Expressing exogenous functional odorant receptors in cultured olfactory sensory neurons
© Chen et al; licensee BioMed Central Ltd. 2008
Received: 22 May 2008
Accepted: 11 September 2008
Published: 11 September 2008
Olfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.
We demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro.
This culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.
It is established that most of the olfactory sensory neurons (OSNs) express only one type of odorant receptor (OR) among a thousand gene choices [1, 2]. Each OSN functions as a specialized sensor for the detection of diverse odorants in the environment. OSNs expressing the same OR converge their axons to the same glomerular pair in the olfactory bulb [3, 4]. Thus, the one receptor-one neuron rule is essential for establishing the discrete sensory map of olfactory neuronal connections.
The ability to discriminate odorants in the environment depends on OR-ligand interactions and signaling capacity within the OSNs. Several experimental approaches for investigating OR functions have been reported [5–8]. Though heterologous systems allow rapid screening of ligand binding specificity of the ORs, validation of these results in the OSNs has been challenging without an efficient OSN culture system . One of the other challenges in olfaction is to understand the regulatory mechanism for OR selection in the OSNs. Transcriptional regulatory elements that control OR expression have been identified [10, 11]. In addition, a negative feedback model for achieving single OR expression in each OSN has been proposed [11–13]. The feedback inhibition of OR expression appears to require OR protein. Recent evidence indicates that the OR coding sequence plays a critical role in the suppression of multiple OR expression. Not only can endogenous OR expression be suppressed, but ectopic OR expression appears to be suppressed as well via the OR coding sequence .
An in vitro system that allows genetic manipulation will be helpful in determining the genes and the functions of their products that are involved in the regulation of OR expression and function. Several primary OSN culture techniques have been reported [15–17]. Studies using cultured OSNs have primarily focused on investigating the regulatory mechanism of neurogenesis and differentiation, though responses to odorants and signaling molecule functions were reported in primary OSN cultures [18, 19]. However, the expression of ORs and the genetic manipulation of OR expression in cultured OSNs have not been reported to date. Here we describe an OSN culture system that allows gene expression manipulation, monitoring of OR expression and testing of OR-ligand specificity. Lentiviral vectors are used to achieve a high percentage of infection of cultured OSNs and to express ectopically genes of interest. Using this system, we observed that OSNs allow expression of two ORs when introduced by lentiviral-mediated gene transfer.
Primary olfactory sensory neurons express olfactory specific markers in vitro
Do the bipolar neurons display molecular markers of OSNs? We observed that all neurons in the cultures, identified by β-tubulin III expression, expressed neuronal cell adhesion molecule (NCAM; Figure 2A), which is expressed by OSNs in vivo . In addition, at 3 DIV, all cultured neurons were also positive for GAP-43 immunostaining, which is a marker for immature OSNs (Figure 2A). Adenylyl cyclase (AC)3 and Gαolf are key players in mediating olfactory signals and are expressed in OSN dendrites and axons during development . Both AC3 and Gαolf expression were detected in cultured OSNs at 3 DIV by both immunostaining and RT-PCR (Figure 2). AC3 and Gαolf protein were distributed throughout the OSNs, including the cell body, and dendrite- and axon-like processes at 3 DIV. At 5 DIV, however, AC3 was primarily concentrated at the tip of the short dendritic like processes, and at low levels in the cell body and their axons. At 5 DIV, Gαolf immunostaining signal was detected in the cell body and concentrated at the tip of the short dendritic-like processes as well (Figure 2C). We observed that AC3 and Gαolf transcript levels were higher at 5 DIV when compared to those at 3 DIV (Figure 2D). Cyclic nucleotide gated channel A2 subunit (CNGA2) was also expressed in the cultured OSNs at 3 DIV and 5 DIV (Figure 2D). Expression of progenitor and neuronal precursor markers were also examined using RT-PCR. Both Mash1, an amplifying progenitor marker, and Ngn1, a neuronal precursor marker, are expressed in OE in vivo . However, we did not detect expression of these two genes in the OSN cultures at 3 DIV and 5 DIV. NeuroD, which is expressed in immature OSNs in vivo , was detected in the OSN cultures at both 3 DIV and 5 DIV (Figure 2D).
