Olfactory Receptor Neurons Exist As Distinct Subclasses of Immature and Mature Cells in Primary Culture

Classes of olfactory receptor neurons in primary cultureNeuroscience Vol. 93, No. 4, pp. 1301–1312, 1301 1999 Copyright ᭧ 1999 IBRO. Published by Elsevier Science Ltd Pergamon Printed in Great Britain. All rights reserved PII: S0306-4522(99)00193-1 0306-4522/99 $20.00+0.00

OLFACTORY RECEPTOR NEURONS EXIST AS DISTINCT SUBCLASSES OF IMMATURE AND MATURE CELLS IN PRIMARY CULTURE

A. M. CUNNINGHAM,*†‡ P. B. MANIS,§k R. R. REED*k and G. V. RONNETT†k¶ *The Howard Hughes Medical Institute, Departments of †Neurology and §Otolaryngology, Head and Neck Surgery, kDepartment of Neuroscience, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, U.S.A.

Abstract—The processes of neuronal differentiation and survival are key questions in neurobiology. The olfactory system possesses unique regenerative capacity, as its neurons are continually replaced throughout adulthood from a maintained population of precursor cells. Primary cultures of olfactory epithelium enriched in olfactory neurons would provide a useful model to study the processes of neurogenesis, differentiation and senescence. To determine whether immature olfactory neurons could be isolated in primary culture and to investigate the mechanisms underlying these processes, culture conditions which selectively favored the presence of immature olfactory neurons were optimized. Using low plating densities, a population of cells was identified which, by reverse transcription–polymerase chain reaction, demonstrated messages for olfactory neuronal markers, including Golf, olfactory cyclic nucleotide-gated channel and olfactory marker protein, as well as the p75 low-affinity nerve growth factor receptor. Immunocytochemical analysis showed that these putative immature olfactory neurons possessed immunoreactivity to Golf, neuron-specific tubulin, neural cell adhesion molecule, synaptophysin and neurofilament. These neurons were defined as olfactory receptor neuron-1 cells. Under these conditions, a separate class of rarely occurring cells with different morphology demonstrated immunoreactivity to mature markers, such as adenylyl cyclase III and olfactory marker protein. Electrophysiologically, these cells displayed properties consistent with those of acutely dissociated olfactory receptor neurons. Another class of rarer cells which represented less than 2% of cells in culture demonstrated immunoreactivity to glial fibrillary acidic protein. These cultures can serve as a model for in vitro analysis of olfactory receptor neuronal development and maintenance, and provide a potential substrate for the development of cell lines. ᭧ 1999 IBRO. Published by Elsevier Science Ltd.

Key words: olfaction, primary culture, neurogenesis, differentiation, immunocytochemistry, electrophysiology.

The olfactory system is unique in the mammalian CNS as it is the neuronal progenitor cells.11 The flattened, horizontal basal a site of continuing neurogenesis in adulthood, replenishing cells are a distinct cell type residing at the base of the neuro- its neuronal cells from a mitotic basal cell layer.27 The factors epithelium adjacent to the basal lamina.25 controlling this process of neurogenesis, differentiation and ORNs die and are replaced by new, immature neurons continual reinnervation of the neuronal target tissue, the arising from cells resident in the basal cell layer.26 A signifi- olfactory bulb, are beginning to be defined.9,10,14,21,22,25,28,36,50 cant proportion of cells dies “precociously” prior to reaching The olfactory system provides a remarkable model in which maturity, and some live considerably longer than 30 days.15 to study the regulation of these processes. Following globose basal cell mitosis, a daughter cell moves Mammalian olfactory receptor neurons (ORNs) are located apically in the epithelium and develops a basally directed in a pseudo-stratified columnar epithelium consisting of axon and an apical dendrite. The axon extends through the essentially four cell types. Sustentacular, or support cells, basement membrane and grows towards the olfactory bulb. functionally resemble glial cells and extend from the epithe- Final maturation takes place when the axon forms a synapse lial surface to the basal lamina; they do not express glial in the bulb, and the dendrite reaches the surface and develops filbrillary acidic protein (GFAP). The initial events of olfac- cilia from its dendritic projection. tory signal transduction occur in the sensory cilia35 of ORNs. In vivo, the phenotype of the cells at various stages of the The cell bodies of the sensory neurons lie at various levels in differentiation process can be characterized by defined the epithelial layer, and mature cells extend dendrites to the markers. The horizontal basal cells are negative for neuronal ciliary surface and axons to their neuronal target cells in markers, but express keratin at high levels.40 Globose basal the olfactory bulb. Globose basal cells are believed to include cells, which are thought to be the precursors of ORNs,11 express GBC-1.23 Immature neurons express a neuronal surface marker, neuronal cell adhesion molecule ¶To whom correspondence should be addressed at: Department of 12,13 51 Neuroscience. Tel.: ϩ1-410-614-6482; fax: ϩ1-410-614-6249. (NCAM), and neuron-specific tubulin (NST). As they E-mail address: [email protected] (G. V. Ronnett) develop, the ORNs express growth-associated protein-43 ‡Present address: The Garvan Institute of Medical Research, 384 Victoria (GAP-43), a protein associated with axonal outgrowth during St., Darlinghurst, NSW 2010, Australia. development,58 and they continue to express NST. Finally, the Abbreviations: EGTA, ethyleneglycolbis(aminoethyl ether)tetra-acetate; cells lose GAP-43 and NST immunoreactivities, 51 and GAP-43, growth-associated protein-43; GFAP, glial fibrillary acidic 0 protein; HEPES, N-2-hydroxyethylpiperazine-N -2-ethanesulfonic acid; express markers typical of mature ORNs, e.g., olfactory 2,47 InsP3, inositol 1,4,5-trisphosphate; MEM, modified Eagle’s medium; marker protein (OMP), and components of the signal trans- NCAM, neuronal cell adhesion molecule; NGF, nerve growth factor; duction cascade, such as type III adenylyl cyclase1 and the NST, neuron-specific tubulin; OcNC1, olfactory cyclic nucleotide- odorant receptor molecules.8,47 There is overlap in the cells gated channel; OMP, olfactory marker protein; ORN, olfactory receptor 51 neuron; p75, low-affinity nerve growth factor receptor; PBS, phosphate- expressing OMP and NST. ORNs express odorant recep- 37 buffered saline; PCR, polymerase chain reaction. tors. The presence of the progenitor cells in the basal part

