George Gomez

Characteristics of Odorant Elicited Calcium Changes in Cultured Human Olfactory Neurons

An important step in establishing and utilizing a cell culture system for the in vitro study of olfaction is assessing whether the cultured cells possess physiological properties similar to those of mature olfactory neurons. Various investigators have successfully established proliferating cell lines from olfactory tissue, but few have demonstrated the characteristics of odor sensitivity of these cells. We successfully established cultured cell lines from adult human olfactory tissue obtained using an olfactory biopsy procedure and measured their ability to respond to odor stimulation using calcium imaging techniques. A subset of the human olfactory cells in culture displayed a distinct morphology and specifically expressed immunocytochemical markers characteristic of mature human olfactory neurons such as OMP, Golf, NCAM and NST. Under defined growth conditions, these cultured cells responded to odorant mixes that have been previously shown to elicit intracellular calcium changes in acutely‐isolated human olfactory neurons. These odorant‐elicited calcium responses displayed characteristics similar to those found in mature human olfactory neurons. First, cultured cells responded with either increases or decreases in intracellular calcium. Second, increases in calcium were abolished by removal of extracellular calcium. Third, inhibitors of the olfactory signal transduction cascades reversibly blocked these odorant‐elicited intracellular calcium changes. Our results demonstrate that cultures of adult human olfactory cells established from olfactory biopsies retain some of the in vivo odorant response characteristics of acutely isolated cells from the adult olfactory epithelium. This work has important ramifications for investigation of olfactory function and dysfunction using biopsy procedures and in vitro assays of odor sensitivity. J. Neurosci. Res. 62:737–749, 2000.

Human olfactory epithelial cells generated in vitro express diverse neuronal characteristics

The olfactory epithelium constitutes the sole source of regenerating neural cells that can be obtained from a living human. As such, primary cultures derived from human olfactory epithelial biopsies can be utilized to study neurobiological characteristics of individuals under different conditions and disease states. Here, using such human cultures, we report in vitro generation of cells that exhibit a complex neuronal phenotype, encompassing receptors and signaling pathways pertinent to both olfaction and other aspects of CNS function. Using in situ hybridization, we demonstrate for the first time the native expression of olfactory receptors in cultured cells derived from human olfactory epithelial tissue. We further establish the presence and function of olfactory transduction molecules in these cells using immunocytochemistry, calcium imaging and molecular methods. Western blot analysis revealed the expression of neurotransmitter receptors for dopamine (D2R), 5-HT (5HT2C) and NMDA subtypes 1 and 2A/2B. Stimulation with dopamine or 5-HT enhanced receptor G protein activation in a subtype specific manner, based on 35S-guanosine triphosphate incorporation assay. Functional characteristics of the cultured cells are demonstrated through enhanced tyrosine phosphorylation of NMDAR 2A/2B and recruitment of signaling partners in response to NMDA stimulation. The array of neuronal characteristics observed here establishes that proliferating cells derived from the human olfactory epithelium differentiate in vitro to express functional and molecular attributes of mature olfactory neurons. These cultured neural cells exhibit neurotransmitter pathways important in a number of neuropsychiatric disorders. Their ready availability from living humans thus provides a new tool to link functional and molecular features of neural cells with clinical characteristics of individual living patients.

Frequency filter properties of lobster chemoreceptor cells determined with high-resolution stimulus measurement

Abstract1.We developed a high resolution, on-line stimulus measurement system for accurate control of chemical stimulus applications for Homarus americanus lateral antennule chemoreceptors. Focal stimulus presentations in an electrophysiological preparation with the receptor sensilla intact were measured at small spatial (30 μm) and time (5 ms) scales.2.We tested 15 receptor cells with ten 100 ms pulses of 104M hydroxyproline at 0.5, 1, 2 and 4 Hz and with a single 8 s square pulse. Individual cells showed differences in their capabilities to resolve pulses (“flicker fusion”). At 2 Hz stimulation, some cells could follow stimulus pulses while others could not. At 4 Hz, 3 cells could still encode individual stimulus pulses accurately. The population resolved pulses up to 2 Hz; at 4 Hz, the population response to a pulse series approximated the response to a square pulse.3.Repetitive stimulation caused a gradual decrease in the number of spikes and a gradual increase in first spike latency (“cumulative adaptation”). Increased stimulation frequency resulted in greater cumulative adaptation.4.Since individual differences in adaptation and disadaptation rates of the receptor cells could not be attributed to measured stimulus variability in situ, lobster chemoreceptor cell populations have intrinsic temporal diversity which, we hypothesize, could be used to analyze pulsatile stimuli that occur in natural turbulent odor plumes.

