昆虫嗅觉受体的研究进展

昆虫的嗅觉对昆虫的栖息地选择、觅食、群集、趋避、繁殖以及信息传递等行为具有重要的影响。对昆虫嗅觉机理的深入研究和嗅觉信号传导途径的完整阐述,是探索农业害虫的专一性防治的基础。嗅觉受体(olfactory receptors,Ors)是G蛋白偶联受体(G protein-coupled receptor)的一种,是嗅觉系统的关键成分。近年来嗅觉受体的研究日益受到关注。本文对昆虫嗅觉的基本过程、基因结构和表达调控特征、蛋白分子结构、生理功能、分布部位和相关配体的研究等进行了综述。     

Basal cells in the mouse olfactory epithelium after axotomy: immunohistochemical and electron-microscopic studies

Summary The olfactory epithelium of mice after axotomy was investigated to clarify the stem cells of olfactory cells by double immunostaining using antikeratin (MA903) and anti-bromodeoxyuridine (BrdU) antibodies and by conventional electron microscopy. When a single dose of BrdU was given to mice 9 days after axotomy, immunostaining for BrdU was found in the globose basal cells which were negative for MA903, but not in the basal cells proper which were positive for MA903. The BrdU-immunoreactive cells increased 3-to 6-fold over the number of these cells in the controls, indicating active cell proliferation. At other postoperative days (4 and 14 days), fewer BrdU-immunoreactive cells were found. Furthermore, three pulses of BrdU resulted in numerous BrdU-immunolabelings in the globose basal cells and a few in the basal cells proper. There was no detectable difference in the number of labeled basal cells proper in operated and unoperated mice. In the electron micrographs 9 days after axotomy, the basal cells proper, flat-shaped in unoperated mice, appeared cylindrical or pyramidal in shape and the globose basal cells often lay between the basal cells proper. In unoperated controls, the globose basal cells were located above the flat-shaped basal cells proper. The results suggest that the stem cells of the olfactory cells are globose basal cells and not basal cells proper, and that the shape of basal cells proper changes in relation to the active proliferation of stem cells.     

Bovine olfactory and nasal respiratory epithelium surfaces

Summary High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 m using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12–13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.     

Cell division in the olfactory epithelium of the lamprey,Lampetra fluviatilis

Summary The turnover of cells within the olfactory epithelium of the lamprey Lampetra fluviatilis was investigated using tritiated thymidine followed by autoradiography. It was found that cell division occurred in three distinct regions of the olfactory lamellae. Two of these regions — a distal lamellar region and a proximal lamellar region occurred outside the sensory area proper, but appeared to contribute cells to the sensory area as well as giving rise to secretory or ciliated cells outside the sensory area. A third region of division occurrred at the base of the sensory area. Division of specialised basal or blastema cells in this region gives rise to cells that are confined to the sensory region of the lamellae. These findings are discussed in the light of previous studies on cell replacement within the olfactory epithelium.     

The cell coat of the developing olfactory epithelium in the chick

Summary During development of the olfactory epithelium in the chick embryo, the cell coat is revealed by treatment with Ruthenium red. On day 4 of incubation the developing sensory epithelium displays a thicker apical and basal cell coat than the neighbouring head ectoderm. The lateral cell coat is of equal thickness in both epithelia. The apical cell coat of the olfactory epithelium increases in thickness from day 4 to day 19 of embryonic life, finally attaining a thickness of about 55 nm.This paper is dedicated to Dr. A.J. ZamoraPortions of this paper were taken from the thesis by the author in fulfilment of the requirement for the degree of Dr. rer. nat. at the University of Essen Supported by the Deutsche Forschungsgemeinschaft (SFB114)     

Effects of reversible nare occlusion on the development of the olfactory epithelium in the rabbit nasal septum

Summary To investigate environmental influences on the development of the olfactory epithelium, semi-thin sections were taken from the nasal septum of newborn and 30-dayold rabbits; the epithelial thickness and the number of olfactory knobs, supporting cells, dark basal cells, and receptor cells were compared. During normal development, a marked increase in epithelial thickness was found, largely because of an increase in the number of receptor cells. Whereas unilateral nare occlusion on day 1 resulted in 10% fewer receptor cells and 25% fewer knobs on the deprived side by day 30, nare occlusion either up to or after day 5 had little effect, and even temporary reopening from days 6–7 was sufficient to stimulate receptor-cell development on the occluded side. Although in these latter cases, a slight deprivation effect of 6% was still found in the number of receptor-cell nuclei, there was no longer a significant difference in the number of knobs between the open and closed sides. Thus, whereas exposure to the environment during the first days of life appears to be sufficient to stimulate sustained growth, the deprived epithelium may retain the capacity to respond to such cues beyond this time. However, as nare occlusion also had an effect on the respiratory epithelium and nasal lymphatic tissue, the nature of the cues stimulating receptor-cell development, whether olfactory or non-olfactory, is not yet clear.     

