GABAB receptor expression and function in olfactory receptor neuron axon growth

Neurotransmitters have been implicated in regulating growth cone motility and guidance in the developing nervous system. Anatomical and electrophysiological studies show the presence of functional GABAB receptors on adult olfactory receptor neuron (ORN) nerve terminals. Using antisera against the GABAB R1a/b receptor isoforms we show that developing mouse olfactory receptor neurons express GABAB receptors from embryonic day 14 through to adulthood. GABAB receptors are present on axon growth cones from both dissociated ORNs and olfactory epithelial explants. Neurons in the olfactory bulb begin to express glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, from E16 through to adulthood. When dissociated ORNs were cultured in the presence of the GABAB receptor agonists, baclofen or SKF97541, neurite outgrowth was significantly reduced. Concurrent treatment of the neurons with baclofen and the GABAB receptor antagonist CGP54626 prevented the inhibitory effects of baclofen on ORN neurite outgrowth. These results show that growing ORN axons express GABAB receptors and are sensitive to the effects of GABAB receptor activation. Thus, ORNs in vivo may detect GABA release from juxtaglomerular cells as they enter the glomerular layer and use this as a signal to limit their outgrowth and find synaptic targets in regeneration and development.     

Metamorphic remodeling of the primary olfactory projection in Xenopus: Developmental independence of projections from olfactory neuron subclasses

In adult Xenopus, the nasal cavity is divided into separate middle (MC) and principal (PC) cavities; the former is used to smell water-borne odorants, the latter air-borne odorants. Recent work has shown that olfactory neurons of each cavity express a distinct subclass of odorant receptors. Moreover, MC and PC axons project to distinct regions of the olfactory bulb. To examine the developmental basis for this specificity in the olfactory projection, we extirpated the developing MC from early metamorphic (stage 54–57) tadpoles and raised the animals through metamorphosis. In most lesioned animals, the MC partly regenerated. Compared with the unlesioned side, reduction of the region of the glomerular layer of the olfactory bulb receiving MC afferents ranged from 70% to 95%. PC afferents did not occupy regions of the olfactory bulb deprived of MC afferents. These results support a model in which intrinsic cues in the olfactory bulb control the projection pattern attained by ingrowing olfactory axons. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 213–222, 1997.     

Stage-specific induction of DNA methyltransferases in olfactory receptor neuron development

DNA methylation-dependent gene silencing, mediated by DNA methyltransferases (DNMTs), is essential for normal mammalian development and its dysregulation has been implicated in neurodevelopmental disorders. Despite this, little is known about DNMTs in the developing or mature nervous system. Here, we show that DNMT1, 3a and 3b are expressed at discrete developmental stages in the olfactory neuron lineage, coincident with key shifts in developmental gene expression. DNMT1 is induced in cycling progenitors and is retained in post-mitotic olfactory receptor neurons (ORNs). DNMT3b is restricted to mitotic olfactory progenitors, whereas DNMT3a is expressed only in post-mitotic immature neurons prior to ORN terminal maturation, coincident with histone deacetylase 2 (HDAC2), a key downstream effector of methylation-dependent chromatin condensation. Similar stage-specific expression of DNMT3b and 3a was also found in other developing sensory and CNS neurons. This suggests that progressive lineage restriction regulated by methylation-dependent silencing could be a highly conserved mechanism shared by multiple lineages in the developing nervous system.     

