Xavier Grosmaitre

Dual functions of mammalian olfactory sensory neurons as odor detectors and mechanical sensors.

Most sensory systems are primarily specialized to detect one sensory modality. Here we report that olfactory sensory neurons (OSNs) in the mammalian nose can detect two distinct modalities transmitted by chemical and mechanical stimuli. As revealed by patch-clamp recordings, many OSNs respond not only to odorants, but also to mechanical stimuli delivered by pressure ejections of odor-free Ringer solution. The mechanical responses correlate directly with the pressure intensity and show several properties similar to those induced by odorants, including onset latency, reversal potential and adaptation to repeated stimulation. Blocking adenylyl cyclase or knocking out the cyclic nucleotide–gated channel CNGA2 eliminates the odorant and the mechanical responses, suggesting that both are mediated by a shared cAMP cascade. We further show that this mechanosensitivity enhances the firing frequency of individual neurons when they are weakly stimulated by odorants and most likely drives the rhythmic activity (theta oscillation) in the olfactory bulb to synchronize with respiration.

SR1, a Mouse Odorant Receptor with an Unusually Broad Response Profile

The current consensus model in mammalian olfaction is that the detection of millions of odorants requires a large number of odorant receptors (ORs) and that each OR interacts selectively with a small subset of odorants, which are typically related in structure. Here, we report the odorant response properties of an OR that deviates from this model: SR1, a mouse OR that is abundantly expressed in sensory neurons of the septal organ and also of the main olfactory epithelium. Patch-clamp recordings reveal that olfactory sensory neurons (OSNs) that express SR1 respond to many, structurally unrelated odorants, and over a wide concentration range. Most OSNs expressing a gene-targeted SR1 locus that lacks the SR1 coding sequence do not show this broad responsiveness. Gene transfer in the heterologous expression system Hana3A confirms the broad response profile of SR1. There may be other mouse ORs with such broad response profiles.

Olfactory Signal Transduction in the Mouse Septal Organ

The septal organ, a distinct chemosensory organ observed in the mammalian nose, is essentially a small island of olfactory neuroepithelium located bilaterally at the ventral base of the nasal septum. Virtually nothing is known about its physiological properties and function. To understand the nature of the sensory neurons in this area, we studied the mechanisms underlying olfactory signal transduction in these neurons. The majority of the sensory neurons in the septal organ express olfactory-specific G-protein and adenylyl cyclase type III, suggesting that the cAMP signaling pathway plays a critical role in the septal organ as in the main olfactory epithelium (MOE). This is further supported by patch-clamp recordings from individual dendritic knobs of the sensory neurons in the septal organ. Odorant responses can be mimicked by an adenylyl cyclase activator and a phosphodiesterase inhibitor, and these responses can be blocked by an adenylyl cyclase inhibitor. There is a small subset of cells in the septal organ expressing a cGMP-stimulated phosphodiesterase (phosphodiesterase 2), a marker for the guanylyl cyclase-D subtype sensory neurons identified in the MOE. The results indicate that the septal organ resembles the MOE in major olfactory signal transduction pathways, odorant response properties, and projection to the main olfactory bulb. Molecular and functional analysis of the septal organ, which constitutes ∼1% of the olfactory epithelium, will provide new insights into the organization of the mammalian olfactory system and the unique function this enigmatic organ may serve.

Modulation of Spontaneous and Odorant-Evoked Activity of Rat Olfactory Sensory Neurons by Two Anorectic Peptides, Insulin and Leptin

In mammals, the sense of smell is modulated by the status of satiety, which is mainly signaled by blood-circulating peptide hormones. However, the underlying mechanisms linking olfaction and food intake are poorly understood. Here we investigated the effects of two anorectic peptides, insulin and leptin, on the functional properties of olfactory sensory neurons (OSNs). Using patch-clamp recordings, we analyzed the spontaneous activity of rat OSNs in an in vitro intact epithelium preparation. Bath perfusion of insulin and leptin significantly increased the spontaneous firing frequency in 91.7% (n = 24) and 75.0% (n = 24) of the cells, respectively. When the activity was electrically evoked, both peptides shortened the latency to the first action potential by approximately 25% and decreased the interspike intervals by approximately 13%. While insulin and leptin enhanced the electrical excitability of OSNs in the absence of odorants, they surprisingly reduced the odorant-induced activity in the olfactory epithelium. Insulin and leptin decreased the peak amplitudes of isoamyl acetate-induced electroolfactogram (EOG) signals to 46 and 38%, respectively. When measured in individual cells by patch-clamp recordings, insulin and leptin decreased odorant-induced transduction currents and receptor potentials. Therefore by increasing the spontaneous activity but reducing the odorant-induced activity of OSNs, an elevated insulin and leptin level (such as after a meal) may result in a decreased global signal-to-noise ratio in the olfactory epithelium, which matches the smell ability to the satiety status.

