Neurestin: putative transmembrane molecule implicated in neuronal development.

We have cloned a novel cDNA encoding a putative transmembrane protein, neurestin, from the rat olfactory bulb. Neurestin was identified based on a sequence similar to that of the second extracellular loops of odorant receptors in the cysteine-rich CC box located immediately after EGF-like motifs. Neurestin shows homology to a neuregulin gene product, human gamma-heregulin, a Drosophila receptor-type pair-rule gene product, Odd Oz (Odz) / Ten(m), and Ten(a), suggesting a possible function in synapse formation and morphogenesis. Recently, a mouse neurestin homolog has independently been cloned as DOC4 from the NIH-3T3 cell line. Northern blot analysis showed that neurestin is highly expressed in the brain and also in other tissues at much lower levels. In situ hybridization studies showed that neurestin is expressed in many types of neurons, including pyramidal cells in the cerebral cortex and tufted cells in the olfactory bulb during development. In adults, neurestin is mainly expressed in olfactory and hippocampal granule cells, which are known to be generated throughout adulthood. Nonetheless, in adults the expression of neurestin was experimentally induced in external tufted cells during regeneration of olfactory sensory neurons. These results suggest a role for neurestin in neuronal development and regeneration in the central nervous system.     

Expression of odorant receptor family, type 2 OR in the aquatic olfactory cavity of amphibian frog Xenopus tropicalis

Recent genome wide in silico analyses discovered a new family (type 2 or family H) of odorant receptors (ORs) in teleost fish and frogs. However, since there is no evidence of the expression of these novel OR genes in olfactory sensory neurons (OSN), it remains unknown if type 2 ORs (OR2) function as odorant receptors. In this study, we examined expression of OR2 genes in the frog Xenopus tropicalis. The overall gene expression pattern is highly complex and differs depending on the gene and developmental stage. RT-PCR analysis in larvae showed that all of the OR2η genes we identified were expressed in the peripheral olfactory system and some were detected in the brain and skin. Whole mount in situ hybridization of the larval olfactory cavity confirmed that at least two OR2η genes so far tested are expressed in the OSN. Because tadpoles are aquatic animals, OR2η genes are probably involved in aquatic olfaction. In adults, OR2η genes are expressed in the nose, brain, and testes to different degrees depending on the genes. OR2η expression in the olfactory system is restricted to the medium cavity, which participates in the detection of water-soluble odorants, suggesting that OR2ηs function as receptors for water-soluble odorants. Moreover, the fact that several OR2ηs are significantly expressed in non-olfactory organs suggests unknown roles in a range of biological processes other than putative odorant receptor functions.     

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod(+/lacZ) mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for beta-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

Neurons with choline acetyltransferase immunoreactivity and mRNA are present in the human cerebral cortex

 We examined the cerebral cortex of five autopsied individuals without neurological and psychiatric diseases by immunohistochemistry using an anti-human recombinant choline acetyltransferase (ChAT) polyclonal antibody and in situ hybridization with ³⁵S-labeled human ChAT riboprobes. The immunohistochemistry detected positive neurons which were medium-sized or large pyramidal neurons located predominantly in layers III and V. The density of such neurons was higher in the motor and secondary sensory areas than in other cortical areas; the immunoreactive neurons in layer V were more densely distributed in the motor area and those in layer III were distributed in the secondary sensory areas. Positively stained, non-pyramidal neurons were observed in the superficial layer of the cingulate gyrus and parahippocampus. No immunoreactive neurons were found in the primary sensory areas. The in situ hybridization detected some neurons with signals for ChAT mRNA in the cerebral cortex, most of which were distributed in layer V of the motor area and in layer III of the secondary visual area. These results indicate that the human cerebral cortex contains cholinergic neurons and displays regional and laminal variations in their distribution. Accepted: 17 November 1998     

