All procedures involving animals were carried out in accordance with NIH standards and approved by the Harvard University Institutional Animal Care and Use Committee (IACUC). Unless otherwise indicated, values are presented as the mean ± standard error of the mean.
Transgenic and gene targeted mice
The Flag-H2be transgenic mouse line (), which expresses FLAG-H2BE under control of the H2be
promoter, was generated based on a described protocol (Yang et al., 1997
). Briefly, a FLAG-encoding DNA sequence was inserted through homologous-recombination immediately upstream of the H2be
CDS within BAC RP23-16G3, which contains a 200-kb region of mouse genomic sequence surrounding the H2be
gene. The modified BAC was amplified in E. coli
, confirmed by sequencing, and injected (Harvard Genome Modification Facility) into fertilized mouse zygotes. Transgenic founders were crossed to C57Bl/6 mice to establish the Flag-H2be line. Heterozygous Flag-H2be mice contain a single genomic copy of the transgene that is expressed in a pattern and at a level indistinguishable from that of the endogenous gene (see ).
The H2be-KO mouse line (), in which the endogenous H2be CDS is replaced with a sequence encoding GAP43-mCherry (an N-terminal fusion of the first 20 amino acids of GAP43 to mCherry), was generated through homologous recombination of the endogenous H2be locus in mouse embryonic stem cells (ESCs) using standard methods. Following selection, ESCs were screened for the desired recombination events, confirmed by sequencing, and injected (Harvard Genome Modification Facility) into mouse blastocysts. Founders were crossed to C57Bl/6 mice to establish the H2be-KO line, in which Gap43-mCherry is expressed in a pattern indistinguishable from that of H2be (see ).
The H2be-GF transgenic mouse line (), which expresses Flag-H2be under control of the olfactory marker protein (Omp
) promoter, was generated by complete replacement of the Omp
CDS in plasmid pJOMP (Danciger et al., 1989
) with a sequence encoding FLAG-H2BE, followed by pronuclear injection (Harvard Genome Modification Facility) of the linearized construct into fertilized mouse zygotes. Transgenic founders were crossed to C57Bl/6 mice to establish the H2be-GF line. Heterozygous mice contain approximately 12 genomic copies of the transgene, which are expressed in mature olfactory neurons throughout the MOE with the exception of a band of neurons near zone 2 (see ), a pattern that was reproducibly observed in all individuals examined (n
The P2-IRES-Tau-LacZ mouse line (Mombaerts et al., 1996
), which was used to identify possible axon guidance defects in H2be-KO mice (), and the Cnga2-null/Tau-LacZ (Zhao and Reed, 2001
) and Adcy3-null (Wong et al., 2000
) mouse lines, which were used to identify second messengers affecting H2be
expression () were described previously.
Primary antibodies used
- Active-CASP3 (rabbit polyclonal; Promega, Madison, WI, USA; G7481)
- β-GAL (rabbit polyclonal; MP Biomedicals, Solon, OH, USA; 55976)
- BrdU (mouse monoclonal Bu20a; Dako North America, Carpinteria, CA, USA; M0744)
- FLAG (mouse monoclonal M2; Sigma-Aldrich, St. Louis, MO, USA; F1804)
- FLAG (rabbit polyclonal; Sigma-Aldrich; F7425)
- GAP43 (rabbit polyclonal; Novus Biologicals, Littleton, CO, USA; NB300-143A4)
- H2B (rabbit polyclonal; EMD Millipore, Billerica, MA, USA; 07-371)
- H2B-Lys5-Me (rabbit polyclonal; Abcam, Cambridge, MA, USA; ab12929)
- H2B-Lys5-Ac (rabbit monoclonal; Abcam; ab40886)
- H2B-Lys120-Ub (mouse monoclonal NRO3; Medimabs, Montréal, Québec, Canada; MM-0029)
- Histone H3 (rabbit polyclonal; Abcam; ab1791)
- NEUROD1 (goat polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc1084)
- OMP (goat polyclonal; Wako Chemicals USA, Richmond, VA, USA; 344-10001)
- Tyrosine hydroxylase (rabbit polyclonal; Millipore; AB152)
All histological OR gene expression analyses were performed using fluorescent in situ hybridization (ISH). With the exception of H2be mRNA analyses (), which were performed using chromogenic ISH, all other histological analyses were performed using immunofluorescence (IF) or immunohistochemistry (IHC; for Active-CASP3, ). Unless noted, all images are of coronal tissue sections.
