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FM1-43 is a permeant blocker of mechanosensitive ion channels in sensory neurons and inhibits behavioural responses to mechanical stimuli

FM1-43 is a permeant blocker of mechanosensitive ion channels in sensory neurons and inhibits... The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated 2+ currents. Blockade and dye uptake is voltage dependent and regulated by external Ca . The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them. mechanosensitive channels and showed that the dye per- Background FM1-43 is a cationic styryl pyridinium dye used to fluores- meated the non-selective cation channels TRPV1 and cently label biological membranes. It is used to study P2X . When AM1-43, a fixable analogue of FM1-43, was endocytosis, exocytosis and endosome trafficking and, in injected subcutaneously, after 24–48 hours labelling of a particular, synaptic vesicle recycling [1,2]. Gale et al [3] number of sensory organs was observed. Interestingly, recently showed that FM1-43 acts as a permeant blocker of most of these structures had mechanosensory functions; mechanotransducing ion channels in murine hair cells. they included hair cells in the cochlea and vestibular FM1-43 applied to the extracellular surface of hair cells organs, Merkel cells and their neurites, muscle spindles, was taken up by the cell (resulting in fluorescent labelling corneal nociceptors and enteric neurons. Fluorescence of the cytoplasm) at a site close to the proposed site of also accumulated (to varying degrees) in the cell bodies of transduction in a manner dependent on mechanical stim- all sensory neurons of the dorsal root ganglia (DRG) and ulation. FM1-43 was also shown to inhibit mechanically other sensory ganglia [4]. In experiments using local dye activated currents in a voltage-dependent manner where injections and nerve ligation it was shown that dye entry the lowest IC was 1.2 µM at -4 mV. Meyers et al [4] also into sensory neurons was via uptake at their peripheral, reported that FM1-43 is a permeant blocker of hair cell sensory terminals [4]. Page 1 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 Mechanically activated (MA) cationic currents in cultured Mechanical stimulation and drug application DRG display properties that correspond to their in vivo A heat-polished glass pipette was used to mechanically physiological function [5-7]. In this study the effects of stimulate neurons [5,6]. The probe was controlled by a FM1-43 on MA currents in cultured sensory neurons and piezo-electric crystal drive (Burleigh) (tip diameter ≈5 on behavioural responses to noxious mechanical stimuli µm) and was positioned at an angle of 70° to the surface were investigated. The effects of the related dye FM3-25, of the culture dish. The probe was moved at a speed of 0.5 which has two 18 carbon chains, rather than 2 butyryl µm/msec and the stimulus was applied for 200 msec groups on either side of the polar head, were also analysed (unless stated otherwise). Cells that showed a reproduci- in these two systems. The results show that FM1-43 is a ble response to mechanical stimuli (>200 pA stimulated permeant blocker of DRG neuron mechanosensitive ion at 20 sec intervals) were selected for further experimenta- channels, whereas FM3-25 is a non-permeant antagonist tion. Action potentials were recorded in the current-clamp of these channels. In behavioural tests, these dyes inhib- configuration and were evoked by 1 msec square waves of ited responses in two assays of mechanosensitivity. depolarising current. Capsaicin (1 µM) and low pH (pH 5.3) were applied for 4 sec using a multi-barrel rapid solu- Materials and methods tion changer (Biologic). Experiments were done at room Cell culture temperature. All reagents from Sigma unless stated otherwise. Neonatal (P1) Sprague Dawley rats were decapitated, and 25–30 FM1-43 was visualised by excitation at 479 nm and DRG were taken from each and digested in a solution of through FITC filters. Images were acquired using Openlab collagenase (Type XI, 0.6 mg/ml), dispase (3 mg/ml) and software and further analysed using MCID Basic software. 2+ 2+ glucose (1.8 mg/ml) in Ca /Mg free PBS (Gibco) for 25 Samples of pixel intensity were taken from 5 random minutes prior to mechanical trituration. Cells were cul- areas within the neuronal cytoplasm and 6 from the back- tured in Dulbecco's modified Eagle medium containing ground region adjacent to the cells and the mean of the 10% foetal bovine serum (Gibco), 2 mM glutamine latter was subtracted from that of the former. (Gibco), 10,000 IU/ml penicillin/streptomycin (Gibco) and 100 ng/ml nerve growth factor on poly-L-lysine- and Behavioural testing For all experiments, male, 6–8 week, 20–25 g mice were laminin-coated dishes and used the day after preparation. used and the experimenter was blind to the treatment Electrophysiology given to each animal. Withdrawal thresholds to punctate Whole-cell, perforated patch recordings were made using mechanical stimuli were tested using von Frey hairs an Axopatch 200B amplifier (Axon Instruments) control- applied to the plantar surface of the left hind paw. Mice led by pCLAMP 9 (Axon Instruments). Patch pipettes were were habituated to the testing conditions for 1.5 hours. made from thin-walled glass (Harvard Apparatus) and Then, following acquisition of control data, intraplantar had an initial resistance of 2–3 MΩ when filled with inter- injections of drug or vehicle (20 µl, 5% DMSO in standard nal solution. Seals had a series resistance of 4–10 MΩ external solution, see above) were given and withdrawal compensated for by 40–60% (feedback lag; 18 µs). Volt- thresholds were retested 20–40 minutes post-injection. age-clamp recordings were made at a holding potential of 50% withdrawal thresholds were calculated using the "up- -70 mV unless otherwise stated. down" method [8,9]. The standard intracellular solution contained (in mM): For experiments using the Randall-Sellito device, mice 110methanesulfonic acid, 30 KCl, 1 MgCl and 10 HEPES, were placed in a restraining tube and pressure was applied pH 7.35(pH was corrected using KOH; final K concentra- to a point midway along the tail. An ascending pressure tion ≈140 mM); 200 µg/ml amphotericin B was added ramp was applied until the animal showed obvious signs immediately before recording. When testing the blocking of discomfort and this pressure was taken as the pain efficiency of FM1-43 on inward and outward currents a threshold. Three control recordings were taken prior to caesium based internal solution was used (in mM): 110 injection of vehicle or drug (80 µl was given on the dorsal caesium methane sulfonate, 30 CsCl, 1 MgCl , 10 HEPES, and ventral side of the tail) and then 4 further measure- pH 7.3 (adjusted using CsOH). The