1301 1302 A. M. Cunningham et al. of the epithelium and the occurrence of this repeated cycle of tubulin isotype III (NST; Sigma) at 1:400 dilution; monoclonal anti- neuronal differentiation make this a unique tissue source for GAP-43 antibody at 1:200 dilution (Boehringer Mannheim); monoclonal anti-synaptophysin antibody at 1:25 dilution (Boehringer establishing primary cultures and examining the characteris- Mannheim); monoclonal anti-NCAM (NCAM16.2) at 1:200 dilution tics of cells at various stages of neuronal commitment in vitro. (gift from Dr J. Hemperly, Becton Dickinson, NC); monoclonal anti- Olfactory sensory neurons have proved difficult to culture. neurofilament antibody (68,000 mol. wt form) at 1:20 dilution Embryonic explant cultures facilitated the characterization (Boehringer Mannheim); polyclonal anti-GFAP (DAKO-Patts) at of the role of several growth factors in olfactory neuro- 1:800 dilution; polyclonal anti-OMP antibody at 1:1000 dilution (gift 13,14,59 from Dr F. Margolis, University of Maryland School of Medicine, genesis; more recent systems have employed astrocyte Baltimore, MD); an affinity-purified polyclonal antibody made to a feeder layers.41 The difficulties in obtaining dissociated cells degenerate peptide corresponding to the C-terminal cytoplasmic region in culture are, in major part, likely to be due to the require- of the putative odorant receptor family (AC529) at 1:100 dilution;17,46 a ments of these small, sensory neurons for specific growth polyclonal antibody to type III adenylyl cyclase at 1:100 dilution;1 polyclonal antibodies made in rabbit or goat to the purified cerebellar factors. In addition, the primary dissociated culture dissection 18,39,54,55 inositol 1,4,5-trisphosphate (InsP3) receptor at 1:40 and 1:50 technique is traumatic to the neuronal cell, as all mature dilutions, respectively (a gift from Dr A. Sharp, Johns Hopkins Univer- neurons which have an axon terminating in the olfactory sity, Baltimore, MD). bulb will be axotomized by the procedure, an insult likely For double-labeling immunofluorescence studies, methanol-fixed to compromise neuronal survival. Here, we report a culture cells were permeabilized as above and blocked in 10% normal donkey serum prior to incubation with the two primary antibodies: affinity- technique that is a variation of that described previously, to purified goat polyclonal antibody to InsP receptor at 1:50 final dilution 49 3 culture mature ORNs, using enzymatic dissociation and rabbit polyclonal Golf antibody at 1:100 final dilution, for 1 h at followed by gentle mechanical disruption and cultivation room temperature. Secondary antibodies were fluorescein (DTAF)- with selective media and substrates. This method differen- conjugated affinipure donkey anti-goat immunoglobulin G (HϩL) at 1:50 dilution and lissamine rhodamine-conjugated affinipure donkey tially favors the survival of immature cells, and provides a anti-rabbit immunoglobulin G (HϩL) at 1:50 dilution (Jackson potentially invaluable model for studying the characteristics Immunoresearch Laboratories). Control cells were incubated with of these immature neurons. Preliminary reports of some preimmune sera in place of the primary antibodies. After washing, aspects of this work have been presented.16 sections were mounted in 90% glycerol in PBS with 1 mg/ml p-phenyl- enediamine dihydrochloride.

