Isolation and Characterization of the Olfactory Epithelial Cells of the Catfish

A preparation enriched in olfactory receptor cells has beenobtained from the olfactory mucosa of the catfish (Ictaluruspunctatus). The tissue was treated successively with trypsin,DNase, trypsin inhibitor, EDTA in Ca^{+ +} , Mg^{+ +} free mediumaccording to a method derived from that of Cohen, et al.⁽¹⁾.After mechanical disruption of the isolated olfactory lamellae,the cells were isolated by centrifugation on a Ficoll gradient.Each type of cell was morphologically identified by comparingin situ and in vitro preparations by SEM. Small round cellswere collected on 10% Ficoll. The nature of these cells is notknown but part of them are certainly basal cells which havebeen shown⁽²⁾ to be the precursors of the constantly regeneratingolfactory neurons. Respiratory cells settled mainly on 20% Ficoll.A fraction containing 60% sustentacular cells was collectedon 33% Ficoll. Olfactory cells characterized by an axon, a dendriteand several cilia, were found on 37% Ficoll. This fraction alsocontains up to 40% sustentacular cells. A yield of 20% was measuredfor olfactory cell isolation. Vital staining and ability tosynthesize RNA indicate a viability of the final preparationof 70% to 80%. Further identification of the cells was performedby measuring the binding activity of a series of amino acidsto a preparation enriched in olfactory cells. A good correlationwas determined between the extent of the binding and the reportedelectrophysiological activities of these amino acids recordedin vivo. Although the final olfactory cell suspension is notpure, it constitutes the first step in the study of the olfactoryreceptor sites.     

Taste Responses to Binary Mixtures of Amino Acids in the sea catfish, Arius felis

In vivo electrophysiological recordings in the sea catfish,Arius felis, showed that the magnitude of the integrated facialtaste responses to binary mixtures of amino acids was predictablewith knowledge obtained from previous cross-adaptation studiesof the relative independence of the respective binding sitesof the component stimuli. Each component from which equal aliquotswere drawn to form the mixtures was adjusted in concentrationto provide for approximately equal reponse magnitudes. The magnitudeof the taste responses to binary mixtures whose component aminoacids showed minimal cross-adaptation was significantly greaterthan that to binary mixtures whose components exhibited considerablecross-reactivity. There was no evidence for mixture suppression.The relative magnitude of the taste responses in the sea catfishto stimulus mixtures is similar to that previously reportedfor olfactory receptor responses in the freshwater channel catfishand chorda tympani taste responses in the hamster. Chem. Senses21: 45–53, 1996.     

Evidence for Ca(2+)-permeable AMPA receptors in the olfactory bulb

-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (AMPARs), a subtype of glutamate receptor, contribute to olfactory processing in the olfactory bulb (OB). These ion channels consist of various combinations of the subunits GluR1–GluR4, which bestow certain properties. For example, AMPARs that lack GluR2 are highly permeable to Ca²⁺ and generate inwardly rectifying currents. Because increased intracellular Ca²⁺ could trigger a host of Ca²⁺-dependent odor-encoding processes, we used whole cell recording as well as histological and immunocytochemical (ICC) techniques to investigate whether AMPARs on rat OB neurons flux Ca²⁺. Application of 1-naphthylacetyl spermine (NAS), a selective antagonist of Ca²⁺-permeable AMPARs (CP-AMPARs), inhibited AMPAR-mediated currents in subsets of interneurons and principal cells in cultures and slices. The addition of spermine to the electrode yielded inwardly rectifying current-voltage plots in some cells. In OB slices, olfactory nerve stimulation elicited excitatory responses in juxtaglomerular and mitral cells. Bath application of NAS with D,L-2-amino-5-phosphonovaleric acid (AP5) to isolate AMPARs suppressed the amplitudes of these synaptic responses compared with responses obtained using AP5 alone. Co²⁺ staining, which involves the kainate-stimulated influx of Co²⁺ through CP-AMPARs, produced diverse patterns of labeling in cultures and slices as did ICC techniques used with a GluR2-selective antibody. These results suggest that subsets of OB neurons express CP-AMPARs, including functional CP-AMPARs at synapses. Ca²⁺ entry into cells via these receptors could influence odor encoding by modulating K⁺ channels, N-methyl-D-aspartate receptors, and Ca²⁺-binding proteins, or it could facilitate synaptic vesicle fusion. GluR2; polyamines; cobalt; glutamate receptor; olfaction     

