
ilia are microtubule-based structures that project from the
surface of most cells in humans and are implicated in
in olfactory cilia, little is known regarding the mechanisms of
their trafficking and subcellular localization.
Cilia of OSNs lack the necessary machinery for protein
synthesis. Therefore, nascent proteins must be transported from
the cell body into the cilium. Movement along the ciliary
axoneme is tightly regulated and most likely involves evolution-
arily conserved intraflagellar transport (IFT) proteins, whereas
multiprotein complexes at the basal body act as a barrier to
diffusion and restrict access to the cilium (1, 12). Importantly,
appears to be a selective gate at the base that regulates entry.
Despite ever-increasing knowledge of IFT components,
the mechanisms regulating protein sorting/entry are poorly
Genetic mutations in ciliary, basal body, and centrosomal
proteins lead to pleiotropic human diseases, including polycystic
kidney disease, Bardet–Biedl syndrome, Senior–Loken syn-
drome, Meckel syndrome, and retinitis pigmentosa (RP) (2, 7,
13–15). Nephrocystins are a family of ciliary proteins that are
likely involved in cargo sorting during transport from the basal
body to the ciliary axoneme (16–20). Loss-of-function mutations
in CEP290 are associated with Joubert syndrome, which is
characterized by nephronophthisis, retinal degeneration, and
cerebellar vermis hypoplasia (21, 22). Interestingly, an in-frame
deletion of exons 35–39 in CEP290 causes early-onset retinal
degeneration in the rd16 mouse, without associated cerebellar or
kidney abnormalities (23). This deletion led to the identification
of hypomorphic CEP290 mutations in ?20% of patients with
Leber congenital amaurosis (LCA), a severe retinal dystrophy
characterized by visual impairment from birth (24, 25). As in the
rd16 mouse, these LCA patients did not exhibit cerebellar or
renal abnormalities. CEP290 is localized to the connecting
cilium of retinal photoreceptors, the basal body of an inner
medullary collecting duct cell line (IMCD-3), and the centro-
Author contributions: R.K.K. and H.K. contributed equally to this work; D.P.M., A.S., and
J.R.M. designed research; D.P.M., R.K.K., H.K., P.M.J., and I.L. performed research; D.P.M.,
R.K.K., H.K., P.M.J., A.S., and J.R.M. analyzed data; and D.P.M., R.K.K., H.K., P.M.J., A.S.,
and J.R.M. wrote the paper.
The authors declare no conflict of interest.
Abbreviations: OSN, olfactory sensory neuron; IFT, intraflagellar transport; RP, retinitis
pigmentosa; LCA, Leber congenital amaurosis; GEF, guanine–nucleotide exchange factor;
ACIII, adenylyl cyclase type III.
¶To whom correspondence may be addressed at: Ophthalmology and Visual Sciences
and Human Genetics, 537, Kellogg Eye Center, 1000 Wall Street, University of Michigan,
Ann Arbor, MI 48105. E-mail: firstname.lastname@example.org.
?To whom correspondence may be addressed at: Department of Pharmacology, University
of Michigan, 1301 MSRB III, 1150 W. Medical Center, Ann Arbor, MI 48109-5632. E-mail:
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2007 by The National Academy of Sciences of the USA
October 2, 2007 ?
vol. 104 ?
no. 40 ?
some of mitotic cells (21–23). Furthermore, the basal body of
mammalian olfactory cilia is enriched in ?-tubulin and resembles
the Caenorhabditis elegans transition zone, which is enriched in
nephrocystin proteins (7, 16–18). Hence, mutations in CEP290
may alter ciliary microtubule transport.
We therefore hypothesized that CEP290 participates in reg-
ulating the transport of specific signaling proteins involved in
odorant detection and that olfactory function may be impaired
in blind individuals with CEP290 mutations. Here, we show that
CEP290–LCA patients and the rd16 mouse exhibit severe ol-
factory dysfunction, likely caused by altered ciliary localization
of olfactory G proteins in sensory neurons. This work describes
a mechanism by which G protein trafficking along the ciliary
axoneme is selectively regulated at the dendritic knobs by
Impaired Olfactory Function in LCA Patients with CEP290 Mutations.
We investigated olfactory function in a French–Canadian LCA
family, which originally revealed the CEP290 mutation, a splice-
site change resulting in a premature stop codon (Cys998X) (24).
