T-type Ca2+

T-type Ca 2+channels contribute to IBMX/forskolin-and K +-induced Ca 2+transients in porcine olfactory receptor neuronsShree Hari Gautam a ,Ken-ichi Otsuguro b ,Shigeo Ito b ,Toshiyuki Saito c ,Yoshiaki Habara a ,*aLaboratory of Physiology,Department of Biomedical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,JapanbLaboratory of Pharmacology,Department of Biomedical Sciences,Graduate School of Veterinary Medicine,Hokkaido University,Sapporo 060-0818,JapancLaboratory of Neurobiology,National Institute of Agrobiological Sciences,Tsukuba 305-0901,JapanReceived 3August 2006;accepted 25September 2006Available online 30October 2006AbstractT-type Ca 2+channels are low-voltage-activated Ca 2+channels that control Ca 2+entry in excitable cells during small depolarization above resting ing Ca 2+imaging with a laser scanning confocal microscope we investigated the involvement of T-type Ca 2+channels in IBMX/forskolin-and sparingly elevated extracellular K +-induced Ca 2+transients in freshly isolated porcine olfactory receptor neurons (ORNs).In the presence of mibefradil (10m M)or Ni 2+(100m M),the selective T-type Ca 2+channel inhibitors,IBMX/forskolin-induced Ca 2+transients in the soma were either strongly (>60%)inhibited or abolished completely.However,the Ca 2+transients in the knob were only partially (<60%)inhibited.Ca 2+transients induced by 30mM K +were also partially ($60%)inhibited at both the knob and soma.Furthermore,ORNs responded to as little as a 2.5mM increase in the extracellular K +concentration (7.5mM K +),and such responses were completely inhibited by mibefradil or Ni 2+.These results reveal functional expression of T-type Ca 2+channels in porcine ORNs,and suggest a role for these channels in the spread Ca 2+transients from the knob to the soma during activation of the cAMP cascade following odorant binding to G-protein-coupled receptors on the cilia/knob of ORNs.#2006Elsevier Ireland Ltd and the Japan Neuroscience Society.All rights reserved.Keywords:Olfaction;Signal transduction;Ca 2+dynamics;cAMP;Mibefradil;Ni 2+;Pig1.IntroductionIn most mammalian species,odor signals play critical roles in several vital functions such as feeding,reproduction,and social organization.These behaviors require the fundamental operation of odor detection and discrimination.Olfactory receptor neurons (ORNs),the sensory neurons present in the nasal cavity,are primarily involved in coding sensory information from odorous stimuli.An understanding of the mechanism of signal transduction in ORNs is thus fundamental for an appreciation of how odor signals affect animal behavior.Based on the evidence available so far,cyclic AMP is known as the universal second messenger of olfactory signaltransduction in vertebrates (Schild and Restrepo,1998;Chen et al.,2000;Takeuchi and Kurahashi,2003;Delay and Restrepo,2004;Madrid et al.,2005).Other possible messengers such as InsP 3,NO,and CO are now believed to play modulatory roles for some odorants (Schild and Restrepo,1998;Gold,1999;Chen et al.,2000;Takeuchi and Kurahashi,2003).According to the cAMP-mediated signal transduction cascade,odorant binding to G-protein-coupled receptors stimulates an adenylyl cyclase (type III),and the resulting increase in cAMP directly activates cyclic nucleotide-gated (CNG)channels,leading to an influx of Ca 2+(Schild and Restrepo,1998;Ronnett and Moon,2002).The subsequent rise in the Ca 2+concentration activates Ca 2+-dependent Cl Àconductance,giving depolarizing receptor potential during excitatory responses (Kurahashi and Yau,1993;Lowe and Gold,1993;Reuter et al.,1998;Reisert et al.,2003)or Ca 2+-dependent K +conductance,giving hyperpolarizing receptor/locate/neuresNeuroscience Research 57(2007)129–139*Corresponding author.Tel.:+81117065199;fax:+81117065202.E-mail address:habara@vetmed.hokudai.ac.jp (Y .Habara).0168-0102/$–see front matter #2006Elsevier Ireland Ltd and the Japan Neuroscience Society.All rights