EXPERIMENTAL PROCEDURES Primary culture of neonatal olfactory tissue Polymerase chain reaction assays Approximately four litters of rat pups, or 40–45 animals, were used Total RNA was prepared from cells after five days in culture and per preparation. The 24-h-old rat pups were decapitated, and olfactory from the following tissues: neonatal olfactory turbinate tissue (post- tissue was dissected and placed in modified Eagle’s medium (MEM) natal day 1), olfactory turbinate tissue, olfactory bulb, forebrain and × liver from 150 g adult rats.4 First strand cDNA was prepared from 2 mg containing 4.8 g/l HEPES buffer. Tissue was centrifuged at 700 g for m 7 min. The supernatant was poured off and the tissue minced with fine total RNA in a 50- l reaction [50 mM Tris, pH 8.3, 8 mM MgCl2, scissors to produce small fragments about 0.5 mm in size. Tissue was 40 mM KCl, 4 mM dithiothreitol, 1 mM deoxynucleotide tripho- resuspended in 30 ml MEM/HEPES containing 1% (w/v) bovine sphates, 500 ng random hexamers (Pharmacia), 1 U RNasin (Promega), 18 units AMV reverse transcriptase (Life Sciences)]. serum albumin, radioimmunoassay grade (Sigma), 1 mg/ml hyaluro- m m nidase (Sigma), 50 mg/ml DNase (Sigma), 1 mg/ml collagenase Each of the resultant cDNAs (1 l) was used in a 25- l polymerase (Boehringer Mannheim) and 5 mg/ml dispase (Boehringer Mannheim) chain reaction (PCR) under the following conditions: 10 mM Tris (pH for 1 h at 37ЊC. The cell suspension was triturated 10 times, and then 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.1% (w/v) gelatin, 0.2 mM deoxy- m nucleotide triphosphates, 1 mM of each oligonucleotide. For simpli- passed through a 150- m wire mesh (Small Parts) and recentrifuged at Њ 500 × g for 5 min. The cell pellet was resuspended in plating medium city, an annealing temperature of 48 C was chosen to allow the composed of MEM with d-valine (Gibco), containing 10% (v/v) dialysed reactions with all primer pairs to be performed simultaneously. Reac- m tions were denatured for 5 min at 94ЊC and then annealed for 1 min at fetal calf serum (Gibco), 5% NU serum (Collaborative Research), 10 M Њ Њ cytosine arabinoside (Sigma) and 25 ng/ml nerve growth factor (NGF; 48 C. Extensions were for 1 min at 72 C, followed by denaturation at 94ЊC for 45 s. The reactions were continued for 35 cycles, after which a Collaborative Research). After resuspension, cells were filtered sequen- Њ tially through 50- and 10-mm Nylon mesh filters (Small Parts), and plated single cycle with a 5-min, 72 C extension was performed. Oligo- at densities varying from 0.2 to 1 × 106 cells per cm2 into Labtek tissue nucleotide primers designed to the following sequences were used. OMP, the olfactory-specific marker protein: AMC61 and AMC62;29 culture chamber slides (Nunc) coated with MEM/d-valine containing 33 m Golf, the olfactory specific G-protein: BL11 and AMC63; OcNC1, the laminin at 25 g/ml (Collaborative Research). Cultures were grown at 19 37ЊCin5%CO and were fed on day 2 and subsequent days with olfactory-specific nucleotide-gated channel: RR210 and RR217; p75, 2 the low-affinity NGF receptor:44 AMC71 and AMC72; a set of degen- MEM/d-valine containing 15% dialysed fetal calf serum, gentamycin, 34 kanamycin, NGF and cytosine arabinoside. erate primers designed to odorant receptors: NL61 and NL63; and primers to actin, which served as a control: NL9 and NL10. Immunocytochemistry Oligonucleotide sequences: OMP: 0 0 After five days in culture, slides were rinsed briefly in phosphate- AMC61: 5 -CAGCAGCTGGATATG-3 buffered saline (PBS) at 37ЊC and then fixed in methanol at Ϫ20ЊC for 0 0 20 min. Following fixation, cells were permeabilized in 0.1% (v/v) AMC62: 5 -GGTGATGAGGAAATACAT-3 Triton X-100 for 15 min. The same fixation procedure was used throughout these studies. Slides were then rinsed three times with Golf: 0 0 PBS and incubated with primary antibodies for 15 min at room BL11: 5 -GCGCCTGGCTTACAAA-3 0 0 temperature. After three rinses with PBS, slides were immunostained AMC63: 5 -GGCTAGGGGGATATGC-3 using either the Vectastain Elite ABC kit (Vector Laboratories) with diaminobenzidine as the chromogen or a fluorescein (DTAF)-conju- OcNC1: gated affinipure goat anti-rabbit secondary antibody (Jackson Immuno- 0 0 RR210: 5 -AATTGCGAGACAACTATA-3 research Laboratories). For chromogen visualization, endogenous 0 0 peroxidases were quenched by incubation in 0.3% hydrogen peroxide RR217: 5 -TTCTTGAGGACTTCTCGT-3 in PBS. Slides were mounted in Aquamount (BDH) and photographed p75: with a Zeiss Axiophot photomicroscope. 0 0 Antibodies were used in the following dilutions: polyclonal G AMC71: 5 -TACTCAGACGAAGCCAAC-3 olf 0 0 antibody (DJ6.3APl) at 1:100 dilution;32 monoclonal anti-b-specific AMC72: 5 -CTGCCAGATGTCGCCAGG-3 Classes of olfactory receptor neurons in primary culture 1303