Expression Patterns of Odorant Receptors and Response Properties of Olfactory Sensory Neurons in Aged Mice

The sense of smell deteriorates in normal aging, but the underling mechanisms are still elusive. Here we investigated age-related alterations in expression patterns of odorant receptor (OR) genes and functional properties of olfactory sensory neurons (OSNs)-2 critical factors that define the odor detection threshold in the olfactory epithelium. Using in situ hybridization for 9 representative OR genes, we compared the cell densities of each OR in coronal nose sections at different ages (3-27 months). The cell density for different ORs peaked at different time points and a decline was observed for 6 of 9 ORs at advanced ages. Using patch clamp recordings, we then examined the odorant responses of individual OSNs coexpressing a defined OR (MOR23) and green fluorescent protein. The MOR23 neurons recorded from aged animals maintained a similar sensitivity and dynamic range in response to the cognate odorant (lyral) as those from younger mice. The results indicate that although the cell densities of OSNs expressing certain types of ORs decline at advanced ages, individual OSNs can retain their sensitivity. The implications of these findings in age-related olfactory deterioration are discussed.

G protein-coupled odorant receptors underlie mechanosensitivity in mammalian olfactory sensory neurons

Significance Mechanical stimuli (pressure, shear stress, membrane stretch, etc.) are a basic form of stimulation that can induce physiological responses in many body organs (skin, muscle, ear, lung, airway, kidney, blood vessels, etc.). The current dogma in sensory systems is that mechanical stimuli are mainly transduced by force-gated ion channels. Our study reveals a previously unidentified cascade for mechanotransduction in neurons and suggests that G protein-coupled receptors may have an overlooked function as mechanical sensors. This finding establishes a molecular mechanism through which the nose sends an afferent signal of breathing to the brain to facilitate integration of orofacial sensation and synchronize delta/theta-band activity in certain brain regions with respiration. Mechanosensitive cells are essential for organisms to sense the external and internal environments, and a variety of molecules have been implicated as mechanical sensors. Here we report that odorant receptors (ORs), a large family of G protein-coupled receptors, underlie the responses to both chemical and mechanical stimuli in mouse olfactory sensory neurons (OSNs). Genetic ablation of key signaling proteins in odor transduction or disruption of OR–G protein coupling eliminates mechanical responses. Curiously, OSNs expressing different OR types display significantly different responses to mechanical stimuli. Genetic swap of putatively mechanosensitive ORs abolishes or reduces mechanical responses of OSNs. Furthermore, ectopic expression of an OR restores mechanosensitivity in loss-of-function OSNs. Lastly, heterologous expression of an OR confers mechanosensitivity to its host cells. These results indicate that certain ORs are both necessary and sufficient to cause mechanical responses, revealing a previously unidentified mechanism for mechanotransduction.

Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth Mamestra brassicae.

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.

The β2-adrenergic receptor as a surrogate odorant receptor in mouse olfactory sensory neurons.

In the mouse, mature olfactory sensory neurons (OSNs) express one allele of one of the ~1200 odorant receptor (OR) genes, which encode G-protein coupled receptors (GPCRs). Axons of OSNs that express the same OR coalesce into homogeneous glomeruli at conserved positions in the olfactory bulb. ORs are involved in OR gene choice and OSN axonal wiring, but the mechanisms remain poorly understood. One approach is to substitute an OR genetically with another GPCR, and to determine in which aspects this GPCR can serve as a surrogate OR under experimental conditions. Here, we characterize a novel gene-targeted mouse strain in which the mouse β2-adrenergic receptor (β2AR) is coexpressed with tauGFP in OSNs that choose the OR locus M71 for expression (β2AR→M71-GFP). By crossing these mice with β2AR→M71-lacZ gene-targeted mice, we find that differentially tagged β2AR→M71 alleles are expressed monoallelically. The OR coding sequence is thus not required for monoallelic expression - the expression of one of the two alleles of a given OR gene in an OSN. We detect strong β2AR immunoreactivity in dendritic cilia of β2AR→M71-GFP OSNs. These OSNs respond to the β2AR agonist isoproterenol in a dose-dependent manner. Axons of β2AR→M71-GFP OSNs coalesce into homogeneous glomeruli, and β2AR immunoreactivity is detectable within these glomeruli. We do not find evidence for expression of endogenous β2AR in OSNs of wild-type mice, also not in M71-expressing OSNs, and we do not observe overt differences in the olfactory system of β2AR and β1AR knockout mice. Our findings corroborate the experimental value of the β2AR as a surrogate OR, including for the study of the mechanisms of monoallelic expression.

