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.     

Expression of trace amine-associated receptors in the Grueneberg ganglion

The Grueneberg ganglion (GG) in the vestibule of the anterior nasal cavity is considered as an olfactory subcompartment based on expression of the olfactory marker protein (OMP) and axonal projection to the olfactory bulb. Searching for olfactory receptors present in the GG, it has been observed recently that V2r83, a member of the V2R class of olfactory receptors, is expressed in numerous cells in the GG of mice. However, no other olfactory receptors have been found to be present in a considerable number of GG neurons so far. Here, we report that GG neurons express trace amine-associated receptors (TAARs) that have most recently been described as a novel class of olfactory receptors. It was observed that several TAAR subtypes are expressed by defined subpopulations of GG neurons distinct from the V2r83-positive cells. Analyzing the time course of TAAR expression during pre- and postnatal development revealed that TAARs are expressed by a substantial portion of GG neurons in late embryonic and neonatal stages, whereas in juveniles and adults, the number of TAAR-positive cells in the GG was significantly decreased.     

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.     

Receptor guanylyl cyclases in mammalian olfactory function

The contributions of guanylyl cyclases to sensory signaling in the olfactory system have been unclear. Recently, studies of a specialized subpopulation of olfactory sensory neurons (OSNs) located in the main olfactory epithelium have provided important insights into the neuronal function of one receptor guanylyl cyclase, GC-D. Mice expressing reporters such as β-galactosidase and green fluorescent protein in OSNs that normally express GC-D have allowed investigators to identify these neurons in situ, facilitating anatomical and physiological studies of this sparse neuronal population. The specific perturbation of GC-D function in vivo has helped to resolve the role of this guanylyl cyclase in the transduction of olfactory stimuli. Similar approaches could be useful for the study of the orphan receptor GC-G, which is expressed in another distinct subpopulation of sensory neurons located in the Grueneberg ganglion. In this review, we discuss key findings that have reinvigorated the study of guanylyl cyclase function in the olfactory system.     

Localization of cGMP immunoreactivity and of soluble guanylyl cyclase in antennal sensilla of the hawkmoth Manduca sexta

The intracellular messenger cGMP (cyclic guanosine monophosphate) has been suggested to play a role in olfactory transduction in both invertebrates and vertebrates, but its cellular location within the olfactory system has remained elusive. We used cGMP immunocytochemistry to determine which antennal cells of the hawkmoth Manduca sexta are cGMP immunoreactive in the absence of pheromone. We then tested which antennal cells increase cGMP levels in response to nitric oxide (NO) and to long pheromonal stimuli, which the male encounters close to a calling female moth. In addition, we used in situ hybridization to determine which antennal cells express NO-sensitive soluble guanylyl cyclase. In response to long pheromonal stimuli with NO donors present, cGMP concentrations change in at least a subpopulation of pheromone-sensitive olfactory receptor neurons. These changes in cGMP concentrations in pheromone-dependent olfactory receptor neurons cannot be mimicked by the addition of NO donors in the absence of pheromone. NO stimulates sensilla chaetica type I and II, but not pheromone-sensitive trichoid sensilla, to high levels of cGMP accumulation as detected by immunocytochemistry. In situ hybridizations show that sensilla chaetica, but not sensilla trichodea, express detectable levels of mRNA coding for soluble guanylyl cyclase. These results suggest that intracellular rises in cGMP concentrations play a role in information processing in a subpopulation of pheromone-sensitive sensilla in Manduca sexta antennae, mediated by an NO-sensitive mechanism, but not an NO-dependent soluble guanylyl cyclase.     

Guanylyl cyclase structure, function and regulation

Nitric oxide, bicarbonate, natriuretic peptides (ANP, BNP and CNP), guanylins, uroguanylins and guanylyl cyclase activating proteins (GCAPs) activate a family of enzymes variously called guanyl, guanylyl or guanylate cyclases that catalyze the conversion of guanosine triphosphate to cyclic guanosine monophosphate (cGMP) and pyrophosphate. Intracellular cyclic GMP is a second messenger that modulates: platelet aggregation, neurotransmission, sexual arousal, gut peristalsis, blood pressure, long bone growth, intestinal fluid secretion, lipolysis, phototransduction, cardiac hypertrophy and oocyte maturation. This review briefly discusses the discovery of cGMP and guanylyl cyclases, then nitric oxide, nitric oxide synthase and soluble guanylyl cyclase are described in slightly greater detail. Finally, the structure, function, and regulation of the individual mammalian single membrane-spanning guanylyl cyclases GC-A, GC-B, GC-C, GC-D, GC-E, GC-F and GC-G are described in greatest detail as determined by biochemical, cell biological and gene-deletion studies.     