Patients homozygous for the mutation and heterozygous carriers
were evaluated by using the Brief Smell Identification Test. All
LCA patients examined exhibited severely abnormal olfactory
function (Fig. 1), whereas CEP290 Cys998X heterozygous car-
riers exhibited mild to severe microsmia (data not shown).
Retinopathy patients with mutations in RPGR, RPGRIP, and
USH2A, three other cilia and/or centrosomal proteins, demon-
strated normal olfactory function (Fig. 1). These data have been
stratified by age as shown in supporting information (SI) Fig. 7.
Olfactory Dysfunction in rd16 Mice Carrying In-Frame Deletion of
CEP290.To investigate the mechanism of anosmia associated with
hypomorphic CEP290 alleles in LCA, we assessed olfactory
function in 1-month-old rd16 mice by electro-olfactograms,
which measure the odorant-induced, summated generator po-
tential of sensory neurons from the mucosal surface. At all doses
of amyl acetate or other odorants tested, the rd16 mice showed
significantly reduced electro-olfactogram responses compared
with those of WT mice (Fig. 2 A and B), validating the results
from our LCA patients.
Structural Integrity of Olfactory Cilia in rd16 Mice. The anosmic
phenotype in the Bbs4-null mice (7, 26) is caused by the
disruption of the microtubule network and a deficiency in the
number of olfactory cilia (7). Hence, one possible mechanism for
olfactory dysfunction caused by CEP290 mutations can be the
loss of cilia from OSNs. In the rd16 mice, both the cilia, as
measured by acetylated ?-tubulin staining (Fig. 3 Center), and
the dendritic knobs, marked by ?-tubulin (Fig. 3A), appeared
unaltered. The localization of mutant CEP290 to the dendritic
knobs also was unaffected in the rd16 OSNs (Fig. 3 B and C).
Further examination by scanning electron microscopy of the
olfactory epithelium surface from WT and rd16 mice demon-
strated an intact cilia layer from which no discernible differences
in the ultrastructure of the cilia could be detected (Fig. 3D). The
dendritic endings with proximal parts of the olfactory cilia for
both WT and rd16 mice are shown in SI Fig. 8 A and B. In
addition, we measured the width of the ciliary layer as demar-
cated by acetylated tubulin staining and found no statistical
difference between WT and rd16 mice (SI Fig. 8C). We,
however, observed that select regions of the epithelium showed
signs of dendritic microtubule disorganization. The normal
parallel tracks of acetylated ?-tubulin staining are observed in
the dendrites of WT mice; these tracks were disordered in small
clusters in the rd16 OSNs (SI Fig. 9 A and B). TUNEL staining
revealed that this population of neurons may be in the early
stages of neuronal apoptosis and/or epithelial degeneration (SI
Fig. 9C), whereas other regions of the rd16 olfactory epithelium
appear normal (SI Fig. 9D) compared with WT mice (SI Fig.
9E). Overall, our results do not provide any evidence of defects
in ciliogenesis or global integrity of olfactory cilia in the rd16
Mislocalization of Olfactory G Proteins in rd16 Mice. In the absence
of any overt structural defects in the cilia of OSNs, we examined
whether the CEP290 mutation altered its expression or ability to
interact with other transport proteins. Immunoblot analysis of
the olfactory epithelial protein extract revealed that, aside from
the expected molecular mass shift observed in the rd16 mouse,
there was no detectable change in the amount of the CEP290
protein (Fig. 4A). Immunoprecipitations from rd16 olfactory
epithelial tissue showed that CEP290 remains associated with
retinopathies (LCA or RP) were evaluated for olfactory defects with the Brief
scores ?8 were considered abnormal. Patients with LCA (CEP290 Cys998X/
with mutations in other retinopathy genes were unaffected.
Assessment of olfactory function in LCA patients. Individuals with
from WT (n ? 9) and rd16 (n ? 5) mice after exposure of the olfactory
epithelium to three concentrations of amyl acetate. Arrow indicates time of
both WT (empty bars; n ? 9) and rd16 (filled bars; n ? 5) mice showing four
different concentrations of amyl acetate, along with five other odorants all
tested at a concentration of 10?3M of the respective odorant. All responses
were normalized to a pulse of pure amyl acetate given during the same trace.