The extremely broad odorant response profile of mouse olfactory sensory neurons expressing the odorant receptor MOR256-17 includes trace amine-associated receptor ligands.

The mouse olfactory system employs ~1100 G‐protein‐coupled odorant receptors (ORs). Each mature olfactory sensory neuron (OSN) is thought to express just one OR gene, and the expressed OR determines the odorant response properties of the OSN. The broadest odorant response profile thus far demonstrated in native mouse OSNs is for OSNs that express the OR gene SR1 (also known as Olfr124 and MOR256‐3). Here we showed that the odorant responsiveness of native mouse OSNs expressing the OR gene MOR256‐17 (also known as Olfr15 and OR3) is even broader than that of OSNs expressing SR1. We investigated the electrophysiological properties of green fluorescent protein (GFP)+ OSNs in a MOR256‐17‐IRES‐tauGFP gene‐targeted mouse strain, in parallel with GFP+ OSNs in the SR1‐IRES‐tauGFP gene‐targeted mouse strain that we previously reported. Of 35 single chemical compounds belonging to distinct structural classes, MOR256‐17+ OSNs responded to 31 chemicals, compared with 10 for SR1+ OSNs. The 10 compounds that activated SR1+ OSNs also activated MOR256‐17+ OSNs. Interestingly, MOR256‐17+ OSNs were activated by three amines (cyclohexylamine, isopenthylamine, and phenylethylamine) that are typically viewed as ligands for chemosensory neurons in the main olfactory epithelium that express trace amine‐associated receptor genes, a family of 15 genes encoding G‐protein‐coupled receptors unrelated in sequence to ORs. We did not observe differences in membrane properties, indicating that the differences in odorant response profiles between the two OSN populations were due to the expressed OR. MOR256‐17+ OSNs appear to be at one extreme of odorant responsiveness among populations of OSNs expressing distinct OR genes in the mouse.

Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse

Tracing of mono- and polysynaptic afferent connections between the main olfactory bulb and higher-order brain regions in the mouse Processing of odors in the main olfactory bulb (MOB) is modulated by higher brain afferents depending on the internal state, motivation, memory and emotions. For example satiety or hunger are known to change the perception of food odors. To shed light on this modulation a greater understanding of the underlying circuitry is required. To this aim we conducted tracing experiments in mice. First, stereotaxic injections of monosynaptic retrograde DiI and choleratoxin subunit B (CTb) into the dorsal olfactory bulb (focusing on the granular layer) were performed. In a second approach, we injected the pseudorabies virus 152 (PRV152) (kindly provided by Prof L. Enquist; Princeton University). As a neuronal tracer, this neurotropic virus can spread in synaptically connected neurons, dissecting the entire circuitry. The temporal analysis of the viral distribution allows to determine the number of synapses crossed. Both DiI and CTb confirm all the main centrifugal afferents to the MOB which were previously described in the literature. Thus, except the olfactory tubercle, all regions belonging to the olfactory primary cortex were labeled. Moreover, in the piriform cortex, labeling was mainly located in its dorsal part, confirming the topographical anatomical organization of cortico-bulbar projections. Direct projections arising from orexinergic neurons in the lateral hypothalamus were also observed. Regarding polysynaptic tracing, mice were sacrificed one, two and three days after PRV152 injections. After one day, only some of the direct neuromodulatory afferents were labelled. Thus, the locus coeruleus, which has a very caudal location in the brain close to the forth ventricle, showed already staining. In contrast, other neuromodulatory afferents (arising from raphe nuclei, ventral tegmental area, basal forebrain) were not labeled at this stage. Two days after injection, comparison with DiI/CTb results indicate that all the first-order connections were labeled. At this stage, second-order projections started to appear (for example, the midline thalamic nuclei as reuniens and rhomboid nuclei). Lastly, after three days, an extensive brain labeling occurred although some brain regions still showed a lack of staining. In conclusion, using DiI/CTb tracers that do not cross synapses allow to stain all primary afferents. In contrast, viral tracer migration depends on timing as well as number of connecting synapses. Thus, labeling occurring already one day after injection indicates a strong connection. Accordingly, our results suggest that the stained cells in the locus coeruleus send strong projections to the MOB. Furthermore this method allows to follow up the circuits involved in olfactory modulation. Data analysis of all the other labeled regions, focusing on hypothalamic nuclei and brain areas involved in arousal and food intake, are in progress.