CNS glial cells support in vitro survival, division, and differentiation of dissociated olfactory neuronal progenitor cells.

Olfactory receptor neurons (ORNs) are replaced and differentiate in adult animals, but differentiation in dissociated cell culture has not been demonstrated. To test whether contact with the CNS regulates maturation, neonatal rat olfactory cells were grown on a culture substrate or on CNS astrocytes. Mature ORNs, immunopositive for olfactory marker protein (OMP), disappeared rapidly from both systems. Neurons positive for neuron-specific tubulin (immature and mature) disappeared from substrate-only cultures, but remained abundant in the cocultures. OMP-positive neurons reappeared after 10 days in vitro. Pulse labeling with [3H]thymidine showed extensive neurogenesis of both immature and mature olfactory neurons. This demonstrates, in vitro, both division and differentiation of olfactory progenitor cells.     

Postembryonic proliferation in the spiny lobster antennular epithelium: rate of genesis of olfactory receptor neurons is dependent on molt stage

Olfactory systems undergo continuous growth and turnover in many animals. Many decapod crustaceans, such as lobsters and crayfish, have indeterminate growth, and in these animals, turnover of both peripheral and central components of the olfactory system occurs continuously throughout life. In this study, we examine the dynamics of olfactory receptor neuron (ORN) proliferation in the antennule of the Caribbean spiny lobster, Panulirus argus, using in vivo incorporation of the cell proliferation marker BrdU. We show that addition of ORNs occurs in a "proximal proliferation zone" (PPZ), which exists on the proximo-lateral margin of the existing ORN population. The PPZ is spatially and temporally dynamic in that it travels as a wave in the proximal and lateral directions in the antennule. This wave results in continuous addition of ORNs throughout the molt cycle. The rate of proliferation, as measured by the size and shape of the PPZ, changes depending on the animal's molt stage. The rate is highest during premolt and lowest during intermolt. ORNs are the most prominent cell-type produced in the PPZ, but other cell types, including glia, are also produced. Patches of proliferating epithelial cells occur immediately proximal to the PPZ, suggesting that neuronal and glial precursors reside in this region. Possible mechanisms for peripheral and central modulation of ORN development are discussed.     

GABAB receptor expression and function in olfactory receptor neuron axon growth

Neurotransmitters have been implicated in regulating growth cone motility and guidance in the developing nervous system. Anatomical and electrophysiological studies show the presence of functional GABAB receptors on adult olfactory receptor neuron (ORN) nerve terminals. Using antisera against the GABAB R1a/b receptor isoforms we show that developing mouse olfactory receptor neurons express GABAB receptors from embryonic day 14 through to adulthood. GABAB receptors are present on axon growth cones from both dissociated ORNs and olfactory epithelial explants. Neurons in the olfactory bulb begin to express glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, from E16 through to adulthood. When dissociated ORNs were cultured in the presence of the GABAB receptor agonists, baclofen or SKF97541, neurite outgrowth was significantly reduced. Concurrent treatment of the neurons with baclofen and the GABAB receptor antagonist CGP54626 prevented the inhibitory effects of baclofen on ORN neurite outgrowth. These results show that growing ORN axons express GABAB receptors and are sensitive to the effects of GABAB receptor activation. Thus, ORNs in vivo may detect GABA release from juxtaglomerular cells as they enter the glomerular layer and use this as a signal to limit their outgrowth and find synaptic targets in regeneration and development.     

A presynaptic gain control mechanism fine-tunes olfactory behavior

Early sensory processing can play a critical role in sensing environmental cues. We have investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. Olfactory receptor neurons (ORNs) express the GABA(B) receptor (GABA(B)R), and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, each ORN channel has a unique baseline level of GABA(B)R expression. ORNs that sense the aversive odorant CO(2) do not express GABA(B)Rs and do not have significant presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABA(B)Rs and exhibit strong presynaptic inhibition. Furthermore, pheromone-dependent mate localization is impaired in flies that lack GABA(B)Rs in specific ORNs. These findings indicate that different olfactory receptor channels employ heterogeneous presynaptic gain control as a mechanism to allow an animal's innate behavioral responses to match its ecological needs.     

Differential spatial and temporal expression of two type III intermediate filament proteins in olfactory receptor neurons

Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons.     

Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.     

Axonal targeting of olfactory receptor neurons in Drosophila is controlled by Dscam

Different classes of olfactory receptor neurons (ORNs) in Drosophila innervate distinct targets, or glomeruli, in the antennal lobe of the brain. Here we demonstrate that specific ORN classes require the cell surface protein Dscam (Down Syndrome Cell Adhesion Molecule) to synapse in the correct glomeruli. Dscam mutant ORNs frequently terminated in ectopic sites both within and outside the antennal lobe. The morphology of Dscam mutant axon terminals in either ectopic or cognate targets was abnormal. Target specificity for other ORNs was not altered in Dscam mutants, suggesting that different ORNs use different strategies to regulate wiring. Multiple forms of Dscam RNA were detected in the developing antenna, and Dscam protein was localized to developing ORN axons. We propose a role for Dscam protein diversity in regulating ORN target specificity.     

Extremely Sparse Olfactory Inputs Are Sufficient to Mediate Innate Aversion in Drosophila

Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction.     

Afferent induction of olfactory glomeruli requires N-cadherin

Drosophila olfactory receptor neurons (ORNs) elaborate a precise internal representation of the external olfactory world in the antennal lobe (AL), a structure analagous to the vertebrate olfactory bulb. ORNs expressing the same odorant receptor innervate common targets in a highly organized neuropilar structure inside the AL, the glomerulus. During normal development, ORNs target to specific regions of the AL and segregate into subclass-specific aggregates called protoglomeruli prior to extensive intermingling with target dendrites to form mature glomeruli. Using a panel of ORN subclass-specific markers, we demonstrate that in the adult AL, N-cadherin (N-cad) mutant ORN terminals remain segregated from dendrites of target neurons. N-cad plays a crucial role in protoglomerulus formation but is largely dispensible for targeting to the appropriate region of the AL. We propose that N-cad, a homophilic cell adhesion molecule, acts in a permissive fashion to promote subclass-specific sorting of ORN axon terminals into protoglomeruli.     

Olfactory Coding in Antennal Neurons of the Malaria Mosquito, Anopheles gambiae

Olfactory receptor neurons (ORNs) in the antenna of insects serve to encode odors in action potential activity conducted to the olfactory lobe of the deuterocerebrum. We performed an analysis of the electrophysiological responses of olfactory neurons in the antennae of the female malaria mosquito Anopheles gambiae s.s. and investigated the effect of blood feeding on responsiveness. Forty-four chemicals that are known to be present in human volatile emanations were used as odor stimuli. We identified 6 functional types of trichoid sensilla and 5 functional types of grooved-peg sensilla (GP) based on a hierarchical cluster analysis. Generalist ORNs, tuned to a broad range of odors, moderate specialist ORNs and 2 ORNs tuned to only one odor were identified in different sensilla types. Neurons in GP were tuned to more polar compounds including the important behavioral attractant ammonia and its synergist L-lactic acid, responses to which were found only in GP. Combinatorial coding is the most plausible principle operating in the olfactory system of this mosquito species. We document for the first time both up- and downregulation of ORN responsiveness after blood feeding. Modulation of host-seeking and oviposition behavior is associated with both qualitative and quantitative changes in the peripheral sensory system.     

The role of <Emphasis Type="Italic">Dlx</Emphasis> homeogenes in early development of the olfactory pathway

Development of the olfactory pathway requires interaction between cells and signals of different origin. Olfactory receptor neurons (ORN) in the olfactory placodes (OP) extend axons towards the forebrain (FB); with innervation taking place at a later time following degradation of the basement membrane. Cells from the OP migrate along ORN axons and differentiate into various elements, including ensheathing and Gonadotropin Releasing Hormone (GnRH)+ cells. The importance of the olfactory connection and migration is highlighted by the severe endocrine phenotype in Kallmann’s patients who lack this migratory pathway. Little is known about the genetic control of intrinsic ORN properties. Inactivation of the distalless-related Dlx5 prevents connections between ORNs and FB. Using a grafting approach we show that misguidance and lack of connectivity is due to intrinsic defects in ORN neurites and migratory cells (MgC), and not to environmental factors. These data point to a cell-autonomous function of Dlx5 in providing ORN axons with their connectivity properties. Dlx5 also marks a population of early MgC that partly overlaps with the GnRH+ population. In the absence of Dlx5 MgCs of the Dlx5+ lineage migrate, associated with PSA-NCAM+ axons, but fail to reach the FB as a consequence of the lack of axonal connection and not an inability to migrate. These data suggests that Dlx5 is not required to initiate migration and differentiation of MgCs. An erratum to this article can be found at     