UMBONIUM VESTIARIUM (L.) (GASTROPODA, TROCHACEA) AS THE FOOD SOURCE FOR NATICID GASTROPODS AND A STARFISH ON A MALAYSIAN SANDY SHORE

Umbonium vestiarium (L.) forms virtually the entire diet of3 (possibly 4) species of naticid snails and the starfish Astropectenvappa Mueller annd Troschel on some north Penang sandy shores.Umbonium comprises about 99% of numbers and tissue of macrofauna.Predation totalled some 1.75 Umbonium (ca. 33 mg dry tissue).m^-2.day^-1 across much of the downshore sand flats rising to 2.3Umbonium (ca. 45 mg). m^-2. day^-1 near MLWS. Natica maculosaLamarck comprised > 80% of the predators and took 77–94%of the Umbonium eaten. Natica antonii Phillippi alone addedto this toll on the upper reaches of the zone while Polinicesspp and Astropecten appear to have taken 12–14% of thetotal toll of Umbonium near MLWS. Total predation is indicatedat 237–327 kJ.m^-2. year^-1 across the shore and this representsalmost the total flow of energy from primary consumers to intertidalbenthic predators on such shores and accounts for some 15.6%(lower shore)—20.5% (upper shore) of total Umbonium production. (Received 10 September 1982;     

Bimodal (Taste/Tactile) Fibers Innervate the Maxillary Barbel in the Channel Catfish

Analysis of single fibers isolated from a branch of the facial/trigeminalcomplex innervating the maxillary barbel of the channel catfish,Ictalurus punctatus, indicated the existence of bimodal (taste/tactile)fibers. Of the 60 single fibers recorded, 14 (23%) respondedto both taste (amino acid) and tactile stimulation, 43 (72%)were responsive to only tactile stimulation and three (5%) respondedonly to taste stimulation. Quinine hydrochloride at a concentrationof 1.0 mM suppressed the mechanosensory activity of the bimodalfibers, but had no effect on the tactile-only fibers. Chem.Senses 22: 477–482, 1997. ¹Current address: Department of Otolaryngology, Kagoshima UniversityMedical School, 8-35-1 Sakuragaoka, Kagoshima 890, Japan ²Current address: Department of Oral Physiology, Ohu UniversitySchool of Detistry, 31-1 Misumido, Tomita, Koriyama, Fukushima963, Japan     

Gut evacuation rates and ingestion rates of Eudiaptomus graciloides measured by means of the gut fluorescence method

Journal of Plankton Research, 8, 973–983, 1986 FIg. 2. Time-dependent changes in the gut content (percentageof initial ng pigment) of E. gro.ciloides at different temperaturesunder simultaneous feeding. Fig. 4. The relationship between instantaneous evacuation rateand temperature of E. graciloides. The regresston equation forfeeding animals: y = 0.0044 e(0.141 ) (r² = 0.90). For comparisonthe results of non-feeding animals are indicated with open circles.     

Studies on Mechanoperception in Characean Cells: Pharmacological Analysis

The mechanisms for generating receptor potentials and actionpotentials upon mechanical stimulation were studied in internodalcells of Chara. Receptor potentials and the subsequent actionpotentials could be generated even when the electrogenic protonpump was inhibited, indicating that the proton pump does notplay a central role in generating receptor potentials and actionpotentials. The involvement of Ca²⁺ and/or Cl^– channelsin both receptor and action potentials was suggested, basedon the equilibrium potentials of these ions across the plasmamembrane. Inhibitors of the Ca²⁺ channel, Cl^– channeland stretch-activated channel could not inhibit generation ofthe receptor potential. These findings suggested that the channelsinvolved in generating the receptor potential are insensitiveto these channel inhibitors, although all inhibitors significantlyinhibited the action potential. (Received July 26, 1996; Accepted November 19, 1996)     