Efficient gene transfer using lentiviruses
Functional expression of I7 in cultured OSNs
To examine whether ectopically expressed I7 in cultured OSNs are functional receptors, we tested whether these infected OSNs would respond to octanal (octyl aldehyde), which is one of the documented I7 odorant ligands. It is reported that functional odorant receptors can trigger an increase in [Ca2+]i in OSNs when stimulated by their odorant ligands. To measure [Ca2+]i, cultured OSNs were loaded with fura-2/AM and cells with stable basal levels of [Ca2+]i were selected for recording. As a control, we first examined whether cultured OSNs infected with recombinant GFP-expressing lentivirus respond to the presence of octanal. Octanal at 100 μM rarely elicits responses in a majority of the cultured OSNs expressing GFP alone (Figure 5Bi). Among 64 cells tested, one cell showed a [Ca2+]i increase in response to octanal, suggesting that this cell may express endogenous functional receptors. It has been shown that propionic acid is not a ligand for the I7 receptor . In OSN cultures infected with I7 lentiviral vectors, none of the I7-expressing cells showed responses to 100 μM propionic acid (n = 28; Figure 5Bii); however, all I7-expressing cells tested displayed an increase in [Ca2+]i in response to 100 μM octanal (n = 31; Figure 5Biii). This result indicates that ectopically expressed mouse I7 in cultured OSNs are functional ORs.
Expressing functional I7 does not alter OSN axon extension
ORs play important roles in OSN axon convergence. The site of the axon convergence could be influenced by the level of OR expression suggested by OR knock-in animal models. Expression of the OR has also been shown to be linked to the expression of certain cell surface adhesion molecules . In our culture system, OSN axons migrate on the surface of the feeder layer astrocytes. Whether over-expressing ORs influences the rate of OSN axon extension has not been examined. Utilizing the OSN cultures, we examined this question by comparing the axon length of the OSNs between I7-expressing and control cultures. At 3 DIV, when the average axon length in the control noninfected cultures was normalized to 1, the average axon length of the I7 lentiviral vector-infected culture was 1.00 ± 0.17 (mean ± SE, n = 56, t-test p > 0.9; Figure 4B). Therefore, over-expressing I7 does not alter the extension of the OSN axons. In addition, when OSNs are grown at a density of the 5 × 104/cm2, we did not observe any fasciculation between I7-expressing OSN axons.
Expression of two exogenous ORs is possible in cultured OSNs
In this study we report that cultured primary OSNs express endogenous ORs along with other characteristic odorant signaling molecules. Lentiviral vector-mediated gene transfer allows efficient and successful ectopic expression of functional ORs in culture. The mouse OSN culture system described here will enable future studies of OR ligand specificity and transcription regulation within its intrinsic cellular environment.
In vitro characteristics of primary OSNs resemble their in vivo characteristics
In the OSN culture system reported here, several modifications were made to enhance the survivability and morphology of the OSNs compared to other published studies [15, 28]. Dissociated OE cells were plated on a feeder layer of confluent astrocytes instead of directly on matrix coated coverglass to enhance their attachment, survivability and differentiation. Defined, serum-free media supported the survival of neurons and inhibited cell proliferation and neurogenesis. Consistent with previous reports, OMP expression was present immediately after the plating and was undetectable at 3 DIV . A rapid decrease in OMP positive cells indicated that the culture condition does not support the survival of mature OSNs dissociated from the OE. In addition, a larger numbers of multipolar neurons were present at 1 DIV. The percentage of bipolar neurons increased almost 100% from 1 DIV to 6 DIV, while the number of multipolar neurons decreased. This change in the cell population in the OSN cultures suggests that mature OMP positive neurons and multipolar neurons were not well supported under our culture conditions. We do not have evidence at this point as to whether the multipolar neurons represent certain populations of OSNs. All multipolar neurons were GAP43 and NCAM positive in the cultures. Since all OSNs in the cultures were positive for GAP43 staining at 3 DIV, the OSNs in the cultures are most likely immature neurons. Expression of neuronal progenitor markers Mash1 and Ngn1 was not detected at 3 DIV in the culture. This observation is consistent with the idea that the culture conditions do not support neurogenesis. In addition, it also suggests that the precursors dissociated from the OE either did not survive in the cultures or progressed along their differentiation path to become immature OSNs at 3 DIV. The small number of OMP positive neurons detected at 8 DIV leads us to suggest that OMP positive mature OSNs can be generated under these defined culture conditions.
