The Effect of Electrotonus on the Olfactory Epithelium

The effect of electrotonus on the slow potential of the olfactory epithelium of the frog was studied. The "on"-slow potential induced by a general odor like amyl acetate increased its magnitude in accordance with increase of anodal current, while it decreased its magnitude with increase of cathodal current. Similar relations were also found in the case of the vapors of organic solvents like ethyl ether of low concentrations. Conversely, the on-slow potential induced by the vapors of organic solvents of high concentration decreased its magnitude in accordance with the increase of anodal current, while it increased its magnitude with the increase of cathodal current. The "off"-slow potential induced by the vapors of organic solvents of high concentration showed a potential change under the action of electrotonic currents which is similar to the change of the on-slow potential induced by general odors. It was concluded that there are two receptive processes in the olfactory cell. One is an ordinary excitatory process which produces an electronegative slow potential in response to general odors. The other is a process of a different kind which is activated only by the vapor of an organic solvent of high concentration and which shows an entirely opposite reaction from that generally found in excitable tissues when an electrotonic current is applied.     

The Olfactory System in the Hagfish Myxine glutinosa

The apical part of the olfactory epithelium in Myxine glutinosa was investigated by optical and electron microscopy. This part of the epithelium consists of supporting cells and two types of olfactory receptor cells, i.e., ciliated receptor cells and microvillous receptor cells. The olfactory cilia have a 9 + 0 pattern of the microtubules, occasionally with one pair of the doublets dislocated towards the center of the cilium. Giant cilia were observed. The supporting cells bear microvilli and are rich in tonofilaments. The supporting cells also have a secretory function, their secretion consisting mainly of acid mucopolysaccharides. An asymmetrical type of desmosome was found between the olfactory receptor cells and the supporting cells.     

Spontaneous Gating of Olfactory Cyclic-Nucleotide-Gated Channels

In vertebrates, cilia on the olfactory receptor neurons have a high density of cyclic-nucleotide-gated (CNG) channels. During transduction of odorous stimuli, cyclic AMP is formed. cAMP gates the CNG channels and this initiates the neuronal depolarization. Here it is shown that the ciliary CNG channels also open spontaneously. In the absence of odorants and second messengers, olfactory cilia have a small basal conductance to cations. Part of this conductance is similar to the cAMP-activated conductance in its sensitivity to channel inhibitors and divalent cations. The basal conductance may help to stabilize the neuronal membrane potential while limiting the sensitivity of odorant detection. Received: 30 May 2000/Revised: 8 August 2000     

Ultrastructural Aspects of Olfactory Signaling

The olfactory area of the nasal cavity is lined with olfactoryreceptor cell cilia that come in contract with incoming odormolecules. Ultrastructural immunocytochemical studies in rodentshave shown that these cilia contain all the proteins necessaryto transduce the odorous message into an electrical signal thatcan be transmitted to the brain. These signaling proteins includeputative odor receptors, GTP binding proteins, type III adenylylcyclase and cyclic nucleotide-gated channels. The rest of thecells, including dendrites and dendritic knobs, showed no discerniblelabeling with antibodies to these signaling proteins. Furthermore,freeze-fracture and freeze-etch studies have shown that themembrane morphology of olfactory cilia differs substantiallyfrom that of non-sensory cilia. Olfactory cilia have many moremembrane particles. Transmembrane signaling proteins, such asodor receptors, adenylyl cyclase and cyclic nucleotide-gatedchannels, conceivably appear as membrane particles. Thus, thelong-standing supposition that olfactory cilia are peculiarlyadapted to deal with the reception and initial transductionof odorous messages has now been verified in terms of both ultrastructuralmorphology and cytochemistry. Emerging studies on vomeronasalreceptor cell microvilli indicate that the same is true forthis organ, even though the actual signaling components differfrom those of the main olfactory system. Chem. Senses 22: 295–311,1997.     