Electrophysiological characterization of the node in Chara corallina: functional differentiation for wounding response

Electrical characteristics of the node were analyzed in comparisonwith those of the flank of the internodal cell in Chara corallina.The dependence of the membrane potential of the node on pH andK⁺ concentration was almost the same as that of the flank. Inthe flank, the increase in the Ca²⁺ concentration stopped thedepolarization in the presence of 100 mM KCl. In the node, however,Ca²⁺ could not stop the depolarization induced by 100 mM KCl.It has been reported that the node has a function to tranducethe signal of osmotic shock into a transient depolarization.In combination with osmotic shock, 10 mM K⁺ could induce a long-lastingdepolarization of the node. These electrical characteristicsof the node were suggested to be responsible for the electricalresponse to wounding in Characeae.     

The Olfactory System as a Model for the Analysis of the Contribution of Gene Expression to Programmed Cell Death

The process of programmed cell death is frequently attenuatedby inhibitors of protein and RNA synthesis. This implies thatgene expression is necessary for the active elimination of somecell types. Genes such as bcl-2 and bax have been implicatedin the direct control of cell death, while cellular immediate-earlygenes (clEGs), such as c-fos and c-jun have been repeatedlyassociated with neuronal degeneration. We are using the olfactoryneuroepithelium as a model system to investigate the role thatexpression of such genes might play in cell death. The advantagesof this system is that even in the adult, there is spontaneousdegeneration of olfactory receptor neurons followed by theirreplacement by the division and differentiation of precursors.Futhermore, the receptor neurons can be induced to die synchronouslyby removal of the olfactory bulb or intranasal administrationof toxic agents. We have generated fos-lacZ and jun-lacZ transgenicmice that can be used to assess expression of c-fos and c-junfollowing these various manipulations. In addition, a line oftransgenic mice has been derived that express Bcl-2 under thecontrol of the olfactory receptor protein promoter. These micehave high levels of Bcl-2 selectively in receptor neurons ofthe primary neuro-epithelium and vomeronasal organ. Since insome circumstances, Bcl-2 can protect against programmed celldeath these mice are being assessed for neuronal turnover underbasal conditions and following olfactory bulbectomy.     

Effects of Blue Light Pretreatments on Internode Extension Growth in Mustard Seedlings after the Transition to Darkness: Analysis of the Interaction with Phytochrome

The effects of blue light (B) pretreatments on internode extensiongrowth and their possible interaction with phytochrome mediatedresponses were examined in Sinapis alba seedlings grown for11 d under 280 µmol m^–2 s^–1 of continuousblue-deficient light from low pressure sodium lamps (SOX). SupplementaryB (16 µmol m^–2 s^–1) caused no detectable inhibitionof the first internode growth rate under continuous SOX, butgrowth rate was inhibited after transfer to darkness. This effect,and the growth promotion caused by far-red bend-of-day' lightpulses were additive. The addition of B at 16 µmol m^–2s^–1 during 11 d, or only during the first 9 or 10 d orthe latest 0.75, 1 or 2 d of the SOX pretreatment caused approximatelythe same extent of inhibition after the transition to darkness.A single hour of supplementary B before darkness caused morethan 50% of the maximum inhibition. However, 24 h of lower fluencerates of B (4 or 7 µmol m^–2 s^–1) were ineffective.Covering the internode during the supplementary B period didnot prevent the response to B after the transition to darkness.Far-red light given simultaneously with B (instead of the SOXbackground) reduced the inhibitory effect of B. Above a given threshold fluence rate, B perceived mainly inthe leaves inhibits extension growth in subsequent darkness,provided that high phytochrome photo-equilibria are presentduring the irradiation with B. Once triggered, this effect doesnot interact significantly with the ‘end-of-day’phytochrome effect. Key words: Blue light, extension growth, phytochrome     