Neuron–Glia Communication via Nitric Oxide Is Essential in Establishing Antennal-Lobe Structure in Manduca sexta

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.     

Inhibition of nitric oxide and soluble guanylyl cyclase signaling affects olfactory neuron activity in the moth, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Nitric oxide is emerging as an important modulator of many physiological processes including olfaction, yet the function of this gas in the processing of olfactory information remains poorly understood. In the antennal lobe of the moth, Manduca sexta, nitric oxide is produced in response to odor stimulation, and many interneurons express soluble guanylyl cyclase, a well-characterized nitric oxide target. We used intracellular recording and staining coupled with pharmacological manipulation of nitric oxide and soluble guanylyl cyclase to test the hypothesis that nitric oxide modulates odor responsiveness in olfactory interneurons through soluble guanylyl cyclase-dependent pathways. Nitric oxide synthase inhibition resulted in pronounced effects on the resting level of firing and the responses to odor stimulation in most interneurons. Effects ranged from bursting to strong attenuation of activity and were often accompanied by membrane depolarization coupled with a change in input resistance. Blocking nitric oxide activation of soluble guanylyl cyclase signaling mimicked the effects of nitric oxide synthase inhibitors in a subset of olfactory neurons, while other cells were differentially affected by this treatment. Together, these results suggest that nitric oxide is required for proper olfactory function, and likely acts through soluble guanylyl cyclase-dependent and -independent mechanisms in different subsets of neurons.     

Regulation of intracellular cyclic GMP levels in olfactory sensory neurons

Cyclic AMP is the primary second messenger mediating odorant signal transduction in mammals. A number of studies indicate that cyclic GMP is also involved in a variety of other olfactory signal transduction processes, including adaptation, neuronal development, and long-term cellular responses in the setting of odorant stimulation. However, the mechanisms that control the production and degradation of cGMP in olfactory sensory neurons (OSNs) remain unclear. Here, we investigate these mechanisms using primary cultures of OSNs. We demonstrate that odorants increase cGMP levels in intact OSNs in vitro. Different from the rapid and transient cAMP responses to odorants, the cGMP elevation is both delayed and sustained. Inhibition of soluble guanylyl cyclase and heme oxygenase blocks these odorant-induced cGMP increases, whereas inhibition of cGMP PDEs (phosphodiesterases) increases this response. cGMP PDE activity is increased by odorant stimulation, and is sensitive to both ambient calcium and cAMP concentrations. Calcium stimulates cGMP PDE activity, whereas cAMP and protein kinase A appears to inhibit it. These data demonstrate a mechanism by which odorant stimulation may regulate cGMP levels through the modulation of cAMP and calcium level in OSNs. Such interactions between odorants and second messenger systems may be important to the integration of immediate and long-term responses in the setting odorant stimulation.     

Potential Role of Transient Receptor Potential Channel M5 in Sensing Putative Pheromones in Mouse Olfactory Sensory Neurons

Based on pharmacological studies of chemosensory transduction in transient receptor potential channel M5 (TRPM5) knockout mice it was hypothesized that this channel is involved in transduction for a subset of putative pheromones in mouse olfactory sensory neurons (OSNs). Yet, in the same study an electroolfactogram (EOG) in the mouse olfactory epithelium showed no significant difference in the responses to pheromones (and odors) between wild type and TRPM5 knockout mice. Here we show that the number of OSNs expressing TRPM5 is increased by unilateral naris occlusion. Importantly, EOG experiments show that mice lacking TRPM5 show a decreased response in the occluded epithelia to putative pheromones as opposed to wild type mice that show no change upon unilateral naris occlusion. This evidence indicates that under decreased olfactory sensory input TRPM5 plays a role in mediating putative pheromone transduction. Furthermore, we demonstrate that cyclic nucleotide gated channel A2 knockout (CNGA2-KO) mice that show substantially decreased or absent responses to odors and pheromones also have elevated levels of TRPM5 compared to wild type mice. Taken together, our evidence suggests that TRPM5 plays a role in mediating transduction for putative pheromones under conditions of reduced chemosensory input.     