AA, amyl acetate; 8-AL, octanal; 7-AL, heptaldehyde; 6-AL, hexanal; Eug,
eugenol; Car, carvone.*, P ? 0.05 as determined by an unpaired t test.
www.pnas.org?cgi?doi?10.1073?pnas.0704140104McEwen et al.
ciliary, basal body, and microtubule transport proteins, including
p150Glued(a dynein motor protein), KIF3A (a kinesin motor
involved in IFT), RPGRORF15[a putative guanine–nucleotide
exchange factor (GEF)], and ?-tubulin (Fig. 4B), as observed in
WT retina (16). We did not detect an interaction between
CEP290 and an intraflagellar transport protein, IFT88, or a
Bardet–Biedl syndrome protein, BBS4 (Fig. 4B). No change was
detected in the interaction of any of these proteins in the rd16
mice compared with WT. We, however, discovered that both
also immunoprecipitate with CEP290 in WT and rd16 mice (Fig.
4C), suggesting that CEP290 may regulate the transport of
olfactory signal proteins.
To further dissect the mechanism of impaired olfactory func-
tion, we examined the localization of olfactory signaling mole-
cules in OSNs of the rd16 mouse. Prominent ciliary staining of
G?13and Golfwas detected in the WT, but these G proteins were
undetectable in the OSNs cilia of the rd16 mice (Fig. 5 A–D).
Although the G?13 protein exhibited mislocalization to cell
bodies and dendrites (see Fig. 5 A and B), Golf could not be
detected in the extra-ciliary layers of the rd16 OSNs (Fig. 5 C and
D). Interestingly, type III adenylyl cyclase (ACIII; isoform
expressed in OSNs) and the ?-subunit of the cyclic nucleotide-
gated channel, CNGA2, remained enriched in olfactory cilia and
colocalized with acetylated tubulin, consistent with the observed
maintenance of ciliary integrity in rd16 mice (Fig. 5 E–H). Fig.
5I shows the averaged data in which the fluorescence signal from
the immunostained images was quantitated. These results indi-
cate that the olfactory phenotype in LCA patients and rd16 mice
is likely caused by the defective transport of olfactory G proteins.
We then tested the subcellular localization of G protein-
coupled odorant receptors by immunohistochemical analysis.
Unlike the G proteins, mOR28 remained localized to OSNs cilia
of rd16 mice (Fig. 6 A–C), showing a similar staining pattern to
WT mouse olfactory epithelium (SI Fig. 10). In addition, anti-
bodies against two other odorant receptors, mOR256 and
mOR262, marked cilia in OSNs of rd16 mice (SI Fig. 11). These
data indicate that olfactory signaling components do not get
transported as a preassembled complex and that the entry of G
protein signaling complex into cilia is regulated by at least two
Our study shows that CEP290 participates in regulating the
detection and that olfactory function is impaired in blind indi-
pattern of CEP290 in vivo. Olfactory epithelial slices were subject to immuno-
histochemistry by using either CEP290 antiserum or an anti-?-tubulin anti-
body. (A) ?-Tubulin localizes to dendritic knobs. (B and C) Staining patterns
indicate that CEP290 localization is similar to ?-tubulin in dendritic knobs in
both the WT (B) and rd16 (C) mice. (Scale bar, 10 ?m.) (D) Scanning electron
microscopy revealed no difference in the olfactory epithelium between WT
Immunohistochemistry from WT and rd16 mice showing the staining
(A) Western blot analysis of olfactory epithelium shows CEP290 expression in
297-aa deletion in CEP290. (B) Lysates from WT and rd16 mouse olfactory
epithelium were immunoprecipitated with, and blotted for the indicated
antibodies. Molecular mass in kilodaltons is shown to the left of each blot.
Input indicates 10% of the immunoprecipitate starting material. (C) Lysates
were immunoprecipitated with the indicated antibodies and separated by
SDS/PAGE. Immunoblots were probed with CEP290 antiserum, and the arrow
the immunoprecipitate starting material in both WT and rd16 mice. IP, im-
munoprecipitation; IB, immunoblotting.
McEwen et al.