Odorant receptors: The modification used in this current method and study 0 0 NL61: 5 -CGGAATTCCCXATGTA(C/T)(C/T)TXTT(C/T)CT-3 relied on use of the polyclonal antibody to Golf (DJ6.3APl), 0 NL63: 5 -ATAAGCTTAG(G/A)TGX(G/C)(T/A)X(G/C)C(G/A) to identify olfactory neurons in culture, as this antibody had 0 CAXGT-3 been shown to be a highly specific marker for ORNs in vivo.33 Actin: In vivo,Golf immunoreactivity is enriched in the olfactory 0 0 ciliary layer, but is also expressed by ORN cell bodies and NL9: 5 -TCATGTTTGAGACCTTC-3 0 0 axons. In vitro, we found that rounded, clustered cells with NL10: 5 -CCAGGAAGGAAGGCTGGA-3 XG, A, T, C. fine processes were immunoreactive for Golf over their entire cell bodies and processes. In an effort to enrich these Golf- The reactions were extracted with chloroform and separated by electro- positive cells, we manipulated the culture conditions, includ- phoresis on 5% polyacrylamide gels visualized with ethidium bromide. ing the age of the neonatal animals at time of dissection, concentration of NGF in the plating media and the plating Electrophysiology densities. Cultures were subsequently evaluated for any resul- Whole-cell tight seal recordings were made from olfactory epithe- tant changes in the abundance of cells expressing Golf lium cells maintained in culture for two to five days using standard immunoreactivity. We found that plating density was the methods. Electrodes were prepared from TW150-F glass (WPI), pulled critical factor, and by decreasing the cell plating density to to have resistances of 5–10 MV. The standard electrode filling solution as low as 0.1 × 106 cells per cm2, we obtained a four-fold was (in mM): 140 KCl, 1.1 EGTA, 0.1 CaCl2, 0.2 Mg2ATP, 2 MgSO4, 4 NaCl and 10 HEPES (pH 7.2). In some experiments, KCl was enrichment in this cell type. Figure 1A and B illustrate that, replaced with equimolar CsF or CsCl. The electrodes also contained under these sparse culture conditions, the clusters of Golf- caged compounds (InsP3, cyclic GMP, cyclic AMP and ATP), all at immunoreactive cells comprised 80–85% of the total cells 1 mM (Calbiochem). The data reported here were collected prior to any exposure to the ultraviolet light used to release these compounds in culture at day 5. These cells have small oval cell bodies from the cage. The extracellular solution contained (in mM): 145 with slightly eccentric nuclei and are phase bright (Fig. 1A). NaCl, 2.5 KCl, 2.0 CaCl2, 1.0 MgCl2,10d-glucose, 20 sucrose, 10 They are bipolar with fine long processes and tend to grow in HEPES (pH 7.35). The bath volume was ϳ250 ml and was superfused clusters, their processes intertwined. They have one small at 0.5 ml/min. The bath was not recirculated. Recordings were made process which is sometimes difficult to visualize and a slender, with Axopatch 1C and Axopatch 200 amplifiers (Axon Instruments) in voltage-clamp mode. The relatively small currents observed in these much longer (50–100 cell body lengths) process emanating cells (usually less than 0.5 nA) and the low electrode resistance result from the opposite pole. These cells stain uniformly for Golf in a voltage error of Ͻ5 mV due to series resistance drops; therefore, (Fig. 1B). This cell type will be referred to as ORN-1, to the amplifiers were usually not compensated. Data were digitized as distinguish it from other cell types in the cultures. 2048-point records at 5–10 kHz and low pass filtered at 2–5 kHz using the internal filters of the amplifiers. Cells were maintained at a standard Ϫ holding potential of 60 mV between voltage pulses. Polymerase chain reaction characterization The standard protocol used to evaluate ionic currents in the cells consisted of 100-ms voltage steps to levels between Ϫ100 and To determine whether the cells in culture expressed well- ϩ 50 mV, in 10-mV increments, delivered once per second. Averages defined components of mature, differentiated olfactory of four records were collected at each voltage level. Data are plotted after correction for leakage resistance, but without correction for series sensory neurons, OMP, Golf, OcNC and p75, we performed resistance. Capacitative transients have been subtracted using an PCR assays on RNA isolated from day 5 primary cultures appropriately scaled version of averaged hyperpolarizing steps. To using specific oligonucleotide pairs for these four components test responses to odorants, a cocktail consisting of 1 mM cineole, (Fig. 2). In addition, we used a pair of degenerate primers acetophenone, amyl acetate and 2-isobutyl-3-methoxypyrazine dissolved in the normal extracellular solution was applied via a puffer based on the sequences of the putative olfactory receptor gene pipette (tip diameter ϳ2 mm) placed 10–20 mm from the cell body. family to ascertain whether they were detectable in vitro. Pairs of puffs separated by 500 and 50 ms in duration were applied. In PCRs were performed simultaneously on the panel of these experiments, the recording electrodes contained CsCl instead of cDNA templates made from the primary cultures, neonatal KCl, and also 2 mM Mg2ATP and 0.4 mM GTP. olfactory tissue, adult olfactory tissue, olfactory bulb, forebrain, liver and water (to serve as a control). The aim of this RESULTS comparison of tissues was to help to validate the reaction product obtained in the primary lane by comparison with Morphology of primary cultures and selection for enrichment the product or products obtained from endogenous tissues. of G -immunoreactive cells olf The PCR products (expected size 437 bp) obtained using a Several features of this modified primary culture system are primer pair for actin showed that cDNA primed in all lanes similar to a culture technique described previously.49 This except for the water control (Fig. 2F), and that the amount of method yields cells which begin to extend processes within product was similar across tissues. 24 h of plating, and by 72–96 h in culture the cells have In Fig. 2A, a product of the expected size (415 bp) for the reached maximal density. Process outgrowth was generally classical marker for mature ORNs, OMP, is found in the bipolar and in some cells was very extensive, 50–100 body primary lane, as well as olfactory epithelium and bulb. lengths. By day 4 in culture, the cells were morphologically There is also message detectable in the forebrain, and this is heterogeneous, but the predominant cell type (approximately consistent with reports by others in vivo.2 Product of the 70%) was a bipolar, slightly fusiform cell with long tapering expected size (1112 bp) was seen in the primary culture processes. The next most frequent cell type (comprising lane with Golf primers (Fig. 2B). Golf was also apparent in approximately 20% of the total) was a small cell with round adult olfactory tissue, bulb and forebrain, with lesser amounts to oval nucleus and fine bipolar processes which tended to in neonatal tissue. Although we do not propose that this assay grow in clusters. Most of the remaining cells were isolated, is rigorously quantitative, it is of note that these results again having multiple processes and flattened, polygonal epithelial- would be in keeping with observations about the distribution 30 like shape. of Golf in the rat, including the forebrain localization. 1304 A. M. Cunningham et al.

and under these culture conditions, without the addition of other growth factors. Subsequently, the PCR product was isolated, cloned and sequenced, and this conﬁrmed that odorant receptor sequences, corresponding to previously cloned cDNAs and novel members of the receptor family, were able to be ampliﬁed from the primary cultures.5 In summary, we detected products of the expected size in the primary cultures with sets of primer pairs for Golf, OMP, OcNC1, p75 and actin.