OSNs are bipolar neurons with their dendritic process extending towards the surface of the nasal cavity. At the tip of the dendrite, the ending swells into a knob-like morphology and ciliary processes extend out from the dendritic-knob. OR proteins and signaling proteins mediating the odorant activity are found to be enriched in the ciliary processes [19, 29]. In cultured OSNs, signaling molecules exhibit preferential localization at the tip of the dendritic-like process. Gαolf and particularly AC3 were both initially distributed throughout the cell body, axon and dendritic processes, and eventually accumulated at the tips of the dendrites. The concentrated subcellular localization of AC3 and Gαolf at the dendritic tip is evident at 5 DIV, and resembles their in vivo distribution [30, 31]. Though olfactory signaling molecules and several OSN specific markers were present, the cultured OSNs did not recapitulate all cellular and molecular characteristics of mature OSNs in vivo. Only a small proportion of OMP positive neurons differentiated from immature neurons and only at 8 DIV or later in the culture. It is possible that OMP expression may be partially dependent upon the presence of the cellular targets of the OSNs. Overall, this culture technique provided an efficient method to culture OSNs, whose morphology, molecular characteristics and maturation progression in culture resembled those observed in vivo.
Odorant receptor expression and function in cultured OSNs
Based on the developmental timing and the distribution of OR expression in the epithelium, it was concluded that OR expression precedes OMP expression . As it is not practical to examine the expression of all ORs, we selected five ORs that represent OR genes located on different chromosomes that are turned on at different developmental time points [24, 23]. All ORs examined were expressed in our OSN culture. Since cultured OSNs are obtained from embryonic day (E)17-P0 mouse OE, it is possible that all ORs are expressed by P0. A recent study of the septal organ demonstrated clearly that OR expression is temporally asynchronous . The onset of OR expression in the septal organ can span several days from E16 to P0. Similar expression onset asynchrony is also reported in the main OE . The density and distribution of OR expressing OSNs also vary within the OE. Therefore, it is possible that not all ORs can be detected in the cultured OSNs.
Investigations of OR ligand specificity have been challenging due to the difficulty of expressing ORs in heterologous cells. Amino-terminal modification by adding a rhodopsin tag to the OR coding sequence enhanced the ability of OR to be transported to the plasma membrane . However, this extracellular modification may alter ligand receptor binding specificity . Accessory proteins, including odorant receptor transporting proteins and Ric8b, are known to aid in the efficiency of OR expression . Combinations of these accessory factors and signal transduction molecules allow expression of a subpopulation of ORs and subsequent high throughput screening for their ligand. However, many intrinsic differences within the heterologous systems, including availability and subcellular localization of the signaling molecules and the concentration of OR expressed, could all influence the experimental outcome. ORs expressed in their intrinsic environment by OSNs presumably behave most closely to their in vivo state .
In this study, we report an efficient technique for ectopic expression of ORs in OSNs. Using lentiviral vectors, we report close to 100% infection of the cultured OSNs. This high infection efficiency allows not only easy access to the OR-expressing cells but also monitoring of changes using molecular and biochemical approaches. In addition, we provide evidence that the ectopically expressed mouse I7 is functional. Though an earlier report indicated that mouse I7 is more sensitive to heptanol, later evidence argued for mouse I7 to respond to multiple aldehyde compounds . To define the functionality of ectopic mouse I7, but without the intent to discriminate ligand-receptor specificity among aldehydes, we used a higher concentration of octanal (100 μM) as an agonist for receptor-induced odorant responses. Calcium imaging experiments confirmed that I7-expressing OSNs responded to odorant stimulation. Consistent with the finding that endogenous ORs are expressed by cultured OSNs, we detected occasional responsive cells among control cultures. This observation suggests that endogenous ORs are functional receptors as well. The cultured OSN system reported here provides an efficient way to express functional ORs and to study ligand specificity in their intrinsic cellular environment.