reserved.doi:10.1016/j.neures.2006.09.016potential during inhibitory responses(Delgado et al.,2003; Delay and Restrepo,2004;Madrid et al.,2005).Notably,all of these major transduction events are localized to the cilia and knob,the apical portion of the dendrite exposed to the mucus layer in the nasal cavity(Menco et al.,1992;Menco,1997; Lowe and Gold,1991;Schild et al.,1994;Hallani et al.,1998; Kaur et al.,2001;Morales et al.,1997;Gautam et al.,2006). The signal generated at the cilia/knob has to propagate to the soma,the site of action potential generation located deep within the olfactory epithelium,for the transmission of the sensory information from odorous stimuli to the second-order neurons in the specific glomeruli in the olfactory bulb(Narusuye et al., 2003;Lowe,2003).However,the mechanisms underlying the propagation of chemoelectrical signals from the cilia/knob to the soma remain unclear.Experiments carried out in the last decade have also indicated that Ca2+is the sole‘third messenger’of olfactory signal transduction regardless of the possible involvement of multiple second messengers(Schild and Restrepo,1998). However,the role of Ca2+in ORNs is not limited to being the third messenger of signal transduction mediating receptor potential generation,it is,rather involved in the entire sensory process taking place from the cilia to the soma,including negative feedback actions on various stages of the odor transduction mechanism and initiation of the repolarization process(by activating Ca2+-dependent K+channels)after the generation of action potentials(Menini,1999;Matthews and Reisert,2003).Consistent with the complex actions of Ca2+,it has been suggested that Ca2+is tightly regulated in a compartmentalized manner within ORNs(Restrepo et al., 1993;Schild et al.,1994;Tareilus et al.,1995;Leinders-Zufall et al.,1997,1998;Gautam et al.,2006).However,the mechanism underlying the spread of Ca2+transients from the cilia/knob to the soma is not fully understood.So far,based on thefindings from a number of vertebrate species,it is known that Ca2+transients in the soma involve activation of high-voltage-activated(HVA)Ca2+channels(Schild and Restrepo, 1998).In salamander ORNs it has also been reported that store-operated Ca2+release in the soma may amplify the Ca2+ transients primarily evoked by the activation of HVA Ca2+ channels(Zufall et al.,2000).In a recent report,we have shown in rat ORNs that the spread of Ca2+transients from the cilia/knob to the soma can also be a function of the intensity of stimulation(Gautam et al.,2006).Based on the electro-physiological properties low-voltage-activated(LVA)(T-type)Ca2+channels have been reported in newt ORNs (Kawai et al.,1996;Kawai and Miyachi,2001).However,no information is yet available about their localization or on how the presence of T-type Ca2+channels may influence the spatiotemporal dynamics of Ca2+during activation of the signal transduction cascade.The relevance of T-type Ca2+ channels in the ORNs of mammalian species also remains to be investigated.In the present study,using a combination of IBMX(a phosphodiesterase inhibitor)and forskolin(an adenylyl cyclase activator)as the pharmacological activator of the cAMP cascade,and mibefradil and Ni2+as selective inhibitors of T-type Ca2+channels(Perez-Reyes,2003),we herein show how T-type Ca2+channels contribute to the spread of Ca2+ transients from the knob to the soma during activation of the cAMP cascade in isolated pig ORNs.Additionally,the data from the elevated extracellular K+-induced responses are shown to facilitate understanding of the role of T-type Ca2+ channels during activation of the cAMP cascade by IBMX/ forskolin.2.Materials and methods2.1.Isolation of porcine ORNsAll protocols for experiments on animals described in this work were approved by the Animal Research Committee of the Graduate School of Veterinary Medicine,Hokkaido University.Crossbred male piglets(Sus scrofa domestica,LW,4–6weeks