Immunocytochemical characterization of olfactory receptor neuron-1 cells with antibodies to defined neuronal and olfactory markers We used the well-characterized marker for neurons, b- specific tubulin isotype 3, or NST, in order to determine which cells in the culture were of the neuronal phenotype.3 NST is expressed early in immature ORNs and expression continues in mature ORNs, overlapping with OMP expression, as determined by double-labeling immunohistochemistry.51 Figure 3A shows that the ORN-1 cells are immunoreactive with this neuronal marker, and Fig. 3B (higher magnification) illustrates their particular morphological features, as was seen with the antibody to Golf. Based on NST immunoreactivity and this characteristic morphology, we were able to establish that a large percentage of the neurons in culture was ORN-1 cells, usually in excess of 80%. We observed occasional multi-processed cells expressing NST, which were clearly not ORN-1 cells (data not shown), and another class of cells described in detail Fig. 1. Phase-contrast microscopy and immunocytochemistry for Golf of low-density cultures. Cells were prepared and plated at low density, and below. fixed for microscopy and immunocytochemistry as described in the Experi- A control slide (Fig. 3C) was included in which non- mental Procedures. The same fixation protocol was used for all immuno- immune mouse serum was used in place of the primary anti- cytochemistry. (A) Culture conditions favored small, bipolar cells, which were clustered into groups. Neurons had fine bipolar processes, one process body, and this supports the specificity of our immuno- often being very long. (B) Golf immunoreactivity was visualized after addi- reactivity. As other controls for the polyclonal antibodies, tion of conjugated secondary antibody and chromogen. Up to 80% of cells we used both preimmune (where available) and non-immune in culture demonstrated Golf immunoreactivity along their fine processes rabbit sera in place of the primary antibodies (data not × × and in their cell bodies. Magnifications: 400 (A), 200 (B). shown). Staining with the Golf antibody also identified some cells Figure 2C shows that the product obtained with primers distinct from ORN-1 cells which also possessed a neuronal designed to OcNC1 resulted in a band in the primary cultures phenotype. A characteristic example is illustrated in Fig. 3D of the predicted size (610 bp), and in vivo this message was (arrow). These cells had round cell bodies and nuclei about confined to the olfactory epithelium. It is of interest that p75 20–30% larger than those of the ORN-1 cells and had appeared to be up-regulated in the primary cultures (Fig. 2D), multiple processes (usually five or six). These cells were a large amount of product being seen in these primaries with- quite rare, comprising less that 2% of the Golf-, NST-immuno- out significant product being detectable at the predicted size reactive cells in the cultures. This panel also illustrates another (600 bp) in olfactory epithelium. Product was seen in the unusual result, which was a Golf-immunoreactive, binucleated olfactory bulb, and this is a known site of significant p75 in cell which may represent a cell in the process of division. vivo.24,50 Immunoreactivity with the monoclonal antibody to the We were interested in determining whether the large family immature marker for growth cones and axonal outgrowth, of related putative G-protein-coupled odorant receptor mole- GAP-43, produced a surprising result. Only a subset of the cules first reported by Buck and Axel8 was expressed in our in ORN-1 population was GAP-43 immunoreactive (Fig. 3E), vitro culture system. This PCR assay used a set of degenerate and a clearly non-immunoreactive ORN-1 cell is seen in this primers designed against transmembrane regions 2 and 6 in figure. GAP-43 immunoreactivity was distributed in the cell the receptor family, which would produce a product of body and along both neuritic processes, without any evidence 550 bp. This product is easily visible in the adult olfactory of polarity of distribution. On average, about 40% of the tissue lane (Fig. 2E), but is not easily appreciated on this ORN-1 cells were GAP-43 immunoreactive. This suggested particular gel in the primary lane. We were able to detect a that there was heterogeneity in the state of differentiation faint band in the primary lane when we lowered the annealing amongst the ORN-1 cells in culture. temperature to 46ЊC and increased the number of cycles to 40 Synaptophysin is the major protein component of (data not shown). This result would indicate that ORN-1 cells presynaptic vesicles, and as such is an excellent marker for did not express significant amounts of odorant receptor synapses both in vivo and in vitro.31,56 All ORN-1 cells mRNAs as assessed using this particular set of PCR primers expressed synaptophysin in a pattern that was most distinctive. Classes of olfactory receptor neurons in primary culture 1305

Fig. 2. Reverse transcription–PCR analysis of primary cultures and related tissues for olfactory markers, p75 and actin. RNA was reverse transcribed and PCR performed as described in the Experimental Procedures from primary cultures (cultures), neonatal olfactory turbinate tissue (neonatal olfactory neuroepithelium, ON), adult olfactory turbinate tissue (adult olfactory neuroepithelium), olfactory bulb, forebrain, liver and water control. Products with size corresponding to the predicted lengths of products are indicated by the arrowheads at sizes 415, 1112 610, 600, 550 and 437 bp, respectively. (A) PCR products using primers for OMP. Products are easily visualized in lanes containing cDNA from cultures, adult olfactory neuroepithelium tissue, olfactory bulb and forebrain, but rarer in neonatal olfactory neuroepithelium tissue, and absent in liver and water control. (B) Products for the reaction using Golf primers are shown. Golf was present in cultures and adult olfactory neuroepithelium tissues, as well as olfactory bulb and forebrain, but absent from liver and water control; only a very faint band was detectable in neonatal olfactory neuroepithelium tissue. (C) PCR products using primers designed to amplify OcNC1 demonstrate products in cultures, and neonatal and adult tissue. Interestingly, the product is far more abundant in neonatal than in adult olfactory neuroepithelium tissue. (D) PCR products obtained using primers for the p75 neurotrophin receptor demonstrate an up-regulation of mRNA in cultures and just detectable bands in adult olfactory bulb tissue. (E) PCR products using a set of degenerative primers designed against transmembrane regions 2 and 6 of the odorant receptor family only demonstrated a robust band in adult olfactory tissue. Under some conditions, a faint band could be observed in cultures (data not shown). (F) PCR products obtained using primers designed to amplify actin. Bands are seen in all lanes, except for the water control.

Immunoreactivity was concentrated in punctate accumula- immunoreactive for two other neuronal markers: NCAM tions, which were most dense when ORN-1 cell bodies and neurofilament. These patterns of immunoreactivity are were clustered together (Fig. 3F). These accumulations, shown in Fig. 3G and H, respectively. which appeared superficially around the cell bodies, also Immunocytochemistry with markers for cells of the glial occurred in relation to the ORN-1 processes and when phenotype confirmed that the majority of cells, including the processes from neighboring cells were seen in close contact ORN-1 population, were not glial in nature. In general, the (data not shown). The ORN-1 cells were uniformly cells which stained with anti-GFAP antibody were isolated 1306 A. M. Cunningham et al.