Preparation of ISH probes
ISH target sequences were amplified by PCR and inserted into the pCRII-TOPO vector (Life Technologies, Grand Island, NY, USA). OR antisense probes were designed to span 500–1000 base pairs, to target CDS or UTR gene regions, and to have <70% identity to any other sequence in the mouse genome. Probes were generated from 1 μg of linearized plasmid template using T7 or Sp6 polymerases (Promega) and digoxigenin or fluorescein RNA labeling mixes (Roche Applied Science, Indianapolis, IN, USA), treated with DNaseI (Promega) and ethanol precipitation, and dissolved in a 30-μL volume of water.
Whole tissues were carefully dissected from surrounding bones, frozen immediately in OCT compound (Sakura Finetek USA, Torrance, CA, USA) on dry ice, and stored at −80°C. Tissue blocks were cut into 12-μm thick cryo-sections, placed onto slides, and stored at −80°C. Chromogenic ISH experiments were performed essentially as described (Schaeren-Wiemers and Gerfin-Moser, 1993
One-color fluorescent ISH
Fluorescent ISH experiments were performed using a modified version of the chromogenic ISH method. Briefly, slide-mounted sections were warmed (37°C, 10 min), equilibrated in phosphate-buffered saline (PBS; pH 7.2; 5 min, room temperature [RT]), fixed in paraformaldehyde (PFA; 4% in PBS; 10 min, RT), washed in PBS (3 min, RT), permeabilized with Triton-X-100 (0.5% in PBS; 10 min, RT) followed by sodium dodecyl sulfate (1% in PBS; 5 min, RT), washed in PBS (3 × 3 min, RT), incubated in acetylation solution (triethanolamine [0.1 M; pH 7.5], acetic anhydride [0.25%]; 10 min, RT), washed in PBS (3 × 3 min, RT), incubated in hybridization solution (formamide [50%], SSC [2×], Denhardts [5×], yeast tRNA [250 μg/mL], herring sperm DNA [200 μg/mL], EDTA [1 mM], sodium phosphate [0.05 M; pH 7]; 30 min, RT), hybridized with a digoxigenin-labeled antisense RNA probe (1:1000 in hybridization solution; 16 hr, 42°C), washed with SSC (2×; 5 min, 42°C), washed with SSC (0.2×; 3 × 30 min, 42°C), incubated in H2O2 (3% in TN [Tris–HCl (0.1 M; pH 7.5), 0.15 M NaCl]; 30 min, RT), washed in TNT (Tween-20 [0.05%] in TN; 3 × 3 min, RT), incubated in TNB (Blocking Reagent [Perkin Elmer, Waltham, MA, USA; 0.05% in TN]; 30 min, RT), incubated with anti-digoxigenin-POD antibody (Roche; 1:1000 in TNB; 12 hr, 4°C), and washed in TNT (3 × 20 min, RT). Fluorescent signals were generated using the Tyramide Signal Amplification (TSA) Plus Fluorescein Kit (Perkin Elmer) according to the manufacturer's instructions. Slides were mounted using Vectashield (Vector Laboratories, Burlingame, CA, USA) containing DAPI (5 μg/mL).
Two-color fluorescent ISH
Two-color ISH was performed as described for one-color ISH, with the following modifications: Tissue sections were simultaneously hybridized with both digoxigenin- and fluorescein- or dinitrophenyl-labeled antisense RNA probes (1:1000 each in hybridization solution). Following incubation in TNB (30 min, RT), sections were incubated with anti-fluorescein-POD antibody (Roche; 1:1000 in TNB; 12 hr at 4°C) or anti-dinitrophenyl-HRP antibody (Perkin Elmer; 1:350 in TNB; 3 hr at 25°C) and washed in TNT (3 × 20 min, RT). Fluorescent signals corresponding to the fluorescein- or dinitrophenyl-labeled probes were generated using the TSA Plus Fluorescein Kit, after which sections were washed in TNT (2 × 3 min, RT), incubated in H2O2 (3% in TN; 1 hr, RT), washed in TNT (3 × 3 min, RT), incubated with anti-digoxigenin-POD antibody (1:1000 in TNB; 12 hr, 4°C), and washed in TNT (3 × 20 min, RT). Fluorescent signals corresponding to the digoxigenin-labeled probe were generated using the TSA Plus Cyanine5 Kit (Perkin Elmer) according to the manufacturer's instructions. Slides were mounted using Vectashield containing DAPI (5 μg/mL).
