Joerg Fleischer

Olfactory Receptor Proteins in Axonal Processes of Chemosensory Neurons

Olfactory receptors are supposed to act not only as molecular sensors for odorants but also as cell recognition molecules guiding the axons of olfactory neurons to their appropriate glomerulus in the olfactory bulb. This concept implies that olfactory receptor proteins are located in sensory cilia and in the axons. To approach this critical issue, antibodies were generated against two peptides, one derived from olfactory receptor mOR256-17, one derived from the “mOR37” subfamily. By means of immunohistochemistry and double-labeling studies using transgenic mouse lines as well as Western blot analyses, it was demonstrated that the newly generated antibodies specifically recognized the receptor proteins. To scrutinize the hypothesis that olfactory receptor proteins may also be present in the axonal processes and the nerve terminals, serial sections through the olfactory bulb were probed with the antibodies. Two glomeruli in each bulb were stained by anti-mOR256-17, one positioned in the medial, one in the lateral hemisphere. Fiber bundles approaching the glomeruli through the outer nerve layer also displayed intense immunofluorescence. A similar picture emerged for the antibody anti-mOR37, a small number of glomeruli in the ventral domain of the bulb was stained. On serial sections through the olfactory bulb of mOR37-transgenic mouse lines, double-labeling experiments demonstrated that distinct immunoreactive glomeruli corresponded to glomeruli that were targeted by neurons expressing a particular member of the mOR37 receptor subfamily. These data indicate that olfactory receptor (OR) proteins are indeed present in the axonal processes and nerve terminals of olfactory sensory neurons, thus supporting the notion that ORs may participate in the molecular processes underlying the fasciculation and targeting of olfactory axons.

The sense of smell: multiple olfactory subsystems

Abstract.The mammalian olfactory system is not uniformly organized but consists of several subsystems each of which probably serves distinct functions. Not only are the two major nasal chemosensory systems, the vomeronasal organ and the main olfactory epithelium, structurally and functionally separate entities, but the latter is further subcompartimentalized into overlapping expression zones and projection-related subzones. Moreover, the populations of ‘OR37’ neurons not only express a unique type of olfactory receptors but also are segregated in a cluster-like manner and generally project to only one receptor-specific glomerulus. The septal organ is an island of sensory epithelium on the nasal septum positioned at the nasoplatine duct; it is considered as a ‘mini-nose’ with dual function. A specific chemosensory function of the most recently discovered subsystem, the so-called Grueneberg ganglion, is based on the expression of olfactory marker protein and the axonal projections to defined glomeruli within the olfactory bulb. This complexity of distinct olfactory subsystems may be one of the features determining the enormous chemosensory capacity of the sense of smell.

Signaling in the Chemosensory Systems

Abstract.The mammalian olfactory system is not uniformly organized but consists of several subsystems each of which probably serves distinct functions. Not only are the two major nasal chemosensory systems, the vomeronasal organ and the main olfactory epithelium, structurally and functionally separate entities, but the latter is further subcompartimentalized into overlapping expression zones and projection-related subzones. Moreover, the populations of ‘OR37’ neurons not only express a unique type of olfactory receptors but also are segregated in a cluster-like manner and generally project to only one receptor-specific glomerulus. The septal organ is an island of sensory epithelium on the nasal septum positioned at the nasoplatine duct; it is considered as a ‘mini-nose’ with dual function. A specific chemosensory function of the most recently discovered subsystem, the so-called Grueneberg ganglion, is based on the expression of olfactory marker protein and the axonal projections to defined glomeruli within the olfactory bulb. This complexity of distinct olfactory subsystems may be one of the features determining the enormous chemosensory capacity of the sense of smell.