Primary structure of cAMP-gated channel from bovine olfactory epithelium

The complete amino-acid sequence of the bovine olfactory epithelium adenosine 3',5'cyclic monophosphate (cAMP)-gated channel has been determined by cloning and sequencing its cDNA. It exhibits a high degree of sequence homology with the cGMP-gated channel of rod photoreceptors, suggesting that cyclic nucleotide-gated channels fall into a new family of genetically related proteins.     

Muscarinic receptors binding in retinal pigment epithelium during rat development

[³H]Quinuclidinyl benzylate (³H-QNB) specific binding of the developing rat retinal pigment epithelium (RPE) and neural retina has been examined. The binding of³H-QNB to RPE was saturable and displaced by the antagonist pirenzepine. Scatchard analysis of³H-QNB binding showed two high affinity sites to RPE, with K_B=2.6nM and 45 nM. Specific³H-QNB binding membranes from neural retina exhibited a characteristic developmental profile. RPE showed a high density of³H-QNB binding sites through all developmental periods studied. The major onset of binding sites is at the time of RPE differentiation. Our data open the possibility of muscarinic receptors being involved in differentiation and/or proliferation of RPE.     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog,ox, rat,and dog

Summary A comparative study using freeze-fracturing has been made of surface structures of olfactory and nasal respiratory epithelia of frog, ox, rat and dog. Special attention has been paid to cilia and microvilli present at these surfaces, although the observations include various other structures such as small intracellular vacuoles present in the olfactory receptor endings and infrequent brush cells. Within the mucus overlying the olfactory epithelium membranous vesicles, often attached to olfactory cilia, are seen. Some of these show intramembranous particle distributions similar to those of the rest of the cilia, whereas others are devoid of particles. Smooth vesicles are also found in the mucus of other types of epithelium (respiratory epithelium and Bowman's glands). The freeze-fracture morphology of intracellular secretory vacuoles present in olfactory supporting, Bowman's and respiratory glandular cells of the frog is similar in all these epithelia. Quantitative comparisons are made of the different structures of interest. When corrected for cilia which were not observed, mammalian receptor endings bear 17 cilia on average, whereas frog receptor endings have 6 cilia. The relative magnitudes of the diameters of the cilia and microvilli are, except for frog, the same for all species studied. Dimensions of other structures e.g., axons, dendrites and dendritic endings are compared in the various species. Freeze-fracture diameters are usually larger than those seen by techniques using dehydration. Dendritic ending densities range from 4.5 × 10⁶ (frog) to 8.3 × 10⁶ (dog) endings per cm². Possible sex-dependent differences are only found for these densities and dendritic ending diameters.     

Ultrastructural studies on the epithelia of the olfactory organ of cyprinodonts (teleostei,cyprinodontoidea)

Summary The epithelia of the olfactory organ of two cyprinodontoid fish species were studied both by transmission and scanning electron microscopy. The relatively flat floor of the organ is covered by sensory and nonsensory epithelia. The latter is distributed in the form of bands or ridges separating distinct areas of sensory epithelium. Differences between the olfactory organs of the two species investigated related only to the topography and quantitative distribution of the epithelia. Their ultrastructural features are very similar. The nonsensory stratified squamous epithelium contains numerous goblet cells and surface cells provided with microridges. A hypothetical function of the microridges is discussed. The sensory epithelium consists mainly of basal, supporting, and two types of sensory cells, i.e., ciliated and microvillous receptor cells. The cilia exhibit a predominant 9+0 microtubule pattern. Both epithelia are covered by a mucus layer in which all surface structures seem to be embedded. The possible nature, origin, and movement mechanisms of the mucus are discussed.This work was supported by the Deutsche ForschungsgemeinschaftDedicated to Prof. Dr. med. W. Bargmann on the occasion of his 70th birthday     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary A comparison of the tight-junctions of various cell types in the nasal epithelia of frog, ox, rat and dog shows that Bowman's gland cells have lowest number of strands (4–8), whereas olfactory receptor and supporting, and ciliated respiratory cells show no conspicuous differences and have 6–11 strands. Tight-junctional strand numbers show slight species-dependent variations. In regions where three cells join (observed for receptor and respiratory cells), fracture faces show two parallel strands which fuse at certain points. These strands run perpendicularly to the rest of the tight-junctional belt, which also shows an increased number of strands (13–16) in this region.Tight-junctions of mammalian olfactory dendritic endings usually show strands composed of particles, whereas those of the other three epithelial cell types consist of continuous or discontinuous bars. Tight-junctions of dendritic endings of the frog also conform to the latter type. Differences in strand density are only slight and range from 16–27 strands/m. Small angular gap-junctions were observed only within the tight-junctions of supporting cells in the rat.     