standard external ments were made. The first control and first post injection solution contained (in mM): 140 NaCl, 4 KCl, 2 CaCl , 1 tests were discarded from analysis, to control for effects of MgCl and 10HEPES, pH 7.4 (adjusted with NaOH). Low learning and stress, respectively. pH solutions were made with 10 mM morpholi- noethansulfonic acid in place of HEPES. FM1-43 (5/10 Data were analysed using Sigmaplot 8 and Sigmastat 4 mM), FM3-25 (3 mM) and capsaicin (10 mM) stock solu- software. Data are presented as mean ± standard error tions were made in DMSO. (unless otherwise stated). Page 2 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 icant labelling of the neuronal cytoplasm was observed Results To determine whether FM1-43 inhibited mechanically (Figs. 2c,d), suggesting that the molecule cannot permeate activated (MA) currents in DRG neurons, currents were the channel and that FM1-43 uptake is not via non-spe- recorded in the presence of extracellular FM1-43 at a range cific membrane binding. Finally, FM1-43 uptake did not of concentrations from 0.6 to 15.0 µM. Cultured neurons occur when mechanically stimulated neurons were held at generate MA currents that vary in magnitude and kinetics +35 mV showing that dye influx is voltage-dependent according to neuronal subtype. A subpopulation of noci- (Figs. 2c,d). ceptive neurons generates slowly adapting currents (<25% adaptation) whereas in cultures from neonatal animals, The voltage dependence of current inhibition by per- most currents show mainly rapid adaptation (RA); RA cur- meant blockers is complex; as the membrane potential rents in capsaicin insensitive (Caps-) are typically faster becomes more negative there is greater binding of the than in capsaicin sensitive (Caps +) neurons but the dis- compound to the channel pore (increases blockade) but tinction is not as clear as in adult neurons [[5,6] manu- also greater passage through the channel (decreases block- script submitted]. In this study, FM1-43 blocked rapidly- ade) (see Ref. 3). The ability of FM1-43 to inhibit MA cur- and slowly-adapting MA currents in both Caps- and rents was assessed at 3 membrane potentials (-70, -35 and Caps+ neurons. The effect was relatively similar on all +35 mV). In these experiments FM1-43 was an equally three classes of currents (Figs. 1a,b) although overall the efficient blocker of MA currents at -70 and -35 mV (69.7 ± inhibitory effect was greater on SA currents than either RA 4.3% versus 70.3 ± 2.1% inhibition, n = 4–5) but its population (2-way ANOVA, P < 0.001, Figs. 1a,b). From blocking activity was significantly reduced at the positive the concentration-inhibition plots it can be seen that 50% holding potential (34.1 ± 2.1%, n = 7, P = 0.01, Fig. 3a). inhibition is achieved at around 3 µM for SA currents and This is again consistent with it permeating and blocking 2+ around 5 µM for RA currents in Caps- and Caps+ neurons the underlying ion channel. Both Ca [10] and FM1-43 (Fig. 1a). The related dye FM3-25 was also tested; in con- act as permeant blockers of auditory mechanotransduc- 2+ trast to the inactivity displayed by the compound at hair tion ion channels [3,4] and Ca is also a permeant cell mechanotransduction channels, 5 µM FM3-25 inhib- blocker of DRG mechanosensitive ion channels [5,11]. ited DRG MA currents by 30.3 ± 4.2% (n = 9, Fig. 1c). Therefore, to determine if the two molecules interact, the inhibitory effect of FM1-43 on currents was tested when 2+ Permeation of FM1-43 through ion channels results in the Ca concentration was either reduced to nominally 2+ fluorescent labelling of the cytoplasm [3,4]. Therefore we zero or doubled to 4 mM. Raising Ca levels to 4 mM compared cytoplasmic dye accumulation to the total inhibited MA currents by 32.0 ± 1.1% whilst inhibition of amount of MS channel activity evoked in the presence of MA currents by 5 µM FM1-43 fell from 62.4 ± 0.6% to FM1-43 or FM3-25. (This was enabled by the use of the 39.1 ± 6.1% (n = 3, Caps-, RA currents, P < 0.05, Fig. 3b). 2+ perforated patch technique, as the dye is unable to pass Conversely, removing external Ca (no chelator through the pores formed by amphotericin B.) When included) increased the effect of FM1-43 to 142.2 ± 4.5% mechanical stimuli were applied in the presence of 5 µM of control levels (n = 3, Student's paired t-test, P < 0.05). FM1-43, cytoplasmic labelling was correlated with chan- nel activity. Three neurons were mechanically stimulated We have proposed that the mechanosensitive ion chan- th th th 30 times (at 9 µm) and after the 10 , 20 and 30 stimuli nels expressed on the cell bodies of cultured neurons are cytoplasmic florescence was measured; in each cell fluo- normally present on the sensory nerve terminal in vivo rescence was seen to increase with the number of stimuli where they mediate mechanosensation. To test this, we (Figs. 2a,2b). Moreover, comparison of 18 neurons (with first examined whether peripheral application of FM1-43 widely varying MA current amplitudes) showed that the to the stimulated region of mouse paws affected mechan- intensity of fluorescent labelling after mechanical stimu- ically evoked paw withdrawal behaviour. Using von Frey lation (10 × 9 µm) was strongly correlated to the total hairs to assess withdrawal threshold in response to punc- charge transfer (r = 0.82, P = 0.002, Pearson's product tate mechanical stimuli, intraplantar injection of FM1-43 moment, Fig. 2c). FM1-43 can be internalised through (5 nMoles) led to an increase in the 50% paw withdrawal 2+ endocytosis [1,2] so Ca influx through mechanogated threshold of over 100% (1.3 ± 0.2 g to 2.9 ± 0.4 g, P < ion channels could induce vesicular trafficking in neurons 0.05, n = 7 Fig. 4a). Interestingly, in 3 animals tested in proportional to the extent of channel activity. To exclude using von Frey hairs, intraplantar FM3-25 (5 nMoles) this possibility, it was shown that cytoplasmic loading increased the 50% withdrawal threshold by approxi- was indistinguishable from that in control conditions mately 75% (1.1 ± 0.3 g to 1.9 ± 0.5 g, n = 3, P < 0.05, Fig. 2+ when MA currents were evoked in FM1-43 in Ca free 4a). Secondly, we also found FM1-43 to have an inhibi- extracellular solution (Fig. 2c). Consistently, when 3 neu- tory action in the Randall-Selitto test, in which ascending rons were mechanically stimulated in the presence of 5 pressure levels are applied to the tail until a pain-related µM FM3-25 (which also binds cell membranes) no signif- behaviour is evoked. Here, local injection of 40 nMoles Page 3 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 RA, Caps - SA, Caps - RA, Caps + 0 2 4 6 8 10 12 14 16 [FM1-43] (microM) RA, Caps- SA Con Con FM3-25; RA, Caps- Caps+ Con Con Inhibition of MA cur Figure 1 rents by the styryl dyes FM1-43 and FM3-25 Inhibition