Mammalian olfactory receptors

Perception of chemical stimuli from the environment is essential to most animals; accordingly, they are equipped with a complex olfactory system capable of receiving a nearly unlimited number of odorous substances and pheromones. This enormous task is accomplished by olfactory sensory neurons (OSNs) arranged in several chemosensory compartments in the nose. The sensitive and selective responsiveness of OSNs to odorous molecules and pheromones is based on distinct receptors in their chemosensory membrane; consequently, olfactory receptors play a key role for a reliable recognition and an accurate processing of chemosensory information. They are therefore considered as key elements for an understanding of the principles and mechanisms underlying the sense of smell. The repertoire of olfactory receptors in mammals encompasses hundreds of different receptor types which are highly diverse and expressed in distinct subcompartments of the nose. Accordingly, they are categorized into several receptor families, including odorant receptors (ORs), vomeronasal receptors (V1Rs and V2Rs), trace amine-associated receptors (TAARs), formyl peptide receptors (FPRs), and the membrane guanylyl cyclase GC-D. This large and complex receptor repertoire is the basis for the enormous chemosensory capacity of the olfactory system.

Grueneberg ganglion neurons respond to cool ambient temperatures

The Grueneberg ganglion (GG) – a neuronal cell cluster of unknown function localized to the vestibule of the anterior nasal cavity – is considered as a chemosensory compartment based on the expression of olfactory receptors and the olfactory marker protein. Axonal projection of GG neurons to so‐called ‘necklace glomeruli’ in the olfactory bulb of the brain, which are thought to be important for suckling behaviour in rodent pups, has led to the hypothesis that the GG might be involved in mother/child interactions. To survey potential activation of GG neurons in living animals during the course of mother/child interactions, expression of the activity‐dependent gene c‐Fos in the GG of neonatal mouse pups was monitored in the presence and absence of the dam. It was found that GG neurons were only activated in the absence of the mother. Moreover, GG activation was independent from olfactory cues as revealed by naris occlusion. Searching for stimuli eliciting GG activity in pups separated from the dam, cool ambient temperatures were found to induce strong c‐Fos expression in GG neurons whereas warmer temperatures did not. These coolness‐induced responses were only observed in a distinct subset of GG neurons characterized by the expression of the olfactory receptor V2r83. Finally, GG responsiveness to coolness was remarkably reduced in older stages. In summary, these findings suggest that the GG of neonatal mice is activated by cool ambient temperatures to which they are exposed in the absence of their dam, indicating that the GG might function as a thermosensor.

Expression of cGMP signaling elements in the Grueneberg ganglion

The Grueneberg ganglion (GG) is a cluster of neurons localized to the vestibule of the anterior nasal cavity. Based on axonal projections to the olfactory bulb of the brain, as well as expression of olfactory receptors and the olfactory marker protein, it is considered a chemosensory subsystem. Recently, it was observed that in mice, GG neurons respond to cool ambient temperatures. In mammals, coolness-induced responses in highly specialized neuronal cells are supposed to rely on the ion channel TRPM8, whereas in thermosensory neurons of the nematode worm Caenorhabditis elegans, detection of environmental temperature is mainly mediated by cyclic guanosine monophosphate (cGMP) pathways, in which cGMP is generated by transmembrane guanylyl cyclases. To unravel the molecular mechanisms underlying coolness-induced responses in GG neurons, potential expression of TRPM8 in the murine GG was investigated; however, no evidence was found that this ion channel is present in the GG. By contrast, a substantial number of GG neurons was observed to express the transmembrane guanylyl cyclase subtype GC-G. In the nose, GC-G expression appears to be confined to the GG since it was not detectable in other nasal compartments. In the GG, coolness-stimulated responses are only observed in neurons characterized by the expression of the olfactory receptor V2r83. Interestingly, expression of GC-G in the GG was found in this V2r83-positive subpopulation but not in other GG neurons. In addition to GC-G, V2r83-positive GG cells also co-express the phosphodiesterase PDE2A. Thus, in summary, coolness-sensitive V2r83-expressing GG neurons are endowed with a cGMP cascade which might underlie thermosensitivity of these cells, similar to the cGMP pathway mediating thermosensation in neurons of C. elegans.

Grueneberg ganglion neurons are activated by a defined set of odorants.