October 2, 2007 ?
vol. 104 ?
no. 40 ?
viduals with CEP290 mutations. CEP290 is expressed in OSNs
and is localized to dendritic knobs, which contain multiple basal
bodies that form the base of extending cilia. In OSNs, hypomor-
phic mutations in CEP290 specifically result in the mislocaliza-
tion of olfactory G proteins but not of other components of the
signaling cascade tested. These results suggest that the entire
complement of OSN transduction proteins does not move into
cilia as a single preassembled complex. The odorant receptors
traffic into cilia independently of olfactory G proteins and,
therefore, may assemble into a signaling complex within the
Our studies suggest that OSNs use different mechanisms for
the control of protein entry into the cilium. One interesting
hypothesis is that there may be differences in the way that
peripheral and transmembrane proteins localize to cilia. Al-
though anchored, peripheral membrane proteins exist predom-
inantly in the cytoplasm and are more likely to interact with the
basal body complex found beneath the plasma membrane. As an
integral component of this complex, CEP290 is positioned to
regulate movement of cargo into and out of the cilium. The basal
body has been proposed to act as a highly selective barrier
regulating protein entry into the cilium (27). Located adjacent to
the basal body is a protein-rich region of the ciliary membrane,
termed the ‘‘ciliary necklace,’’ which has been compared func-
tionally with the nuclear pore complex (1, 27). Selective entry
through the nuclear pore requires cargo binding to a GTP-
binding protein and guanine–nucleotide exchange involving a
GEF to release cargo into the nucleus (27). We showed that
RPGRORF15, a putative GEF (28), is a cilia/centrosomal protein
bodies distally along the ciliary axoneme in the photoreceptor
connecting cilium (14, 29). We now show that RPGRORF15is in
complex with CEP290 in OSNs of both WT and rd16 mice,
suggesting a possible role in regulating protein entry into the
olfactory cilium. Furthermore, recent reports have identified
Ric8B as a putative GEF, which interacts with Golfand increases
functional expression of odorant receptors (30, 31). Therefore,
it is tempting to hypothesize a role for GEF activity acting in
complex at the basal body with CEP290 to regulate protein entry
into the cilia. These results, together with our finding that cilia
remain intact in rd16 OSNs, suggest that CEP290 plays multiple
regulatory roles in protein entry into the cilium. It may act as a
docking zone for IFT motors, such as KIF3A, while simulta-
neously orchestrating peripheral membrane protein trafficking
It has been shown previously that, in rd16 mice, mutations in
CEP290 result in retinal degeneration of rod, but not cone,
photoreceptors (23, 32). Unlike OSNs, where G protein-coupled
receptors remain localized to cilia, in rod photoreceptors both
rhodopsin and arrestin are mislocalized before any degeneration
molecules enriched in cilia (green) and costained with acetylated ?-tubulin
(red). (A) G?13is enriched in cilia of WT mouse OSNs and colocalizes with
acetylated ?-tubulin (merged image). (B) G?13is undetectable in the ciliary
layer and is mislocalized to dendrites and cell bodies in rd16 mice. (C) Similar
to G?13staining, Golfis enriched in olfactory cilia and colocalizes with acety-
lated ?-tubulin. (D) In the rd16 mice, Golfis absent from olfactory cilia and,
unlike G?13, does not appear to redistribute within OSNs. (E and F) ACIII
remains enriched in both WT and rd16 mice and colocalizes with acetylated
mice and colocalizes with acetylated ?-tubulin in OSN cilia. (Scale bar, 10 ?m.)
(I) Fluorescence quantitation of immunohistochemical images represented in
A–H. Data are averages of at least 30 images per protein from three discon-
tinuous regions per olfactory epithelium. Four different mice were analyzed
for both WT (filled bars) and rd16 (empty bars).***, P ? 0.001 as determined
by an unpaired t test.
Defects in localization of olfactory G proteins in rd16 mice. Olfactory
olfactory epithelial slices were stained by using antibodies directed against
mOR28. (A) A compressed confocal image showing multiple mOR28-
expressing olfactory sensory neurons. (Scale bar, 20 ?m.) (B) A ?2 zoomed
image of the boxed neuron in A showing mOR28 expression in the cell body,
dendrite, and dendritic knob. (Scale bar, 5 ?m.) (C) A ?2.5 zoomed image of
the boxed region in B showing receptor expression in OSNs cilia. Arrows
indicate individual cilia. (Scale bar, 2 ?m.)
