Mammalian odorant receptor tuning breadth persists across distinct odorant panels.

The molecular receptive range (MRR) of a mammalian odorant receptor (OR) is the set of odorant structures that activate the OR, while the distribution of these odorant structures across odor space is the tuning breadth of the OR. Variation in tuning breadth is thought to be an important property of ORs, with the MRRs of these receptors varying from narrowly to broadly tuned. However, defining the tuning breadth of an OR is a technical challenge. For practical reasons, a screening panel that broadly covers odor space must be limited to sparse coverage of the many potential structures in that space. When screened with such a panel, ORs with different odorant specificities, but equal tuning breadths, might appear to have different tuning breadths due to chance. We hypothesized that ORs would maintain their tuning breadths across distinct odorant panels. We constructed a new screening panel that was broadly distributed across an estimated odor space and contained compounds distinct from previous panels. We used this new screening panel to test several murine ORs that were previously characterized as having different tuning breadths. ORs were expressed in Xenopus laevis oocytes and assayed by two-electrode voltage clamp electrophysiology. MOR256-17, an OR previously characterized as broadly tuned, responded to nine novel compounds from our new screening panel that were structurally diverse and broadly dispersed across an estimated odor space. MOR256-22, an OR previously characterized as narrowly tuned, responded to a single novel compound that was structurally similar to a previously known ligand for this receptor. MOR174-9, a well-characterized receptor with a narrowly tuned MRR, did not respond to any novel compounds in our new panel. These results support the idea that variation in tuning breadth among these three ORs is not an artifact of the screening protocol, but is an intrinsic property of the receptors.

cAMP and IP3 signaling pathways in HEK293 cells transfected with canine olfactory receptor genes.

Olfactory receptors (ORs) expressed at the cell surface of olfactory sensory neurons lining the olfactory epithelium are the first actors of events leading to odor perception and recognition. As for other mammalian ORs, few dog OR have been deorphanized, mainly because of the absence of good methodology and the difficulties encountered to express ORs at the cell surface. Within this work, our aim was 1) to deorphanize a large subset of dog OR and 2) to compare the implication of the 2 main pathways, namely the cAMP and inositol 1,4,5-triphosphate (IP3) pathways, in the transduction of the olfactory message. For this, we used 2 independent tests to assess the importance of each of these 2 pathways and analyzed the responses of 47 canine family 6 ORs to a number of aliphatic compounds. We found these ORs globally capable of inducing intracellular calcium elevation through the IP3 pathway as confirmed by the use of specific inhibitors and/or a cAMP increase in response to aldehyde exposure. We showed that the implication of the cAMP or/and IP3 pathway was dependent upon the ligand-receptor combination rather than on one or the other partner. Finally, by exposing OR-expressing cells to the 21 possible pairs of C6-C12 aliphatic aldehydes, we confirmed that some odorant pairs may have an inhibitory or additive effect. Altogether, these results reinforce the notion that odorant receptor subfamilies may constitute functional units and call for a more systematic use of 2 complementary tests interrogating the cAMP and IP3 pathways when deorphanizing ORs.

Zinc alleviates pain through high-affinity binding to the NMDA receptor NR2A subunit.

Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.

Study of smell and reproductive organs in a mouse model for CHARGE syndrome.

CHARGE syndrome is a multiple congenital anomaly syndrome characterised by Coloboma, Heart defects, Atresia of choanae, Retardation of growth and/or development, Genital hypoplasia, and Ear anomalies often associated with deafness. It is caused by heterozygous mutations in the CHD7 gene and shows a highly variable phenotype. Anosmia and hypogonadotropic hypogonadism occur in the majority of the CHARGE patients, but the underlying pathogenesis is unknown. Therefore, we studied the ability to smell and aspects of the reproductive system (reproductive performance, gonadotropin-releasing hormone (GnRH) neurons and anatomy of testes and uteri) in a mouse model for CHARGE syndrome, the whirligig mouse (Chd7(Whi/+)). We showed that Chromodomain Helicase DNA-binding protein 7 (Chd7) is expressed in brain areas involved in olfaction and reproduction during embryonic development. We observed poorer performance in the smell test in adult Chd7(Whi/+) mice, secondary either to olfactory dysfunction or to balance disturbances. Olfactory bulb and reproductive organ abnormalities were observed in a proportion of Chd7(Whi/+) mice. Hypothalamic GnRH neurons were slightly reduced in Chd7(Whi/+) females and reproductive performance was slightly less in Chd7(Whi/+) mice. This study shows that the penetrance of anosmia and hypogonadotropic hypogonadism is lower in Chd7(Whi/+) mice than in CHARGE patients. Interestingly, many phenotypic features of the Chd7 mutation showed incomplete penetrance in our model mice, despite the use of inbred, genetically identical mice. This supports the theory that the extreme variability of the CHARGE phenotype in both humans and mice might be attributed to variations in the fetal microenvironment or to purely stochastic events.