Cyclic AMP diffusion coefficient in frog olfactory cilia

Cyclic AMP (cAMP) is one of the intracellular messengers that mediate odorant signal transduction in vertebrate olfactory cilia. Therefore, the diffusion coefficient of cAMP in olfactory cilia is an important factor in the transduction of the odorous signal. We have employed the excised cilium preparation from the grass frog (Rana pipiens) to measure the cAMP diffusion coefficient. In this preparation an olfactory cilium is drawn into a patch pipette and a gigaseal is formed at the base of the cilium. Subsequently the cilium is excised, allowing bath cAMP to diffuse into the cilium and activate the cyclic nucleotide-gated channels on the plasma membrane. In order to estimate the cAMP diffusion coefficient, we analyzed the kinetics of the currents elicited by step changes in the bath cAMP concentration in the absence of cAMP hydrolysis. Under such conditions, the kinetics of the cAMP-activated currents has a simple dependence on the diffusion coefficient. From the analysis we have obtained a cAMP diffusion coefficient of 2.7 +/- 0.2. 10(-6) cm2 s-1 for frog olfactory cilia. This value is similar to the expected value in aqueous solution, suggesting that there are no significant diffusional barriers inside olfactory cilia. At cAMP concentrations higher than 5 microM, diffusion slowed considerably, suggesting the presence of buffering by immobile cAMP binding sites. A plausible physiological function of such buffering sites would be to prolong the response of the cell to strong stimuli.     

Oscillatory Electric Potential on the Olfactory Epithelium Observed during the Breeding Migration Period in the Japanese Toad, Bufo japonicus

Japanese toad (Bufo japonicus) tracks the route to and from the breeding sites using the olfactory cues from the migration route and not from the destination (). We recorded a slow extracellular potential change (electro-olfactogram or EOG) evoked on the olfactory epithelium by applying an olfactory stimulus with an air stream. In September toads, only a simple typical EOG that is common in various vertebrate species was observed. Oscillatory potential changes (OSC) superimposed on the typical EOG were observed in the breeding season when studied throughout a year. There were no sexual differences in the occurrence and the amplitude of the OSC. Oscillatory potentials were observed also from the olfactory nerve of the brain. The OSC in the olfactory epithelium remained even after denervation. In addition, it was suggested that there are multiple sites of OSC initiation in the olfactory epithelium. These results suggest an intimate relationship between OSC appearance and the breeding migration in the toad.     

Prion Shedding from Olfactory Neurons into Nasal Secretions

This study investigated the role of prion infection of the olfactory mucosa in the shedding of prion infectivity into nasal secretions. Prion infection with the HY strain of the transmissible mink encephalopathy (TME) agent resulted in a prominent infection of the olfactory bulb and the olfactory sensory epithelium including the olfactory receptor neurons (ORNs) and vomeronasal receptor neurons (VRNs), whose axons comprise the two olfactory cranial nerves. A distinct glycoform of the disease-specific isoform of the prion protein, PrP^Sc, was found in the olfactory mucosa compared to the olfactory bulb, but the total amount of HY TME infectivity in the nasal turbinates was within 100-fold of the titer in the olfactory bulb. PrP^Sc co-localized with olfactory marker protein in the soma and dendrites of ORNs and VRNs and also with adenylyl cyclase III, which is present in the sensory cilia of ORNs that project into the lumen of the nasal airway. Nasal lavages from HY TME-infected hamsters contained prion titers as high as 10^3.9 median lethal doses per ml, which would be up to 500-fold more infectious in undiluted nasal fluids. These findings were confirmed using the rapid PrP^Sc amplification QuIC assay, indicating that nasal swabs have the potential to be used for prion diagnostics. These studies demonstrate that prion infection in the olfactory epithelium is likely due to retrograde spread from the olfactory bulb along the olfactory and vomeronasal axons to the soma, dendrites, and cilia of these peripheral neurons. Since prions can replicate to high levels in neurons, we propose that ORNs can release prion infectivity into nasal fluids. The continual turnover and replacement of mature ORNs throughout the adult lifespan may also contribute to prion shedding from the nasal passage and could play a role in transmission of natural prion diseases in domestic and free-ranging ruminants.     

