Jörg Strotmann

Expression of odorant receptors in spatially restricted subsets of chemosensory neurones

From a rat olfactory library a cDNA clone (OR37) which is supposed to encode an odorant receptor protein has been isolated and characterized. Specific antisense RNA and in situ hybridization techniques have been employed to monitor the olfactory epithelium for the distribution of olfactory neurones expressing the OR37-gene. The OR37-transcripts were detected only in a subset of receptor cells segregated in two restricted areas of the olfactory epithelium. The clusters of reactive cells appear symmetrically in both nasal cavities. Within a reactive region only a subset of the cells expressed the receptor. The segregation of neurones expressing a distinct receptor supports the notion that a spatial component may be involved in coding odour quality.

A novel brain receptor is expressed in a distinct population of olfactory sensory neurons.

Three novel G‐protein‐coupled receptor genes related to the previously described RA1c gene have been isolated from the mouse genome. Expression of these genes has been detected in distinct areas of the brain and also in the olfactory epithelium of the nose. Developmental studies revealed a differential onset of expression: in the brain at embryonic stage 17, in the olfactory system at stage E12. In order to determine which cell type in the olfactory epithelium expresses this unique receptor type, a transgenic approach was employed which allowed a coexpression of histological markers together with the receptor and thus visualization of the appropriate cell population. It was found that the receptor‐expressing cells were located very close to the basal membrane of the epithelium; however, the cells extended a dendritic process to the epithelial surface and their axons projected into the main olfactory bulb where they converged onto two or three glomeruli in the dorsal and posterior region of the bulb. Thus, these data provide evidence that this unique type of receptor is expressed in mature olfactory neurons and suggests that it may be involved in the detection of special odour molecules.

Olfactory receptors in the mouse septal organ

In this study we have identified a repertoire of chemosensory receptors expressed in the septal organ (SO). The results suggest that septal organ neurons are specified to express receptor genes belonging to class II olfactory receptors that are also expressed in the main olfactory epithelium. We found no evidence for the expression of members from the vomeronasal receptor gene families. In the SO, no topography analogous to the receptor expression zones of the main olfactory epithelium was evident. The majority of identified receptors corresponds to genes with restricted expression in the medial and lateral zones of the main olfactory epithelium. This coincides with the expression of olfactory cell adhesion molecule (OCAM) throughout the SO, which is considered as a marker for the medial‐lateral zones. In contrast, NADPH:quinone oxidoreductase 1 expression, a characteristic marker for the dorsal zone, was lacking in the SO. Most of the receptor types were found to be expressed in rather few SO neurons; as an exception, the receptor mOR244‐3 was observed in a very high proportion of cells. Although a very high fraction of SO neurons expressed mOR244‐3, we found no evidence for the coexpression of different receptors in individual cells.

Brain targeting and glomerulus formation of two olfactory neuron populations expressing related receptor types

Olfactory sensory neurons expressing different members of the mOR37 odourant receptor subfamily send their axons to distinct glomeruli located in the immediate vicinity in the olfactory bulb [Strotmann, J., Conzelmann, S., Beck, A., Feinstein, P., Breer, H. & Mombaerts, P. (2000) J. Neurosci., 20, 6927–6938]. In this study, the potential of transgenic mouse lines was used to explore the onset of receptor expression, the outgrowth of axons as well as the glomerulus formation for two neuron populations expressing different mOR37 subtypes. The data indicate a synchronous time course of these features for both neuron populations. From E15 until the day of birth, the axons of the two mOR37 populations terminate in a common, small area of the presumptive olfactory bulb. During a short postnatal phase, the two axon populations segregate into distinct, protoglomerular structures; some aberrant fibers can still be observed during this period.

Projection pattern of nerve fibers from the septal organ: DiI-tracing studies with transgenic OMP mice

The septal organ represents one of the three chemosensory subsystems found in most vertebrate species. Analyzing the projection pattern of septal organ neurons using the OMP-GFP transgenic mouse line revealed that axons navigate in highly variable fiber tracks across the main olfactory epithelium toward the main olfactory bulb. All septal organ axons cross through the cribriform plate at a spatially defined site and terminate exclusively in the posterior, ventromedial aspect of the bulb. Here, one portion of axons forms a dense network on the medial side where they apparently enter glomeruli which are mainly innervated by axons of olfactory sensory neurons from the main olfactory epithelium. Another significant portion of the axons targets a few glomeruli which appear to receive input exclusively from the septal organ neurons.

Retinoic Acid Receptor-Dependent Survival of Olfactory Sensory Neurons in Postnatal and Adult Mice

To address the hypothesis that retinoids produced by synthesizing enzymes present in the primary olfactory system influence the mouse olfactory sensory map, we expressed a dominant-negative retinoic acid receptor selectively in olfactory sensory neurons. We show that neurons deficient in nuclear retinoid signaling are responsive to odors and form correct odorant receptor-specific axonal projections to target neurons in the olfactory bulb of the brain. Subsequent to the formation of the map, the neurons die prematurely by retrograde-driven caspase-3 activation, which resembles the previously described mechanism of neural death after olfactory bulb ablation. This neurodegenerative event is initiated the second postnatal week and occurs in the adult animal without a compensatory increase of progenitor cell proliferation. In addition, we find that nuclear retinoid signaling is required for the expression of a retinoic acid-degrading enzyme, Cyp26B1, in a small fraction of mature neurons. Collectively, the results provide evidence for a role of locally regulated retinoid metabolism in neuroprotection and in determining population size of neurons at a late stage of neural circuit formation.