of MA currents by the styryl dyes FM1-43 and FM3-25. (A) Concentration-inhibition functions for FM1-43 for inhibition of 3 classes of MA currents (each data point, n = 3–10). FM1-43 inhibited SA currents (Caps- neurons) at lower concentrations than RA currents in either Caps- or Caps+ neurons (2-way ANOVA, P < 0.001). At 0.6 µM FM1-43 had a slight facilitatory effect on currents. (B) Example traces showing inhibition of 3 classes of MA currents by 2, 6 and 15 µM FM1-43. (C) Inhibition of a RA current (Caps- neuron) by 5 µM FM3-25. All vertical scale bars: 0.2 nA. Horizontal scale bars: (B) 50 msec, (C) 100 msec. Page 4 of 8 (page number not for citation purposes) Percent Control Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 20 14 Neuron A Neuron B Neuron C 0 0.5 1.0 1.5 2.0 Current density (nC) B D 2 Light- 10 + 35 Field mV 0 Stimuli 0 0.5 1.0 1.5 2.0 Current density (nC) 2+ FM1-43 : Control FM1-43 : 0 mM Ca 30 FM FM1-43 : + 35 mV FM3-25 : Control Stimuli Stimuli 3-25 FM1-43 : Unstimulated Cytoplasmic ac Figure 2 cumulation of FM1-43 via permeation through MS ion channels Cytoplasmic accumulation of FM1-43 via permeation through MS ion channels. Using the perforated patch configu- ration allowed dye accumulation in the cytoplasm to be measured. (A) Cytoplasmic fluorescence through FM1-43 uptake increased in 3 neurons after 10, 20 and 30 mechanical stimuli; shown is Intensity of fluorescent labelling against cumulative charge transfer. (B) Example images of a neuron (light field image, top left) following 10, 20 and 30 mechanical stimuli. N.B. the dye does not enter the nucleus. (C) Accumulation of FM1-43 is dependent on MS channel activity. In control conditions (stand- ard external solution, membrane potential; -70 mV, ● solid line) fluorescent intensity is correlated with the amount of channel 2+ activity (as total charge transfer)(n = 18, Spearman's ranked order, r = 0.83, P < 0.001, fit: solid line). Removal of external Ca () had no apparent effect on dye uptake (n = 8, Spearman's ranked order, r = 0.83, P < 0.001, fit dotted line, partly occluded). Neither application of FM1-43 when the neuron was held at +35 mV (n = 5, ▼) nor application of FM3-25 (at -70 mV, n = 3, š ) resulted in significant cytoplasmic fluorescence. Also shown is average background labelling after FM1-43 exposure in the absence of mechanical stimulation (n = 10, standard deviation indicated, ■). D. Examples of neurons stimulated in FM1-43 at +35 mV (top) and in FM3-25 at -70 mV (bottom). FM1-43 into the tail increased pain thresholds by approx- the membrane potential (-57.5 to -55.8 mV, n = 6, P < imately 50% (effect significant relative to vehicle effect P 0.05), but did not affect action potential threshold, dura- < 0.05, n = 6, Fig 4b). tion, amplitude or the maximal rate of depolarisation (data not shown). FM1-43 does not block acid sensing ion channel (ASIC) mediated currents in DRG neurons; at 20 µM FM1-43 Discussion slightly potentiated transient proton-gated currents from We have demonstrated that FM1-43 acts as a permeant 3.81 ± 0.22 nA to 4.10 ± 0.32 nA (107.0 ± 3.1%, n = 5, P blocker of mechanosensitive ion channels expressed by < 0.05). When cells were exposed to low pH (pH5.4) in cultured DRG neurons and that when given peripherally the presence of 5 µM FM1-43 staining was observed in it inhibits behavioural responses to mechanical stimuli. It some neurons and not others (0.077 ± 0.019 arbitrary had previously been shown that FM1-43 blocks and per- intensity units, n = 5) but there was no correlation meates mechanosensitive ion channels of hair cells [3,4]; between labelling and total charge transfer (r = 0.394, P = although these two channel types have a number of dis- 0.512, data not shown). In 4 cells action potentials were tinct properties [5] the action of FM1-43 was similar. Both elicited in the presence of 5 µM FM1-43; cytoplasmic channels were blocked by low micromolar concentrations labelling was not significantly above background after 50 of the drug and in both cases fluorescent labelling of the action potentials (0.026 ± 0.007 vs 0.020 ± 0.002). This cytoplasm was induced by mechanical stimulation. In this concentration of FM1-43 caused a slight depolarisation of study, using the perforated patch technique, the degree of Page 5 of 8 (page number not for citation purposes) Flourescence (a.u.) Flourescence (a.u.) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 1nA 100 msec Veh FM FM DMSO 1-43 3-25 -70 mV -35 mV -35 mV 0 mM 2 mM 4 mM Vehicle FM1-43 Con Post Con Post 2+ inhibitory a Figure 3 Membrane ctions of FM1-43 potential and external Ca concentration affect 2+ Membrane potential and external Ca concentration affect inhibitory actions of FM1-43. (A) Inhibition of MA FM1-43 in stimuli Figure 4 hibits behavioural responses to noxious mechanical currents at -70, -35 and +35 mV holding potentials (Caps- FM1-43 inhibits behavioural responses to noxious neurons, RA currents, n = 7). Activity was indistinguishable at mechanical stimuli. (A) 5 nMoles FM1-43 (P < 0.05, n = 7, -70 mV (69.7 ± 4.3% inhibition) and -35 mV (70.3 ± 2.1%) but average increase above pre-injection control 200.7 ± 76.6%) was significantly lower at +35 mV (34.1 ± 2.1%, Student's and FM3-25 (P < 0.05, n = 3, 80.9 ± 11.6%) both increased paired t-test, P < 0.01). Top, example traces from the same the 50% withdrawal threshold in the von Frey test. Injection 2+ cell. (B) Effect of Ca on FM1-43 activity. Increasing external of the vehicle had no significant effect. (B) Using the mouse 2+ Ca from 2 to 4 mM decreased the inhibitory activity of tail to test FM1-43 in the Randall-Selitto assay it was found FM1-43 by 37.4 ± 9.8% (n = 3, Student's paired t-test, P < that FM1-43 increased pain thresholds by 55.2 ± 23.9 % (2 × 2+ 0.05) whereas removing external Ca (no chelator included) 20 nMoles top and bottom of tail injected, n = 6, Student's increased the effect of FM1-43 to 142.2 ± 4.5% of control unpaired t-test versus vehicle effect, P < 0.05) whilst vehicle levels (n = 6, Student's paired t-test, P < 0.05). injection had no significant effect on thresholds (n = 6). Injec- tion of the 5% DMSO vehicle (n = 9) had no significant effect. Page 6 of 8 (page number not for citation purposes) % Control Pain Threshold (g) Relative block (% of 2 mM) % Inhibition Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 staining was found to correlate with the amount of mech- nals of DRG fibres where they mediate sensory mechan- anosensitive ion channel activity. The efficacy of FM1-43 otransduction. in inhibiting slowly adapting currents was slightly but sig- nificantly higher than that for rapidly adapting currents, Following systemic administration of the FM1-43 ana- suggesting that distinct ion channels may be responsible logue AM1-43 primary receptor