Combined ISH and IF
Combined ISH and IF experiments were performed as described for one-color ISH, with the following modifications: Acetylation, which dramatically reduces detection of the FLAG epitope, was omitted. Following incubation in TNB (30 min, RT), sections were incubated with a mixture of anti-digoxigenin-POD and mouse anti-FLAG antibodies (each 1:1000 in TNB; 12 hr, 4°C) and washed in TNT (3 × 20 min; RT). Fluorescent signals corresponding to the digoxigenin-labeled RNA probe were generated using the TSA Plus Fluorescein Kit, after which sections were washed in TNT (2 × 3 min, RT), incubated with anti-mouse-Alexa647 antibody (Invitrogen; 1:1000 in TNB; 12 hr, 4°C), and washed in TNT (3 × 20 min, RT). Slides were mounted using Vectashield containing DAPI (5 μg/mL).
One- or two-color IF
Animals were anesthetized with ketamine and perfused transcardially on ice with ice-cold PBS (25 mL) followed by ice cold PFA (4% in PBS; 25 mL). Whole tissues were carefully dissected from surrounding bones and immersed in ice-cold PFA (4% in PBS; 1 hr [OB] or overnight [MOE]). MOE tissue was decalcified in EDTA (250 mM in PBS, pH 8.5; 2 days, 4°C), and all tissues were cryoprotected in sucrose (10, 20, and 30% in PBS; 2 hr, 2 hr, and overnight, respectively). Tissues were frozen in OCT on dry ice and stored at −80°C. Tissue blocks were cut into 12-μm thick cryo-sections, placed onto slides, and stored at −80°C.
IF experiments were performed as follows: briefly, slide-mounted sections were warmed (37°C, 10 min), equilibrated in PBS (5 min, RT), fixed in PFA (4% in PBS; 10 min, RT), washed in PBS (3 min, RT), permeabilized with Triton X-100 (0.5% in PBS; 10 min, RT) followed by SDS (1% in PBS; 5 min, RT; omitted if preservation of intrinsic mCherry was necessary), washed in TNT (3 × 5 min, RT), blocked in fetal bovine serum (FBS; 10% in TN; 30 min, RT), incubated with primary antibodies (typically diluted 1:500–1:1000 in 10% FBS; 12 hr, 4°C), washed in TNT (3 × 5 min, RT), incubated with secondary antibodies (typically, Alexa488-labeled [or Alexa488- and Alexa647-labeled, for two-color IF]; Invitrogen; 1:1000 in 10% FBS; 12 hr, 4°C), and washed in TNT (3 × 15 min, RT). Slides were mounted using Vectashield containing DAPI (5 μg/mL).
Combined IHC (active-CASP3) and IF (OMP)
Mice were perfused and the MOE tissue processed as described for IF with the following modifications: After fixation in PFA and PBS washes, slide-mounted sections were permeabilized with Triton X-100 (0.5% in PBS; 30 min, RT), washed with PBS (3 × 3 min, RT), incubated in H2O2 (3% in TN buffer; 30 min, RT), washed with TNT (3 × 3 min, RT), blocked in TNB (30 min, RT), incubated with a mixture of anti-active-CASP3 and anti-OMP antibodies (each 1:300 in TNB; 12 hr, 4°C), washed with TNT (3 × 3 min, RT), and incubated with anti-rabbit-HRP (Jackson; 1:500 in TNB) for 12 hr, 4°C. Fluorescent signals corresponding to active-CASP3 were generated using the TSA Plus Fluorescein Kit, after which sections were washed in TNT (3 × 5 min, RT), incubated with anti-goat-Cy5 (Jackson ImmunoResearch Laboratories, West Grove, PA, USA; 1:500 in TNB; 12 hr, 4°C), and washed in TNT (3 × 15 min, RT). Slides were mounted using Vectashield containing DAPI (5 μg/mL).
Mice were injected intraperitoneally with BrdU (3 × 50 mg/kg in PBS; injections spaced 30 min apart) and sacrificed at the indicated timepoints (). The T = 0 timepoint, defined as 15 days post-injection, was chosen to avoid analysis of immature neurons, a large fraction of which are known to die prior to maturity (Kondo et al., 2010
Mice were perfused and the MOE tissue processed as described for IF with the following modifications: After permeabilization with Triton X-100 and SDS, slide-mounted sections were washed with PBS (3 × 3 min, RT) and water (3 min at RT), incubated in HCl (2 N; 1 hr, 37°C), and washed with TNT (3 × 3 min, RT). Sections were blocked and further processed as described. Primary antibodies were used at concentrations of 1:50 (BrdU) and 1:300 (OMP).
