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. 2012 Jul 4:7:17.
doi: 10.1186/1749-8104-7-17.

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance

Thomas K Knott  1 , Pasil A Madany, Ashley A Faden, Mei Xu, Jörg Strotmann, Timothy R Henion, Gerald A Schwarting
Affiliations

Olfactory discrimination largely persists in mice with defects in odorant receptor expression and axon guidance

Thomas K Knott et al. Neural Dev. .

Abstract

Background: The defining feature of the main olfactory system in mice is that each olfactory sensory neuron expresses only one of more than a thousand different odorant receptor genes. Axons expressing the same odorant receptor converge onto a small number of targets in the olfactory bulb such that each glomerulus is made up of axon terminals expressing just one odorant receptor. It is thought that this precision in axon targeting is required to maintain highly refined odor discrimination. We previously showed that β3GnT2(-/-) mice have severe developmental and axon guidance defects. The phenotype of these mice is similar to adenylyl cyclase 3 (AC3) knockout mice largely due to the significant down-regulation of AC3 activity in β3GnT2(-/-) neurons.

Results: Microarray analysis reveals that nearly one quarter of all odorant receptor genes are down regulated in β3GnT2(-/-) mice compared to controls. Analysis of OR expression by quantitative PCR and in situ hybridization demonstrates that the number of neurons expressing some odorant receptors, such as mOR256-17, is increased by nearly 60% whereas for others such as mOR28 the number of neurons is decreased by more than 75% in β3GnT2(-/-) olfactory epithelia. Analysis of axon trajectories confirms that many axons track to inappropriate targets in β3GnT2(-/-) mice, and some glomeruli are populated by axons expressing more than one odorant receptor. Results show that mutant mice perform nearly as well as control mice in an odor discrimination task. In addition, in situ hybridization studies indicate that the expression of several activity dependent genes is unaffected in β3GnT2(-/-) olfactory neurons.

Conclusions: Results presented here show that many odorant receptors are under-expressed in β3GnT2(-/-) mice and further demonstrate that additional axon subsets grow into inappropriate targets or minimally innervate glomeruli in the olfactory bulb. Odor evoked gene expression is unchanged and β3GnT2(-/-) mice exhibit a relatively small deficit in their ability to discriminate divergent odors. Results suggest that despite the fact that β3GnT2(-/-) mice have decreased AC3 activity, decreased expression of many ORs, and display many axon growth and guidance errors, odor-evoked activity in cilia of mutant olfactory neurons remains largely intact.