after birth,8–12kg)purchased from Nishihara Farm(Rusutsu,Hokkaido,Japan)were housed with food and water ad libitum at room temperature(20–268C)for2–4days before use.Piglets were deeply anaesthetized with pentobarbital sodium(30mg/kg,i.v.,Dainippon Pharma-ceutical Co.Ltd.,Osaka,Japan)after sedation with a combination of mid-azolam(0.5mg/kg,i.m.,Dormicum,Astellas,Tokyo,Japan)and ketamine (10mg/kg,i.m.,Ketalar,Sankyo,Tokyo,Japan);and then sacrificed by exsanguination from the carotid arteries and jugular veins.All efforts were made to minimize animal suffering.The head was quickly separated from the torso,de-skinned and sawed sagittally into two halves to expose the nasal cavity.The nasal septum and turbinates were quickly removed and transferred to ice-cold divalent cation-free Ringer’s solution.Olfactory epithelium was dissected out of the underlying septal and turbinate bone/cartilage followed by mincing with dissecting scissors(1–2mm3)and then digested with 10U per ml papain in divalent cation-free Ringer’s solution for10min at room temperature.The tissue was then transferred to normal Ringer’s solution, washed several times and gently triturated with a disposable plastic Pasteur pipette to dissociate the cells.The cell suspension wasfiltered through a nylon mesh,centrifuged at60Âg for5min and resuspended in1–2ml of normal Ringer’s solution.The data presented in this study were obtained from a total of34isolations(one piglet per isolation).In general,responses from3to4 cells were recorded per isolation and each experiment employed cells from multiple isolations.2.2.Dye loadingTwelve microliters of1mM Fluo-4AM(Molecular Probes,Eugene,OR, USA)in DMSO was mixed with990m l of normal Ringer’s solution by sonication for40–50s.Then1ml of the cell suspension was mixed with the Fluo-4solution to give afinal concentration of6m M Fluo-4AM and loading proceeded for1h at room temperature.The cell suspension was then gently centrifuged,thoroughly washed and resuspended in2ml of normal Ringer’s solution.To begin the experiment,100m l of the suspension of Fluo-4-loaded cells was transferred to Cell-Tak(Becton Dickinson Labware,Bedford,MA,USA)-coated coverslips that were attached to the bottoms of recording chambers (volume100m l)with odourless dental wax.The chambers containing the cell suspension were left undisturbed for about10min and centrifuged at60Âg for 5min.This method was sufficient to ensurefirm attachment of the dendritic knob and soma of each isolated ORN onto the coverslip of the chamber.2.3.Calcium imagingWe recorded changes in the intracellular Ca2+concentration,[Ca2+]i,using a confocal laser scanning imaging system(Fluoview FV500,Olympus,Tokyo, Japan),which allowed recording of the stimulus-induced spatiotemporal dynamics of Ca2+transients in individual ORNs.The recording chamber was set on the stage of an inverted microscope with a perfusion system and the cells were continuously perfused with normal Ringer’s solution at aflow rateS.H.Gautam et al./Neuroscience Research57(2007)129–139 130of2ml per min for10–15min prior to and throughout the experiments.A forced air-cooled argon ion laser beam(488nm)with a total laser output of10mWand linear polarization was used,and the emittedfluorescence(>505nm)was guided through aÂ40water immersion objective to a pinhole diaphragm. Photodamage was minimized by operating the laser at its lowest power setting and by attenuating the laser intensity by interposing a neutral densityfilter(1% transmission)into the illumination path.Isolated individual cells were imaged atÂ4–5magnification with the electronic zoom setting of the imaging software. The resting levelfluorescence intensity was generally lower at the dendrite and knob than at the soma,possibly due to the differences in their optical thicknesses and[Ca2+]i(Restrepo et al.,1993;Leinders-Zufall et al.,1997). The restingfluorescence signal was adjusted to the minimum by changing the PMT sensitivity in