Imaging and quantitative fluorescence microscopy
Images were obtained using LSM710 and AxioImager Z2 (Carl Zeiss, Oberkochen, Germany) microscopes. Confocal images (1–5-μm thick optical sections) were used to quantify fluorescence signals, with care taken to ensure that exposures not exceed the instrument's dynamic range. Intensities were quantified using Zen software (Zeiss). For quantification of nuclear fluorescence in the MOE, circular regions encompassing individual nuclei were defined by DAPI fluorescence. Within each quantified image, a region surrounding the neuronal population (excluding immature and sustentacular cells) was defined to allow normalization to average neuronal nuclear fluorescence. For quantification of fluorescence in the OB, circular regions encompassing individual glomeruli were defined based on surrounding periglomerular cells, which were identified by morphology and DAPI fluorescence. p-Values corresponding to relative H2BE expression variances associated with specific ORs were calculated using a one-tailed F
-test with FDR correction for multiple comparisons (Benjamini and Hochberg, 1995
Quantification of OR expression, apoptosis, and BrdU frequencies in the MOE
Fluorescent olfactory neuron counts corresponding to MOE tissue from an individual mouse were determined from a series of 10–12 stained coronal sections located approximately 400 μm apart and spanning the anterior–posterior length of the organ. Fluorescent cell counting was performed using Velocity software (Perkin Elmer) or, when necessary due to difficulties in resolving individual olfactory neurons, manually. Epithelial volumes were calculated from areas determined using Velocity software, based on OMP and DAPI signals.
Analysis of the effects of H2be or Flag-H2be expression in cell culture
Coding sequences for H2BE, FLAG-H2BE, and consensus H2B were inserted into the pLNCX2 vector (Clontech Laboratories, Mountain View, CA, USA). Retroviruses carrying the resulting clones were generated and used according to the Retroviral Gene Transfer and Expression User Manual (Clontech). NIH-3T3 and HEK-293 cells were transduced by retroviral infection and cell lines stably expressing high levels of each transgene were selected using Geneticin (500 µg/mL; Invitrogen). Expression of transgenes in selected lines was verified by quantitative RT-PCR. Transgenic and non-transgenic cell lines were analyzed for cell viability using a Vi-Cell XR Cell Viability Analyzer (Beckman Coulter, Brea, CA, USA).
Laser-capture micro-dissection and microarray gene expression analyses
For experiments leading to the initial identification of H2be, RNA was obtained by laser-capture micro-dissection (LCM) of apical and basal neurons in the VNO. Briefly, whole VNOs from 8-week old CD1 male mice were carefully dissected from surrounding bones and immediately frozen in OCT. Tissue blocks were cut into 12-μm thick cryo-sections, placed alternately onto Superfrost and Superfrost plus slides (VWR, Radnor, PA, USA). Sections on Superfrost plus slides were stained by ISH for the Gnai2 and Gnao genes, which mark the apical and basal zones, respectively, and used as guides for LCM. Sections on Superfrost slides were stained with toluidine blue and used for LCM of approximately 100 apical and 100 basal neurons per section using a PixCell II LCM system (Arcturus, now Life Technologies, Grand Island, NY, USA). Samples were immediately frozen and pooled into groups of 10 (approximately 1000 cells per group), from which the RNA was extracted using the Arcturus Picopure Kit (Life Technologies, Grand Island, NY, USA). LCM RNA samples were amplified in parallel with whole VNO RNA samples using the Arcturus RiboAmp OA RNA Amplification Kit (Applied Biosystems) and analyzed using Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) according to the manufacturer's instructions.
For all other data reported in this study, RNA was prepared from whole MOE tissue or, in the case of UNO expression analyses, from MOE halves that had been carefully removed from the medial bone. RNA was isolated using Trizol Reagent (Invitrogen) and purified using an RNeasy Miniprep Kit (Qiagen, Valencia, CA, USA). Experiments were performed using 3–6 biological replicates per condition or genotype and MOE tissue from 2–4 individual mice per replicate. Samples were processed and applied to Affymetrix Mouse Gene 1.0 ST Arrays according to the manufacturer's instructions.