Molecular cloning of a lobster Gβ subunit and Gβ expression in olfactory receptor neuron dendrites and brain neuropil

We have isolated from the olfactory organ of the American lobster (Homarus americanus) two cDNA clones with homology to β subunits of G proteins. LobG_β1 contained a complete open reading frame that predicted an amino acid sequence with >80% identity to G_β sequences from other species. LobG_β2 was a fragment of an open reading frame whose predicted amino acid sequence had 65–69% identity to other G_β sequences. LobG_β2 mRNA was not detectable in the brain, eye plus eyestalk, leg, dactyl, olfactory organ, or tail muscle. In contrast, lobG_β1 was expressed in all these tissues as a single mRNA species of 6.4 kb and a protein of 37 kD. In the brain and olfactory organ, G_β immunoreactivity was almost exclusively confined to neurites: the neuropil regions of the brain and the outer dendrites of the olfactory receptor neurons. Coimmunoprecipitation revealed that lobster G_β interacted with both G_αs and G_αq. LobG_β1 is likely to be involved in a wide range of signaling events including olfactory transduction and synaptic transmission in the brain. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 525–536     

Olfactory response termination involves Ca2+-ATPase in vertebrate olfactory receptor neuron cilia

In vertebrate olfactory receptor neurons (ORNs), odorant-induced activation of the transduction cascade culminates in production of cyclic AMP, which opens cyclic nucleotide–gated channels in the ciliary membrane enabling Ca²⁺ influx. The ensuing elevation of the intraciliary Ca²⁺ concentration opens Ca²⁺-activated Cl⁻ channels, which mediate an excitatory Cl⁻ efflux from the cilia. In order for the response to terminate, the Cl⁻ channel must close, which requires that the intraciliary Ca²⁺ concentration return to basal levels. Hitherto, the extrusion of Ca²⁺ from the cilia has been thought to depend principally on a Na⁺–Ca²⁺ exchanger.In this study, we show using simultaneous suction pipette recording and Ca²⁺-sensitive dye fluorescence measurements that in fire salamander ORNs, withdrawal of external Na⁺ from the solution bathing the cilia, which incapacitates Na⁺–Ca²⁺exchange, has only a modest effect on the recovery of the electrical response and the accompanying decay of intraciliary Ca²⁺ concentration. In contrast, exposure of the cilia to vanadate or carboxyeosin, a manipulation designed to block Ca²⁺-ATPase, has a substantial effect on response recovery kinetics. Therefore, we conclude that Ca²⁺-ATPase contributes to Ca²⁺ extrusion in ORNs, and that Na⁺–Ca²⁺exchange makes only a modest contribution to Ca²⁺ homeostasis in this species.     

Opposing effects of bone morphogenetic proteins on neuron production and survival in the olfactory receptor neuron lineage

In olfactory epithelium (OE) cultures, bone morphogenetic proteins (BMPs) can strongly inhibit neurogenesis. Here we provide evidence that BMPs also promote, and indeed are required, for OE neurogenesis. Addition of the BMP antagonist noggin inhibited neurogenesis in OE-stromal cell co-cultures. Bmp2, Bmp4 and Bmp7 were expressed by OE stroma, and low concentrations of BMP4 (below the threshold for inhibition of neurogenesis) stimulated neurogenesis; BMP7 did not exhibit a stimulatory effect at any concentration tested. Stromal cell conditioned medium also stimulated neurogenesis; part of this effect was due to the presence within it of a noggin-binding factor or factors. Studies of the pro-neurogenic effect of BMP4 indicated that it did not increase progenitor cell proliferation, but rather promoted survival of newly generated olfactory receptor neurons. These findings indicate that BMPs exert both positive and negative effects on neurogenesis, depending on ligand identity, ligand concentration and the particular cell in the lineage that is responding. In addition, they reveal the presence of a factor or factors, produced by OE stroma, that can synergize with BMP4 to stimulate OE neurogenesis.     

Efficient olfactory coding in the pheromone receptor neuron of a moth

The concept of coding efficiency holds that sensory neurons are adapted, through both evolutionary and developmental processes, to the statistical characteristics of their natural stimulus. Encouraged by the successful invocation of this principle to predict how neurons encode natural auditory and visual stimuli, we attempted its application to olfactory neurons. The pheromone receptor neuron of the male moth Antheraea polyphemus, for which quantitative properties of both the natural stimulus and the reception processes are available, was selected. We predicted several characteristics that the pheromone plume should possess under the hypothesis that the receptors perform optimally, i.e., transfer as much information on the stimulus per unit time as possible. Our results demonstrate that the statistical characteristics of the predicted stimulus, e.g., the probability distribution function of the stimulus concentration, the spectral density function of the stimulation course, and the intermittency, are in good agreement with those measured experimentally in the field. These results should stimulate further quantitative studies on the evolutionary adaptation of olfactory nervous systems to odorant plumes and on the plume characteristics that are most informative for the ‘sniffer’. Both aspects are relevant to the design of olfactory sensors for odour-tracking robots.     

