Olfactory ensheathing cells are the main phagocytic cells that remove axon debris during early development of the olfactory system

During development of the primary olfactory system, axon targeting is inaccurate and axons inappropriately project within the target layer or overproject into the deeper layers of the olfactory bulb. As a consequence there is considerable apoptosis of primary olfactory neurons during embryonic and postnatal development and axons of the degraded neurons need to be removed. Olfactory ensheathing cells (OECs) are the glia of the primary olfactory nerve and are known to phagocytose axon debris in the adult and postnatal animal. However, it is unclear when phagocytosis by OECs first commences. We investigated the onset of phagocytosis by OECs in the developing mouse olfactory system by utilizing two transgenic reporter lines: OMP-ZsGreen mice which express bright green fluorescent protein in primary olfactory neurons, and S100β-DsRed mice which express red fluorescent protein in OECs. In crosses of these mice, the fate of the degraded axon debris is easily visualized. We found evidence of axon degradation at embryonic day (E)13.5. Phagocytosis of the primary olfactory axon debris by OECs was first detected at E14.5. Phagocytosis of axon debris continued into the postnatal animal during the period when there was extensive mistargeting of olfactory axons. Macrophages were often present in close proximity to OECs but they contributed only a minor role to clearing the axon debris, even after widespread degeneration of olfactory neurons by unilateral bulbectomy and methimazole treatment. These results demonstrate that from early in embryonic development OECs are the primary phagocytic cells of the primary olfactory nerve.

Encoding of mixtures in a simple olfactory system.

Natural odors are usually mixtures; yet, humans and animals can experience them as unitary percepts. Olfaction also enables stimulus categorization and generalization. We studied how these computations are performed with the responses of 168 locust antennal lobe projection neurons (PNs) to varying mixtures of two monomolecular odors, and of 174 PNs and 209 mushroom body Kenyon cells (KCs) to mixtures of up to eight monomolecular odors. Single-PN responses showed strong hypoadditivity and population trajectories clustered by odor concentration and mixture similarity. KC responses were much sparser on average than those of PNs and often signaled the presence of single components in mixtures. Linear classifiers could read out the responses of both populations in single time bins to perform odor identification, categorization, and generalization. Our results suggest that odor representations in the mushroom body may result from competing optimization constraints to facilitate memorization (sparseness) while enabling identification, classification, and generalization.

Encoding of mixtures in a simple olfactory system

Natural odors are usually mixtures; yet, humans and animals can experience them as unitary percepts. Olfaction also enables stimulus categorization and generalization. We studied how these computations are performed with the responses of 168 locust antennal lobe projection neurons (PNs) to varying mixtures of two monomolecular odors, and of 174 PNs and 209 mushroom body Kenyon cells (KCs) to mixtures of up to eight monomolecular odors. Single-PN responses showed strong hypoadditivity and population trajectories clustered by odor concentration and mixture similarity. KC responses were much sparser on average than those of PNs and often signaled the presence of single components in mixtures. Linear classifiers could read out the responses of both populations in single time bins to perform odor identification, categorization, and generalization. Our results suggest that odor representations in the mushroom body may result from competing optimization constraints to facilitate memorization (sparseness) while enabling identification, classification, and generalization

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Sensing danger through the olfactory system: The role of the hypothalamic dorsal premammillary nucleus

The dorsal premammillary nucleus (PMd) has a critical role on the expression of defensive responses to predator odor. Anatomical evidence suggests that the PMd should also modulate memory processing through a projecting branch to the anterior thalamus. By using a pharmacological blockade of the PMd with the NMDA-receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5), we were able to confirm its role in the expression of unconditioned defensive responses, and further revealed that the nucleus is also involved in influencing associative mechanisms linking predatory threats to the related context. We have also tested whether olfactory fear conditioning, using coffee odor as CS, would be useful to model predator odor. Similar to cat odor, shock-paired coffee odor produced robust defensive behavior during exposure to the odor and to the associated context. Shock-paired coffee odor also up-regulated Fos expression in the PMd, and, as with cat odor, we showed that this nucleus is involved in the conditioned defensive responses to the shock-paired coffee odor and the contextual responses to the associated environment. (C) 2008 Elsevier Ltd. All rights reserved.