Probe cell intensity files (CEL) were analyzed for potential outliers using the Bioconductor software package arrayQualityMetrics (Kauffmann et al., 2009
). CEL files were processed with the Affymetrix Expression Console software to generate probe level summarization files (CHP) using the iterative Probe Logarithmic Intensity Error Estimation (IterPLIER) and sketch-quantile normalization algorithms. Statistical analyses of differential expression between groups were carried out using the Bioconductor limma package (Smyth, 2004
) implemented through the Bioconductor affylmGUI software (Wettenhall et al., 2006
) to generate unadjusted p-values and false discovery rate (FDR) corrections for multiple comparisons. For analyses of gene expression defects in H2be-KO MOE tissue, FDR corrections were made based on all represented genes with log2
expression levels above five. For analyses of OR expression defects in H2be-GF mice and expression differences following UNO, FDR corrections were made based on all represented OR genes with log2
expression levels above seven or five, respectively.
Quantitative PCR (qPCR) analysis of gene expression
cDNA samples for qPCR analysis were prepared using the QuantiTect Reverse Transcription Kit (Qiagen) starting from whole MOE RNA prepared using Trizol Reagent and purified using an RNeasy Miniprep Kit. Single-plex experiments () were performed using the QuantiTect SYBR Green PCR Kit (Qiagen) with an MJ Opticon 2 instrument (Bio-Rad Laboratories, Hercules, CA, USA). Multiplex experiments () were performed using the QuantiTect Multiplex PCR Kit (Qiagen) with an ABI 7900HT instrument (Applied Biosystems). Primer pairs (Integrated DNA Technologies, Coralville, IA, USA) and fluorophore/quencher-labeled probes (Eurofins MWG Operon, Huntsville, AL, USA; Integrated DNA Technologies) were designed using the Primer-BLAST tool (NCBI). Primer efficiencies were assessed using standard curves and pairs exhibiting efficiencies of >99% were used for analysis.
Unilateral naris occlusion
14-day old mice were administered Buprenorphine (0.05 mg/kg), anesthetized using isoflurane (confirmed through a tail pinch), and subjected immediately to electrocautery for approximately 5 s on the right nostril under a dissecting microscope (with care taken to avoid contact of the electrocautery unit with any non-superficial tissues). Mice were administered additional doses of Buprenorphine 12 and 24 hr after the procedure and examined on a daily basis to ensure complete blockage of the right nostril through scar formation (typically approximately 3–5 days after the procedure) and normal mouse development and activity.
Chronic odor exposure
Odors were presented as mixtures in mineral oil (octanal [0.31%], heptanal [0.068%], eugenol [2.65%]) or propylene glycol (lyral [10%]) continuously for 21 days at concentrations designed to achieve a vapor pressure of approximately 1 Pa per odorant. Odorants were presented in 100-µL volumes of each solution applied to a cotton pad and inserted into a metal mesh tea ball, which was suspended in the middle of the mouse cage by a metal chain. Odorants were exchanged every 24 hr.
Nuclear fractionation, immunoprecipitation of native mononucleosomes, and mass spectrometry
Preparation of soluble nuclear proteins, chromatin, and mononucleosomes from MOE tissue and immunoprecipitation of FLAG-H2BE-containing mononucleosomes were performed as described (Okada and Fukagawa, 2006
; Okada et al., 2006
), with modifications. Briefly, MOE tissue from four 8-week old Flag-H2be and four 8-week old WT mice were dissected and immediately minced and mechanically homogenized. Nuclei were filtered through a cell strainer, pelleted, washed, and disrupted by sonication. Chromatin was separated from soluble nucleoplasm by centrifugation, washed, and digested with micrococcal nuclease to mononucleosomes. Mononucleosomes were solubilized in 350 mM KCl buffer and affinity-purified using Anti-FLAG M2 affinity gel (Sigma-Aldrich). Proteins were eluted with FLAG peptide (Sigma-Aldrich), TCA-precipitated, run on a 15% SDS-PAGE gel, and separated into the following molecular weight fractions: >100, 35–100, 18–35, and <18 kDa. Gel fractions were submitted for MS/MS analysis and protein identification within the Harvard University Microchemistry and Proteomics Analysis Facility.