such a way that thefluorescence at the knob was still visible. Images were acquired every2s by normal XYT scanning.All the scan settings were kept constant over the series of experiments.To reduce photobleaching, scanning was paused during washings between two successive stimulus appli-cations.All the cells used in this study were clearly identifiable as ORNs by their characteristic morphology,having an oval to round soma with a slender dendrite that ended in a small knob-like swelling.Cilia were too thin to be seen clearly unless magnified above the normal experimental level.Only isolated ORNs maintaining morphological integrity were used for the experi-ments.We discarded the cells in which the knob and soma lay in different focal planes,those lacking proper attachment onto the coverslips of the recording chamber,cells showing a relatively higher level of restingfluor-escence intensity and those with retracted dendrites.Bath solution and the stimulus solutions were superfused by gravity feed from the reservoirs. Stimulus solutions were pulse-applied by switching the bath solution;this allowed a complete change of bath solution within3–4s.For the experiments involving repetitive stimulations the interval between the successive stimula-tions was set at more than3min to ensure that the cells were not desensitized by the previous stimulation.2.4.ChemicalsNormal Ringer’s solution contained(in mM)138NaCl,5KCl,2CaCl2,1 MgCl2,10HEPES,10glucose and1Na-pyruvate supplemented with0.1% BSA(pH7.4).For elevated extracellular K+solutions,NaCl was replaced with an equimolar concentration of KCl.For divalent cation-free isolation solution, CaCl2and MgCl2were omitted and replaced with1mM EDTA(pH7.4). Forskolin,Na-pyruvate,3-isobutyl-1-methyl-xanthine(IBMX),and mibefradil were purchased from Sigma Chemical Co.(St.Louis,MO,USA).Other chemicals were obtained from Wako Pure Chemicals(Osaka,Japan)unless otherwise specified.2.5.Data analysisInitially the time courses of the changes influorescence intensities at the selected regions of ORNs were recorded in arbitrary units using Fluoview4.2 with Tiempo(Olympus,Tokyo,Japan).These data were then converted to relative changes influorescence intensity(%D F/F),where D F and F refer to the stimulus-induced change in thefluorescence and the resting-levelfluores-cence,respectively.The averagefluorescence intensity during20–30s prior to the stimulus application was taken as the resting-levelfluorescence.The magnitude of the response was assessed by taking the peak%D F/F values during stimulation.The average results are expressed as meansÆS.E.of independent experiments(n),where n refers to the number of ORNs.Statistical analysis was done using the paired and unpaired Student’s t-tests.Results were considered significant at P<0.05.3.Results3.1.Dissociated porcine ORNsThe population of dissociated pig ORNs consisted of cells with dendrites of various lengths(Fig.1),probably representing their stage of maturity and/or position of the soma within the olfactory epithelium.Since the cilia were hardly visible under the normal scanning mode set for Ca2+imaging,the brightness and contrast of these images were adjusted later using Adobe Photoshop to assure the visibility of the cilia.While the majority of the cells were relatively small in size(Fig.1A–C), some of them were unusually large(Fig.1D).We could not record responses from such large cells since none of them was viable at the time of imaging.As ORNs senesce and die at a regular rate,the large cells possibly represented old cells approaching degeneration,and hence their vulnerability to the experimental manipulations.3.2.IBMX/forskolin-and slightly elevated extracellularK+-induced Ca2+transientsFig.2A(a)shows a transmission image of an isolated pig ORN used for an experiment and Fig.2A(b–h)pseudocolour images of this cell at various time points during the course of the experiment as indicated in Fig.2B.Fig.2B shows the time courses of