Temporal resolution in olfaction III: flicker fusion and concentration-dependent synchronization with stimulus pulse trains of antennular chemoreceptor cells in the American lobster

Abstract To understand how chemoreceptor organs may extract temporal information from odor plumes, we investigated the frequency filter properties of lobster chemoreceptor cells. We used rapid stimulation and high-resolution stimulus measurement for accurate stimulus control and recorded extracellular responses from chemoreceptors in the lobster lateral antennule in situ. We tested 16 hydroxyproline-sensitive cells with a series of ten 100-ms pulses at 10, 100 and 1000 μmol l−1 at stimulation frequencies from 0.5 Hz to 4 Hz. Receptor cell responses could accurately encode 10 μmol l−1, but not 100 or 1000 μmol l−1 pulses, delivered at rates of 4 Hz. Flicker-fusion frequency and synchronization with the stimulus pulse train were concentration dependent: performance rates above 1 Hz became poorer both with increasing pulse amplitude and frequency. Flicker fusion frequency was 3 Hz for 100 μmol l−1 pulses and 2 Hz for 1000 μmol l−1 pulses. Individual cells showed differences in their stimulus pulse following capabilities, as measured by the synchronization coefficient. These individual differences may form a basis for coding temporal features of an odor plume in an across-fiber pattern.

The Superior Turbinate as a Source of Functional Human Olfactory Receptor Neurons

Objectives The function of human olfactory receptor neurons (ORNs) remains incompletely understood, in part because of the difficulty of obtaining viable olfactory tissue for study. During endoscopic sphenoidotomy, a portion of the superior turbinate is often removed to achieve wide and safe access to the sphenoid sinus. The purpose of this study was to determine whether functional olfactory mucosa could be obtained from such superior turbinate tissue.

The peripheral olfactory system of the domestic chicken: Physiology and development

Olfaction is a ubiquitous sensory system found in all terrestrial vertebrates. Birds use olfaction for several important activities such as feeding and mating; thus, understanding bird biology would also require the systematic study olfaction. In addition, the olfactory system has several unique features that are useful for the study of nervous system function and development, including a large multigene family for olfactory receptor expression, peripheral neurons that regenerate, and a complex system for sensory innervation of the olfactory bulb. We focused on physiological, anatomical and behavioral approaches to study the chick olfactory neurons and the olfactory bulb. Chick olfactory neurons displayed some properties similar to those found in mature neurons of other vertebrate species, and other properties that were unique. Since information from these neurons is initially processed in the olfactory bulb, we also conducted preliminary studies on the developmental timeline of this structure and showed that glomerular structures are organized in ovo during a critical time period, during which embryonic chicks can form behavioral associations with odorants introduced in ovo. Lastly, we have shown that chick olfactory neurons can grow and mature in vitro, allowing their use in cell culture studies. These results collectively demonstrate some of the features of the olfactory system that are common to all vertebrates, and some that are unique to birds. These highlight the potential for the use of the physiology and development of the olfactory system as a model system for avian brain neurobiology.

Modulation of odor-induced increases in [Ca2+]i by inhibitors of protein kinases A and C in rat and human olfactory receptor neurons

Protein kinases A and C have been postulated to exert multiple effects on different elements of signal transduction pathways in olfactory receptor neurons. However, little is known about the modulation of olfactory responses by protein kinases in intact olfactory receptor neurons. To further elucidate the details of the modulation of odorant responsiveness by these protein kinases, we investigated the action of two protein kinase inhibitors: H89, an inhibitor of protein kinase A, and N-myristoylated EGF receptor, an inhibitor of protein kinase C, on odorant responsiveness in intact olfactory neurons. We isolated individual olfactory neurons from the adult human and rat olfactory epithelium and measured responses of the isolated cells to odorants or biochemical activators that have been shown to initiate cyclic AMP or inositol 1,4,5-trisphospate production in biochemical preparations. We employed calcium imaging techniques to measure odor-elicited changes in intracellular calcium that occur over several seconds. In human olfactory receptor neurons, the protein kinase A and C inhibitors affected the responses to different sets of odorants. In rats, however, the protein kinase C inhibitor affected responses to all odorants, while the protein kinase A inhibitor had no effect. In both species, the effect of inhibition of protein kinases was to enhance the elevation and block termination of intracellular calcium levels elicited by odorants. Our results show that protein kinases A and C may modulate odorant responses of olfactory neurons by regulating calcium fluxes that occur several seconds after odorant stimulation. The effects of protein kinase C inhibition are different in rat and human olfactory neurons, indicating that species differences are an important consideration when applying data from animal studies to apply to humans.

