Comparison of turnover in the olfactory organ of early juvenile stage and adult Caribbean spiny lobsters

Proliferation and turnover of neurons occurs in the olfactory systems of many animals. In this study, we examined developmental changes in turnover in the olfactory organ of the Caribbean spiny lobster Panulirus argus by examining two life-history stages—early juveniles and young adults. Turnover was compared using external morphology of the olfactory organ before and after molting to determine addition and loss of aesthetascs and other chemosensilla, and BrdU labeling to identify newly proliferated cells. The number of olfactory receptor neurons (ORNs) innervating each aesthetasc increased only slightly over development, but there was a net increase of olfactory sensory units (i.e. aesthetascs and their ORNs) at each molt. This increase was similar in early juveniles and young adults when expressed as absolute number of ORNs neurons but greater in early juveniles when expressed as a proportion of existing ORNs. The net increase in olfactory sensory units in early juveniles is due solely to addition, since virtually no aesthetascs are lost. In contrast, the net increase in olfactory sensory units in adults reflects addition of new units accompanied by considerable loss of old units. These developmental changes result in expansive enlargement of the olfactory organ without turnover in early juveniles, and a more modest growth combined with continuous turnover and replenishment of ORNs in adults.     

Amputation-induced activity of progenitor cells leads to rapid regeneration of olfactory tissue in lobsters

Lobsters have a self-renewing olfactory system and, like many animals, continuously replace old or dying olfactory receptor neurons. In addition, lobsters are able to regenerate the peripheral olfactory system even after complete loss. The olfactory sensors in lobsters are located distally on a pair of antennules. These antennules are often damaged, but this has little impact on the lobster's sense of smell because damaged olfactory tissue is rapidly replaced. In this study, we investigated damage-induced regeneration of the olfactory system by measuring cell proliferation following controlled amputation. We show that amputation-induced regeneration occurs as a result of up-regulating the normal development of olfactory sensors. A unique feature of up-regulated development is the formation of patches of proliferating cells within the antennular epithelium. Epithelial patches were typically formed between 3 and 10 days postamputation on the amputated side. They were characterized by their: proximal position with respect to developing clusters of olfactory receptor neurons (ORNs); tendency to form two discrete patches within the borders of each existing annulus; cell size, which was approximately twice that of mature ORNs; and location within the ventral epithelium. The development of epithelial patches was immediately followed by proliferation of clusters of ORNs and associated glial cells, and the level of this proliferation increased significantly during the premolt stage of the lobster's molt cycle. These epithelial patches may represent populations of precursor cells, because they develop in response to amputation and immediately precede development of cell clusters composed of ORNs and glia. Possible regulatory signals controlling epithelial patch development are discussed.     

Amputation‐induced activity of progenitor cells leads to rapid regeneration of olfactory tissue in lobsters

Lobsters have a self‐renewing olfactory system and, like many animals, continuously replace old or dying olfactory receptor neurons. In addition, lobsters are able to regenerate the peripheral olfactory system even after complete loss. The olfactory sensors in lobsters are located distally on a pair of antennules. These antennules are often damaged, but this has little impact on the lobster's sense of smell because damaged olfactory tissue is rapidly replaced. In this study, we investigated damage‐induced regeneration of the olfactory system by measuring cell proliferation following controlled amputation. We show that amputation‐induced regeneration occurs as a result of up‐regulating the normal development of olfactory sensors. A unique feature of up‐regulated development is the formation of patches of proliferating cells within the antennular epithelium. Epithelial patches were typically formed between 3 and 10 days postamputation on the amputated side. They were characterized by their: proximal position with respect to developing clusters of olfactory receptor neurons (ORNs); tendency to form two discrete patches within the borders of each existing annulus; cell size, which was approximately twice that of mature ORNs; and location within the ventral epithelium. The development of epithelial patches was immediately followed by proliferation of clusters of ORNs and associated glial cells, and the level of this proliferation increased significantly during the premolt stage of the lobster's molt cycle. These epithelial patches may represent populations of precursor cells, because they develop in response to amputation and immediately precede development of cell clusters composed of ORNs and glia. Possible regulatory signals controlling epithelial patch development are discussed. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 97–114, 2003     

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.     