the changes in the intracellular Ca2+concentration in response to30s pulse stimulation with10m M IBMX plus 1m M forskolin(Fig.2B,left)and10mM K+(Fig.2B,right). The IBMX/forskolin-induced increase in thefluorescence intensity appearedfirst at the knob(Fig.2A(c)and B),and then extended to the dendrite followed by the soma(Fig.2B).The elevated extracellular K+-induced response,however,showed simultaneous onset of the responses at the knob,dendrite and soma(Fig.2B,right).3.3.Effects of T-type Ca2+channel inhibitors on IBMX/ forskolin-induced Ca2+transients3.3.1.Effect of Ni2+Micromolar concentrations of Ni2+(such as100m M)are known to selectively inhibit T-type Ca2+channels in various mammalian cell types(Perez-Reyes,2003).Kawai et al.(1996) also demonstrated the specificity of Ni2+on T-type Ca2+ channels in newt ORNs.We used100m M Ni2+to test the involvement of T-type Ca2+channels in the spatiotemporal dynamics of[Ca2+]i during IBMX/forskolin-induced activation of the cAMP cascade in pig ORNs(Fig.3).The presence of Ni2+in the bath solution1min prior to and during the test stimulations inhibited IBMX/forskolin-induced Ca2+transients in two different ways:(1)In5of12cells,the presence of Ni2+ almost completely abolished the response at the dendrite and soma,resulting in a knob-confined response with reduced magnitude(Type-I inhibition).(2)In the remaining7cells, however,the Ca2+transients were only partially inhibited throughout the knob,dendrite and soma(Type-II inhibition). Fig.3A(a)shows an example of Ni2+-induced Type-I inhibition, and Fig.3A(b)summarizes the results fromfive such experiments in which the extents of inhibition in the knob and soma were54Æ9%(P<0.05)and95Æ2%(P<0.001), respectively.The extent of inhibition at the dendrite was similar to that at the soma(data not shown).Likewise,Fig.3B(a)shows an example of Ni2+-induced Type-II inhibition,and Fig.3B(b)S.H.Gautam et al./Neuroscience Research57(2007)129–139131summarizes the results from seven such experiments where the extents of inhibition in the knob and soma were 48Æ9%(P <0.01)and 64Æ4%(P <0.001),respectively.Taking all 12cells together,Ni 2+inhibited the IBMX/forskolin-induced Ca 2+transients by 50Æ6%and 77Æ5%in the knob and soma,respectively.3.3.2.Effect of mibefradilMibefradil was basically developed as a specific blocker of T-type Ca 2+channels (Clozel et al.,1997),and now is a well-known selective nondihydropyridine blocker of native as well as recombinant T-type channels (Mishra and Hermsmeyer,1994;Cribbs et al.,1998;Martin et al.,2000;Michels et al.,2002;Perez-Reyes,2003).While T-type Ca 2+channels may differ considerably in their pharmacological properties depending on the tissue or cell type (Huguenard,1996),10m M mibefradil has been shown to completely inhibit LVA Ca 2+currents/transients in dissociated rat retinal bipolar cells (Pan et al.,2001).In the present study also,10m M mibefradil was sufficient to almost completely abolish 7.5or 10mM K +-induced Ca 2+transients (Section 3.4.1).Hence,we examined the effect of 10m M mibefradil on the spatiotemporal dynamics of [Ca 2+]i during IBMX/forskolin-induced activation of the cAMP cascade (Fig.4).As in the case of Ni 2+,the presence ofmibefradil in the bath solution 1min prior to and during the test stimulations caused Type-I inhibition in 7cells and Type-II inhibition in the other 6of the 13cells tested.Fig.4A(a)shows an example of mibefradil-induced Type-I inhibition,and Fig.4A(b)summarizes the results from seven such experi-ments where the extents of inhibition in the knob and soma were 49Æ8%(P <0.01)and 93Æ2%(P <0.001),respec-tively.In a similar way,Fig.4B(a)shows an example of mibefradil-induced Type-II inhibition,and Fig.4B(b)sum-marizes the results from six such experiments where the extents of inhibition in the knob and soma were 52Æ8%(P <0.01)and 66Æ7%(P <0.001),respectively.Taking all 13experiments together,mibefradil inhibited IBMX/forsko-lin-induced Ca 2+transients