Cytochrome oxidase staining reveals functionally important activity bands in the olfactory epithelium of newborn rat

We used cytochrome oxidase (CytOx) staining intensity, which is correlated with neuronal functional activity, to evaluate maturity and functionality of newborn rat olfactory epithelium (OE) and olfactory receptor neurons (ORNs). Nasal olfactory tissue of neonatal rats was stained with CytOx and analyzed qualitatively and quantitatively. Results revealed that newborn OE shows six differentially stained horizontal bands. Bands run parallel to the OE surface and were categorized as very light, medium or darkly stained. A narrow and pale Band 1 overlapped with horizontal basal cells. Next, a wide and lightly stained Band 2 was observed that coincides with the globose basal cell layer and immature ORNs, deep in OE. Next apically, a medium-staining Band 3 overlapped with ORN perikarya. Closer to the surface, a medium to light Band 4 was discerned where dendrites of mature ORNs normally occur. This band was interrupted with lighter areas due to the presence of supporting cells nuclei. Next, a superficial but dark Band 5 occurred, populated by the apical portions of ORN dendrites and their ciliated knobs and by supporting cell apices; mitochondria in apices of supporting cells contribute predominantly to dense staining of this Band 5. Apical to Band 5, a thin and fairly light Band 6 was observed which overlaps with the mucus layer that contains part of the ORN knobs, their cilia and supporting cell microvilli. Along the length of ORN dendrites, apical segments just below the ORN knobs, and wide basal segments showed a darker staining than the middle segments implying “microzones” of higher neural activity within the most apical and basal regions of dendrites. Our findings agree with ultrastructural studies showing a presence of mitochondria in knobs, basal portions of ORN dendrites and in OE supporting cell apices, suggesting that apical regions of both olfactory and supporting cells near the surfaces are metabolically most active, in odorant detection, signal processing, and detoxification, the latter for supporting cells.     

Modeling the response of a population of olfactory receptor neurons to an odorant

We modeled the firing rate of populations of olfactory receptor neurons (ORNs) responding to an odorant at different concentrations. Two cases were considered: a population of ORNs that all express the same olfactory receptor (OR), and a population that expresses many different ORs. To take into account ORN variability, we replaced single parameter values in a biophysical ORN model with values drawn from statistical distributions, chosen to correspond to experimental data. For ORNs expressing the same OR, we found that the distributions of firing frequencies are Gaussian at all concentrations, with larger mean and standard deviation at higher concentrations. For a population expressing different ORs, the distribution of firing frequencies can be described as the superposition of a Gaussian distribution and a lognormal distribution. Distributions of maximum value and dynamic range of spiking frequencies in the simulated ORN population were similar to experimental results.     

Neurogenesis and cell death in olfactory epithelium

The olfactory epithelium (OE) of the mammal is uniquely suited as a model system for studying how neurogenesis and cell death interact to regulate neuron number during development and regeneration. To identify factors regulating neurogenesis and neuronal death in the OE, and to determine the mechanisms by which these factors act, investigators studied OE using two major experimental paradigms: tissue culture of OE; and ablation of the olfactory bulb or severing the olfactory nerve in adult animals, procedures that induce cell death and a subsequent surge of neurogenesis in the OE in vivo. These studies characterized the cellular stages in the olfactory receptor neuron (ORN) lineage, leading to the realization that at least three distinct stages of proliferating neuronal precursor cells are employed in generating ORNs. The identification of a number of factors that act to regulate proliferation and survival of ORNs and their precursors suggests that these multiple developmental stages may serve as control points at which cell number is regulated by extrinsic factors. In vivo surgical studies, which have shown that all cell types in the neuronal lineage of the OE undergo apoptotic cell death, support this idea. These studies, and the possible coregulation of neuronal birth and apoptosis in the OE, are discussed. © 1996 John Wiley & Sons, Inc.     