cells in a number of for the two currents. mechanosensory systems were labelled with this dye [4]. Given the absence of cell labelling following action poten- Calcium is also a permeant blocker of hair cell channels tial trains, we speculate that permeation through mech- [10,11] and mechanosensitive ion channels in DRG neu- anosensitive ion channels is a major entry point for styryl rons [5,12]. Here increasing external calcium levels dyes into sensory neurons when they are given systemi- reduced the potency with which FM1-43 blocked MA cur- cally. In the auditory system FM1-43 has become an rents. Hence, the data in the two auditory papers and this important tool in the study of mechanotransduction; it work are consistent with the divalent cation FM1-43 inter- has been used in knockdown studies [13,14] and for acting with the channel pore in a manner similar to cal- investigation of the maturation of this system [15,16]. cium. Permeation of FM1-43 is further supported by the This study has characterised the action of FM1-43 on DRG observation that its inhibitory action was not reduced by mechanosensitive ion channels, demonstrated that this depolarising the neuron to -35 mV, but it was reduced by compound can inhibit behavioural responses and sug- holding it at +35 mV. For permeant blockers, the voltage gests that this dye is a useful tool for the study of somato- dependence is determined by the membrane potential sensory mechanotransduction. drawing the compound into the channel pore and subse- quently encouraging expulsion into the cytoplasm; thus at Acknowledgements We thank Mark Baker for discussions and comments on the manuscript and more negative potentials the compound enters the chan- Caroline Stirling and Mohammad Nasser for help with behavioural testing. nel more readily but also passes through it more rapidly We also thank Jonathan Gale for discussion and provision of FM3-25. The (See Ref. 3). At +35 mV the dye did not significantly enter MRC and the Wellcome Trust supported this work. the cell following mechanical stimulation suggesting that at this membrane potential there was an insufficient elec- References trochemical gradient for FM1-43 to permeate the channel. 1. Betz WJ, Mao F, Smith CB: Imaging exocytosis and endocytosis. Curr Opin Neurobiol 1996, 6:365-371. 2. Ryan TA: Presynaptic imaging techniques. Curr Opin Neurobiol FM3-25 inhibited MA currents in DRG neurons by around 2001, 11:544-549. 30% at 5 µM, in contrast to auditory transduction chan- 3. Gale JE, Marcotti W, Kennedy HJ, Kros CJ, Richardson GP: FM1-43 dye behaves as a permeant blocker of the hair-cell mechan- nels where it is inactive (up to 30 µM) [3]. However, as in otransducer channel. J Neurosci 2001, 21:7013-7025. hair cells, FM3-25 was not taken up by the cell in response 4. Meyers JR, MacDonald RB, Duggan A, Lenzi D, Standaert DG, Corwin to mechanical stimulation. These data suggest that the JT, Corey DP: Lighting up the senses: FM1-43 loading of sen- sory cells through nonselective ion channels. J Neurosci 2003, polar head group of FM3-25 is able to block the DRG 23:4054-4065. channel but the larger size of FM3-25 makes it imper- 5. Drew LJ, Wood JN, Cesare P: Distinct mechanosensitive prop- meant. erties of capsaicin-sensitive and -insensitive sensory neurons. J Neurosci 2002, 22:RC228. 6. Drew LJ, Rohrer DK, Price MP, Blaver KE, Cockayne DA, Cesare P, Local application of FM1-43 and FM3-25 blocked behav- Wood JN: ASIC2 and ASIC3 do not contribute to mechani- cally activated currents in mammalian sensory neurons. J ioural responses to noxious mechanical stimuli as Physiol 2004, 556:691-710. assessed by von Frey hairs and the Randall-Selitto device. 7. Di Castro A, Drew LJ, Wood JN, Cesare P: Modulation of sensory Both compounds increased paw withdrawal thresholds in neuron mechanotransduction by PKC- and nerve growth factor-dependent pathways. Proc Natl Acad Sci USA 2006, response to punctate stimulation with von Frey hairs and 103:4699-4704. it is notable that the degree of behavioural inhibition 8. Dixon WJ: Efficient analysis of experimental observations. appeared directly related to the compounds' efficiency in Annu Rev Pharmacol Toxicol 1980, 20:441-462. 9. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantita- blocking MA currents. Whereas this test measures the tive assessment of tactile allodynia in the rat paw. J Neurosci acute withdrawal from a punctate stimulus the Randall- Methods 1994, 53:55-63. 10. Ricci AJ, Fettiplace R: Calcium permeation of the turtle hair cell Selitto device measures a response to high levels of pres- mechanotransducer channel and its relation to the composi- sure related to C-fibre activation. FM1-43 did not affect tion of endolymph. J Physiol 1998, 506:159-173. action potential generation so the effect of the dye is likely 11. McCarter GC, Levine JD: Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons. Mol Pain dependent on inhibition of transduction. This is the first 2006, 2:28. demonstration of a mechanosensitive ion channel antag- 12. Lumpkin EA, Marquis RE, Hudspeth AJ: The selectivity of the hair cell's mechanoelectrical-transduction channel promotes onist inhibiting a mammalian behavioural response to 2+ 2+ Ca flux at low Ca concentrations. Proc Natl Acad Sci USA mechanical stimulation. The results are consistent with 1997, 94:10997-11002. the ion channels we have characterised on the somata of 13. Sidi S, Friedrich RW, Nicolson T: NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Sci- cultured neurons being expressed at the peripheral termi- ence 2003, 301:96-99. Page 7 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 14. Corey DP, Garcia-Anoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, Amalfitano A, Cheung EL, Derfler BH, Duggan A, Geleoc GS, Gray PA, Hoffman MP, Rehm HL, Tamasauskas D, Zhang DS: TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature 2004, 432:723-730. 15. Geleoc GS, Holt JR: Developmental acquisition of sensory transduction in hair cells of the mouse inner ear. Nat Neurosci 2003, 6:1019-1020. 16. Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, Zhang DS, Garcia-Anoveros J, Hinds PW, Corwin JT, Corey DP, Chen ZY: Pro- liferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 2005, 307:1114-1118. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 8 of 8 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecular Pain Springer Journals

FM1-43 is a permeant blocker of mechanosensitive ion channels in sensory neurons and inhibits behavioural responses to mechanical stimuli

Drew, Liam; Wood, John
Molecular Pain , Volume 3 (1) – Jan 6, 2007
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Springer Journals
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Copyright © 2007 by Drew and Wood; licensee BioMed Central Ltd.