Slow potentials of the olfactory bulb in the carp

The slow negative potentials evoked in carp olfactory bulb (OB) by some odorants and slow positive potentials evoked by nonspecific irritation (water stream, NaCl solutions) of olfactory epithelium have been studied. The slow potentials of both types were not inverted in deep layers of OB and were resistant to blockade of synaptic transmission by manganese ions. The negative slow potentials were not also affected by hypoxia and associated with local increase of OB tissue resistance. Positive slow potentials were affected by hypoxia and associated with local decrease of OB tissue resistance. The electrical tetanization of local zones of olfactory epithelium evoked in OB steady potential shifts of negative polarity, but diffuse tetanization of olfactory nerve evoked shifts of positive polarity. The results support the hypothesis of glial origin of slow potentials. Possible mechanisms of slow negative and positive potential generation are discussed.     

The Olfactory System in the Pacific Hagfishes Eptatretus stoutii,Eptatretus deani,and Myxine circifrons

In front of the olfactory organ in the northeastern Pacific hagfishes Eptatretus stoutii, E. deani, and Myxine circifrons there is a valve that may function to direct water in between the olfactory laminae. In Myxine circifrons the well developed valve is supposed to act alone, whereas the smaller valve in the two species of Eptatretus studied is supposed to act together with the horizontal extensions of the median olfactory lamina. No significant differences were found between the investigated species by ultrastructural examination. In the olfactory epithelium the supporting cells are provided with microvilli and generally contain a great amount of light secretory granules. Both ciliated olfactory receptor cells and microvillous olfactory receptor cells are present. The cilia show a 9 + 0 arrangement of the microtubules with a tendency for a dislocation of one pair of the microtubules toward the center of the cilium. These remarkable features of the olfactory receptor cells, not yet seen in other vertebrates, appear to be a character common to the myxinoid cyclostomes.     

Cartilaginous torus epithelium of the vomeronasal organ of swine and sheep]

The vomeronasal organ consists of receptor cells of microvillous type, supporting and basal cells. According to their ultrastructural organization the microvillar cells are analogous to those in the main olfactory organ in the pig and have all signs of the receptor cell: microvilli at the top and centrioles in cytoplasm, as well as the central process getting off the cell body. Both in the pig and in the sheep the supporting cells contain in their apical region a number of basal bodies with cilia, getting them off. In the receptor zones of epithelium albuminous glands predominate, in the respiratory zones--mucous ones. A great amount of liquid mucus, excreted on the surface of the epithelium by numerous glands and supporting cells, apparently, facilitates adsorption and desorption of odorous molecules from the receptor cells after their stimulation. The cilia of the supporting cells probably from the stream of the vomeronasal mucus. The cartilagenous torus epithelium of the vomeronasal organ of the pig and sheep has in general a similar structural organization. This demonstrates general for Vertebrata receptor mechanisms of odorous substances, evidently connected with perception of feramones or contact olfaction.     

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.     

Odorant-induced responses recorded from olfactory receptor neurons using the suction pipette technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert & Zhao, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca(2+)-activated Cl(-) channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body. In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors. The suction pipette technique was later adapted to also record from ORNs. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.     

OLFACTORY CILIA IN THE FROG

Olfactory epithelium from the frog was examined in the living state by light microscopy and in the fixed state by electron microscopy. Particular attention was paid to the layer of cilia and mucus which covers the surface of the epithelium. The olfactory cilia differed from typical cilia in that they (a) arose from bipolar neurons and had centrioles near their basal bodies, (b) were up to 200 microns in length, of which the greater part was a distal segment containing an atypical array of ciliary fibers, (c) were often immotile, (d) had their distal segments arranged in parallel rows near the surface of the mucus, and (e) had many vesicles along their shafts and had splits in the array of fibers in their distal segments. These specializations make the olfactory cilia similar to cilia found on other sensory cells and support the theory that they are the locus where electrical excitation in the olfactory organ is initiated by contact with odorous substances.     