Olfactory receptor expressed in ganglia of the autonomic nervous system

Certain members of the olfactory receptor superfamily appear to be expressed not only in chemosensory neurons of the nasal epithelium. Analyzing the transgenic mouse line MOL2.3‐IGITL, the olfactory receptor subtype MOL2.3 was found to be expressed in distinct subpopulations of cells within a cranial, a cervical as well as within a thoracic ganglion. By means of coexpressed markers, the axonal processes of MOL2.3 expressing cells could be visualized and thus the target tissues innervated by these ganglionic neurons identified. Stained fibers, but no stained cell bodies were visible in distinct head regions, notably in the lateral nasal gland and in the so‐called Harderian gland; staining was also observed on distinct segments of blood vessels, especially within the tongue. In the thoracic region, the heart and a small segment of the aorta as well as a distinct population of lung alveoli were labeled by incoming blue fibers. Expression of MOL2.3 in cells of the autonomic nervous system supports the idea that at least some of the multiple olfactory receptor types serve functions others than odorant detection.

Subzonal organization of olfactory sensory neurons projecting to distinct glomeruli within the mouse olfactory bulb

Olfactory sensory neurons located in the nasal neuroepithelium send their axons directly into the olfactory bulb, where they contact the dendrites of second‐order neurons in specialized spherical structures called glomeruli; each sensory neuron projects to a single glomerulus. All neurons expressing the same odorant receptor gene are confined to distinct zones within the epithelium and converge their axons onto a small number of common glomeruli. In the present study, we analyzed transgenic mouse lines in which the projection of a neuron population expressing a particular receptor gene can be visualized as a result of axonal markers that are coexpressed. The target glomeruli could thus reproducibly be identified and allowed to deposit retrograde tracers precisely. After an appropriate incubation time, olfactory sensory neurons within distinct areas of the olfactory epithelium were labeled. The two subpopulations of neurons retrogradely stained by differently colored fluorescent dyes deposited at the dorsal and the dorsomedial glomerulus, respectively, were found to be segregated within distinct areas of the expression zone, where the cells expressing the same receptor type displayed a stochastic distribution. J. Comp. Neurol. 458:209–220, 2003.

Internalization of odorant-binding proteins into the mouse olfactory epithelium

The detection of odorants in vertebrates is mediated by chemosensory neurons that reside in the olfactory epithelium of the nose. In land-living species, the hydrophobic odorous compounds inhaled by the airstream are dissolved in the nasal mucus by means of specialized globular proteins, the odorant-binding proteins (OBPs). To assure the responsiveness to odors of each inhalation, a rapid removal of odorants from the microenvironment of the receptor is essential. In order to follow the fate of OBP/odorant complexes, a recombinant OBP was fluorescently labeled, loaded with odorous compounds, and applied to the nose of a mouse. Very quickly, labeled OBP appeared inside the sustentacular cells of the epithelium. This uptake occurred only when the OBP was loaded with appropriate odorant compounds. A search for candidate transporters that could mediate such an uptake process led to the identification of the low density lipoprotein receptor Lrp2/Megalin. In the olfactory epithelium, megalin was found to be specifically expressed in sustentacular cells and the Megalin protein was located in their microvilli. In vitro studies using a cell line that expresses megalin revealed a rapid internalization of OBP/odorant complexes into lysosomes. The uptake was blocked by a Megalin inhibitor, as was the internalization of OBPs into the sustentacular cells of the olfactory epithelium. The results suggest that a Megalin-mediated internalization of OBP/odorant complexes into the sustentacular cells may represent an important mechanism for a rapid and local clearance of odorants.

Mammalian-specific OR37 receptors are differentially activated by distinct odorous fatty aldehydes.

The capacity of the mammalian olfactory system to detect an enormous collection of different chemical compounds is based on a large repertoire of odorant receptors (ORs). A small group of these ORs, the OR37 family, is unique due to a variety of special features. Members of this subfamily are exclusively found in mammals, they share a high degree of sequence homology and are highly conserved during evolution. It is still elusive which odorants may activate these atypical receptors. We have reasoned that compounds from skin, hairs, or skin glands might be potential candidates. We have exposed mice to such compounds and monitored activation of glomeruli through the expression of the activity marker c-fos in juxtaglomerular cells surrounding ventrally positioned glomeruli in the olfactory bulb (OB). Employing this methodology it was found that stimulation with long-chain alkanes elicits activation in the ventral part of the OB, however, none of the OR37 glomeruli. Analyses of long-chain hydrocarbon compounds with different functional groups revealed that long-chain aliphatic aldehydes elicited an activation of defined OR37 glomeruli, each of them responding preferentially to an aldehyde with different chain lengths. These results indicate that OR37 receptors may be tuned to distinct fatty aldehydes with a significant degree of ligand specificity.