Genome-wide location analysis of FLAG-H2BE
Chromatin immunoprecipitation (ChIP) and ligation-mediated PCR amplification of the purified DNA were performed essentially as described (Lee et al., 2006
). Input chromatin was prepared from whole MOE tissue dissected from 5-week old male Flag-H2be mice using mouse anti-FLAG and rabbit anti-histone H3 antibodies. Amplified DNA samples from the FLAG and H3 ChIPs were separately fragmented and analyzed in duplicate (2 mice per replicate) using GeneChip Mouse Promoter 1.0R Arrays (Affymetrix), which tile 10 kb of DNA surrounding the transcript start site of approximately 25,500 mouse genes. CEL files were processed using Tiling Analysis Software (Affymetrix) to generate BAR files.
ChIP signals were analyzed at each position in the genome as a ratio of FLAG to H3 and assigned to a specific gene promoter region using Galaxy (http://main.g2.bx.psu.edu/
). Transcripts were grouped based on their expression level in OMP+
olfactory neurons, using expression values downloaded from (Sammeta et al., 2007
), or based on their gene family. Within each group, signals at each position relative to the transcript or CDS start site were averaged and plotted to obtain signal profiles for each gene group.
Quantitative PCR analyses of relative FLAG-H2BE levels in the protein-coding (CDS) regions of histone and OR genes were performed on ligation-mediated PCR-amplified DNA from FLAG or H3 ChIP samples. Experiments were performed in triplicate for each input (FLAG or H3) and primer pair combination starting from 0.2 ng of input DNA per reaction. Reactions were performed using the QuantiTect SYBR Green PCR Kit (Qiagen) and an MJ Opticon 2 instrument (Bio-Rad). Primer pairs (Integrated DNA Technologies) were designed using the Primer-BLAST tool (NCBI). Primer efficiencies were assessed using standard curves and pairs exhibiting efficiencies of >99% were used for analysis.
One and two-color fluorescent western blot analyses
Protein samples were separated by electrophoresis on a TRIS-glycine-SDS polyacrylamide gel (10–20% gradient; Bio-Rad), and transferred to nitrocellulose membranes. Membranes were washed in TN (5 min, RT), blocked in Blotto (Santa Cruz Biotechnology; 5% in TN; 1 hr, RT), incubated with primary antibodies (each diluted 1:2000 in 5% Blotto [in TNT]; 12 hr, 4°C), washed in TNT (3 × 5 min, RT), incubated with a mixture of Alexa488-conjugated anti-rabbit and Alexa647-conjugated anti-mouse secondary antibodies (Invitrogen; 1:2000 in 5% Blotto [in TNT]; 1–2 hr, RT), and washed in TNT (3 × 10 min, RT). Blots were scanned using a Typhoon Trio Imager and analyzed using ImageQuant software (GE Healthcare Biosciences, Pittsburgh, PA, USA).
Eight-well Streptavidin High Binding Capacity strips (Thermo-Fisher Scientific, Waltham, MA, USA) were washed (Tris [25 mM, pH 7.5], NaCl [150 mM], BSA [1%], and Tween-20 [0.05%]). Custom-synthesized C-terminally-biotinylated peptides (Abgent, San Diego, CA, USA) corresponding to the N-terminal 13 amino acids of H2BE or the mouse consensus H2B and containing a methyl-modified lysine residue at position five were immobilized within the well strips (1 μg per well in block buffer [0.2 µg mouse IgG in wash buffer]; 3 hr, RT), washed, incubated with anti-H2B-Lys5-Me antibody (1:5000–1:5,000,000 in block buffer; 12 hr; 4°C), washed, incubated with anti-rabbit-HRP (1:2000 in block buffer; 1 hr, RT), and washed. ELISA signals were developed using the 1-Step Ultra TMB-ELISA reagent (Thermo Scientific) according to the manufacturer's instructions.
Olfactory odor discrimination training
Ten 4-month old H2be-KO and heterozygous littermates were subjected to odor discrimination training using water restriction for motivation, under a behavioral paradigm similar to that described (Uchida and Mainen, 2003
), but adapted for mice. In an initial experiment, mice were challenged to discriminate between hexanol and hexanoic acid, and in a second experiment, were challenged with the two stereoisomers of carvone. Prior to the initial experiment, mice were trained to obtain water from two ports unconditionally (day 1), after first poking an odor port (days 2–5), after poking an odor port presenting isoamyl-acetate odor (days 6–13), and after only a single poke of an odor port presenting isoamyl-acetate odor (days 14–20). Odor discrimination training began on day 21. Odors streams, controlled by an olfactometer, were generated from air passed through a filter containing 10% of the concentrated odorant in mineral oil and diluted 1:20 into a stream of clean air.