Formation and maturation of olfactory cilia monitored by odorant receptor-specific antibodies

The responsiveness of olfactory sensory neurons (OSNs) is based on odorant receptors (ORs) residing in the membrane of chemosensory cilia. It is still elusive as to when and how olfactory cilia are equipped with OR proteins rendering them responsive to odorants. To monitor the appearance of OR proteins in sensory compartments of OSNs, the olfactory epithelium of mice at various stages of prenatal development (lasting 19 days from conception) was investigated using immunohistochemical approaches and antibodies specific for different OR subtypes. These experiments uncovered that OR proteins accumulated in dendritic knobs of OSNs before the initiation of ciliogenesis (embryonic stage E12). As the first cilia were formed (E13), immunostaining in the knobs diminished. Cilia extended uprightly into the nasal cavity and were immunoreactive along the entire length, and particularly intense labeling was observed in expanded tips of cilia. During this phase of development (up to E18), the number of cilia per knob continuously increased. In the course of perinatal stages, longer cilia began to bend off and lie flat on the epithelial surface. The multiple cilia of a knob extended in length, and eventually the ciliary meshwork reached the characteristic complex pattern. In all stages, OR immunostaining was visible along the entire cilium. Thus, OR-specific antibodies allowed, for the first time, monitoring at the level of light microscopy the generation, outgrowth, and maturation of cilia in OSNs.     

Olfactory metamorphosis in the Coastal Giant Salamander (Dicamptodon tenebrosus)

This study examined the gross morphology and ultrastructure of the olfactory organ of larvae, neotenic adults, and terrestrial adults of the Coastal Giant Salamander (Dicamptodon tenebrosus). The olfactory organ of all aquatic animals (larvae and neotenes) is similar in structure, forming a tube extending from the external naris to the choana. A nonsensory vestibule leads into the main olfactory cavity. The epithelium of the main olfactory cavity is thrown into a series of transverse valleys and ridges, with at least six dorsal and nine ventral valleys lined with olfactory epithelium, and separated by ridges of respiratory epithelium. The ridges enlarge with growth, forming large flaps extending into the lumen in neotenes. The vomeronasal organ is a diverticulum off the ventrolateral side of the main olfactory cavity. In terrestrial animals, by contrast, the vestibule has been lost. The main olfactory cavity has become much broader and dorsoventrally compressed. The prominent transverse ridges are lost, although small diagonal ridges of respiratory epithelium are found in the lateral region of the ventral olfactory epithelium. The posterior and posteromedial wall of the main olfactory cavity is composed of respiratory epithelium, in contrast to the olfactory epithelium found here in aquatic forms. The vomeronasal organ remains similar to that in large larvae, but is now connected to the mouth by a groove that extends back through the choana onto the palate. Bowman's glands are present in the main olfactory cavity at all stages, but are most abundant and best developed in terrestrial adults. They are lacking in the lateral olfactory epithelium of the main olfactory cavity. At the ultrastructural level, in aquatic animals receptor cells of the main olfactory cavity can have cilia, short microvilli, a mix of the two, or long microvilli. Supporting cells are of two types: secretory supporting cells with small, electron-dense secretory granules, and ciliated supporting cells. Receptor cells of the vomeronasal organ are exclusively microvillar, but supporting cells are secretory or ciliated, as in the main olfactory cavity. After metamorphosis two distinct types of sensory epithelium occur in the main olfactory cavity. The predominant epithelium, covering most of the roof and the medial part of the floor, is characterized by supporting cells with large, electron-lucent vesicles. The epithelium on the lateral floor of the main olfactory cavity, by contrast, resembles that of aquatic animals. Both types have both microvillar and ciliated receptor cells. No important changes are noted in cell types of the vomeronasal organ after metamorphosis. A literature survey suggests that some features of the metamorphic changes described here are characteristic of all salamanders, while others appear unique to D. tenebrosus.     

Olfactory Evoked Potential Produced by Electrical Stimulation of the Human Olfactory Mucosa

Most physiological studies of the human olfactory system haveconcentrated on the cortical level; the olfactory bulbar levelhas been studied rarely. We attempted to stimulate the humanolfactory mucosa by electrical pulse to detect the bulbar potentials.Electrical stimulation (2 mA, 0.5 ms) of the human olfactorymucosa evoked a change in potential recorded from the frontalsector of the head. A negative peak of the evoked potentialthat occurred at 19.4 ms (grand means, n = 5) after stimulationwas the clearest. The highest amplitude of the potential wasrecorded from the frontal sector of the head on the stimulatedside. Our findings were similar to the experimental resultsobtained from the olfactory bulbs of animals. This evoked potentialwas considered to be the human olfactory bulbar potential. Whenthe subjects were stimulated by applying electricity to theolfactory mucosa, no sensation of smell occurred even thoughevoked potentials were recorded. Evoked potentials were recordedonly when the stimulating electrode was located in the olfactorycleft. When the stimulating electrode was outside the olfactorycleft, the stimulation caused pain. The trigeminal nerve seemedto be stimulated by electricity. Olfactory evoked potentialsproduced by the electrical stimulation of the human olfactorymucosa should aid the research on human olfactory physiology,and may be applicable to clinical tests of olfactory dysfunction.Chem. Senses 22: 77–81, 1997.     