Subject
Medicine & Public Health; Pain Medicine; Molecular Medicine; Neurobiology
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1744-8069
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1744-8069
DOI
10.1186/1744-8069-3-1
pmid
17207285
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Abstract

The molecular identity and pharmacological properties of mechanically gated ion channels in sensory neurons are poorly understood. We show that FM1-43, a styryl dye used to fluorescently label cell membranes, permeates mechanosensitive ion channels in cultured dorsal root ganglion neurons, resulting in blockade of three previously defined subtypes of mechanically activated 2+ currents. Blockade and dye uptake is voltage dependent and regulated by external Ca . The structurally related larger dye FM3-25 inhibited mechanically activated currents to a lesser degree and did not permeate the channels. In vivo, FMI-43 decreases pain sensitivity in the Randall-Selitto test and increases the withdrawal threshold from von Frey hairs, together suggesting that the channels expressed at the cell body in culture mediate mechanosensation in the intact animal. These data give further insight into the mechanosensitive ion channels expressed by somatosensory neurons and suggest FM dyes are an interesting tool for studying them. mechanosensitive channels and showed that the dye per- Background FM1-43 is a cationic styryl pyridinium dye used to fluores- meated the non-selective cation channels TRPV1 and cently label biological membranes. It is used to study P2X . When AM1-43, a fixable analogue of FM1-43, was endocytosis, exocytosis and endosome trafficking and, in injected subcutaneously, after 24–48 hours labelling of a particular, synaptic vesicle recycling [1,2]. Gale et al [3] number of sensory organs was observed. Interestingly, recently showed that FM1-43 acts as a permeant blocker of most of these structures had mechanosensory functions; mechanotransducing ion channels in murine hair cells. they included hair cells in the cochlea and vestibular FM1-43 applied to the extracellular surface of hair cells organs, Merkel cells and their neurites, muscle spindles, was taken up by the cell (resulting in fluorescent labelling corneal nociceptors and enteric neurons. Fluorescence of the cytoplasm) at a site close to the proposed site of also accumulated (to varying degrees) in the cell bodies of transduction in a manner dependent on mechanical stim- all sensory neurons of the dorsal root ganglia (DRG) and ulation. FM1-43 was also shown to inhibit mechanically other sensory ganglia [4]. In experiments using local dye activated currents in a voltage-dependent manner where injections and nerve ligation it was shown that dye entry the lowest IC was 1.2 µM at -4 mV. Meyers et al [4] also into sensory neurons was via uptake at their peripheral, reported that FM1-43 is a permeant blocker of hair cell sensory terminals [4]. Page 1 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 Mechanically activated (MA) cationic currents in cultured Mechanical stimulation and drug application DRG display properties that correspond to their in vivo A heat-polished glass pipette was used to mechanically physiological function [5-7]. In this study the effects of stimulate neurons [5,6]. The probe was controlled by a FM1-43 on MA currents in cultured sensory neurons and piezo-electric crystal drive (Burleigh) (tip diameter ≈5 on behavioural responses to noxious mechanical stimuli µm) and was positioned at an angle of 70° to the surface were investigated. The effects of the related dye FM3-25, of the culture dish. The probe was moved at a speed of 0.5 which has two 18 carbon chains, rather than 2 butyryl µm/msec and the stimulus was applied for 200 msec groups on either side of the polar head, were also analysed (unless stated otherwise). Cells that showed a reproduci- in these two systems. The results show that FM1-43 is a ble response to mechanical stimuli (>200 pA stimulated permeant blocker of DRG neuron mechanosensitive ion at 20 sec intervals) were selected for further experimenta- channels, whereas FM3-25 is a non-permeant antagonist tion. Action potentials were recorded in the current-clamp of these channels. In behavioural tests, these dyes inhib- configuration and were evoked by 1 msec square waves of ited responses in two assays of mechanosensitivity. depolarising current. Capsaicin (1 µM) and low pH (pH 5.3) were applied for 4 sec using a multi-barrel rapid solu- Materials and methods tion changer (Biologic). Experiments were done at room Cell culture temperature. All reagents from Sigma unless stated otherwise. Neonatal (P1) Sprague Dawley rats were decapitated, and 25–30 FM1-43 was visualised by excitation at 479 nm and DRG were taken from each and digested in a solution of through FITC filters. Images were acquired using Openlab collagenase (Type XI, 0.6 mg/ml), dispase (3 mg/ml) and software and further analysed using MCID Basic software. 2+ 2+ glucose (1.8 mg/ml) in Ca /Mg free PBS (Gibco) for 25 Samples of pixel intensity were taken from 5 random minutes prior to mechanical trituration. Cells were cul- areas within the neuronal cytoplasm and 6 from the back- tured in Dulbecco's modified Eagle medium containing ground region adjacent to the cells and the mean of the 10% foetal bovine serum (Gibco), 2 mM glutamine latter was subtracted from that of the former. (Gibco), 10,000 IU/ml penicillin/streptomycin (Gibco) and 100 ng/ml nerve growth factor on poly-L-lysine- and Behavioural testing For all experiments, male, 6–8 week, 20–25 g mice were laminin-coated dishes and used the day after preparation. used and the experimenter was blind to the treatment Electrophysiology given to each animal. Withdrawal thresholds to punctate Whole-cell, perforated patch recordings were made using mechanical stimuli were tested using von Frey hairs an Axopatch 200B amplifier (Axon Instruments) control- applied