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Figures

Figure 1
Figure 1
Quantitative PCR analysis for relative expression of ORs. In β3GnT2−/− mice the expression of mOR256-17 (Olfr15), mOR125-1 (Olfr43), and mOR256-7 (Olfr136) increased while that of mOR13-4 (Olfr640), mOR16 (Olfr1264), and mOR28 (Olfr 1507) decreased compared to WT mice. Results are the average relative value of three animals normalized to an RNA polymerase 2 reference target. Statistics: Student’s t test, **P < 0.001. * < 0.05; mean ± SEM (n = 3).
Figure 2
Figure 2
In situhybridization of ORs in the olfactory epithelium of β3GnT2−/−mice. The expression of some individual odorant receptor mRNAs is decreased in the OE of β3GnT2−/− mice compared to controls. A, B. The number of P2 positive cells decreased significantly in β3GnT2−/− mice. C, D. The number and position of OR37 expressing cells (arrows) was unchanged in β3GnT2−/− mice. E, F. The number and position of M72 expressing cells (arrows) was also unchanged in β3GnT2−/− mice. Scale bar = 200 μM in A-D and 100 μM in E, F.
Figure 3
Figure 3
In situhybridization of ORs in the olfactory epithelium of β3GnT2−/−mice. Several mRNAs for OR genes are increased in expression in the olfactory epithelium of β3GnT2−/− mice compared to controls. These include mOR256-14, (A,B), mOR256-17 (C,D) and mOR125-1 (E,F). Scale bar in all panels = 250 μM.
Figure 4
Figure 4
mOR256-17 Immunocytochemical analysis of the adult olfactory bulb. In WT mice antibodies to the mOR256-17 OR identify two lateral glomeruli (arrows in A) and two medial glomeruli in the extreme caudal OB (arrows in B). In β3GnT2−/− mice there are four lateral glomeruli. The most rostral mutant glomerulus (arrow in C) is found in its approximate normal position compared to controls. Three additional lateral glomeruli in the null OB are located in an aberrant caudal and ventral position (arrows in D, E and F) compared to WT glomeruli. In β3GnT2−/− mice, there are three diffuse medial glomeruli, one of which (arrow in G) is in the approximate correct position. The other three medial glomeruli in the null OB are in either more ventral or more dorsal positions (arrows in G and H) compared to control glomeruli. Scale bar = 500 μM.
Figure 5
Figure 5
Composite map of mOR256-17 glomerular positions in adult β3GnT2 mice. The position of glomeruli in one OB of WT (A) and β3GnT2−/−(B) mice was plotted as a two dimensional map. The OB was flattened and unrolled from the dorsal midline (arrows), and the location of mOR256-17 glomeruli plotted as the relative circumferential distance from the dorsal midline in serial 50 mm coronal sections (horizontal lines) beginning at the anterior extent of the OB. The distance in microns from the anterior tip of the OB is at the right of each plot. Note that the plot of the mutant OB is smaller than that of the WT OB along the anterior-posterior axis.
Figure 6
Figure 6
Reduction in OR expression does not prevent axons from correctly targeting the OB. I7+ axons normally form a medial glomerulus in the OB near the ventral midline (arrow in a). In β3GnT2−/− mice, there are insufficient numbers of I7+ axons to form a glomerulus but individual axon targets an identically positioned medial glomerulus (arrow). Synaptophysin (red) staining in A and B suggest that WT and null glomeruli contain functionally active connections. mOR28 is expressed on clusters of neurons in the ventral OE and is heavily expressed on dendrites on the luminal surface of the OE. In null OE’s (D), the number of mOR28+ neurons is greatly reduced compared to OEs (C). mOR28+ axons normally form a medial glomerulus in the caudal OB near the ventral midline (arrow in E). Very few mOR28+ axons can be found targeting this glomerulus in β3GnT2−/− OBs (arrow in F, see inset). Scale bar = 50 μM in all panels.
Figure 7
Figure 7
In situ hybridization of odor-evoked activity dependent genes in the OE.A. The expression of genes that are regulated by activity-dependent processes were analyzed in adult WT and β3GnT2−/− OEs. Calretinin, which is expressed in immature neurons and is greatly enriched in CNGA2−/− mice is unchanged in expression in β3GnT2−/− OEs. The Leucine Rich Repeat Containing (Lccr3b), S100A5, and kirrel2 genes, which are all expressed in mature neurons in WT OEs and down-regulated in CNGA2−/− mice, are also unchanged in levels of mRNA expression. Note that increased cell death in β3GnT2−/− OEs results in a thinner layer of mature neurons [8]. Scale bar = 50 μM in all panels. B. Real-time quantitative PCR analysis of activity-dependent gene expression in microdissected adult wild type and β3GnT2−/− OE samples. Relative levels of Lrrc3b, S100A5 and kirrel2 mRNA in null mice decreases in parallel with the mature neuron marker OMP. Immature neurons expressing GAP-43 correspondingly increase in proportion in the null OE. Consistent with this, calretinin expression in the basal OE is preserved in null mice. Thus, the expression of activity-dependent genes fluctuates only in accordance with the dynamics of the cell population they are expressed in, suggesting that odor-evoked activity is not as severely compromised in β3GnT2−/− mice relative to CNGA2 mutants. Error bars are the mean +/− SEM (n = 3 for GAP-43; n = 6 for all others).
Figure 8
Figure 8
β3GnT2−/−mice retain the majority of their olfactory discriminatory ability. Mice were tested against pairs of odors for their ability to associate odor with a food reward. β3GnT2−/− mice can discriminate two of the odors, citralva and β-ionone as well as wild type controls. There is a significant (asterisks) reduction in the ability of null mice to discriminate geraniol, citronellal, decanal, and 3-n-hexylpyridine, but the differences are small and highly variable.
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