Classes and narrowing selectivity of olfactory receptor neurons of Xenopus laevis tadpoles

In olfactory receptor neurons (ORNs) of aquatic animals amino acids have been shown to be potent stimuli. Here we report on calcium imaging experiments in slices of the olfactory mucosa of Xenopus laevis tadpoles. We were able to determine the response profiles of 283 ORNs to 19 amino acids, where one profile comprises the responses of one ORN to 19 amino acids. 204 out of the 283 response profiles differed from each other. 36 response spectra occurred more than once, i.e., there were 36 classes of ORNs identically responding to the 19 amino acids. The number of ORNs that formed a class ranged from 2 to 13. Shape and duration of amino acid-elicited [Ca2+]i transients showed a high degree of similarity upon repeated stimulation with the same amino acid. Different amino acids, however, in some cases led to clearly distinguishable calcium responses in individual ORNs. Furthermore, ORNs clearly appeared to gain selectivity over time, i.e., ORNs of later developmental stages responded to less amino acids than ORNs of earlier stages. We discuss the narrowing of ORN selectivity over stages in the context of expression of olfactory receptors.     

Temporally staggered glomerulus development in the moth Manduca sexta

Glomeruli, neuropilar structures composed of olfactory receptor neuron (ORN) axon terminals and central neuron dendrites, are a common feature of olfactory systems. Typically, ORN axons segregate into glomeruli based on odor specificity, making glomeruli the basic unit for initial processing of odorant information. Developmentally, glomeruli arise from protoglomeruli, loose clusters of ORN axons that gradually synapse onto dendrites. Previous work in the moth Manduca sexta demonstrated that protoglomeruli develop in a wave across the antennal lobe (AL) during stage 5 of the 18 stages of metamorphic adult development. However, ORN axons from the distal segments of the antenna arrive at the AL for several more days. We report that protoglomeruli present at stage 5 account for only approximately two or three of adult glomeruli with the number of structures increasing over subsequent stages. How do these later arriving axons incorporate into glomeruli? Examining the dendritic projections of a unique serotonin-containing neuron into glomeruli at later stages revealed glomeruli with immature dendritic arbors intermingled among more mature glomeruli. Labeling ORN axons that originate in proximal segments of the antenna suggested that early-arriving axons target a limited number of glomeruli. We conclude that AL glomeruli form over an extended time period, possibly as a result of ORNs expressing new odorant receptors arriving from distal antennal segments.     

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.     

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.     

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.     

Olfactory receptor neurons in partially purified epithelial cell cultures: comparison of techniques for partial purification and identification of insulin as an important survival factor

Olfactory neurons have the rare property of being replaced throughoutlife. Factors regulating different developmental stages of olfactoryreceptor neurons (ORNs) are of great interest, because suchfactors might be used to extend regeneration in the post-developmentalbrain and spinal cord. Also, these factors may potentially beexploited to treat various smell disorders arising from changesin the olfactory epithelium. Characterization of trophic factorsfor ORNs requires cell culture systems that are simple and easyto manipulate. We have compared four different cell culturepreparations, using two different enzymes and two differentmedia to develop a simple culture system of olfactory epithelialcells. Our preferred preparation, which produces partially purifiedolfactory epithelial cultures, uses trypsin dissociation anda serum-free keratinocyte growth medium (KGM) supplemented withinsulin. These conditions support ORN survival up to 1 week.They also supported other elements of the olfactory epitheliumsuch as Bowman’s gland cells and horizontal basal cells.Olfactory epithelial cells predominate, while contaminatingmesenchymal cells (glia and fibroblasts) are present in lownumbers. Using these cultures, it was determined that insulinwas required for ORN survival in vitro. The simplicity of theepithelial cultures will be useful for further studies of insulinand other ORN trophic factors.     