Materials and methods
C57/BL6 mice were purchased from Charles River Laboratories (Wilmington, MA). Embryonic day 0 (E0) was defined by the day when the copulation plug was detected in the morning. The day of birth was defined as P0. All experimental procedures were conducted according to institutional and NIH guidelines and were approved by the Institution Animal Care and Use Committee.
Cortical astrocyte feeder layer
Primary astrocytes were isolated from P0 mouse neocortex and cultured according to a published protocol . Before plating OSNs, astrocytes were plated and allowed to reach confluency on poly-D-lysine and laminin coated glass coverslips. The purity of the astrocytes was evaluated by immunostaining a sample of the astrocyte culture for glial fibrillary acidic protein to identify the astrocyte population and β-tubulin III to determine the presence of neurons. When no neurons were observed, astrocytes were used for OSN culture.
Dissociated primary OSN culture
E17 to P0 OE were dissected from the nasal cavity. After 40 minutes' incubation in 2 mg/ml dispase at room temperature, OE were separated from the underlining stroma using fine tungsten needles. The OE were incubated in Waymouth's MB 752/1 medium with N2 supplement (Invitrogen, Carlsbad, CA, USA) at 37°C for 2 hours before treatment with 0.05% trypsin for dissociation. After trypsinization, dissociated cells were plated at a density of 5 × 104 cells/cm2 on the confluent astrocyte feeder layer. Dissociated OSNs were maintained in Waymouth's MB 752/1 medium with N2 supplement for up to 10 days.
Production of lentiviral vectors
Primer sequences for this study
Cultured cells were fixed with 4% paraformaldehyde for 15 minutes and immunostained as described previously . The sources and the dilutions of the primary antibodies were: monoclonal mouse anti-β-tubulin III 1:300 (Sigma, Saint Louis, MO, USA); rabbit anti-GAP-43 1:200 (Chemicon, Temecula, CA, USA); mouse anti-NCAM 1:100 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA); chicken anti-OMP 1:1000 ; rabbit anti-AC3 1:100 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit anti-Gαolf 1:100 (Santa Cruz Biotechnology); rabbit anti-GFP 1:3000 (Invitrogen); rabbit anti-RFP 1:250 (MBL International Corp., Woburn, MA, USA); rabbit anti-MOR42-3 1:2000 (Osenses Pty Ltd, Flagstaff Hill, SA, Australia)
Total RNAs were extracted from the OSN cultures, including astrocyte feeder layer cells and astrocyte feeder layer only cultures, using TRIZOL reagent (Invitrogen). Template cDNAs were obtained by reverse transcription (RT) using oligo dT primers. Detection of cDNAs was done by PCR reactions using primers listed in Table 1. All RT-PCR products were confirmed by sequence analysis.
Single-cell intracellular calcium imaging
Before each experiment, cultured OSNs were loaded with 1 μM fura-2/AM (Molecular Probes/Invitrogen, Eugene, OR, USA) for 5 minutes in Ringer's solution containing 0.02% (v/v) pluronic acid (Molecular Probes/Invitrogen). After washing, cells were then incubated for an additional 30 minutes at 37°C. Coverslips with fura-2- loaded OSNs were then placed into the recording chamber continuously perfused with Ringer's solution and mounted on a Zeiss Axiovert 200 inverted microscope (Zeiss, Thornwood, NY, USA). Solution exchange was accomplished via a gravity-driven perfusion system, which allowed complete solution exchange in the recording area within 2 to 5 seconds. Fluorescence images of fura-2- loaded OSNs were acquired using a SenSys CCD camera (Roper Scientific, Tucson, AZ, USA). A lambda DG-4 filter changer (Sutter Instrument, Novato, CA, USA) was used for switching between 340 nm and 380 nm excitation wavelengths. Changes in intracellular calcium concentrations ([Ca2+]i) were monitored as changes of the ratio of the fura-2- fluorescence intensity recorded at 340 nm and 380 nm excitation wavelengths (R340/360). All experiments were performed at room temperature. Data acquisition and analysis were performed using MetaFluor v7.0 software (Universal Imaging, Downingtown, PA, USA). In each experiment transfected cells were identified by GFP fluorescence and time courses of R340/360 were simultaneously recorded from five to seven transfected cells.