Induction of differentiation of human olfactory neuroblastoma cells into odorant-responsive cells

Olfactory neuroblastoma is a rare malignancy of the olfactory mucosa that may be derived from the olfactory epithelium. To characterize this tumor, we cultured olfactory neuroblastoma cells in the presence or absence of growth factors (transforming growth factor alpha and basic fibroblast growth factor) known to affect olfactory tissue and assessed their responsiveness to known odorants by measuring changes in intracellular calcium. Untreated cells did not respond to odorants. Basic fibroblast growth factor treatment had cytotoxic effects, and treated cells did not respond to odorants. Transforming growth factor alpha treatment resulted in the induction of odor responsiveness in these cells. Cells responded to odorants at 100 nM to 100 microM concentrations and responded with both increases and decreases in intracellular calcium. Increases in intracellular calcium were mediated by a calcium influx and were reversibly blocked by compounds known to inhibit second messenger pathways in olfactory receptor neurons. The calcium responses of the olfactory neuroblastoma cells were thus specific to the odorants and similar to those found in olfactory receptor neurons. The results support the notion that olfactory neuroblastoma cells may be of olfactory origin and thus they can be used as a model cell line to study human olfaction.

Altered olfactory epithelial structure and function in feline models of mucopolysaccharidoses I and VI.

The mucopolysaccharidoses (MPS) are a family of lysosomal storage diseases resulting in developmental defects and, in some types, mental retardation and other neurological symptoms. To gain insight into the neurological dysfunction in MPS, we examined the morphology of olfactory epithelia (OE) and physiology of olfactory receptor neurons (ORNs) in cat models of MPS I, a type in which neuronal lesions are prominent, and MPS VI, in which they are essentially absent. Histopathology showed that both groups of MPS‐affected cats had significantly thinner OE than controls. Although immature and mature ORNs were present in both MPS I and VI affected OE, the OE of MPS I‐affected cats was structurally disorganized. ORN function was assessed with calcium imaging and patch‐clamp recording. Few viable ORNs were recovered from MPS VI cats, but these exhibited normal responses to odors and pharmacological stimuli. In contrast, viable ORNs were as prevalent in MPS I as in controls but were significantly less likely to respond to odor stimuli, although other responses were normal. Disrupted OE organization and impaired ORN function in MPS I, but not MPS VI, corresponds to the central nervous system lesions found in MPS I but not MPS VI. These data represent the first neurophysiological correlate of this correspondence and have implications both for understanding the role of glycosaminoglycans in maintenance of the OE and for targeting further research into the basis for and treatment of the neurological consequences of MPS disorders. J. Comp. Neurol. 511:360–372, 2008.

Chronic odorant exposure upregulates acquisition of functional properties in cultured embryonic chick olfactory sensory neurons

Neuronal development and differentiation is modulated by activity‐dependent mechanisms that stimulate endogenous neurogenesis and differentiation to promote adaptive survival of the organism. Studies on bird odor imprinting have shown how sensory stimuli or environmental influences can affect neonatal behavior, presumably by remodeling the developing nervous system. It is unclear whether these changes originate from the sensory neurons themselves or from the brain. Thus, we attempted to address this by using an in vitro system to separate the peripheral neurons from their central connections. Olfactory neurons from embryonic day 17 Gallus domesticus chicks were isolated, cultured, and exposed to 100 µM amyl acetate or phenethyl alcohol in 12‐hr bouts, alternated with periods of no‐odor exposure. On days 4 and 5 in vitro, cells were immunostained for olfactory marker protein, neuron‐specific tubulin, and olfactory GTP‐binding protein, and tested for odorant sensitivity using calcium imaging. While odorant exposure did not result in a significant increase in the overall number of neurons, it promoted neuron differentiation: a larger proportion of odorant‐exposed cells expressed olfactory marker protein and the olfactory GTP‐binding protein. When cell responsiveness was tested using calcium imaging, a greater proportion of odorant‐exposed cells responded to stimulation with 100 µM amyl acetate or phenethyl alcohol. Thus, odorant exposure during development modulated the developmental trajectories of individual neurons, resulting in changes in protein expression associated with odorant signaling. This suggests that the neuronal changes in the periphery have an important contribution to the overall long‐term functional changes associated with odor imprinting.