Discrimination of bile acids by the rainbow trout olfactory system: evidence as potential pheromone

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reassembling a system from the sensor to cerebral representation: the olfactory system in vitro.

An odorant's code is represented by activity in a dispersed ensemble of olfactory sensory neurons in the nose, activation of a specific combination of groups of mitral cells in the olfactory bulb and is considered to be mapped at divergent locations in the olfactory cortex. We present here an in vitro model of the mammalian olfactory system developed to gain easy access to all stations of the olfactory pathway. Mouse olfactory epithelial explants are cocultured with a brain slice that includes the olfactory bulb and olfactory cortex areas and maintains the central olfactory pathway intact and functional. Organotypicity of bulb and cortex is preserved and mitral cell axons can be traced to their target areas. Calcium imaging shows propagation of mitral cell activity to the piriform cortex. Long term coculturing with postnatal olfactory epithelial explants restores the peripheral olfactory pathway. Olfactory receptor neurons renew and progressively acquire a mature phenotype. Axons of olfactory receptor neurons grow out of the explant and rewire into the olfactory bulb. The extent of reinnervation exhibits features of a postlesion recovery. Functional imaging confirms the recovery of part of the peripheral olfactory pathway and shows that activity elicited in olfactory receptor neurons or the olfactory nerves is synaptically propagated into olfactory cortex areas. This model is the first attempt to reassemble a sensory system in culture, from the peripheral sensor to the site of cortical representation. It will increase our knowledge on how neuronal circuits in the central olfactory areas integrate sensory input and counterbalance damage.

Expression and role of complex carbohydrates in axon guidance in the olfactory system

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood groups Sda (or CT1 antigen) and H are expressed by primary sensory neurons in the olfactory system, while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons only in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the blood group H and A carbohydrates were not expressed in the olfactory systems which caused delayed development of the nerve fibre and glomerular layers in the main olfactory bulb. In contrast, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice perturbed the ability of vomeronasal axons to terminate in the accessory olfactory bulb and affected the selective targeting of axons in the main olfactory bulb. During regeneration following bulbectomy, vomeronasal axons were unable to effectively sort out from the main olfactory axons when blood group A was misexpressed. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development and regeneration of the olfactory nerve pathways.

Spatial and developmental heterogeneity of calcium signaling in olfactory ensheathing cells

Olfactory ensheathing cells (OECs) are specialized glial cells in the mammalian olfactory system supporting growth of axons from the olfactory epithelium into the olfactory bulb. OECs in the olfactory bulb can be subdivided into OECs of the outer nerve layer and the inner nerve layer according to the expression of marker proteins and their location in the nerve layer. In the present study, we have used confocal calcium imaging of OECs in acute mouse brain slices and olfactory bulbs in toto to investigate physiological differences between OEC subpopulations. OECs in the outer nerve layer, but not the inner nerve layer, responded to glutamate, ATP, serotonin, dopamine, carbachol, and phenylephrine with increases in the cytosolic calcium concentration. The calcium responses consisted of a transient and a tonic component, the latter being mediated by store-operated calcium entry. Calcium measurements in OECs during the first three postnatal weeks revealed a downregulation of mGluR(1) and P2Y(1) receptor-mediated calcium signaling within the first 2 weeks, suggesting that the expression of these receptors is developmentally controlled. In addition, electrical stimulation of sensory axons evoked calcium signaling via mGluR(1) and P2Y(1) only in outer nerve layer OECs. Downregulation of the receptor-mediated calcium responses in postnatal animals is reflected by a decrease in amplitude of stimulation-evoked calcium transients in OECs from postnatal days 3 to 21. In summary, the results presented reveal striking differences in receptor responses during development and in axon-OEC communication between the two subpopulations of OECs in the olfactory bulb.