to the plantar surface of the left hind paw. Mice led by pCLAMP 9 (Axon Instruments). Patch pipettes were were habituated to the testing conditions for 1.5 hours. made from thin-walled glass (Harvard Apparatus) and Then, following acquisition of control data, intraplantar had an initial resistance of 2–3 MΩ when filled with inter- injections of drug or vehicle (20 µl, 5% DMSO in standard nal solution. Seals had a series resistance of 4–10 MΩ external solution, see above) were given and withdrawal compensated for by 40–60% (feedback lag; 18 µs). Volt- thresholds were retested 20–40 minutes post-injection. age-clamp recordings were made at a holding potential of 50% withdrawal thresholds were calculated using the "up- -70 mV unless otherwise stated. down" method [8,9]. The standard intracellular solution contained (in mM): For experiments using the Randall-Sellito device, mice 110methanesulfonic acid, 30 KCl, 1 MgCl and 10 HEPES, were placed in a restraining tube and pressure was applied pH 7.35(pH was corrected using KOH; final K concentra- to a point midway along the tail. An ascending pressure tion ≈140 mM); 200 µg/ml amphotericin B was added ramp was applied until the animal showed obvious signs immediately before recording. When testing the blocking of discomfort and this pressure was taken as the pain efficiency of FM1-43 on inward and outward currents a threshold. Three control recordings were taken prior to caesium based internal solution was used (in mM): 110 injection of vehicle or drug (80 µl was given on the dorsal caesium methane sulfonate, 30 CsCl, 1 MgCl , 10 HEPES, and ventral side of the tail) and then 4 further measure- pH 7.3 (adjusted using CsOH). The standard external ments were made. The first control and first post injection solution contained (in mM): 140 NaCl, 4 KCl, 2 CaCl , 1 tests were discarded from analysis, to control for effects of MgCl and 10HEPES, pH 7.4 (adjusted with NaOH). Low learning and stress, respectively. pH solutions were made with 10 mM morpholi- noethansulfonic acid in place of HEPES. FM1-43 (5/10 Data were analysed using Sigmaplot 8 and Sigmastat 4 mM), FM3-25 (3 mM) and capsaicin (10 mM) stock solu- software. Data are presented as mean ± standard error tions were made in DMSO. (unless otherwise stated). Page 2 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 icant labelling of the neuronal cytoplasm was observed Results To determine whether FM1-43 inhibited mechanically (Figs. 2c,d), suggesting that the molecule cannot permeate activated (MA) currents in DRG neurons, currents were the channel and that FM1-43 uptake is not via non-spe- recorded in the presence of extracellular FM1-43 at a range cific membrane binding. Finally, FM1-43 uptake did not of concentrations from 0.6 to 15.0 µM. Cultured neurons occur when mechanically stimulated neurons were held at generate MA currents that vary in magnitude and kinetics +35 mV showing that dye influx is voltage-dependent according to neuronal subtype. A subpopulation of noci- (Figs. 2c,d). ceptive neurons generates slowly adapting currents (<25% adaptation) whereas in cultures from neonatal animals, The voltage dependence of current inhibition by per- most currents show mainly rapid adaptation (RA); RA cur- meant blockers is complex; as the membrane potential rents in capsaicin insensitive (Caps-) are typically faster becomes more negative there is greater binding of the than in capsaicin sensitive (Caps +) neurons but the dis- compound to the channel pore (increases blockade) but tinction is not as clear as in adult neurons [[5,6] manu- also greater passage through the channel (decreases block- script submitted]. In this study, FM1-43 blocked rapidly- ade) (see Ref. 3). The ability of FM1-43 to inhibit MA cur- and slowly-adapting MA currents in both Caps- and rents was assessed at 3 membrane potentials (-70, -35 and Caps+ neurons. The effect was relatively similar on all +35 mV). In these experiments FM1-43 was an equally three classes of currents (Figs. 1a,b) although overall the efficient blocker of MA currents at -70 and -35 mV (69.7 ± inhibitory effect was greater on SA currents than either RA 4.3% versus 70.3 ± 2.1% inhibition, n = 4–5) but its population (2-way ANOVA, P < 0.001, Figs. 1a,b). From blocking activity was significantly reduced at the positive the concentration-inhibition plots it can be seen that 50% holding potential (34.1 ± 2.1%, n = 7, P = 0.01, Fig. 3a). inhibition is achieved at around 3 µM for SA currents and This is again consistent with it permeating and blocking 2+ around 5 µM for RA currents in Caps- and Caps+ neurons the underlying ion channel. Both Ca [10] and FM1-43 (Fig. 1a). The related dye FM3-25 was also tested; in con- act as permeant blockers of auditory mechanotransduc- 2+ trast to the inactivity displayed by the compound at hair tion ion channels [3,4] and Ca is also a permeant cell mechanotransduction channels, 5 µM FM3-25 inhib- blocker of DRG mechanosensitive ion channels [5,11]. ited DRG MA currents by 30.3 ± 4.2% (n = 9, Fig. 1c). Therefore, to determine if the two molecules interact, the inhibitory effect of FM1-43 on currents was tested when 2+ Permeation of FM1-43 through ion channels results in the Ca concentration was either reduced to nominally 2+ fluorescent labelling of the cytoplasm [3,4]. Therefore we zero or doubled to 4 mM. Raising Ca levels to 4 mM compared cytoplasmic dye accumulation to the total inhibited MA currents by 32.0 ± 1.1% whilst inhibition of amount of MS channel activity evoked in the presence of MA currents by 5 µM FM1-43 fell from 62.4 ± 0.6% to FM1-43 or FM3-25. (This was enabled by the use of the 39.1 ± 6.1% (n = 3, Caps-, RA currents, P < 0.05, Fig. 3b). 