Altered olfactory receptor neuron responsiveness in rare Ostrinia nubilalis males attracted to the O. furnacalis pheromone blend

Three percent of E-strain Ostrinia nubilalis males fly upwind in response to the Ostrinia furnacalis pheromone blend [a 40:60 ratio of (E)-12-tetradecenyl acetate to (Z)-12-tetradecenyl acetate (E12-14:OAc to Z12-14:OAc)], in addition to their own pheromone blend [a 99:1 ratio of (E)-11-tetradecenyl acetate to (Z)-11-tetradecenyl acetate) (E11-14:OAc to Z11-14:OAc)]. We assessed the olfactory receptor neuron (ORN) responses of these behaviorally "rare" males versus those of normal males. For the three ORNs housed within each sensillum, we tested responsiveness to Z12-14:OAc, E12-14:OAc, Z11-14:OAc, E11-14:OAc, and the behavioral antagonist (Z)-9-tetradecenyl acetate (Z9-14:OAc). Z11-14:OAc, E11-14:OAc, and Z9-14:OAc stimulated ORNs exhibiting distinct small, large, and medium spike sizes, respectively. For rare and normal males, both Z12-14:OAc and E12-14:OAc usually elicited responses from the largest-spiking ORN. In many ORNs of normal males, Z12-14:OAc or E12-14:OAc stimulated the smaller-spiking ORN that is responsive to Z11-14:OAc. In rare males, detectable ORN responses from the smaller-spiking ORN in response to Z12- and E12-14:OAc were virtually non-existent. These differences in ORN tuning in rare males will tend to create an ORN firing ratio between the large- and small-spiking ORNs in response to the O. furnacalis blend that is similar to that elicited by the O. nubilalis blend.     

Identification of E2/E3 ubiquitinating enzymes and caspase activity regulating Drosophila sensory neuron dendrite pruning

Ubiquitin-proteasome system (UPS) is a multistep protein degradation machinery implicated in many diseases. In the nervous system, UPS regulates remodeling and degradation of neuronal processes and is linked to Wallerian axonal degeneration, though the ubiquitin ligases that confer substrate specificity remain unknown. Having shown previously that class IV dendritic arborization (C4da) sensory neurons in Drosophila undergo UPS-mediated dendritic pruning during metamorphosis, we conducted an E2/E3 ubiquitinating enzyme mutant screen, revealing that mutation in ubcD1, an E2 ubiquitin-conjugating enzyme, resulted in retention of C4da neuron dendrites during metamorphosis. Further, we found that UPS activation likely leads to UbcD1-mediated degradation of DIAP1, a caspase-antagonizing E3 ligase. This allows for local activation of the Dronc caspase, thereby preserving C4da neurons while severing their dendrites. Thus, in addition to uncovering E2/E3 ubiquitinating enzymes for dendrite pruning, this study provides a mechanistic link between UPS and the apoptotic machinery in regulating neuronal process remodeling.     