Maintenance of mOR28 localization to cilia in rd16 mice. rd16 mouse
www.pnas.org?cgi?doi?10.1073?pnas.0704140104 McEwen et al.
in rd16 retina (23). This may reflect differences between the rod
photoreceptor connecting cilium vs. the terminal cilia of OSNs.
Despite these differences, it is apparent that sensory cells are
much more susceptible to mutations in CEP290 than other
ciliary systems. Nonsense mutations of CEP290 result in Joubert
syndrome, characterized by retinal degeneration, cerebellar ver-
mis aplasia, and nephronophthisis, and Meckel syndrome, an
autosomal recessive lethal condition characterized by CNS,
kidney, and liver malformations (15, 21, 22, 25). Missense
mutations (such as those detected in LCA and the rd16 mice),
however, appear to only affect sensory systems, resulting in
retinal degeneration and olfactory dysfunction in both humans
and mice, with no overt kidney or cerebellar defects.
Although mutations in different ciliary proteins can result in
analogous sensory phenotypes, the underlying mechanisms ap-
pear divergent. Similar to our studies presented here, null
mutations in Bbs1, Bbs2, or Bbs4 are reported to result in sensory
defects, including anosmia and retinal degeneration (7, 33, 34).
However, a striking difference between rd16 and Bbs-null mice
is that olfactory cilia remain intact with hypomorphic mutations
in CEP290. This is consistent with earlier studies in C. elegans,
in which mutations in nephrocystins specifically affected che-
mosensation and sensory behavior without altering ciliary as-
sembly or maintenance (18, 19, 35). Furthermore, the odorant
receptors ACIII and CNGA2 remained localized to OSNs cilia,
suggesting that in-frame deletion of CEP290 selectively alters
protein transport into the cilia. The fact that cilia remain intact
makes the rd16 mouse a powerful model to further investigate
the regulatory mechanisms of protein trafficking into cilia.
We postulate that olfactory dysfunction may represent an
underappreciated clinical manifestation of ciliary disease. In
humans, mutations in members of the nephrocystin family of
proteins result in pleiotropic phenotypes, including nephron-
ophthisis, Senior–Loken syndrome, retinitis pigmentosa, hepatic
fibrosis, and situs inversus (36–38). However, olfactory function
has not been reported in these populations. Importantly, LCA
represents a disease in which patients exhibit previously unchar-
acterized impaired olfactory function unrelated to age or head
trauma. Intriguingly, in our study, all CEP290–LCA patients
queried before olfactory testing presented with a self-assumed
normal olfactory function. This false supposition indicates that
olfactory dysfunction may be much more prevalent in patients
with ciliary diseases. We suggest that assessment of olfactory
function may be a useful screening tool for early diagnosis of
Materials and Methods
Individuals with CEP290 Mutations. LCA patients with CEP290
mutations are described in ref. 24.
Antibodies. Antibodies used in this study were procured as
follows: monoclonal anti-acetylated ?-tubulin antibody and a
polyclonal anti-?-tubulin antibody (Sigma–Aldrich, St. Louis,
MO), anti-Kif3A and anti-p150Glued(BD Biosciences, San Jose,
CA), anti-adenylyl cyclase III, anti-Golf polyclonal antibodies,
and HRP-conjugated secondary antibodies (Santa Cruz Bio-
technology, Santa Cruz, CA), polyclonal anti-mOR28 (R. Axel,
Columbia University, New York, NY; see ref. 39), polyclonal
anti-mOR256–14 and mOR262 (H. Breer, University of Ho-
henheim, Stuttgart, Germany; see ref. 40), polyclonal anti-G?13
(R. Margolskee, Mount Sinai School of Medicine of New York
University, New York, NY; see ref. 41), polyclonal IFT88
antibody (B. Yoder University of Alabama, Birmingham, AL;
see ref. 42), and secondary antibodies conjugated to Alexa Fluor
dyes (Invitrogen, Carlsbad, CA). The polyclonal anti-CEP290
and polyclonal anti-RPGR-ORF15CPantibodies are described in
ref. 23. Anti-CNGA2 antibody was purchased from Alomone
Labs (Jerusalem, Israel).