Roles of odorant receptors in projecting axons in the mouse olfactory system

In the mouse olfactory epithelium, there are about ten million olfactory sensory neurons, each expressing a single type of odorant receptor out of approximately 1000. Olfactory sensory neurons expressing the same odorant receptor converge their axons to a specific set of glomeruli on the olfactory bulb. How odorant receptors play an instructive role in the projection of axons to the olfactory bulb has been one of the major issues of developmental neurobiology. Recent studies revealed previously overlooked roles of odorant receptor-derived cAMP signals in the axonal projection of olfactory sensory neurons; the levels of cAMP and neuronal activity appear to determine the expression levels of axon guidance/sorting molecules and thereby direct the axonal projection of olfactory sensory neurons. These findings provide new insights as to how peripheral inputs instruct neuronal circuit formation in the mammalian brain.     

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod^+/lacZ mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for β-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

Interchromosomal interactions and olfactory receptor choice

The expression of a single odorant receptor (OR) gene from a large gene family in individual sensory neurons is an essential feature of the organization and function of the olfactory system. We have used chromosome conformation capture to demonstrate the specific association of an enhancer element, H, on chromosome 14 with multiple OR gene promoters on different chromosomes. DNA and RNA fluorescence in situ hybridization (FISH) experiments allow us to visualize the colocalization of the H enhancer with the single OR allele that is transcribed in a sensory neuron. In transgenic mice bearing additional H elements, sensory neurons that express OR pseudogenes also express a second functional receptor. These data suggest a model of receptor choice in which a single trans-acting enhancer element may allow the stochastic activation of only one OR allele in an olfactory sensory neuron.     

A contextual model for axonal sorting into glomeruli in the mouse olfactory system

No models fully account for how odorant receptors (ORs) function in the guidance of axons of olfactory sensory neurons (OSNs) to glomeruli in the olfactory bulb. Here, we use gene targeting in mice to demonstrate that the OR amino acid sequence imparts OSN axons with an identity that allows them to coalesce into glomeruli. Replacements between the coding regions of the M71 and M72 OR genes reroute axons to their respective glomeruli. A series of M71-M72 hybrid ORs uncover a spectrum of glomerular phenotypes, leading to the concept that the identity of OSN axons is revealed depending on what other axons are present. Naturally occurring amino acid polymorphisms in other ORs also produce distinct axonal identities. These critical amino acid residues are distributed throughout the protein and reside predominantly within transmembrane domains. We propose a contextual model for axon guidance in which ORs mediate homotypic interactions between like axons.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

5-HT<Subscript>1A</Subscript> receptor expression in pyramidal neurons of cortical and limbic brain regions

We studied expression of the 5-HT_1A receptor in cortical and limbic areas of the brain of the tree shrew. In situ hybridization with a receptor-specific probe and immunocytochemistry with various antibodies was used to identify distinct neurons expressing the receptor. In vitro receptor autoradiography with ³H-8-OH-DPAT (³H-8-hydroxy-2-[di-n-propylamino]tetralin) was performed to visualize receptor-binding sites. In the prefrontal, insular, and occipital cortex, 5-HT_1A receptor mRNA was expressed in pyramidal neurons of layer 2, whereas ³H-8-OH-DPAT labeled layers 1 and 2 generating a columnar-like pattern in the prefrontal and occipital cortex. In the striate and ventral occipital cortex, receptor mRNA was present within layers 5 and 6 in pyramidal neurons and Meynert cells. Pyramid-like neurons in the claustrum and anterior olfactory nucleus also expressed the receptor. Principal neurons in hippocampal region CA1 expressed 5-HT_1A receptor mRNA, and ³H-8-OH-DPAT labeled both the stratum oriens and stratum radiatum. CA3 pyramidal neurons displayed low 5-HT_1A receptor expression, whereas granule neurons in the dentate gyrus revealed moderate expression of this receptor. In the amygdala, large pyramid-like neurons in the basal magnocellular nucleus strongly expressed the receptor. Immunocytochemistry with antibodies against parvalbumin, calbindin, and gamma aminobutyric acid (GABA) provided no evidence for 5-HT_1A receptor expression in GABAergic neurons in cortical and limbic brain areas. Our data agree with previous findings showing that the 5-HT_1A receptor mediates the modulation of glutamatergic neurons. Expression in the limbic and cortical areas suggested an involvement of 5-HT_1A receptors in emotional and cognitive processes.This work was supported by the German Science Foundation (SFB 406; C4 to G.F.).     