NO/cGMP signalling: <Emphasis Type="SmallCaps">L</Emphasis>-citrulline and cGMP immunostaining in the central complex of the desert locust <Emphasis Type="Italic">Schistocerca gregaria</Emphasis>

Nitric oxide (NO) is a gaseous messenger molecule formed during conversion of L-arginine into L-citrulline by the enzyme NO synthase (NOS), which belongs to a group of NADPH diaphorases. Because of its gaseous diffusion properties, NO differs from classical neurotransmitters in that it is not restricted to synaptic terminals. In target cells, NO activates soluble guanylyl cyclase leading to an increase in cGMP levels. In insects, this NO/cGMP-signalling pathway is involved in development, memory formation and processing of visual, olfactory and mechanosensory information. We have analysed the distribution of putative NO donor and target cells in the central complex, a brain area involved in sky-compass orientation, of the locust Schistocerca gregaria by immunostaining for L-citrulline and cGMP. Six types of citrulline-immunostained neurons have been identified including a bilateral pair of hitherto undescribed neurons that connect the lateral accessory lobes with areas anterior to the medial lobes of the mushroom bodies. Three-dimensional reconstructions have revealed the connectivity pattern of a set of 18 immunostained pontine neurons of the central body. All these neurons appear to be a subset of previously mapped NADPH-diaphorase-positive neurons of the central complex. At least three types of central-complex neurons show cGMP immunostaining including a system of novel columnar neurons connecting the upper division of the central body and the lateral triangle of the lateral accessory lobe. Our results provide the morphological basis for further studies of the function of the labelled neurons and new insights into NO/cGMP signalling. This work was supported by DFG grant HO 950/16-2.     

It's cold,mom! It's cyclic GMP

Cyclic GMP is the signal transducer of a family of transmembrane, particulate guanylyl cyclase (GC) receptors with key roles in physiology and disease. GC‐G, the last member of the membrane GCs identified in mammals, is an orphan receptor and its regulation and function have remained largely unknown. In this issue of The EMBO Journal, Chao et al ( 2015 ) show that the GC‐G/cGMP pathway, which is expressed in a specific cluster of olfactory neurons of neonatal mice, functions as a cold‐induced thermosensor, which triggers protective maternal care.     

Cloning and localization of the cGMP-specific phosphodiesterase type 9 in the rat brain

In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.     

The Receptor-Bound Guanylyl Cyclase DAF-11 Is the Mediator of Hydrogen Peroxide-Induced Cgmp Increase in Caenorhabditis elegans

Adenosine 3′, 5′-cyclic monophosphate (cAMP) and guanosine 3′, 5′-cyclic monophosphate (cGMP) are well-studied second messengers that transmit extracellular signals into mammalian cells, with conserved functions in various other species such as Caenorhabditis elegans (C. elegans). cAMP is generated by adenylyl cyclases, and cGMP is generated by guanylyl cyclases, respectively. Studies using C. elegans have revealed additional roles for cGMP signaling in lifespan extension. For example, mutants lacking the function of a specific receptor-bound guanylyl cyclase, DAF-11, have an increased life expectancy. While the daf-11 phenotype has been attributed to reductions in intracellular cGMP concentrations, the actual content of cyclic nucleotides has not been biochemically determined in this system. Similar assumptions were made in studies using phosphodiesterase loss-of-function mutants or using adenylyl cyclase overexpressing mutants. In the present study, cyclic nucleotide regulation in C. elegans was studied by establishing a special nematode protocol for the simultaneous detection and quantitation of cyclic nucleotides. We also examined the influence of reactive oxygen species (ROS) on cyclic nucleotide metabolism and lifespan in C. elegans using highly specific HPLC-coupled tandem mass-spectrometry and behavioral assays. Here, we show that the relation between cGMP and survival is more complex than previously appreciated.     

A role for GCAP2 in regulating the photoresponse. Guanylyl cyclase activation and rod electrophysiology in GUCA1B knock-out mice

Cyclic GMP serves as the second messenger in visual transduction, linking photon absorption by rhodopsin to the activity of ion channels. Synthesis of cGMP in photoreceptors is supported by a pair of retina-specific guanylyl cyclases, retGC1 and -2. Two neuronal calcium sensors, GCAP1 and GCAP2, confer Ca(2+) sensitivity to guanylyl cyclase activity, but the importance and the contribution of each GCAP is controversial. To explore this issue, the gene GUCA1B, coding for GCAP2, was disrupted in mice, and the capacity for knock-out rods to regulate retGC and generate photoresponses was tested. The knock-out did not compromise rod viability or alter outer segment ultrastructure. Levels of retGC1, retGC2, and GCAP-1 expression did not undergo compensatory changes, but the absence of GCAP2 affected guanylyl cyclase activity in two ways; (a) the maximal rate of cGMP synthesis at low [Ca(2+)] dropped 2-fold and (b) the half-maximal rate of cGMP synthesis was attained at a higher than normal [Ca(2+)]. The addition of an antibody raised against mouse GCAP2 produced similar effects on the guanylyl cyclase activity in wild type retinas. Flash responses of GCAP2 knock-out rods recovered more slowly than normal. Knock-out rods became more sensitive to flashes and to steps of illumination but tended to saturate at lower intensities, as compared with wild type rods. Therefore, GCAP2 regulation of guanylyl cyclase activity quickens the recovery of flash and step responses and adjusts the operating range of rods to higher intensities of ambient illumination.     

