Several groups have examined neuronal characteristics of primary cultures derived from olfactory epithelial biopsy or cadaver tissue (Feron et al., 1999a
; Othman et al., 2005; Wolozin et al., 1992
; Zhang et al., 2006; Zhang et al., 2004
) but the degree to which these cells attain neuronal maturity has not been fully elucidated. The results of our study demonstrate that a subgroup of these cells mature to express odorant receptors, providing a physiological basis for odorant responsiveness of these cells and further validating their functional maturity. In addition, the expression of neurotransmitter receptors and their active signaling mechanisms qualify these cells as a model system to study dysregulated receptor signaling in specific individuals, as might occur in neuropsychiatric illnesses.
Our results, generated from multiple experimental approaches, establish the presence of each component of the odorant detection cascade – from receptor to calcium signal – in cultured cells derived from the adult human olfactory epithelium. The morphology of immunocytochemically labeled cells varied from rounded cells with one or more processes to larger cells with a more varied shape. Cells in culture may vary in shape depending on their age, substrate, proximity to other cells, media and other culture conditions (Wolozin et al 1992
., Vawter et al 1996, MacDonald et al 1996, Gomez et al 2000
). The single cell functional assays enable comparison of morphology with response to odor or other stimuli. Our experiments and others with these cells indicate no consistent correlation between functional characteristics and a specific morphological cell type in these cell cultures (Gomez et al., 2000
; Bryant B., unpublished observations). The observed low frequency of responses to single odorants is consistent with previous studies of freshly dissociated OSNs (Rawson et al., 1997
), and with the notion that human OSNs appear to be relatively narrowly tuned. These cultures provide a tool for further studies of selectivity and response profiles where the availability of freshly dissociated OSNs is limited.
Our functional and molecular data suggest that the selectivity in odor responsiveness reported in ex vivo
human olfactory neurons (Rawson et al., 1997
) is retained in vitro
. The in situ
hybridization data supports the expression of these two ORs in these cultured cells. The frequency of cells expressing each OR is not unexpected in view of studies of OR expression in rodents suggesting that each OR is expressed in approximately 1% of rodent OSNs in vivo
(Buck and Axel, 1991
; Buck, 2004
). Given the roughly 3-fold fewer number of functional OR genes in human compared to rat, one might predict a somewhat higher frequency of OSNs expressing any given OR. We cannot rule out the possibility that our probes cross-hybridized with other OR mRNAs that have very similar sequences differing only in a few nucleotides. In this context, the OR3A1 probe sequence is 90% identical to a region of the OR3A3 sequence (ACC no. NM_012373) and 89% identical to the OR3A2 sequence (ACC no. NM_002551), and the OR1A1 probe was 83.2% identical to the OR1A2 sequence (ACC no. NM_012352). It is possible that these particular ORs are both represented in the two cultures we examined because of the location of the biopsy from which they were derived, or because of some aspect of the cell culture process. The frequency of cells responsive to helional in a comparable culture is consistent with the OR expression frequency observed in the in situ
hybridization experiments. Some difference would be expected as the cells assayed functionally, although from the same parent culture, were not the identical cells used for in situ
hybridization. These data suggest a number of issues warranting further study, such as the number of different ORs expressed in cultures, their stability over multiple passages, and consistency among cultures derived from the same or different subjects.
Taken together, the results of the present study demonstrate that in our system, a substantial degree of differentiation, including OR expression, can occur in the absence of olfactory bulb connectivity. These hOE cultures thereby provide an in vitro system to study such fundamental questions as how OR gene expression is regulated in living OSNs, how the odorant response pathways develop during functional maturation of the cell, and how these processes may be modulated by the environment.