Sensitivity-dependent hierarchical receptor codes for odors.

In order to comprehend the strategy of odor encoding by odorant receptors, we isolated 2740 mouse receptor neurons from four olfactory epithelial zones and classified them in terms of their sensitivities and tuning specificities to a chiral pair of odorants, S(+)-carvone (caraway-like odor) and R(-)-carvone (spearmint-like odor). Our approach revealed that the majority of receptors at the lowest effective stimulus concentration represented the principal odor qualities characteristic of each enantiomer by means of the principal odor qualities of the odorants for which the receptors were most sensitive. The chiral-non-discriminating receptors were newly recruited 3.7 times of R(-)-carvone-sensitive receptors and totally became 2.8 times (39/14) of R(-)carvone-sensitive receptors in the subpopulations when the stimulus concentration was increased 10-fold [corrected]. More than 80% of the responsive receptors (an estimated 70 +/- alpha types) exhibited overlapping sensitivities between the enantiomers. The signals from the non-discriminating receptors may be reduced to decode the characteristic odor identity for R(-)-carvone in the brain over an adequate range of stimulus strengths. The information processing of odors appears to involve the selective weighting of the signals from the most sensitive receptors. An analysis of the overall receptor codes to carvones indicated that the system employs hierarchical receptor codes: principal odor qualities are encoded by the most sensitive receptors and lower-ranked odor qualities by less sensitive receptors.

Male mice lacking the gastrin-releasing peptide receptor (GRP-R) display elevated preference for conspecific odors and increased social investigatory behaviors.

Previously, we generated gastrin-releasing peptide receptor null mutant mice (GRP-R-deficient mice), and found that these animals displayed increased non-aggressive social responses in an ordinary social interaction test using a resident-intruder method. In the present study, we examined in more detail the social behaviors of GRP-R-deficient male mice. In social interaction tests, GRP-R-deficient mice showed more social responses, such as sniffing and nosing, relative to wild-type mice, and similar results were obtained whether GRP-R-deficient mice served as intruders or residents. In the same way, they showed more contact behaviors toward an anesthetized conspecific, and less locomotor activity than wild-type mice in a social investigation test toward an anesthetized male mouse. Since olfactory systems play important roles in the social behavior of rodents, olfactory preference tests were conducted in order to evaluate the olfactory properties of GRP-R-deficient mice. The results suggest that no differences exist between wild-type mice and GRP-R-deficient mice in the preference between a novel sawdust odor and their own odor, or that of other male mice. However, GRP-R-deficient mice preferred the odor of other male mice to their own, in contrast to wild-type mice. Furthermore, the preferences of GRP-R-deficient and wild-type mice were not disrupted by intraperitoneal infusion of diazepam (1.5 mg/kg). These results indicate that neither the motion, nor the behavior of conspecifics, nor reduced anxiety lead to the increased non-aggressive social responses and/or social investigatory behaviors in GRP-R-deficient mice. Rather, these latter behaviors may be a consequence of altered cognition of conspecific odors in the mutant mice.

Lobster GABA receptor subunit expressed in neural tissues.

A cDNA encoding an ionotropic gamma-aminobutyric acid (GABA) receptor subunit was isolated from a lobster (Homarus americanus) cDNA library. A longer version of this cDNA, containing a 108-bp insert, was also detected. The two cDNAs are predicted to encode alternatively spliced proteins of 485 and 521 amino acids, respectively. The sequences were most similar to the Drosophila RDL (resistance to dieldrin) GABA subunit with 54% identity, and 30-35% identity with vertebrate ionotropic GABA receptor subunits. Only the shorter clone formed functional ion channels when transfected into human embryonic kidney (HEK) 293 cells. GABA caused a Cl(-)-selective current in the presence of GABA that was blocked by picrotoxin. The GABA-induced current was weakly sensitive to the GABA(A) antagonist, bicuculline, but was enhanced by pentobarbital. Expression of the GABA receptor mRNA was highest in brain and the olfactory organ, but was not detected in leg muscle. These data suggest that the isolated cDNAs are likely to encode proteins that comprise subunits of native GABA receptors expressed in olfactory receptor neurons and projection neurons of the olfactory deutocerebrum.