Western Blots. Olfactory epithelia were dissected from C57BL/6
WT and rd16 mutant mice and homogenized with a Dounce
homogenizer in 50 mM Tris/5 mM EDTA (pH 8.0) containing
Complete protease inhibitor tablets (Roche Applied Science,
Indianapolis, IN). Insoluble material was removed by centrifu-
gation at 1,000 ? g for 5 min. Soluble protein was quantified, and
equal amounts of protein were analyzed by SDS/PAGE and
immunoblotting using appropriate antibodies. Blots were visu-
alized with the Enhanced Chemiluminescence kit from Pierce
Immunoprecipitation. Olfactory epithelia were dissected from
mice, and coimmunoprecipitation was performed as
Electro-Olfactograms. Vapor-phase odor stimuli were generated
by placing 10 ml of solution in a sealed 50-ml glass bottle.
Odorants were delivered as a 20-ml pulse injected into a
continuous stream of humidified air flowing over the tissue
sample. Electrodes ranging from 4 to 8 M? in resistance were
placed on either turbinate II or IIb for recording. We analyzed
data with Clampfit (Molecular Devices, Sunnyvale, CA) and
determined peak heights from prepulse baseline. Data were
normalized to a pulse of pure amyl acetate given later in that
same trace. For the rd16 mice, data were normalized to the
average response of the WT mouse for a specific dose of the
odorant. At least three mice were tested for each condition.
Sectioning and Immunohistochemistry. After the preparation for
electro-olfactograms, the other hemisphere of the mouse head
was immersion-fixed in 4% paraformaldehyde for 2–12 h. For
cryosections, the tissue was incubated in 30% sucrose in PBS for
24 h at 4°C, frozen in OCT compound (Sakura Finetek, Tor-
rance, CA), and cut into sections (12–14 ?m) on a cryostat.
Immunohistochemistry was performed on adult olfactory
tissue sections after washing the sections with PBS three times
for 5 min each and then incubating them in blocking buffer
containing 10% normal serum/0.1% Triton X-100 (Fisher Sci-
entific, Pittsburgh, PA) in PBS for 1 h at room temperature.
Sections were then incubated with primary antibody for 1 h at
room temperature or overnight (CEP290) at 4°C. We used
primary antibodies at the following dilutions: ACIII at 1:500;
G?13at 1:500; CEP290 at 1:200; ?-tubulin at 1:500; Golfat 1:200;
and acetylated ?-tubulin at 1:1,000. Sections were washed in PBS
three times for 5 min each at room temperature and visualized
by secondary antibody binding with Alexa Fluor-conjugated
secondary antibodies. Coverslips were mounted with ProLong
Gold (Invitrogen). Images from at least three discontinuous
regions from three different mice were collected on an Olympus
(Melville, NY) Fluoview 500 confocal microscope with a ?100
1.35 NA oil objective.
TUNEL Staining. TUNEL staining was performed on at least three
discontinuous sections from three different mice for each ge-
notype tested according to the manufacturer’s instructions (Mil-
lipore, Billerica, MA).
Scanning Electron Microscopy. The samples were fixed in 2.5%
glutaraldehyde in 0.1 M Sorenson’s buffer at 4°C overnight. The
samples were then washed twice in buffer and dehydrated in
ascending concentrations of ethanol, treated with 100% ethanol,
and dried in hexamethyldisilazane overnight. Dried samples
were mounted on the aluminum stubs and coated with gold by
using a Polaron Sputter Coater (Ringmer, U.K.). The samples
were examined with an Amray (Drogheda, Ireland) 1910FE field
emission scanning electron microscope at 5 kV. Images were
recorded digitally with Semicaps software.
McEwen et al.
October 2, 2007 ?
vol. 104 ?
no. 40 ?
We thank Richard Axel, Gilad Barnea (Columbia University), Heinz
Breer, Robert Margolskee, and Bradley Yoder for the generous gift of
antibodies; Sasha Meshinchi at the University of Michigan Microscopy
and Image Analysis Laboratory for help with the scanning electron
microscopy images; and Bo Chang, Frans Cremers, John Heckenlively,
Friedhelm Hildebrandt, and Anneke den Hollander for valuable discus-
sions. This research was supported by National Institutes of Health
Grants EY007961, EY007003, T32 DC00011, and GM07767, Research
to Prevent Blindness, the Foundation Fighting Blindness of USA and
Canada, and the Fonds de la Recherche en Sante ´e du Que ´bec.
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