2+ perforated patch technique, as the dye is unable to pass Conversely, removing external Ca (no chelator through the pores formed by amphotericin B.) When included) increased the effect of FM1-43 to 142.2 ± 4.5% mechanical stimuli were applied in the presence of 5 µM of control levels (n = 3, Student's paired t-test, P < 0.05). FM1-43, cytoplasmic labelling was correlated with chan- nel activity. Three neurons were mechanically stimulated We have proposed that the mechanosensitive ion chan- th th th 30 times (at 9 µm) and after the 10 , 20 and 30 stimuli nels expressed on the cell bodies of cultured neurons are cytoplasmic florescence was measured; in each cell fluo- normally present on the sensory nerve terminal in vivo rescence was seen to increase with the number of stimuli where they mediate mechanosensation. To test this, we (Figs. 2a,2b). Moreover, comparison of 18 neurons (with first examined whether peripheral application of FM1-43 widely varying MA current amplitudes) showed that the to the stimulated region of mouse paws affected mechan- intensity of fluorescent labelling after mechanical stimu- ically evoked paw withdrawal behaviour. Using von Frey lation (10 × 9 µm) was strongly correlated to the total hairs to assess withdrawal threshold in response to punc- charge transfer (r = 0.82, P = 0.002, Pearson's product tate mechanical stimuli, intraplantar injection of FM1-43 moment, Fig. 2c). FM1-43 can be internalised through (5 nMoles) led to an increase in the 50% paw withdrawal 2+ endocytosis [1,2] so Ca influx through mechanogated threshold of over 100% (1.3 ± 0.2 g to 2.9 ± 0.4 g, P < ion channels could induce vesicular trafficking in neurons 0.05, n = 7 Fig. 4a). Interestingly, in 3 animals tested in proportional to the extent of channel activity. To exclude using von Frey hairs, intraplantar FM3-25 (5 nMoles) this possibility, it was shown that cytoplasmic loading increased the 50% withdrawal threshold by approxi- was indistinguishable from that in control conditions mately 75% (1.1 ± 0.3 g to 1.9 ± 0.5 g, n = 3, P < 0.05, Fig. 2+ when MA currents were evoked in FM1-43 in Ca free 4a). Secondly, we also found FM1-43 to have an inhibi- extracellular solution (Fig. 2c). Consistently, when 3 neu- tory action in the Randall-Selitto test, in which ascending rons were mechanically stimulated in the presence of 5 pressure levels are applied to the tail until a pain-related µM FM3-25 (which also binds cell membranes) no signif- behaviour is evoked. Here, local injection of 40 nMoles Page 3 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 RA, Caps - SA, Caps - RA, Caps + 0 2 4 6 8 10 12 14 16 [FM1-43] (microM) RA, Caps- SA Con Con FM3-25; RA, Caps- Caps+ Con Con Inhibition of MA cur Figure 1 rents by the styryl dyes FM1-43 and FM3-25 Inhibition of MA currents by the styryl dyes FM1-43 and FM3-25. (A) Concentration-inhibition functions for FM1-43 for inhibition of 3 classes of MA currents (each data point, n = 3–10). FM1-43 inhibited SA currents (Caps- neurons) at lower concentrations than RA currents in either Caps- or Caps+ neurons (2-way ANOVA, P < 0.001). At 0.6 µM FM1-43 had a slight facilitatory effect on currents. (B) Example traces showing inhibition of 3 classes of MA currents by 2, 6 and 15 µM FM1-43. (C) Inhibition of a RA current (Caps- neuron) by 5 µM FM3-25. All vertical scale bars: 0.2 nA. Horizontal scale bars: (B) 50 msec, (C) 100 msec. Page 4 of 8 (page number not for citation purposes) Percent Control Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 20 14 Neuron A Neuron B Neuron C 0 0.5 1.0 1.5 2.0 Current density (nC) B D 2 Light- 10 + 35 Field mV 0 Stimuli 0 0.5 1.0 1.5 2.0 Current density (nC) 2+ FM1-43 : Control FM1-43 : 0 mM Ca 30 FM FM1-43 : + 35 mV FM3-25 : Control Stimuli Stimuli 3-25 FM1-43 : Unstimulated Cytoplasmic ac Figure 2 cumulation of FM1-43 via permeation through MS ion channels Cytoplasmic accumulation of FM1-43 via permeation through MS ion channels. Using the perforated patch configu- ration allowed dye accumulation in the cytoplasm to be measured. (A) Cytoplasmic fluorescence through FM1-43 uptake increased in 3 neurons after 10, 20 and 30 mechanical stimuli; shown is Intensity of fluorescent labelling against cumulative charge transfer. (B) Example images of a neuron (light field image, top left) following 10, 20 and 30 mechanical stimuli. N.B. the dye does not enter the nucleus. (C) Accumulation of FM1-43 is dependent on MS channel activity. In control conditions (stand- ard external solution, membrane potential; -70 mV, ● solid line) fluorescent intensity is correlated with the amount of channel 2+ activity (as total charge transfer)(n = 18, Spearman's ranked order, r = 0.83, P < 0.001, fit: solid line). Removal of external Ca () had no apparent effect on dye uptake (n = 8, Spearman's ranked order, r = 0.83, P < 0.001, fit dotted line, partly occluded). Neither application of FM1-43 when the neuron was held at +35 mV (n = 5, ▼) nor application of FM3-25 (at -70 mV, n = 3, š ) resulted in significant cytoplasmic fluorescence. Also shown is average background labelling after FM1-43 exposure in the absence of mechanical stimulation (n = 10, standard deviation indicated, ■). D. Examples of neurons stimulated in FM1-43 at +35 mV (top) and in FM3-25 at -70 mV (bottom). FM1-43 into the tail increased pain thresholds by approx- the membrane potential (-57.5 to -55.8 mV, n = 6, P < imately 50% (effect significant relative to vehicle effect P 0.05), but did not affect action potential threshold, dura- < 0.05, n = 6, Fig 4b). tion, amplitude or the maximal rate of depolarisation (data not shown). FM1-43 does not block acid sensing ion channel (ASIC) mediated currents in DRG neurons; at 20 µM FM1-43 Discussion slightly potentiated transient proton-gated currents from We have demonstrated that FM1-43 acts as a permeant 3.81 ± 0.22 nA to 4.10 ± 0.32 nA (107.0 ± 3.1%, n = 5, P blocker of mechanosensitive ion channels expressed by < 0.05). When cells were exposed to low pH (pH5.4) in cultured DRG neurons and that when given peripherally the presence of 5 µM FM1-43 staining was observed in it inhibits behavioural responses to mechanical stimuli. It some neurons and not