Modulation of K+ channels by arachidonic acid in T84 cells. II. Activation of a Ca2+-independent K+ channel

We usedsingle-channel recording techniques to identify and characterize alarge-conductance,Ca²⁺-independentK⁺ channel in the colonicsecretory cell line T84. In symmetric potassium gluconate, this channelhad a linear current-voltage relationship with a single-channelconductance of 161 pS. Channel open probability(P_o) wasincreased at depolarizing potentials. Partial substitution of bathK⁺ withNa⁺ indicated a permeability ratioof K⁺ toNa⁺ of 25:1. ChannelP_o was reduced byextracellular Ba²⁺. Event-durationanalysis suggested a linear kinetic model for channel gating having asingle open state and three closed states: C₃C₂C₁O.Arachidonic acid (AA) increased theP_o of thechannel, with an apparent stimulatory constant(K_s)of 1.39 µM. Neither channel open time (O) nor the fast closed time(C₁) was affected by AA. Incontrast, AA dramatically reduced mean closed time by decreasing bothC₃ andC₂. Thecis-unsaturated fatty acid linoleate increased P_oalso, whereas the saturated fatty acid myristate and thetrans-unsaturated fatty acid elaidatedid not affectP_o. This channelis activated also by negative pressure applied to the pipette duringinside-out recording. Thus we determined the effect of thestretch-activated channel blockers amiloride and Gd³⁺ on theK⁺ channel after activation by AA.Amiloride (2 mM) on the extracellular side reduced single-channelamplitude in a voltage-dependent manner, whereasGd³⁺ (100 µM) had no effect onchannel activity. Activation of this K⁺ channel may be important duringstimulation of Cl secretionby agonists that use AA as a second messenger (e.g., vasoactiveintestinal polypeptide, adenosine) or during the volume regulatoryresponse to cell swelling.

     

Difference in Behavior Between Responses to Forskolin and General Odorants in Turtle Vomeronasal Organ

To elucidate the signal transduction mechanisms in the turtlevomeronasal receptor neurons, the effects of forskolin, changesin mucosal Ca²⁺ concentrations and ruthenium red on the responsesof the accessory olfactory bulb to general odorants were examined.Forskolin elicited a large response, suggesting that there arecAMP-gated channels in the vomeronasal neurons. On the otherhand, the dependence of the responses to general odorants onCa²⁺ concentrations was different from that of the responseto forskolin. A large response to an odorant (n-amyl acetate)appeared after the cAMP-mediated pathway was fully desensitizedby application of 50 µM forskolin. These results suggestthat the cAMP-mediated pathway does not contribute significantlyto generation of the response to general odorants. A concentrationof 50 µM ruthenium red significantly reduced the responsesto n-amyl acetate alone and after 50 µM forskolin desensitization,suggesting that the inositol triphosphate-mediated pathway contributespartly to generation of the responses to general odorants inthe vomeronasal neurons. Chem Senses 21: 763–771, 1996.     

Evidence for electrogenic active ion transport across the frog olfactory mucosa in vitro

We present the first electrophysiological evidence for electrogenicion transport across the frog olfactory mucosa in vitro. Whenthe isolated dorsal mucosa was placed in an Ussing chamber andbathed symmetrically in amphibian Ringer's, the ciliated sidebecame electronegative (V = –5.2 mV ± 0.7 mV).The resistance of the mucosal preparation was 148 ± 4 cm². The true short-circuit current was obtained as the intersectionof the I–V curve with the current axis after correctingfor the series solution resistance. The average value of theshort-circuit current was 35.9 µA/cm². The I–V relationwas linear over the applied potential range of ± 16mV.The magnitude of the specific resistance of the olfactory mucosais comparable to values reported for various actively transportingrespiratory and oral cavity epithelia. Because the geometricalarea of the aperture used to normalize both the short-circuitcurrent and the resistance undoubtedly underestimates the actualarea of the dorsal olfactory epithelium, the specific resistanceand the short-circuit current are probably underestimated andoverestimated, respectively. Therefore, the nominally low resistanceneed not imply a leaky epithelium. Substitution of NO₃^–for Cl^– caused the current to increase and the resistanceto decrease. These results suggest that cation absorption playsa role in the sign of the short-circuit current. The in vitropreparation responded to the odorant ethyl n-butyrate by givingan electro-olfactogram (EOG)-like voltage transient which wassuperimposed on the steady-state potential created by activeion transport. The significance of these results is discussedfrom the perspective of the peripheral events surrounding olfactorytransduction.     