SNP genotypes of olfactory receptor genes associated with olfactory ability in German Shepherd dogs

To find out the relationship between SNP genotypes of canine olfactory receptor genes and olfactory ability, 28 males and 20 females from German Shepherd dogs in police service were scored by odor detection tests and analyzed using the Beckman GenomeLab SNPstream. The representative 22 SNP loci from the exonic regions of 12 olfactory receptor genes were investigated, and three kinds of odor (human, ice drug and trinitrotoluene) were detected. The results showed that the SNP genotypes at the OR10H1‐like:c.632C>T, OR10H1‐like:c.770A>T, OR2K2‐like:c.518G>A, OR4C11‐like:c.511T>G and OR4C11‐like:c.692G>A loci had a statistically significant effect on the scenting abilities (P < 0.001). The kind of odor influenced the performances of the dogs (P < 0.001). In addition, there were interactions between genotype and the kind of odor at the following loci: OR10H1‐like:c.632C>T, OR10H1‐like:c.770A>T, OR4C11‐like:c.511T>G and OR4C11‐like:c.692G>A (P < 0.001). The dogs with genotype CC at the OR10H1‐like:c.632C>T, genotype AA at the OR10H1‐like:c.770A>T, genotype TT at the OR4C11‐like:c.511T>G and genotype GG at the OR4C11‐like:c.692G>A loci did better at detecting the ice drug. We concluded that there was linkage between certain SNP genotypes and the olfactory ability of dogs and that SNP genotypes might be useful in determining dogs' scenting potential.     

Extensive gains and losses of olfactory receptor genes in mammalian evolution

Odor perception in mammals is mediated by a large multigene family of olfactory receptor (OR) genes. The number of OR genes varies extensively among different species of mammals, and most species have a substantial number of pseudogenes. To gain some insight into the evolutionary dynamics of mammalian OR genes, we identified the entire set of OR genes in platypuses, opossums, cows, dogs, rats, and macaques and studied the evolutionary change of the genes together with those of humans and mice. We found that platypuses and primates have <400 functional OR genes while the other species have 800-1,200 functional OR genes. We then estimated the numbers of gains and losses of OR genes for each branch of the phylogenetic tree of mammals. This analysis showed that (i) gene expansion occurred in the placental lineage each time after it diverged from monotremes and from marsupials and (ii) hundreds of gains and losses of OR genes have occurred in an order-specific manner, making the gene repertoires highly variable among different orders. It appears that the number of OR genes is determined primarily by the functional requirement for each species, but once the number reaches the required level, it fluctuates by random duplication and deletion of genes. This fluctuation seems to have been aided by the stochastic nature of OR gene expression.     

Identification of thermosensory and olfactory neuron-specific genes via expression profiling of single neuron types

Most C. elegans sensory neuron types consist of a single bilateral pair of neurons, and respond to a unique set of sensory stimuli. Although genes required for the development and function of individual sensory neuron types have been identified in forward genetic screens, these approaches are unlikely to identify genes that when mutated result in subtle or pleiotropic phenotypes. Here, we describe a complementary approach to identify sensory neuron type-specific genes via microarray analysis using RNA from sorted AWB olfactory and AFD thermosensory neurons. The expression patterns of subsets of these genes were further verified in vivo. Genes identified by this analysis encode 7-transmembrane receptors, kinases, and nuclear factors including dac-1, which encodes a homolog of the highly conserved Dachshund protein. dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein. On thermal gradients, dac-1 mutants fail to suppress a cryophilic drive but continue to track isotherms at the cultivation temperature, representing the first genetic separation of these AFD-mediated behaviors. Expression profiling of single neuron types provides a rapid, powerful, and unbiased method for identifying neuron-specific genes whose functions can then be investigated in vivo.     

Differences in DBA/1J and DBA/2J reveal lipid QTL genes

Recent advances in mouse genomics have revealed considerable variation in the form of single-nucleotide polymorphisms (SNPs) among common inbred strains. This has made it possible to characterize closely related strains and to identify genes that differ; such genes may be causal for quantitative phenotypes. The mouse strains DBA/1J and DBA/2J differ by just 5.6% at the SNP level. These strains exhibit differences in a number of metabolic and lipid phenotypes, such as plasma levels of triglycerides (TGs) and HDL. A cross between these strains revealed multiple quantitative trait loci (QTLs) in 294 progeny. We identified significant TG QTLs on chromosomes (Chrs) 1, 2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 16, and 19, and significant HDL QTLs on Chrs 3, 9, and 16. Some QTLs mapped to chromosomes with limited variability between the two strains, thus facilitating the identification of candidate genes. We suggest that Tshr is the QTL gene for Chr 12 TG and HDL levels and that Ihh may account for the TG QTL on Chr 1. This cross highlights the advantage of crossing closely related strains for subsequent identification of QTL genes.     