cyclic nucleotide gated channel A2 subunit
days in vitro
enhanced green fluorescent protein
neuronal cell adhesion molecule
olfactory marker protein
olfactory sensory neuron
We thank Richard Tucker and the members of the Gong lab for helpful discussions and critical reading of the manuscript. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Program Grant Number C06 RR12088-01 from the National Center for Research Resources, National Institutes of Health. This study is supported by National Science Foundation IBN0324769 and National Institutes of Health DC06015 (to QG).
- Malnic B, Mirono J, Sato T, Buck LB: Combinatorial receptor codes for odors. Cell. 1999, 96: 713-723. 10.1016/S0092-8674(00)80581-4.View ArticlePubMedGoogle Scholar
- Serizawa S, Ishii T, Nakatani H, Tsuboi A, Nagawa F, Asano M, Sudo K, Sakagami J, Sakano H, Ijiri T, Matsuda Y, Suzuki M, Yamamori T, Iwakura Y, Sakano H: Mutually exclusive expression of odorant receptor transgenes. Nat Neurosci. 2000, 3: 687-693. 10.1038/76641.View ArticlePubMedGoogle Scholar
- Ressler KJ, Sullivan SL, Buck LB: Information coding in the olfactory system: evidence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell. 1994, 79: 1245-1255. 10.1016/0092-8674(94)90015-9.View ArticlePubMedGoogle Scholar
- Vassar R, Chao SK, Sitcheran R, Nunez JM, Vosshall LB, Axel R: Topographic organization of sensory projections to the olfactory bulb. Cell. 1994, 79: 981-991. 10.1016/0092-8674(94)90029-9.View ArticlePubMedGoogle Scholar
- Krautwurst D, Yau KW, Reed RR: Identification of ligands for olfactory receptors by functional expression of a receptor library. Cell. 1998, 95: 917-926. 10.1016/S0092-8674(00)81716-X.View ArticlePubMedGoogle Scholar
- Wellerdieck C, Oles M, Pott L, Korsching S, Gisselmann G, Hatt H: Functional expression of odorant receptors of the zebrafish Danio rerio and of the nematode C. elegans in HEK293 cells. Chem Senses. 1997, 22: 467-476. 10.1093/chemse/22.4.467.View ArticlePubMedGoogle Scholar
- Katada S, Tanaka M, Touhara K: Structural determinants for membrane trafficking and G protein selectivity of a mouse olfactory receptor. J Neurochem. 2004, 90: 1453-1463. 10.1111/j.1471-4159.2004.02619.x.View ArticlePubMedGoogle Scholar
- Zhuang H, Matsunami H: Synergism of accessory factors in functional expression of mammalian odorant receptors. J Biol Chem. 2007, 282: 15284-15293. 10.1074/jbc.M700386200.View ArticlePubMedGoogle Scholar
- Zhao H, Ivic L, Otaki JM, Hashimoto M, Mikoshiba K, Firestein S: Functional expression of a mammalian odorant receptor. Science. 1998, 279: 237-242. 10.1126/science.279.5348.237.View ArticlePubMedGoogle Scholar
- Vassalli A, Rothman A, Feinstein P, Zapotocky M, Mombaerts P: Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb. Neuron. 2002, 35: 681-696. 10.1016/S0896-6273(02)00793-6.View ArticlePubMedGoogle Scholar
- Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, Yoshihara Y, Sakano H: Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science. 2003, 302: 2088-2094. 10.1126/science.1089122.View ArticlePubMedGoogle Scholar