Fig. 3. Immunocytochemical characterization of ORN-1 cells with defined olfactory and neuronal markers. Low-density cultures were prepared, maintained for five days in culture and fixed for immunocytochemistry as described in the Experimental Procedures. (A) NST immunoreactivity. The predominant cell in culture demonstrates immunoreactivity for NST in its cell body and along its fine processes. (B) Higher magnification of Golf immunoreactivity of cells. (C) Control using non-immune serum instead of the primary antibody. No immunoreactivity is observed. (D) Immunoreactivity for Golf. Occasional cells are identified (arrow) which have multiple processes. These cells are quite rare in culture. (E) Immunoreactivity to GAP-43. Not all cells demonstrated GAP-43 immunoreactivity. On average, only 40% of ORN-1 cells in culture were immunoreactive for GAP-43. (F) Synaptophysin immunoreactivity was punctate and aggregated in areas where cells appeared to contact, as well as being present in accumulations along the neuritic processes. (G) ORN-1 immunoreactivity for NCAM. (H) Immunoreactivity for neurofilament. Both NCAM and neurofilament immunoreactivities were visualized along processes and in cell bodies. Magnifications: × 200 (A), × 630 (B), × 400 (C–H). Classes of olfactory receptor neurons in primary culture 1307

Fig. 4. Immunocytochemistry of primary cultures for glial and mature olfactory-specific markers. Primary cultures were grown for five days, fixed and prepared for immunocytochemistry as described in the Experimental Procedures. (A) GFAP immunoreactivity. GFAP immunoreactivity identified cells with larger nuclei and distinct, angular processes. These cells were comparatively rare in culture. ORN-1 cells identified morphologically did not demonstrate GFAP immunoreactivity. (B) OMP immunoreactivity. At low densities of plating, OMP-immunoreactive cells were few and in general did not demonstrate processes. (C) Immunoreactivity with an antibody which recognizes the family of putative odorant receptor proteins (AC529). Very few cells were immunoreactive with this antibody to the family of odorant receptors, and generally the positive cells resembled the OMP-immunoreactive cell type. Occasionally, immunoreactivity was found on cells which were more ORN-1-like in morphology, as demonstrated here. (D) Type III cyclase immunoreactivity. Scattered, round, processless cells demonstrated immunoreactivity for adenylyl cyclase type III, and these cells were similar in morphology to but more prevalent than those which were OMP immunoreactive. Magnifications: × 400. larger cells with distinctive morphologies, such as asymme- found to be expressed in single, round cells which tended to trical, non-tapering processes (Fig. 4A). Some multi- occur as isolated cells in culture. As shown in Fig. 4D, these processed cells, which might possibly have been taken to cells morphologically resembled the OMP-immunoreactive resemble neurons based solely on their morphological cells, but they were significantly, i.e. four-fold, more abun- criteria, were GFAP positive. None of the cells with the clas- dant in the cultures. These data suggest that, under culture sical ORN-1 features was found to be immunoreactive for conditions which favor immature cells, as defined by GFAP. These astrocytic cells were rare; generally, we noted immunocytochemical staining, mature ORNs are rare and about six per 20-cm2 chamber tissue culture slide. In addition frequently do not have identifiable neurites. For simplicity to GFAP-immunoreactive glia, a significant number of elon- of discussion, we will refer to round, solitary OMP- or gated, bipolar cells and cells with other phenotypes was S100 cyclase-immunoreactive cells as ORN-2, to designate a rarer, immunoreactive, indicating that they were very likely to be more differentiated neuronal class in culture. It is quite likely olfactory-ensheathing glia (data not shown). Some of these that the culture conditions described here do not promote the cells and their processes are clearly visible in the backgrounds growth and maintenance of the more mature neurons. This is of Fig. 4A and C. evidenced by the comparison with the abundance and vigorous When we performed immunocytochemistry with the clas- growth in clusters of the ORN-1 cells. It is possible that the sical marker for mature olfactory neurons in vivo, OMP, we cultures are lacking in growth factors specifically important for found that, in general, ORN-1 cells were non-immunoreactive the more differentiated neurons, such as neurotrophins.50 for OMP. There were significant numbers of immunoreactive cells, but they occurred as isolated cells or small groups of round cells without obvious processes (Fig. 4B). Similarly, an Co-expression of inositol 1,4,5-trisphosphate receptor and Golf antipeptide antibody (AC529) made to recognize the family As we had shown expression of Golf in the ORN-1 cell of putative odorant receptor proteins was expressed by a simi- population, we were interested to determine whether the lar population of cells with the same morphology and density InsP3 receptor, a component of a second messenger cascade of expression in culture, although the pattern of immuno- associated with odorant signal transduction,6,20,32,48 was reactivity was faint and punctate associated with the soma, expressed. A question of interest was whether individual rather than filling the cell body like OMP. There were very ORN cells express both InsP3 receptor and Golf, although occasional solitary cells with a more neuronal phenotype, the homogeneous distribution of these components across including relatively long processes, which were immuno- the olfactory ciliary layer has indicated that this is the case reactive for AC529, and an example of one of these cells is in vivo.18 This is particularly interesting, as current evidence shown (Fig. 4C). These cells were exceptionally rare. suggests that individual ORNs express only one type of The type III adenylyl cyclase, which in vivo is localized odorant receptor molecule, and the relative importance of 7 immunohistochemically to the cilia of the mature ORNs, was the cyclic AMP and InsP3 pathways remains controversial. 1308 A. M. Cunningham et al.

Fig. 5. Double-labeling immunofluorescence of primary cultures for Golf and InsP3 receptor. Cell cultures were prepared and grown for five days. Slides were fixed in methanol, and double-labeling immunofluorescence was performed as described in the Experimental Procedures. (A) Immunofluorescent detection of InsP3 receptor in cultures revealed that ORN-1 cells express this receptor over cell bodies and processes. Arrows indicate processes. (B, C) Immunofluor- escence using anti-InsP3 receptor (B) with rhodamine-conjugated secondary simultaneously stained for Golf using anti-Golf (C) antibody visualized by fluorescein-conjugated secondary antibody. Golf and InsP3 receptor are co-expressed in cells whose morphology was consistent with the ORN-1 population of neurons. Arrows indicate some of the cells which are double labeled. Magnifications: × 400 (oil immersion).