by 50Æ5%and 81Æ5%in the knob and soma,respectively.parison of the response susceptible to Type-I inhibition and that susceptible to Type-II inhibition by Ni 2+or mibefradilTo understand why the presence of Ni 2+or mibefradil during IBMX/forskolin-induced responses caused some ORNs to exhibit Type-I inhibition while others showed Type-II inhibition,we compared the averaged peak amplitude of the responses susceptible to Type-I inhibition with that susceptibleS.H.Gautam et al./Neuroscience Research 57(2007)129–139132Fig.1.Representative examples of cells from the population of isolated pig olfactory receptor neurons (ORNs).The majority of ORNs had a small soma with dendrites of various lengths (A–C).Scale bar =10m m.Some ORNs were unusually large (D).Contrast and brightness were adjusted using Adobe Photoshop to increase visibility of the cilia.to Type-II inhibition,combining the data from Ni2+and mibefradil(Fig.5).The amplitude of the response susceptible to Type-II inhibition(n=13)was significantly higher than that susceptible to Type-I inhibition(n=12)(P<0.01,knob; P<0.001,soma).It was,thus,revealed that cells showing relatively smaller responses were susceptible to Type-I inhibition whereas those showing larger ones were susceptible to Type-II inhibition.3.4.Ca2+transients induced by7.5mM K+3.4.1.Effect of mibefradilThe responsiveness of pig ORNs to a slightly elevated extracellular K+concentration(10mM K+)has already been shown in Fig.2.We found that even7.5mM K+was enough to induce significant Ca2+transients in pig ORNs.Considering the magnitude of depolarization likely to be induced by such a small increase in the extracellular K+concentration,we suspected that such responses should be mediated,almost exclusively,by T-type Ca2+channels.Hence,we studied the effects of different concentrations of mibefradil on7.5mM K+-induced responses to determine the optimum dose of mibefradil to inhibit T-type Ca2+channels in isolated pig ORNs. Mibefradil,perfused for1min prior to and during the test stimulations,reversibly reduced the Ca2+transients evoked by 7.5mM K+in a dose-dependent manner.An example of the effect of4,7and10m M mibefradil on7.5mM K+-induced Ca2+transients is shown in Fig.6A.The effect of4m M mibefradil was examined in six cells that were also used to observe the effect of7m M mibefradil as shown in Fig.6A(a). The effect of7m M mibefradil was also examined in11other cells.The effect of10m M mibefradil was recorded for16cells. The effects of the different concentrations of mibefradil are summarized in Fig.6B.The extent of inhibition increased from 4m M(40Æ11%,knob;42Æ7%,soma;P<0.01)to7m M (79Æ16%,knob;76Æ12%,soma;P<0.001)and at10m M the responses were almost completely abolished(96Æ2%, knob;94Æ2%,soma;P<0.001).S.H.Gautam et al./Neuroscience Research57(2007)129–139133Fig.2.Confocal Ca2+imaging of a Fluo-4-loaded pig ORN.(A)(a)Transmission image of an isolated ORN.The black-dotted markings over the knob,dendrite and soma correspond to the area analyzed for the time courses shown in(B).Scale bar=5m m.(b–h)Pseudocolourfluorescence images of the cell at various time points as depicted in(B).The IBMX/Fk-induced increase in thefluorescence intensity appearedfirst at the knob(c),followed by the dendrite and soma(d).(B)Time courses of Ca2+transients at the knob(black trace),dendrite(light grey trace)and soma(dark grey trace).Note that unlike the IBMX/forskolin-induced response,the10mM K+-induced response showed simultaneous onset of the responses at the knob,dendrite and soma.Stimulus was applied for30s.IBMX/Fk,10m M IBMX plus1m M forskolin.134S.H.Gautam et al./Neuroscience Research57(2007)129–139Fig.3.Effects of100m M Ni2+on IBMX/forskolin-induced Ca2+transients.(A)Type-I inhibition.(a)A representative example of Type-I inhibition;that is,complete abolishment of Ca2+transients at the soma(grey trace)with partial inhibition at the knob(black trace).(b)Average peak amplitudes of Ca2+transients from5ORNs showing Type-I inhibition.