Molecular, anatomical, and functional organization of the Drosophila olfactory system

BACKGROUND: Olfactory receptor neurons (ORNs) convey chemical information into the brain, producing internal representations of odors detected in the periphery. A comprehensive understanding of the molecular and neural mechanisms of odor detection and processing requires complete maps of odorant receptor (Or) expression and ORN connectivity, preferably at single-cell resolution. RESULTS: We have constructed near-complete maps of Or expression and ORN targeting in the Drosophila olfactory system. These maps confirm the general validity of the "one neuron--one receptor" and "one glomerulus--one receptor" principles and reveal several additional features of olfactory organization. ORNs in distinct sensilla types project to distinct regions of the antennal lobe, but neighbor relations are not preserved. ORNs grouped in the same sensilla do not express similar receptors, but similar receptors tend to map to closely appositioned glomeruli in the antennal lobe. This organization may serve to ensure that odor representations are dispersed in the periphery but clustered centrally. Integrated with electrophysiological data, these maps also predict glomerular representations of specific odorants. Representations of aliphatic and aromatic compounds are spatially segregated, with those of aliphatic compounds arranged topographically according to carbon chain length. CONCLUSIONS: These Or expression and ORN connectivity maps provide further insight into the molecular, anatomical, and functional organization of the Drosophila olfactory system. Our maps also provide an essential resource for investigating how internal odor representations are generated and how they are further processed and transmitted to higher brain centers.     

The neuronal stem cell of the olfactory epithelium

The vertebrate olfactory epithelium (OE) is a system in which behavior of neuronal progenitor cells can be observed and manipulated easily. It is morphologically and functionally similar to embryonic germinal neuroepithelia, but is simpler in that it produces large numbers of a single type of neuron, the olfactory receptor neuron (ORN). The OE is amenable to tissue culture, gene transfer, and in vivo surgical approaches, and these have been exploited in experiments aimed at understanding the characteristics of OE neuronal progenitor cells. This has led to the realization that the ORN lineage contains at least three distinct stages of proliferating neuronal progenitor cells (including a stem cell), each of which represents a point at which growth control can be exerted. Neurogenesis proceeds continually in the OE, and studies in vivo have shown that this is a regulated process that serves to maintain the number of ORNs at a particular level. These studies suggest that OE neuronal progenitors—which are in close physical proximity to ORNs—can “read” the number of differentiated neurons in their environment and regulate production of new neurons accordingly. Putative neuronal stem cells of the OE have been identified in vitro, and studies of these cells indicate that ORNs produce a signal that feeds back to inhibit neurogenesis. This inhibitory signal may be exerted at the level of the stem cell itself. Recent studies to identify this signal, as well as endogenous stimulatory signals that may be important in regulating OE neurogenesis, are also discussed. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 190–205, 1998     

An enhanced olfactory marker protein immunoreactivity in individual olfactory receptor neurons following olfactory bulbectomy may be related to increased neurogenesis

Olfactory marker protein (OMP) is a 19-kD acidic protein found throughout the cytoplasm of mature olfactory receptor neurons (ORNs). Its function remains unknown. Following olfactory bulbectomy, the proportion of ORNs mature enough to express OMP declines greatly. However, in the few remaining mature ORNs, it has been observed that the intensity of OMP immunoreactivity (IR) appears to increase over that of ORNs on the unoperated side. We have now investigated this phenomenon quantitatively in rats subjected to unilateral olfactory bulbectomy. Results show that at all postbulbectomy survival periods examined quantitatively (3 days to 6 months), a significant decrease (19–37%) occurs in the transmission of incident light through OMP(+)-ORNs in bulbectomized versus unoperated olfactory epithelium (OE). Further, we also observed a consistent side-to-side difference in OMP IR in control unoperated animals. Possible explanations for these observations and their relation to the still unknown function of OMP are discussed. To test the possibility that OMP might serve a mitogenic role in the OE, recombinant OMP was added to organotypic explant cultures of fetal olfactory mucosa. Addition of OMP resulted in a dose-dependent increase in the density of bromodeoxyuridine-positive cells in the cultures, with a 50% increase occurring at the plateau OMP concentration of 25 nM. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 377–390, 1998