- Shykind BM, Rohani SC, O'Donnell S, Nemes A, Mendelsohn M, Sun Y, Axel R, Barnea G: Gene switching and the stability of odorant receptor gene choice. Cell. 2004, 117: 801-815. 10.1016/j.cell.2004.05.015.View ArticlePubMedGoogle Scholar
- Lewcock JW, Reed RR: A feedback mechanism regulates monoallelic odorant receptor expression. Proc Natl Acad Sci USA. 2004, 101: 1069-1074. 10.1073/pnas.0307986100.PubMed CentralView ArticlePubMedGoogle Scholar
- Nguyen MQ, Zhou Z, Marks CA, Ryba NJ, Belluscio L: Prominent roles for odorant receptor coding sequences in allelic exclusion. Cell. 2007, 131: 1009-1017. 10.1016/j.cell.2007.10.050.PubMed CentralView ArticlePubMedGoogle Scholar
- Grill RJ, Pixley SK: In vitro generation of adult rat olfactory sensory neurons and regulation of maturation by coculture with CNS tissues. J Neurosci. 1997, 17: 3120-3127.PubMedGoogle Scholar
- Carter LA, MacDonald JL, Roskams AJ: Olfactory horizontal basal cells demonstrate a conserved multipotent progenitor phenotype. J Neurosci. 2004, 24: 5670-5683. 10.1523/JNEUROSCI.0330-04.2004.View ArticlePubMedGoogle Scholar
- Calof AL, Chikaraishi DM: Analysis of neurogenesis in a mammalian neuroepithelium: Proliferation and differentiation of an olfactory neuron precursor in vitro. Neuron. 1989, 3: 115-127. 10.1016/0896-6273(89)90120-7.View ArticlePubMedGoogle Scholar
- Ronnett GV, Hester LD, Snyder SH: Primary culture of neonatal rat olfactory neurons. J Neurosci. 1991, 11: 1243-1255.PubMedGoogle Scholar
- Matsuzaki O, Bakin RE, Cai X, Menco BP, Ronnett GV: Localization of the olfactory cyclic nucleotide-gated channel subunit 1 in normal, embryonic and regenerating olfactory epithelium. Neuroscience. 1999, 94: 131-140. 10.1016/S0306-4522(99)00228-6.View ArticlePubMedGoogle Scholar
- Zou DJ, Chesler AT, Le Pichon CE, Kuznetsov A, Pei X, Hwang EL, Firestein S: Absence of adenylyl cyclase 3 perturbs peripheral olfactory projections in mice. J Neurosci. 2007, 27: 6675-6683. 10.1523/JNEUROSCI.0699-07.2007.View ArticlePubMedGoogle Scholar
- Beites CL, Kawauchi S, Crocker CE, Calof AL: Identification and molecular regulation of neural stem cells in the olfactory epithelium. Exp Cell Res. 2005, 306: 309-316. 10.1016/j.yexcr.2005.03.027.View ArticlePubMedGoogle Scholar
- Nibu K, Li G, Zhang X, Rawson NE, Restrepo D, Kaga K, Lowry LD, Keane WM, Rothstein JL: Olfactory neuron-specific expression of NeuroD in mouse and human nasal mucosa. Cell Tissue Res. 1999, 298: 405-414. 10.1007/s004410050063.View ArticlePubMedGoogle Scholar
- Royal SJ, Key B: Development of P2 olfactory glomeruli in P2-internal ribosome entry site-tau-LacZ transgenic mice. J Neurosci. 1999, 19: 9856-9864.PubMedGoogle Scholar
- Potter SM, Zheng C, Koos DS, Feinstein P, Fraser SE, Mombaerts P: Structure and emergence of specific olfactory glomeruli in the mouse. J Neurosci. 2001, 21: 9713-9723.PubMed CentralPubMedGoogle Scholar
- Moon C, Yoo JY, Matarazzo V, Sung YK, Kim EJ, Ronnett GV: Leukemia inhibitory factor inhibits neuronal terminal differentiation through STAT3 activation. Proc Natl Acad Sci USA. 2002, 99: 9015-9020. 10.1073/pnas.132131699.PubMed CentralView ArticlePubMedGoogle Scholar
- McClintock TS, Landers TM, Gimelbrant AA, Fuller LZ, Jackson BA, Jayawickreme CK, Lerner MR: Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors. Brain Res Mol Brain Res. 1997, 48: 270-278. 10.1016/S0169-328X(97)00099-5.View ArticlePubMedGoogle Scholar