(B)Type-II inhibition.(a)A representative example of Type-II inhibition;that is,partial inhibition of Ca2+transients at both the soma(grey trace)and the knob(black trace).(b)Averaged peak amplitudes of Ca2+transients from7ORNs showing Type-II inhibition.Stimulus was applied for20s.*P<0.05, **P<0.01,***P<0.001paired Student’s t-test vs.control.Fig.4.Effects of10m M mibefradil on IBMX/forskolin-induced Ca2+transients.(A)Type-I inhibition.(a)A representative example of Type-I inhibition;that is, complete abolishment of Ca2+transients at the soma(grey trace)with partial inhibition at the knob(black trace).(b)Average peak amplitudes of Ca2+transients from 7ORNs showing Type-I inhibition.(B)Type-II inhibition.(a)A representative example of Type-II inhibition;that is,partial inhibition of Ca2+transients at both the soma(grey trace)and the knob(black trace).(b)Averaged peak amplitudes of Ca2+transients from6ORNs showing Type-II inhibition.Stimulus was applied for20s. **P<0.01,***P<0.001paired Student’s t-test vs.control.3.4.2.Effect of Ni 2+Like mibefradil,Ni 2+almost completely abolished the 7.5mM K +-induced Ca 2+responses in ORNs (Fig.7).The effect of Ni 2+was examined in 14cells as shown in Fig.7A.On average,Ni 2+reduced the 7.5mM K +-induced response by 93Æ3%and 94Æ2%(P <0.0001)in the knob and soma,respectively (Fig.7B).3.5.Effects of mibefradil and Ni 2+on 30mM K +-induced Ca 2+transientsTo assess the contribution of T-type Ca 2+channels to the Ca 2+transients evoked during moderate depolarization of ORNs we examined the effects of mibefradil and Ni 2+on 30mM K +-induced Ca 2+transients.Fig.8shows repre-sentative examples and the averaged results of the experi-ments performed.Mibefradil (10m M)reduced the peak amplitudes of 30mM K +-induced Ca 2+transients by 64Æ5%and 62Æ3%(n =9;P <0.001)in the knob and soma,respectively (Fig.8A).Likewise,Ni 2+inhibited the peak amplitudes of the response by 60Æ6%and 58Æ3%(n =8;P <0.01)in the knob and soma,respectively (Fig.8B).Cells were exposed to 30mM K +for 20s and the drugs were applied for 1min prior to and during the test stimulations.3.6.Effects of mibefradil and Ni 2+on 100mM K +-induced Ca 2+transientsTo demonstrate the specificities of 10m M mibefradil and 100m M Ni 2+for the low-voltage-activated T-type Ca 2+channels in the isolated pig ORNs,we next examined the effects of these drugs on Ca 2+transients induced by 100mM K +(Fig.9).Consistent with the well-known specificities ofmibefradil and Ni 2+for T-type Ca 2+channels,these drugs had no effect on 100mM K +-induced Ca 2+transients while the same drugs almost completely abolished the 10mM K +-induced Ca 2+transients in the same ORNs.Similar results were obtained with four cells tested for Ni 2+and 5cells tested for mibefradil.Cells were exposed to 10mM K +for 20s and to 100mM K +for 6s.The traces shown in Fig.9represent means of the responses at the knob and soma.The drugs were applied in the same way as in other experiments described above.S.H.Gautam et al./Neuroscience Research 57(2007)129–139135Fig.5.Cells showing relatively small responses were susceptible to Type-I inhibition by Ni 2+or mibefradil.The Averaged peak amplitudes of IBMX/forskolin-induced Ca 2+transients that were susceptible to Type-I inhibition (n =12;5,Ni 2+and 7,mibefradil)were compared with those susceptible to Type-II inhibition (n =13;7,Ni 2+and 6,mibefradil).**P <0.01,***P <0.001unpaired Student’s t-test.Fig.6.Effects of mibefradil on 7.5mM K +-induced Ca 2+transients.(A)Representative time courses corresponding to the knob (black trace)and soma (grey trace)in the presence and absence of 4or 7m M (a),or 10m M (b)mibefradil.(B)Averaged peak amplitudes of Ca 2+transients in the presence and absence of 4m M (n =6),7m M (n =17)or 10m M (n =16)mibefradil.Stimulus was applied for 20s.Cells were bathed in normal Ringer’s solution containing 5mM K +.**P <0.01,***P <0.001paired Student’s t -test vs.control.。