Effect of Nasal Dilators on Perceived Odor Intensity

Subjects wearing nasal dilators rated olfactory stimuli as beingmore intense compared with ratings done without nasal expansion.The results support a perceptual constancy model in olfaction.Chem. Senses 22: 177–180, 1997. ¹Present address: Biology Department, St Lawrence UniversityCanton, NY 13617, USA ²Present address: PO Box 802, Drew University, Madison, NJ 07940,USA     

Lotus Lectin Labels Subpopulation of Olfactory Receptor Cells

Experiments were performed to test the hypothesis that subsetsof olfactory receptor cells could be recognized based on theirlectin binding and that mapping of their projections onto theolfactory bulb would reveal details of anatomic organizationof the olfactory nerve projection to the olfactory bulb. Theresults from one lectin, Lotus, were examined in detail. Olfactoryreceptor cells in the lateral part of the main epithelium werelabeled, as well as scattered cells in the remainder of theepithelium. Glomeruli labled by Lotus were concentrated primarilyin the region of the olfactory bulb that receives its inputfrom the lateral epithelium, although scattered glomeruli couldbe identified in other regions. Within the terminal field ofthese axons there was a mosaic pattern, with some glomerulidensely labeled, some lightly labeled and others unlabeled.These findings support the notion that there are biochemicallydistinct populations of olfactory receptor cells having localizeddistributions in the epithelium, with axons that coalesce toterminate in specific glomeruli, rather than diffusely overtheir projection field. Chem. Senses 21: 13–18, 1996     

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time¹. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)^2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning^4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs^6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode³. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings⁸. We provide details of our experimental setup and present representative recording traces for each of these techniques.     

The Site of Action of General Anaesthetics in Insect Olfactory Receptor Neurons

The effect of volatile anaesthetics such as N₂O, Xe, short-chainalkanes and cyclopropane, at pharmacologically relevant concentrations,on olfactory receptor neurons of insects was tested in electrophysiologicalrecordings. CO₂-receptor neurons in moths and files respondwith increased action potential activity, whereas in adherenceto the Meyer-Overton rule; alkanes of a chain length of 5 andabove are less effective or evoke suppression of action potentials.In olfactiory receptor neurons sensitive to benzoic acid infemale moths of Bombyx mori and in pheromone receptor neuronsof male moths of Antheraea polyphemus, anaesthetics are ineffectiveif applied alone; if superimposed on an excitatory olfactorystimulus, an inhibitory effect occurs, Local stimulation ofonly part of a sensory dendrite reveals that the anaestheticsreversibly block the reception of pheromone or its effect onthe conductance of the receptor cell memebrane. The observedinteractions are consistent with the hypothesis that the anaestheticsdo not interact with the primary transduction process, but ratheraffect a later stage such as the activation of ion channels. ^*Dedicated to H-J. Bestman, on the occasion of his 70th birthday.     

PlasmoGF: an integrated system for comparative genomics and phylogenetic analysis of Plasmodium gene families