Using immunofluorescent detection, we were able to show ϩ50 mV) in CsF than did those with no inward current that the ORN-1 cells expressed InsP3 receptor (Fig. 5A) dif- (compare the examples in Fig. 6D and E). fusely over the cell bodies and processes. Using double-labeling The fast inward current was blocked by 1 mM tetrodotoxin. immunofluorescence with a polyclonal goat anti-InsP3 recep- Figure 6C shows the fast inward current in normal conditions tor antibody and a rabbit polyclonal Golf antibody, individual and 1 mM tetrodotoxin, for a voltage step to Ϫ10 mV. The ORN-1 cells were shown to co-express both InsP3 receptor results of these experiments are consistent with the rapidly (Fig. 5B) and Golf (Fig. 5C). Thus, the same cell expressed inactivating inward current being carried by sodium ions and components of both signaling pathways, although this does the non-inactivating outward current by potassium ions. not prove that the InsP3 pathway is used for odorant detection. Recordings were also made from another cell type, characterized as having larger (20 mm), flattened fusiform somas and thicker bipolar processes. These cells commonly had very Electrophysiological characterization low zero-current potentials, rarely exhibited inward currents To determine whether the physiological properties of the and had weak, if any, outward current (data not shown). These cells in culture were similar to those reported previously for cells often had very high input resistance and larger capaci- acutely isolated receptor neurons, ionic currents were char- tance than the ORN-1 cells. A third cell type was also acterized under voltage clamp. Recordings from ORN-1 cells observed. These cells had high resting potentials, lower (identified as round or oval cells, 8–12 mm in diameter, with input resistances and larger outward currents (up to several bipolar processes) made with KCl-filled electrodes revealed nanoamperes). Usually, three components of current were voltage-dependent inward and outward currents, as shown in evident: a rapidly inactivating inward current, a transient Fig. 6. For hyperpolarizing steps, only a leakage current is inactivating outward current and a non-inactivating outward seen. For depolarizing steps to Ϫ30 mV, a fast inward current current. When recordings were made from these cells with develops, and for steps to Ϫ20 mV and above both a fast cesium electrodes, a large sustained inward current was inward current and a non-inactivating outward current can evident for positive depolarizations, in addition to the rapidly be seen (Fig. 6A). The peak inward current and the steady- inactivating inward current. It is likely that this represented a state outward currents are plotted as a function of voltage in calcium current in these cells. the lower part of Fig. 6A. Although the outward current was Finally, we tested the odorant responses of the cultured usually non-inactivating, in a few cells a rapidly inactivating neurons, using a cocktail of 1 mM cineole, acetophenone, outward current was seen superimposed on the non-inactivat- amyl acetate and 2-isobutyl-3-methoxypyrazine. Of 10 cells ing current. Rapidly inactivating inward currents such as tested, only one responded with slow inward currents, as those shown in Fig. 6A were seen in seven of 22 cells, and shown in Fig. 6F (a second cell had very small, equivocal all of these cells had clear delayed rectifier currents. In 12 of currents). This cell also exhibited fast inward sodium currents 22 cells, only outward currents were seen; one example is in response to depolarizing voltage steps. These results illustrated in Fig. 6B. No significant currents were seen in demonstrate that at least some of the cells in the culture the remaining three cells. Recordings were also made with express functional odorant receptors. electrodes containing CsF instead of KCl. The CsF largely, but not completely, blocked the outward currents in most cells. Fast inward currents were seen in 21 of 42 cells DISCUSSION recorded with CsF (Fig. 6D); it is likely that the larger proportion of rapidly activating currents seen with internal The main finding of this study is that there are two major CsF is the result of decreased delayed rectifier conductances, populations of neurons in dissociated primary olfactory which partially masked the inward currents in internal Kϩ. epithelial cultures prepared in this manner: an abundant Interestingly, those cells which had fast inward currents class of immature neurons, ORN-1 cells, and a rarer popu- tended to have much smaller outward currents (measured at lation of more differentiated neurons. ORN-1 cells are Classes of olfactory receptor neurons in primary culture 1309