Nitric oxide regulates neurogenesis in adult olfactory epithelium in vitro

Nitric oxide regulates neurogenesis in the developing and adult brain. The olfactory epithelium is a site of neurogenesis in the adult and previous studies suggest a role for nitric oxide in this tissue during development. We investigated whether neuronal precursor proliferation and differentiation is regulated by nitric oxide using primary cultures of olfactory epithelial cells and an immortalized, clonal, neuronal precursor cell line derived from adult olfactory epithelium. In these cultures NOS inhibition reduced cell proliferation and stimulated neuronal differentiation, including expression of a voltage-dependent potassium conductance of the delayed rectifier type. In the neuronal precursor cell line, differentiation was associated with a significant decrease in nitric oxide release. In contrast, addition of nitric oxide stimulated proliferation and reduced neuronal differentiation. Nitric oxide regulated olfactory neurogenesis independently of added growth factors. Taken together these results indicate that nitric oxide levels can regulate cell proliferation and neuronal differentiation of olfactory precursor cells.     

Molecular composition of tight and adherens junctions in the rat olfactory epithelium and fila

Tight and adherens junctions (TJs, AJs) between neurons, epithelial and glial cells provide barrier and adhesion properties in the olfactory epithelium (OE), and subserve functions such as compartmentalization and axon growth in the fila olfactoria (FO). Immunofluorescence and immunoelectronmicroscopy were combined in sections of rat OE and FO to document the cellular and subcellular localization of TJ proteins occludin(Occl), claudins(Cl) 1-5 and zonula occludens(ZO) proteins 1-3, and of AJ proteins N-cadherin(cad), E-cad, and alpha-, beta- and p120-catenin(cat). With the exception of Cl2, all TJ proteins were colocalized in OE junctions. Differences in relative immunolabeling intensities were noted between neuronal and epithelial TJs. In the FO, Cl5-reactivity was localized in olfactory ensheathing cell (OEC) junctions, Cl1-reactivity in the FO periphery, with differential colocalization with ZOs. Supporting cells formed N-cad-immunoreactive (ir) AJs with olfactory sensory neurons, E-cad-ir junctions with microvillar and gland duct cells, and both N-cad and E-cad-ir junctions in homotypic contacts. Alpha, beta- and p120-cat were localized in all AJs of the OE. AJs were scarce in the globose basal cell layer. Immature and mature neurons formed numerous contacts. In the FO, AJs were documented between OECs, between OECs and axons, and between axons. Most AJs colocalized N-cad with catenins, occasionally E-cad-ir AJs were found in the FO periphery. Characteristics of molecular composition suggest differential properties of TJs formed by neuronal, epithelial and glial cells in the OE and FO. The presence and molecular composition of AJs are consistent with a role of AJ proteins in neuroplastic processes in the peripheral olfactory pathway.     

Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory epithelial surfaces of frog,ox, rat,and dog

Summary The densities and diameters of intramembranous particles in olfactory and nasal respiratory structures of frog, ox, rat and dog have been compared using the freeze-fracture technique. Dendritic endings and the various segments of the cilia of the olfactory receptor cells of a given species have identical particle densities (700–1,800 particles/m² in P-and 100–600 in E-faces). Densities in P-faces of respiratory cilia are about 1/3 of those in the olfactory cilia. E-face particle densities of these respiratory cilia are often higher than P-face densities. Microvillus P-face densities range from 700–2,000 (respiratory cell microvilli) to 1,800–3,400 particles/m² (olfactory supporting and Bowman's gland microvilli). Microvillus E-faces show no conspicuous mutual differences. Literature comparisons showed that odour concentrations at threshold are considerably lower (10⁵–10¹⁰ times) than the concentrations of olfactory receptor ending intramembranous particles (5 M–30 M) expressed in the same units.Relative differences in particle distributions of the various cell structures studied are usually species-independent. Absolute values vary considerably with the species. Relative P-face particle densities of the supporting cell microvilli tend to correlate with those of dendritic ending structures. Particle diameters are usually similar for corresponding structures and fracture faces in the four species. Apical structures of supporting and Bowman's gland cells in rat and dog show rod-shaped particle aggregates in their P-and pits in their E-faces. Neither sex-dependency nor an influence related to physiological treatments on the particle distributions could be demonstrated.     