Based on a variety of recent findings, the Grueneberg ganglion (GG) in the vestibule of the nasal cavity is considered as an olfactory compartment. However, defined chemical substances that activate GG neurons have not been identified. In this study, the responsiveness of murine GG cells to odorants was examined by monitoring the expression of the activity-dependent gene c-Fos. Testing a number of odorous compounds, cells in the GG were found to respond to dimethylpyrazine (DMP) and a few related substances. These responses were dose-dependent and restricted to early postnatal stages. The DMP-responsive GG cells belonged to the subset of GG neurons that coexpress the signaling elements V2r83, GC-G, and CNGA3. These cells have been previously reported to respond to cool ambient temperatures as well. In fact, cool temperatures enhanced DMP-evoked responses of GG cells. These findings support the concept that the GG of neonatal mice operates as a dual sensory organ that is stimulated by both the odorous compound DMP and cool ambient temperatures.

Receptor guanylyl cyclase-G is a novel thermosensory protein activated by cool temperatures

Transmembrane guanylyl cyclases (GCs), with activity regulated by peptide ligands and/or calcium‐binding proteins, are essential for various physiological and sensory processes. The mode of activation of the GC subtype GC‐G, which is expressed in neurons of the Grueneberg ganglion that respond to cool temperatures, has been elusive. In searching for appropriate stimuli to activate GC‐G, we found that its enzymatic activity is directly stimulated by cool temperatures. In this context, it was observed that dimerization/oligomerization of GC‐G, a process generally considered as critical for enzymatic activity of GCs, is strongly enhanced by coolness. Moreover, heterologous expression of GC‐G in cultured cells rendered these cells responsive to coolness; thus, the protein might be a sensor for cool temperatures. This concept is supported by the observation of substantially reduced coolness‐induced response of Grueneberg ganglion neurons and coolness‐evoked ultrasonic vocalization in GC‐G‐deficient mouse pups. GC‐G may be a novel thermosensory protein with functional implications for the Grueneberg ganglion, a sensory organ responding to cool temperatures.

Chemo- and Thermosensory Responsiveness of Grueneberg Ganglion Neurons Relies on Cyclic Guanosine Monophosphate Signaling Elements

Neurons of the Grueneberg ganglion (GG) in the anterior nasal region of mouse pups respond to cool temperatures and to a small set of odorants. While the thermosensory reactivity appears to be mediated by elements of a cyclic guanosine monophosphate (cGMP) cascade, the molecular mechanisms underlying the odor-induced responses are unclear. Since odor-responsive GG cells are endowed with elements of a cGMP pathway, specifically the transmembrane guanylyl cyclase subtype GC-G and the cyclic nucleotide-gated ion channel CNGA3, the possibility was explored whether these cGMP signaling elements may also be involved in chemosensory GG responses. Experiments with transgenic mice deficient for GC-G or CNGA3 revealed that GG responsiveness to given odorants was significantly diminished in these knockout animals. These findings suggest that a cGMP cascade may be important for both olfactory and thermosensory signaling in the GG. However, in contrast to the thermosensory reactivity, which did not decline over time, the chemosensory response underwent adaptation upon extended stimulation, suggesting that the two transduction processes only partially overlap.

The cyclic nucleotide-gated ion channel CNGA3 contributes to coolness-induced responses of Grueneberg ganglion neurons

Localized to the vestibule of the nasal cavity, neurons of the Grueneberg ganglion (GG) respond to cool ambient temperatures. The molecular mechanisms underlying this thermal response are still elusive. Recently, it has been suggested that cool temperatures may activate a cyclic guanosine monophosphate (cGMP) pathway in the GG, which would be reminiscent of thermosensory neurons in Caenorhabditis elegans. In search for other elements of such a cascade, we have found that the cyclic nucleotide-gated ion channel CNGA3 was strongly expressed in the GG and that expression of CNGA3 was confined to those cells that are responsive to coolness. Further experiments revealed that the response of GG neurons to cool temperatures was significantly reduced in CNGA3-deficient mice compared to wild-type conspecifics. The observation that a cGMP-activated non-selective cation channel significantly contributes to the coolness-evoked response in GG neurons strongly suggests that a cGMP cascade is part of the transduction process.