Our results also demonstrate the presence and function of a number of neurotransmitter pathways important for modulation of sensitivity and/or maturation of OSNs and other neuronal types. Neurotransmitter receptors for dopamine have been reported previously on rodent OSNs and their nerve terminals (Feron et al., 1999b
; Koster et al., 1999
). In the epithelium, dopamine receptors are present on both neuronal and non-neuronal cell populations, where dopamine is thought to trigger terminal differentiation and cell death (Feron et al., 1999b
). In addition, the D2R agonist bromocriptine inhibits adenylyl cyclase in rat OE (Mania-Farnell et al., 1993
), suppresses a hyperpolarization activated cation conductance, modulating OSN activity in short-term cultures (Vargas and Lucero, 1999
), and reduces L-type Ca++
channel activity in ORNs (Okada et al., 2003
). The expression of D2R was also reported in hOE explant cultures where it triggered neuronal maturation (Feron et al., 1999b
). Consistent with these reports, we have identified the presence of D2R by immunoblotting () as well as by radioligand binding assay (data not shown). Further, dopamine induced G protein activation was inhibited either by SCH23390 or sulpiride in an isoform specific manner, which supports the presence of both D1R- like and D2R- like receptor signaling pathways in these cultures. The expression and functionality of dopaminergic receptors are of particular interest in these cells since dopaminergic receptor signaling is thought to be dysregulated in schizophrenia, Parkinson's disease and other neuropsychiatric illnesses (Bonci and Hopf, 2005
Serotonin plays a role in early development of the olfactory pathway (Vitalis et al., 2003) and is a well-established neuromodulator in the olfactory systems of invertebrates (Piomelli and Tota, 1983) and sea lamprey (Zielinski et al., 2000
). Modulation of forskolin-stimulated electrical activity by serotonin has been observed in frog OSNs (Frings, 1993), and the serotonin-reuptake blocker amitryptyline reduced adenylyl cyclase activity in olfactory neurons and inhibits olfactory neurite outgrowth (Mania-Farnell et al., 1993
). These data suggest that 5HT receptors may be present on OSNs, but serotonin receptors have not previously been reported on mature mammalian OSNs. Our study demonstrates that hOE cultures robustly express 5HTR2C and possess subtype specific coupling of 5HTR2A, 5HTR2B and 5HTR2C with G proteins. Although in vivo
expression of these receptors in OSNs and other cell types in human olfactory epithelium is unknown, the presence and activity of these receptors in hOE culture cells further position these cells as viable tools to investigate 5HT receptors of specific individuals in the context of neuropsychiatric research.
The excitatory NMDA receptor plays key roles in neuroplasticity, modulation of synaptic transmission and apoptosis (Sheng and Kim, 2002
; Lynch and Guttmann, 2001
). NMDA receptors have not previously been reported on cells within the olfactory epithelium of any species, although their presence and function in the olfactory bulb is well-established (Friedman and Strowbridge, 2000
). Our results, however, indicate that hOE cultures robustly express NMDAR1 (), 2A and 2B (data not shown). Furthermore, functional assays demonstrate that ligand stimulation induced recruitment of NMDAR-linked signaling molecules and enhancement of intracellular calcium influx. Based on the co-expression of voltage-gated calcium channels in cells responding to NMDA (), some of these NMDA-responsive cells appear to be neurons. The presence of these functionally active NMDARs does not appear to be a tissue culture artifact. When freshly obtained human olfactory biopsy tissues were incubated with NMDA, tyrosine phosphorylation of NMDAR1 and recruitment of signaling partners, PLC-γ and src kinase, were also found to increase (). The current data do not address whether these receptors are co-expressed with the other receptors identified, or whether the culture comprises several subpopulations with different neurophysiological characteristics.
Neuronal characteristics of these culture cells, particularly the functionality of neurotransmitter receptors, offer an opportunity to monitor such parameters as biological characteristics of the donor. It will be tempting to define OE biopsy tissues and these culture cells as a cellular system to identify biomarkers associated with diagnosis, prognostic indicators, or responsiveness to therapeutics. In such approaches, however, it will be critical to establish the stability or consistency of each biological parameter of interest, by assessing intra- and inter- subject variability.
We have assessed the expression of D2R, NMDAR1 and CamKII normalized with respect to β-actin in nine randomly selected olfactory cell lines. Expression levels of these proteins varied between subjects with variability (SEM/mean) ranging from 30% (NR1)-45%(D2R) (data not shown). Such variability is most likely to arise from the heterogeneity of cell types, neuronal and non-neuronal, and from varied cellular composition of each cell line. In this regard, single cell based assay strategies, in conjunction with cell type identification, such as electrophysiological studies or single cell based RNA amplification techniques are promising. For future studies, intra- or inter-subject variability must be assumed and the stability of each measure should be tested.
Neurotransmitter receptors appear to be prevalent among these culture cells, suggesting that they are expressed across a large population of mature neurons and/or may be expressed in other cell types. The cell type specific expression of these neurotransmitter receptors needs to be further studied, although at least a subset of NMDA-responsive cells also exhibited voltage-sensitive calcium influx, suggesting a neuronal phenotype. The robust presence and signaling activity of neurotransmitter receptors demonstrates that these cells exhibit greater similarity to their in vivo counterparts, and to CNS neuronal populations relevant to neuropsychiatric illnesses, than previously known.