Summary: Malaria, one of the world's most common diseases, iscaused by the intracellular protozoan parasite known as Plasmodium.Recently, with the arrival of several malaria parasite genomes,we established an integrated system named PlasmoGF for comparativegenomics and phylogenetic analysis of Plasmodium gene families.Gene families were clustered using the Markov Cluster algorithmimplemented in TribeMCL program and could be searched usingkeywords, gene-family information, domain composition, GeneOntology and BLAST. Moreover, a number of useful bioinformaticstools were implemented to facilitate the analysis of these putativePlasmodium gene families, including gene retrieval, annotation,sequence alignment, phylogeny construction and visualization.In the current version, PlasmoGF contained 8980 sets of genefamilies derived from six malaria parasite genomes: Plasmodium.falciparum, P. berghei, P. knowlesi, P. chabaudi, P. vivax andP. yoelii. The availability of such a highly integrated systemwould be of great interest for the community of researchersworking on malaria parasite phylogenomics. Availability: PlasmoGF is freely available at http://bioinformatics.zj.cn/pgf/ Contact: xiaokunli{at}163.net; baoqy{at}genomics.org.cn; fuz3{at}psu.edu Associate Editor: Jonathan Wren The authors wish it to be known that, in their opinion, thefirst two authors should be regarded as joint First Authors.     

A comparison of meta-analysis methods for detecting differentially expressed genes in microarray experiments

Motivation: The proliferation of public data repositories createsa need for meta-analysis methods to efficiently evaluate, integrateand validate related datasets produced by independent groups.A t-based approach has been proposed to integrate effect sizefrom multiple studies by modeling both intra- and between-studyvariation. Recently, a non-parametric ‘rank product’method, which is derived based on biological reasoning of fold-changecriteria, has been applied to directly combine multiple datasetsinto one meta study. Fisher's Inverse ² method, which only dependson P-values from individual analyses of each dataset, has beenused in a couple of medical studies. While these methods addressthe question from different angles, it is not clear how theycompare with each other. Results: We comparatively evaluate the three methods; t-basedhierarchical modeling, rank products and Fisher's Inverse ²test with P-values from either the t-based or the rank productmethod. A simulation study shows that the rank product method,in general, has higher sensitivity and selectivity than thet-based method in both individual and meta-analysis, especiallyin the setting of small sample size and/or large between-studyvariation. Not surprisingly, Fisher's ² method highly dependson the method used in the individual analysis. Application toreal datasets demonstrates that meta-analysis achieves morereliable identification than an individual analysis, and rankproducts are more robust in gene ranking, which leads to a muchhigher reproducibility among independent studies. Though t-basedmeta-analysis greatly improves over the individual analysis,it suffers from a potentially large amount of false positiveswhen P-values serve as threshold. We conclude that careful meta-analysisis a powerful tool for integrating multiple array studies. Contact: fxhong{at}jimmy.harvard.edu Supplementary information: Supplementary data are availableat Bioinformatics online. Associate Editor: David Rocke Present address: Department of Biostatistics and ComputationalBiology, Dana-Farber Cancer Institute, Harvard School of PublicHealth, 44 Binney Street, Boston, MA 02115, USA.     

Permeability of Lipophilic Cations

The permeability (P) of a lipophilic cation, triphenylmethylphosphonium(TPMP⁺) which is frequently used as a membrane potential probe,has been measured in Chara australis (Charophyceae). P_TPMP+across biological membranes is usually thought to be very highbut this is not the case across the plasmalemma of Chara. Thepermeability of TPMP⁺ across the plasmalemma was found to betypical of inorganic cations, about 1.0 nm s^–1. Estimateswere made of the permeability of lipophilic cations across someother cell membranes, based on previously published work. Thepermeability of TPMP⁺ across the plasma membranes of the redalga, Griffithsia monilis and the blue-green alga, Anabaenavariabilis was about 2–5 nm s^–1. The permeabilityof TPMP⁺ across the plasma membranes of eukaryotes and prokaryotesappears to be similar. The permeability of lipophilic cationsacross the cristae of isolated mitochondria are exceptionallyhigh, about 170 nm s^–1. TPMP⁺ did not behave as a thiamineanalogue in Chara, unlike in the case of yeast. The means ofentry of TPMP⁺ into the Chara cell, driven by the electrochemicalgradient across the plasmalemma, has not been identified. Thepresence of a second lipophilic cation probe, DDA⁺ (dibenzyldimethylammonium),caused a decrease in the uptake flux of TPMP⁺; this suggeststhat the two lipophilic cations compete for the same site atthe surface of the plasmalemma. Key words: Chara australis, TPMP⁺, Permeability, Lipophilic cation