Multiple olfactory pathways contribute to the lure process of Caenorhabditis elegans by pathogenic bacteria

Chemosensation is indispensable for the survival of Caenorhabditis elegans to discriminate food and pathogenic bacteria in their living environment. Food-like odors emitted by the pathogen Bacillus nematocida B16 for trapping its hosts and an olfactory signaling pathway responsible to sense the attractant 2-heptanone were identified in our previous study. Here, we further explore how the worms recognize the attractive molecules indole and 2-ethyl hexanol, which have different chemical properties and modest nematode-luring ability. We show that the chemotaxis toward indole and 2-ethyl hexanol requires the G protein-coupled receptors encoded by str-193 on AWC and str-7 on AWA. In a further genetic screen for downstream effectors in olfactory signaling cascades, the Gα subunit GSA-1, guanylyl cyclase ODR-1 and DAF-11 and the cGMP-gated channel TAX-2/TAX-4 were found to be necessary for indole sensation, whereas the TRPV channels OSM-9/OCR-2 and the PLC pathway activated by GPA-6 are responsible for the detection of 2-ethyl hexanol. Altogether, our current work further clarifies the distinct olfactory signaling pathways through which C. elegans senses different chemicals and is lured by B. nematocida B16, improving our comprehensive understanding of the mechanisms by which bacterial pathogens effectively infect their hosts.

     

Invertebrates yield a plethora of atypical guanylyl cyclases

Invertebrate model systems have a long history of generating new insights into neuronal signaling systems. This review focuses on cyclic GMP signaling and describes recent advances in understanding the properties and functions of guanylyl cyclases in invertebrates. The sequencing of three invertebrate genomes has provided a complete catalog of the guanylyl cyclases in C. elegans, Drosophila, and the mosquito Anopheles gambiae. Using this data and that from cloned guanylyl cyclases in Manduca sexta, C. elegans, and Drosophila, plus predictions and models from vertebrate guanylyl cyclases, evidence is presented that there is a much broader array of properties for these enzymes than previously realized. In addition to the classic homodimeric receptor guanylyl cyclases, C. elegans has at least two receptor guanylyl cyclases that are predicted to require heterodimer formation for activity. Soluble guanylyl cyclases are generally recognized as being obligate heterodimers that are activated by nitric oxide (NO). Some of the soluble guanylyl cyclases in C. elegans may heterodimeric, but all appear to be insensitive to NO. The β2 soluble guanylyl cyclase subunit in mammals and similar ones in Manduca and Drosophila are active in the absence of additional subunits and there is evidence that Drosophila and Anopheles also express an additional subunit that enhances this activity.     

Neuron-glia communication via nitric oxide is essential in establishing antennal-lobe structure in Manduca sexta.

Nitric oxide synthase recently has been shown to be present in olfactory receptor cells throughout development of the adult antennal (olfactory) lobe of the brain of the moth Manduca sexta. Here, we investigate the possible involvement of nitric oxide (NO) in antennal-lobe morphogenesis. Inhibition of NO signaling with a NO synthase inhibitor or a NO scavenger early in development results in abnormal antennal lobes in which neuropil-associated glia fail to migrate. A more subtle effect is seen in the arborization of dendrites of a serotonin-immunoreactive neuron, which grow beyond their normal range. The effects of NO signaling in these types of cells do not appear to be mediated by activation of soluble guanylyl cyclase to produce cGMP, as these cells do not exhibit cGMP immunoreactivity following NO stimulation and are not affected by infusion of a soluble guanylyl cyclase inhibitor. Treatment with Novobiocin, which blocks ADP-ribosylation of proteins, results in a phenotype similar to those seen with blockade of NO signaling. Thus, axons of olfactory receptor cells appear to trigger glial cell migration and limit arborization of serotonin-immunoreactive neurons via NO signaling. The NO effect may be mediated in part by ADP-ribosylation of target cell proteins.