The Molecular Phylogeny of a Nematode-Specific Clade of Heterotrimeric G-Protein α-Subunit Genes

In animal olfactory systems, odorant molecules are detected by olfactory receptors (ORs). ORs are part of the G-protein-coupled receptor (GPCR) superfamily. Heterotrimeric guanine nucleotide binding G-proteins (G-proteins) relay signals from GPCRs to intracellular effectors. G-proteins are comprised of three peptides. The G-protein α subunit confers functional specificity to G-proteins. Vertebrate and insect Gα-subunit genes are divided into four subfamilies based on functional and sequence attributes. The nematode Caenorhabditis elegans contains 21 Gα genes, 14 of which are exclusively expressed in sensory neurons. Most individual mammalian cells express multiple distinct GPCR gene products, however, individual mammalian and insect olfactory neurons express only one functional odorant OR. By contrast C. elegans expresses multiple ORs and multiple Gα subunits within each olfactory neuron. Here we show that, in addition to having at least one member of each of the four mammalian Gα gene classes, C. elegans and other nematodes also possess two lineage-specific Gα gene expansions, homologues of which are not found in any other organisms examined. We hypothesize that these novel nematode-specific Gα genes increase the functional complexity of individual chemosensory neurons, enabling them to integrate odor signals from the multiple distinct ORs expressed on their membranes. This neuronal gene expansion most likely occurred in nematodes to enable them to compensate for the small number of chemosensory cells and the limited emphasis on cephalization during nematode evolution. [Reviewing Editor: Dr. John Oakeshott] Damien M. O’Halloran and David A. Fitzpatrick contributed equally to this work.     

Hedonic taste in Drosophila revealed by olfactory receptors expressed in taste neurons

Taste and olfaction are each tuned to a unique set of chemicals in the outside world, and their corresponding sensory spaces are mapped in different areas in the brain. This dichotomy matches categories of receptors detecting molecules either in the gaseous or in the liquid phase in terrestrial animals. However, in Drosophila olfactory and gustatory neurons express receptors which belong to the same family of 7-transmembrane domain proteins. Striking overlaps exist in their sequence structure and in their expression pattern, suggesting that there might be some functional commonalities between them. In this work, we tested the assumption that Drosophila olfactory receptor proteins are compatible with taste neurons by ectopically expressing an olfactory receptor (OR22a and OR83b) for which ligands are known. Using electrophysiological recordings, we show that the transformed taste neurons are excited by odor ligands as by their cognate tastants. The wiring of these neurons to the brain seems unchanged and no additional connections to the antennal lobe were detected. The odor ligands detected by the olfactory receptor acquire a new hedonic value, inducing appetitive or aversive behaviors depending on the categories of taste neurons in which they are expressed i.e. sugar- or bitter-sensing cells expressing either Gr5a or Gr66a receptors. Taste neurons expressing ectopic olfactory receptors can sense odors at close range either in the aerial phase or by contact, in a lipophilic phase. The responses of the transformed taste neurons to the odorant are similar to those obtained with tastants. The hedonic value attributed to tastants is directly linked to the taste neurons in which their receptors are expressed.     