Development of a glial network in the olfactory nerve: role of calcium and neuronal activity

In adult olfactory nerves of mammals and moths, a network of glial cells ensheathes small bundles of olfactory receptor axons. In the developing antennal nerve (AN) of the moth Manduca sexta, the axons of olfactory receptor neurons (ORNs) migrate from the olfactory sensory epithelium toward the antennal lobe. Here we explore developmental interactions between ORN axons and AN glial cells. During early stages in AN glial-cell migration, glial cells are highly dye coupled, dividing glia are readily found in the nerve and AN glial cells label strongly for glutamine synthetase. By the end of this period, dye-coupling is rare, glial proliferation has ceased, glutamine synthetase labeling is absent, and glial processes have begun to extend to enwrap bundles of axons, a process that continues throughout the remainder of metamorphic development. Whole-cell and perforated-patch recordings in vivo from AN glia at different stages of network formation revealed two potassium currents and an R-like calcium current. Chronic in vivo exposure to the R-type channel blocker SNX-482 halted or greatly reduced AN glial migration. Chronically blocking spontaneous Na-dependent activity by injection of tetrodotoxin reduced the glial calcium current implicating an activity-dependent interaction between ORNs and glial cells in the development of glial calcium currents.     

Heteromorphic antennules protect the olfactory midbrain from atrophy following chronic antennular ablation in freshwater crayfish

Posthatch larval crayfish (Procambarus clarkii) were unilaterally antennulectomized and maintained in the laboratory for a 6-month period, during which time all regenerating antennular stumps were periodically excised. In another group of animals at a similar developmental stage a heteromorphic antennule was induced on the side of the head ipsilateral to the chronically sectioned normal antennule. After 6 months, all experimental animals were sacrificed and their brains were fixed and sectioned. Computer-aided quantitative measurements were obtained for the volumes of the olfactory lobes on both the experimental and control sides of the brains of both groups of crayfish. In the brains of the crayfish group in which only chronic antennualectomy had been performed, the olfactory lobe ipsilateral to the lesion was reduced in volume by about 80% compared to the olfactory lobe on the control side. In animals in which the normal antennule had been chronically ablated, but which possessed a heteromorphic antennule on the same side, the olfactory lobe on the lesioned side differed in volume from the control side by a mean value of only 28%. We conclude that afferent fibers from a heteromorphic antennule ipsilateral to a chronically lesioned normal antennule can assume some of the central trophic functions of the afferents from the normal antennule; thus, the presence of a heteromorphic antennule offers some measure of protection from the dystrophic effects of chronic ablation of the normal antennule during development and growth in crayfish.     

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.     

The Ginkgo biloba extract modulates the balance between proliferation and differentiation in the olfactory epithelium of adult mice following bulbectomy.

Abstract - The adult olfactory receptor neurons (ORNs), located in the olfactory epithelium (OE) are permanently renewed thanks to neuronal progenitors present in the deep part of the OE, the globose basal cells (GBCs). Following the ablation of their synaptic target, the olfactory bulb (OB), ORNs degenerate by apoptosis and a wave of neurogenesis, including proliferation of GBCs and neuronal differentiation of their progeny, restores the olfactory function. The Ginkgo biloba extract (EGb 761) (Beaufour Ipsen, France) was administered to adult mice at the doses of 50 or 100 mg/kg, following bilateral bulbectomy and its effects on the expression of PCNA, reflecting the number of proliferating GBCs and on growth associated protein 43 (GAP-43), expressed by differentiating neurons were measured by Western blotting. PCNA expression peaked 9 days post-bulbectomy in untreated animals, but 7 days post-lesion in EGb 761-treated animals. A simultaneous reduction in GAP-43 expression suggested that EGb 761 may temporarily favor the proliferation of GBCs rather than their entry into the differentiation pathway. Probably as a consequence of the earlier onset of the neurogenetic response to bulbectomy, neuronal differentiation was enhanced in the OE, 3 weeks post-bulbectomy. These data suggest that EGb 761 may have beneficial effects upon neurogenesis in the OE through changing the balance between proliferation and differentiation.     

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.