Identification of olfactory receptor genes in Atlantic salmon Salmo salar

It has been hypothesized that salmonids use olfactory cues to return to their natal rivers and streams. The key components of the molecular pathways involved in imprinting and homing, however, are still unknown. Aquatic chemical cues are received through the nares and into the nasal cavity that contains a single olfactory organ, the olfactory rosette. The olfactory rosette contains sensory neurons, each of which is thought to express only one olfactory receptor. If odorants are involved in salmonid homing migration then olfactory receptors should play a critical role in the dissipation of information from the environment to the fish. Therefore, to understand the molecular basis for imprinting and homing in Atlantic salmon Salmo salar it is important to identify and characterize the repertoire of olfactory receptors in this species. The first public assembly of the S. salar genome was searched for genes encoding three of the superfamilies of fish olfactory receptors: V2R-like (olfc), V1R-like (ora) and main olfactory receptor (mor). A further six ora genes were added to ora1 and ora2, which had been described previously. In addition, 48 putative mors were identified, 24 of which appear to be functional based on their gene structures and predicted amino-acid sequences. Phylogenetic analyses were then used to compare these S. salar olfactory receptor genes with those of zebrafish Danio rerio, two pufferfish species Takifugu rubripes and Tetraodon nigroviridis, medaka Oryzias latipes and three-spined stickleback Gasterosteus aculeatus.     

Degeneration patterns of the olfactory receptor genes in sea snakes

The sense of smell relies on the diversity of olfactory receptor (OR) repertoires in vertebrates. It has been hypothesized that different types of ORs are required in terrestrial and marine environments. Here we show that viviparous sea snakes, which do not rely on a terrestrial environment, have significantly lost ORs compared with their terrestrial relatives, supporting the hypothesis. On the other hand, oviparous sea snakes, which rely on a terrestrial environment for laying eggs, still maintain their ORs, reflecting the importance of the terrestrial environment for them. Furthermore, we found one Colubroidea snake (including sea snakes and their terrestrial relatives)‐specific OR subfamily which had diverged widely during snake evolution after the blind snake–Colubroidea snake split. Interestingly, no pseudogenes are included in this subfamily in sea snakes, and this subfamily seems to have been expanding rapidly even in an underwater environment. These findings suggest that the Colubroidea‐specific ORs detect nonvolatile odorants.     

Pax3/7 genes reveal conservation and divergence in the arthropod segmentation hierarchy

Several features of Pax3/7 gene expression are shared among distantly related insects, including pair-rule, segment polarity, and neural patterns. Recent data from arachnids imply that roles in segmentation and neurogenesis are likely to be played by Pax3/7 genes in all arthropods. To further investigate Pax3/7 genes in non-insect arthropods, we isolated two monoclonal antibodies that recognize the products of Pax3/7 genes in a wide range of taxa, allowing us to quickly survey Pax3/7 expression in all four major arthropod groups. Epitope analysis reveals that these antibodies react to a small subset of Paired-class homeodomains, which includes the products of all known Pax3/7 genes. Using these antibodies, we find that Pax3/7 genes in crustaceans are expressed in an early broad and, in one case, dynamic domain followed by segmental stripes, while myriapods and chelicerates exhibit segmental stripes that form early in the posterior-most part of the germ band. This suggests that Pax3/7 genes acquired their role in segmentation deep within, or perhaps prior to, the arthropod lineage. However, we do not detect evidence of pair-rule patterning in either myriapods or chelicerates, suggesting that the early pair-rule expression pattern of Pax3/7 genes in insects may have been acquired within the crustacean-hexapod lineage.