- Serizawa S, Miyamichi K, Takeuchi H, Yamagishi Y, Suzuki M, Sakano H: A neuronal identity code for the odorant receptor-specific and activity-dependent axon sorting. Cell. 2006, 127: 1057-1069. 10.1016/j.cell.2006.10.031.View ArticlePubMedGoogle Scholar
- Grill RJ, Pixley SK: 2-Mercaptoethanol is a survival factor for olfactory, cortical and hippocampal neurons in short-term dissociated cell culture. Brain Res. 1993, 613: 168-172. 10.1016/0006-8993(93)90470-8.View ArticlePubMedGoogle Scholar
- Menco BPM, Cunningham AM, Qasba P, Levy N, Reed RR: Putative odour receptors localize in cilia of olfactory receptor cells in rat and mouse: a freeze-substitution ultrastructural study. J Neurocytol. 1997, 26: 691-706. 10.1023/A:1018554029186.View ArticlePubMedGoogle Scholar
- Bakalyar HA, Reed RR: Identification of a specialized adenylyl cyclase that may mediate odorant detection. Science. 1990, 250: 1403-1406. 10.1126/science.2255909.View ArticlePubMedGoogle Scholar
- Menco BPM, Bruch RC, Dau B, Danho W: Ultrastructural localization of olfactory transduction components: the G protein subunit Golf alpha and type III adenylyl cyclase. Neuron. 1992, 8: 441-453. 10.1016/0896-6273(92)90272-F.View ArticlePubMedGoogle Scholar
- Sullivan SL, Bohm S, Ressler KJ, Horowitz LF, Buck LB: Target-independent pattern specification in the olfactory epithelium. Neuron. 1995, 15: 779-789. 10.1016/0896-6273(95)90170-1.View ArticlePubMedGoogle Scholar
- Tian H, Ma M: Differential development of odorant receptor expression patterns in the olfactory epithelium: A quantitative analysis in the mouse septal organ. Dev Neurobiol. 2008, 68: 476-486. 10.1002/dneu.20612.PubMed CentralView ArticlePubMedGoogle Scholar
- Hall SE, Floriano WB, Vaidehi N, Goddard WA: Predicted 3-D structures for mouse I7 and rat I7 olfactory receptors and comparison of predicted odor recognition profiles with experiment. Chem Senses. 2004, 29: 595-616. 10.1093/chemse/bjh063.View ArticlePubMedGoogle Scholar
- Weinstein DE: Isolation and purification of primary rodent astrocytes. Curr Protoc Neurosci. Edited by: Taylor GP. 2001, John Wiley & Sons, Inc, 3.5.1-1Google Scholar
- Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D: Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science. 2002, 295: 868-872. 10.1126/science.1067081.View ArticlePubMedGoogle Scholar
- Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY: Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol. 2004, 22: 1567-1572. 10.1038/nbt1037.View ArticlePubMedGoogle Scholar
- Gong Q, Bailey MS, Pixley SK, Ennis M, Liu W, Shipley MT: Localization and regulation of low affinity nerve growth factor receptor expression in the rat olfactory system during development and regeneration. J Comp Neurol. 1994, 344: 336-348. 10.1002/cne.903440303.View ArticlePubMedGoogle Scholar
- Chen H, Kohno K, Gong Q: Conditional ablation of mature olfactory sensory neurons mediated by diphtheria toxin receptor. J Neurocytol. 2005, 34: 37-47. 10.1007/s11068-005-5046-8.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.