Fig. 6. Representative examples of voltage-dependent currents recorded in cultured olfactory receptor neurons. (A) Depolarizing voltage pulses evoked a rapidly inactivating inward current and a sustained outward current in some cultured cells. The lower part of the panel shows the current–voltage relationship of the fast inward current (squares) and the steady-state outward current (triangles). (B) The rapidly inactivating inward current was frequently absent in cells with morphology similar to those in A. Note the absence of the inward current at the start of the current traces. (C) The rapidly inactivating inward current was suppressed by 1 mM tetrodotoxin, indicating that it was generated by sodium channels. (D) The outward current was largely suppressed when the electrode solution contained cesium fluoride (CsF). (E) Some cells lacking fast inward currents showed large outward currents when recorded with CsF electrodes. (F) Responses to an odorant cocktail were present in one of 10 cells tested. The responses of this cell to pairs of puffs are shown here. In all panels except for C and F, leak and capacitative currents have been subtracted. characterized by NST and Golf immunoreactivity and avid, immunocytochemistry (data not shown), and this message is punctate expression of synaptophysin. This population of shown to be markedly up-regulated in vitro by PCR analysis. cells shows some heterogeneity in differentiation in culture, Expression of p75 is not a characteristic of differentiated as only a subset of ORN-1 cells was GAP-43 immuno- ORNs in vivo.24 p75 expression may reflect a response to reactive. Morphologically, however, these cells appeared to stress, loss of synaptic contact, or other features of in vitro be very homogeneous, growing in clusters with long neuritic conditions. As proposed for other neurons, cells may require processes. We hypothesize that ORN-1 cells are derived from NGF occupation of the p75 receptor to prevent initiation of a precursors which were either non-committed neurons or cell death program.42,43 p75 induction is observed following immature neurons at the time of culture preparation. Perhaps lesioning of the olfactory bulb in vivo.50 These data suggest the heterogeneity in vitro reflects at which stage of differen- that p75 may play an important role in ORN turnover in tiation these cells were at the time of culture preparation. response to injury. Other factors that might cause differences between ORN-1 The relatively high expression of OcNC1 in neonates cells could be the proximity in vitro to other cell types compared with adult tissue suggests that OcNC1 may func- which were producing required neuronal growth factors. We tion in other processes, in addition to ORN depolarization in have certainly observed that the apparently most vigorous and response to odorants. We have localized OcNC1 to axons of longest-processed ORN-1 cells were found growing together ORNs of both the adult and embryonic olfactory epithelium in clusters, suggesting that they may have derived in vitro and early in development (Matsuzaki and Ronnett, unpub- from a common precursor, or alternatively, that they were lished observations), suggesting an in vivo role for OcNC1 responding to favorable environmental factors. Further developmentally which correlates with our reverse studies considering the labeling with [3H]thymidine are needed transcription–PCR findings in neonates in this study. Another to specifically address these questions. unusual characteristic of ORN-1 cells is the abundant expres- ORN-1 cells also possess some features suggesting that sion of the presynaptic vesicle component, synaptophysin, they have adapted some quantitative differences in expression both over the soma and along the extent of their neurites. In in vitro. Firstly, they express p75, as demonstrated using vivo, synaptophysin is predominantly confined to the synapse 1310 A. M. Cunningham et al. in the olfactory bulb, and the pattern found in vitro is sugges- Preliminary evidence suggests that these cells can indeed tive of a vigorous cell, attempting to establish multiple areas fire at least one action potential for a depolarization, of synaptic contact. These features suggest that the ORN-1 although under the conditions of these experiments (22ЊC, cells possess a significant degree of plasticity and are vigor- whole-cell recording mode), the action potentials were ous, healthy neurons. somewhat broad. In addition, in some cells, the sodium The co-expression of InsP3 receptors and Golf in the same current did not appear to be well clamped, even though it ORNs, as shown by double-labeling immunofluorescence, was small, because the current had a stereotypic shape with a demonstrates that components of both the InsP3 and cyclic latency that varied with the depolarization. This suggests that AMP signaling cascades are present in the same cells. the active conductance was located distant to the recording Although odorants have been shown to stimulate both cyclic site, perhaps in one of the processes. The electrophysiologi- 6,31,48 AMP and InsP3 production, the role of InsP3 has been cal results serve to confirm that approximately half of the 6 questioned. The function of InsP3 may vary between ORN-1 cells in this culture system have membrane conduc- species,19,38 and may be modulatory as opposed to a direct tances similar to those described previously in acute and depolarization of the cell. The present findings, however, cultured preparations of olfactory receptor cells. It is tempt- provide evidence that these two pathways co-exist in the ing to suggest that differences in the expression of the same ORNs, which has been supported by our previous exam- sodium current might be correlated with the relative maturity ination of the ciliary epithelium by double-labeling confocal of the cells; however, we have no data that bear directly on immunohistochemistry.18 Of course, this does not imply that this issue. One population of cells exhibited large outward InsP3 is used for odorant detection in these cells. currents and no inward currents when recorded with cesium- In contrast to the ORN-1 cells, the less common, differen- filled electrodes. It is likely that the outward currents were tiated neurons, designated as ORN-2, which in culture carried through cesium-permeable potassium channels. Such expressed OMP, type III adenylyl cyclase and the odorant channels have been reported in several cell types, including receptors, were possibly mature neurons at the time of dissec- vestibular type I hair cells,52 lateral dorsal tegmental tion. We would hypothesize that these cells, disrupted from neurons53 and neurons of the chicken nucleus magnocellu- their natural environment in the polarized epithelium, as well laris.45 The expression of a distinct set of potassium channels as suffering axonal damage or axotomy during the procedure, in these cells suggests that these cells represent a separate follow a path of decline and senescence in vitro. This is population from the receptor neurons. Finally, we observed consistent with our observation that these cells rarely possess odorant responses in only one of 10 cells tested. This may significant neurites. This is in contrast to previous culture reflect the limited spectrum of receptors that could be acti- methods established at high plating density, in which neurons vated by the four components of the cocktail we used, or it immunoreactive for OMP predominated.49 may reflect a relatively low level of expression of receptors Another possibility is that the mature ORN-2 cells might in the cultured cells. arise from progenitor cells in vitro. We consider that the This well-characterized culture system now provides a current evidence is against this: their rarity in culture and valuable model in which to address questions regarding their lack of clustering, and the time-point of five days neurogenesis and differentiation of olfactory neurons. One would seem too soon for them to have reached this final can now evaluate the effect of a variety of growth and differ- stage of differentiation. None the less, a related question is entiation factors on cell proliferation, neuronal phenotype and whether it is possible to cause further differentiation of the survival in these dissociated neuron-enriched cultures. The ORN-1 cells in vitro and direct them into a committed ORN-2 existence of both mature and immature neurons in mixed olfactory phenotype, such as would be characterized by culture under these conditions suggests that our culture expression of type III cyclase and the odorant receptors, and system has retained some features of the complex milieu of the loss of GAP-43 immunoreactivity. This possibility is the naturally regenerating and differentiating olfactory epithe- suggested by our recent demonstration that brain-derived lium. These cultures have a wider potential application in neurotrophic factor can increase NCAM-positive cells in future studies of neuronal differentiation, neurotoxicity and culture.51 Further studies are now needed to determine neurodegeneration. whether ORN-1 cells can be differentiated into the ORN-2 phenotype in vitro, and we consider that the application of trophic factor studies to this culture method will yield valu- Acknowledgements—We thank Helen Cho for technical assistance able insights into olfactory neuronal differentiation. with neuronal culture. We are grateful to Frank Margolis for the The ionic currents of ORN-1 cells are similar to those gift of OMP antibody, Alan Sharp for the gift of InsP3 receptor antibodies and John Hemperly for the gift of anti-NCAM antibody. reported in the literature for both acutely isolated olfactory We thank anonymous reviewers for helpful critiques. This work was receptor neurons and cultured olfactory neurons. Indeed, the supported in part by a grant to A.M.C. from The Medical Foundation, amplitudes, voltage dependence and kinetics of the currents The University of Sydney, NSW, Australia, a grant from The reported here closely match those described by Trombley National Institute of Mental Health to R.R.R., a USPHS grant NS- and Westbrook.57 Since these cells exhibit appropriate 02131 and a Johns Hopkins Clinician Scientist Award to G.V.R., NIDCD grants K04-DC00048 and R01 DC00425 to P.B.M., a grant sodium and potassium currents, they should be able to fire from International Flavors and Fragrances, and the W. M. Keck action potentials in response to depolarizing stimuli. Foundation.

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(Accepted 25 March 1999)