Expression and intracellular localisation of odorant receptors in mammalian cell lines using Semliki Forest virus vectors

Odorant receptors are members of the G protein-coupled receptor superfamily. They are expressed on the surface of cilia of olfactory neurons, where they bind ligand (odorant). Studies of the molecular mechanisms of olfaction are complicated by the extremely large number of receptor genes, and difficulties in pairing a particular mammalian receptor to a specific odorant ligand in vivo. Here we report expression and localisation studies of two rat odorant receptor genes (17 and OR5), and C. elegans odr-10, using the Semliki Forest virus (SFV) system. All receptors were epitope-tagged at the N- or C-terminus in order to facilitate their detection in infected cells, and determine the localisation and membrane-orientation of recombinant proteins. The immortalised mouse olfactory neuronal cell line OLF 442, rat cortical and striatal primary neuron cultures, and the baby hamster kidney (BHK) cells, were infected and tested. Immunofluorescence and confocal microscopy studies performed on permeabilised, non-permeabilised and native cells revealed that in BHK cells the rat receptors 17 and OR5 were not targeted to the plasma membrane and remained in the endoplasmic reticulum. In contrast, in the mouse olfactory cell line OLF 442 both rat receptors were correctly inserted into the plasma membrane. Similar results were obtained using primary neurons, indicating that like mature neurons, the immortalised OLF 442 cells are capable of providing for correct odorant receptor processing and targeting.     

Minigenes impart odorant receptor-specific axon guidance in the olfactory bulb

An olfactory sensory neuron (OSN) expresses selectively one member from a repertoire of approximately 1000 odorant receptor (OR) genes and projects its axon to a specific glomerulus in the olfactory bulb. Both processes are here recapitulated by MOR23 and M71 OR minigenes, introduced into mice. Minigenes of 9 kb and as short as 2.2 kb are selectively expressed by neurons that do not coexpress the endogenous gene but coproject their axons to the same glomeruli. Deletion of a 395 bp upstream region in the MOR23 minigene abolishes expression. In this region we recognize sequence motifs conserved in many OR genes. Transgenic lines expressing the OR in ectopic epithelial zones form ectopic glomeruli, which also receive input from OSNs expressing the cognate endogenous receptor. This suggests a recruitment through homotypic interactions between OSNs expressing the same OR.     

Developmental regulation of olfactory circuit formation in mice

In mammals, odorants induce various behavioral responses that are critical to the survival of the individual and species. Binding signals of odorants to odorant receptors (ORs) expressed in the olfactory epithelia are converted to an odor map, a pattern of activated glomeruli, in the olfactory bulb (OB). This topographic map is used to identify odorants for memory-based learned decisions. In the embryo, a coarse olfactory map is generated in the OB by a combination of dorsal-ventral and anterior-posterior targeting of olfactory sensory neurons (OSNs), using specific sets of axon-guidance molecules. During the process of OSN projection, odor signals are sorted into distinct odor qualities in separate functional domains in the OB. Odor information is then conveyed by the projection neurons, mitral/tufted cells, to various regions in the olfactory cortex, particularly to the amygdala for innate olfactory decisions. Although the basic architecture of hard-wired circuits is generated by a genetic program, innate olfactory responses are modified by neonatal odor experience in an activity-dependent manner. Stimulus-driven OR activity promotes post-synaptic events and dendrite selection in the responding glomeruli making them larger. As a result, enhanced odor inputs in neonates establish imprinted olfactory memory that induces attractive responses in adults, even when the odor quality is innately aversive. In this paper, I will provide an overview of the recent progress made in the olfactory circuit formation in mice.     

BIG-2 mediates olfactory axon convergence to target glomeruli

Olfactory sensory neurons expressing a given odorant receptor converge axons onto a few topographically fixed glomeruli in the olfactory bulb, leading to establishment of the odor map. Here, we report that BIG-2/contactin-4, an axonal glycoprotein belonging to the immunoglobulin superfamily, is expressed in a subpopulation of mouse olfactory sensory neurons. A mosaic pattern of glomerular arrangement is observed with strongly BIG-2-positive, weakly positive, and negative axon terminals in the olfactory bulb, which is overlapping but not identical with those of Kirrel2 and ephrin-A5. There is a close correlation between the BIG-2 expression level and the odorant receptor choice in individual sensory neurons. In BIG-2-deficient mice, olfactory sensory neurons expressing a given odorant receptor frequently innervate multiple glomeruli at ectopic locations. These results suggest that BIG-2 is one of the axon guidance molecules crucial for the formation and maintenance of functional odor map in the olfactory bulb.