Localization of Phosphatase Responsible for Hydrolysis of Inositol 1,4,5-Triphosphate in the Olfactory Lining of True Sturgeons

The paper presents results of a cytochemical study of localization of phosphatase responsible for hydrolysis of inositol 1,4,5-triphosphate (ITP) in the olfactory lining of true sturgeons (the sturgeon, starred sturgeon, and sterlet). Reaction products as a dark discrete granules are localized in the apical parts of epithelium, practically in the same manner in all the species studied. The precipitate is found on the plasma membranes of cilia, microvilli, and clava of the olfactory cells. Occasionally, the precipitate is also found in the cilia, basal bodies, and rootlets of microvillar cells. The ITP-hydrolyzing phosphatase is supposed to restrict development of transduction process by removing excess messengers from the operating system. The data obtained indicate that in the true sturgeons, the phospholipase cascade of olfactory transduction is concentrated predominantly in the cilia and microvilli of olfactory cells.     

Structure and Development of the Olfactory Organ in the Garfish Belone belone (L.) (Teleostei,Atheriniformes)

Theisen, B., Breucker, H., Zeiske, E., Melinkat, R. 1980. Structure and development of the olfactory organ in the garfish Belone belone (L.) (Teleostei, Atheriniformes). (Institute of Comparative Anatomy, University of Copenhagen, Denmark; Anatomisches Institut, Universität Hamburg, and Zoologisches Institut und Zoologisches Museum, Universität Hamburg, Federal Republic of Germany.) — Acta zool. (Stockh.) 61(3): 161–170. The structure and development of the olfactory organ in the garfish Belone belone (L.) were studied by light and electron microscopy (SEM and TEM). The olfactory organ has the shape of an open groove with a protruding papilla. In embryos and early juveniles the groove is smooth and is provided with a continuous sensory epithelium. During ontogenesis the papilla develops and the composition of the epithelium is changed as areas of nonsensory epithelium appear and eventually separate the sensory epithelium into islets. In adults the sensory epithelium consists of supporting, basal, and two types of receptor cells, ciliated and microvillous. In juveniles also ciliated nonsensory cells are present. This difference can be correlated with differing locomotory habits of adults and juveniles. The receptor cilia show a 9 + 0 microtubular pattern while the nonsensory cilia have the general 9 + 2 pattern. Deviating dendritic endings were found and are considered an indication of ongoing cell dynamics.     

鲻鱼嗅囊组织形态结构观察及功能探讨

实验用鱼为全长35.5~40.0 cm的野生鲻(Mugil cephalus),采用石蜡切片以及透射电镜技术对鲻的嗅囊以及嗅板细胞进行观察。结果表明:鲻的嗅觉器官由左右两个呈扁平椭球形嗅囊构成,分别由前后两个鼻孔与外界相通。嗅囊长径与眼径之比为0.80,长径与短径之比为2.09。嗅囊的嗅轴左右两边分别有垂直于嗅轴并向上倾斜排列整齐的18~25个披针形嗅板,只有初级嗅板未见次级嗅板。嗅板由中央髓和两侧的嗅上皮两部分构成,中央髓由疏松的结缔组织和毛细血管组成。嗅上皮又分为感觉区和非感觉区,感觉区位于嗅板的内侧,具有发达纤毛,呈连续分布状态,非感觉区位于嗅板边缘,细胞纤毛较少。通过光镜和电镜的综合研究结果显示嗅上皮细胞大致可分为5类:基细胞、支持细胞、纤毛非感觉细胞、纤毛感觉细胞和柱状细胞。文章讨论了鲻的感官活动类型。