A morphometric comparison of the olfactory epithelium of newborn and weanling rabbits

Summary As part of a study of the development of olfactory function in the rabbit, a morphometric analysis of the olfactory epithelium in newborn and 30-day-old animals was carried out. Surface area, thickness and cell densities of the olfactory epithelium were compared in hematoxylin-eosin stained serial sections through the nasal cavities of 4 newborn and 3 weanling rabbits. While the basic structure of the olfactory cavity changed little with age, a large quantitative development in the epithelium was observed. The pattern of growth appeared uniform and resulted in a 3-fold increase in total surface area from about 1 cm² per side in the newborn to about 3 cm² in the weanling, and an increase in thickness from approximately 65 m to about 90 m. The increase in thickness was due mainly to a disproportionate, 5-fold increase in the number of olfactory neurons. This resulted in a total of about 32 million cells per side by day 30, and represented an increase in the ratio of neurons to basal cells of 7:1 to 10:1, and neurons to supporting cells of 2:1 to 4:1. While such an increase in the number of primary neurons presumably improves the animal's perceptual abilities, it nevertheless raises the question as to how perceptual constancy can be maintained during a period of such rapid neural change.     

Involvement of muscarinic acetylcholine receptors in chloride secretion by cultured rat epididymal epithelium

The aim of our present study was to investigate the short-circuit current response to carbachol in cultured rat cauda epididymal epithelia and the signal transduction mechanisms involved. Carbachol added basolaterally induced a concentration-dependent increase in short-circuit current (Isc) across the epididymal epithelium consisting of a rapidly rising phase and a long term sustained response. The response was almost abolished by removing Cl(-) from the extracellular medium and blockable by pretreating the tissues with DPC, indicating a substantial contribution of Cl(-) secretion to the carbachol-induced response. The muscarinic acetylcholine receptor antagonist atropine inhibited the response, but the nicotinic acetylcholine receptors antagonist curarine had no effect, suggesting that only the muscarinic acetylcholine receptors mediated the secretory response of the basolateral side of rat cauda epididymal epithelium to carbachol. Addition of carbachol to the apical side of the tissue was found not to elicit an Isc response. These results suggested that muscarinic receptors are present in the basolateral side of rat cauda epididymal epithelium. Activation of these receptors by acetylcholine released from the nerve endings regulates epididymal transepithelial Cl(-) secretion. Cholinergic stimulation therefore contributes to the formation of luminal fluid microenvironment.     

Six1 is essential for early neurogenesis in the development of olfactory epithelium

The olfactory epithelium (OE) is derived from the olfactory placode (OP) during mouse development. At embryonic day (E) 10.0-E10.5, “early neurogenesis” occurs in the OE, which includes production of pioneer neurons that emigrate out of the OE and other early-differentiated neurons. Around E12.5, the OE becomes organized into mature pseudostratified epithelium and shows “established neurogenesis,” in which olfactory receptor neurons (ORNs) are differentiated from basal progenitors. Little is known about the molecular pathway of early neurogenesis. The homeodomain protein Six1 is expressed in all OP cells and neurogenic precursors in the OE. Here we show that early neurogenesis is severely disturbed despite the unaltered expression of Mash1 at E10.5 in the Six1-deficient mice (Six1^−/−). Expression levels of neurogenin1 (Ngn1) and NeuroD are reduced and those of Hes1 and Hes5 are augmented in the OE of Six1^−/− at E10.5. Pioneer neurons and cellular aggregates, which are derived from the OP/OE and situated in the mesenchyme between the OE and forebrain, are completely absent in Six1^−/−. Moreover, ORN axons and the gonadotropin-releasing hormone-positive neurons fail to extend and migrate to the forebrain, respectively. Our study indicates that Six1 plays critical roles in early neurogenesis by regulating Ngn1, NeuroD, Hes1, and Hes5.