others (0.077 ± 0.019 arbitrary had previously been shown that FM1-43 blocks and per- intensity units, n = 5) but there was no correlation meates mechanosensitive ion channels of hair cells [3,4]; between labelling and total charge transfer (r = 0.394, P = although these two channel types have a number of dis- 0.512, data not shown). In 4 cells action potentials were tinct properties [5] the action of FM1-43 was similar. Both elicited in the presence of 5 µM FM1-43; cytoplasmic channels were blocked by low micromolar concentrations labelling was not significantly above background after 50 of the drug and in both cases fluorescent labelling of the action potentials (0.026 ± 0.007 vs 0.020 ± 0.002). This cytoplasm was induced by mechanical stimulation. In this concentration of FM1-43 caused a slight depolarisation of study, using the perforated patch technique, the degree of Page 5 of 8 (page number not for citation purposes) Flourescence (a.u.) Flourescence (a.u.) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 1nA 100 msec Veh FM FM DMSO 1-43 3-25 -70 mV -35 mV -35 mV 0 mM 2 mM 4 mM Vehicle FM1-43 Con Post Con Post 2+ inhibitory a Figure 3 Membrane ctions of FM1-43 potential and external Ca concentration affect 2+ Membrane potential and external Ca concentration affect inhibitory actions of FM1-43. (A) Inhibition of MA FM1-43 in stimuli Figure 4 hibits behavioural responses to noxious mechanical currents at -70, -35 and +35 mV holding potentials (Caps- FM1-43 inhibits behavioural responses to noxious neurons, RA currents, n = 7). Activity was indistinguishable at mechanical stimuli. (A) 5 nMoles FM1-43 (P < 0.05, n = 7, -70 mV (69.7 ± 4.3% inhibition) and -35 mV (70.3 ± 2.1%) but average increase above pre-injection control 200.7 ± 76.6%) was significantly lower at +35 mV (34.1 ± 2.1%, Student's and FM3-25 (P < 0.05, n = 3, 80.9 ± 11.6%) both increased paired t-test, P < 0.01). Top, example traces from the same the 50% withdrawal threshold in the von Frey test. Injection 2+ cell. (B) Effect of Ca on FM1-43 activity. Increasing external of the vehicle had no significant effect. (B) Using the mouse 2+ Ca from 2 to 4 mM decreased the inhibitory activity of tail to test FM1-43 in the Randall-Selitto assay it was found FM1-43 by 37.4 ± 9.8% (n = 3, Student's paired t-test, P < that FM1-43 increased pain thresholds by 55.2 ± 23.9 % (2 × 2+ 0.05) whereas removing external Ca (no chelator included) 20 nMoles top and bottom of tail injected, n = 6, Student's increased the effect of FM1-43 to 142.2 ± 4.5% of control unpaired t-test versus vehicle effect, P < 0.05) whilst vehicle levels (n = 6, Student's paired t-test, P < 0.05). injection had no significant effect on thresholds (n = 6). Injec- tion of the 5% DMSO vehicle (n = 9) had no significant effect. Page 6 of 8 (page number not for citation purposes) % Control Pain Threshold (g) Relative block (% of 2 mM) % Inhibition Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 staining was found to correlate with the amount of mech- nals of DRG fibres where they mediate sensory mechan- anosensitive ion channel activity. The efficacy of FM1-43 otransduction. in inhibiting slowly adapting currents was slightly but sig- nificantly higher than that for rapidly adapting currents, Following systemic administration of the FM1-43 ana- suggesting that distinct ion channels may be responsible logue AM1-43 primary receptor cells in a number of for the two currents. mechanosensory systems were labelled with this dye [4]. Given the absence of cell labelling following action poten- Calcium is also a permeant blocker of hair cell channels tial trains, we speculate that permeation through mech- [10,11] and mechanosensitive ion channels in DRG neu- anosensitive ion channels is a major entry point for styryl rons [5,12]. Here increasing external calcium levels dyes into sensory neurons when they are given systemi- reduced the potency with which FM1-43 blocked MA cur- cally. In the auditory system FM1-43 has become an rents. Hence, the data in the two auditory papers and this important tool in the study of mechanotransduction; it work are consistent with the divalent cation FM1-43 inter- has been used in knockdown studies [13,14] and for acting with the channel pore in a manner similar to cal- investigation of the maturation of this system [15,16]. cium. Permeation of FM1-43 is further supported by the This study has characterised the action of FM1-43 on DRG observation that its inhibitory action was not reduced by mechanosensitive ion channels, demonstrated that this depolarising the neuron to -35 mV, but it was reduced by compound can inhibit behavioural responses and sug- holding it at +35 mV. For permeant blockers, the voltage gests that this dye is a useful tool for the study of somato- dependence is determined by the membrane potential sensory mechanotransduction. drawing the compound into the channel pore and subse- quently encouraging expulsion into the cytoplasm; thus at Acknowledgements We thank Mark Baker for discussions and comments on the manuscript and more negative potentials the compound enters the chan- Caroline Stirling and Mohammad Nasser for help with behavioural testing. nel more readily but also passes through it more rapidly We also thank Jonathan Gale for discussion and provision of FM3-25. The (See Ref. 3). At +35 mV the dye did not significantly enter MRC and the Wellcome Trust supported this work. the cell following mechanical stimulation suggesting that at this membrane potential there was an insufficient elec- References trochemical gradient for FM1-43 to permeate the channel. 1. Betz WJ, Mao F, Smith CB: Imaging exocytosis and endocytosis. Curr Opin Neurobiol 1996, 6:365-371. 2. Ryan TA: Presynaptic imaging techniques. Curr Opin Neurobiol FM3-25 inhibited MA currents in DRG neurons by around 2001, 11:544-549. 30% at 5 µM, in contrast to auditory transduction chan- 3. Gale JE, Marcotti W, Kennedy HJ, Kros CJ, Richardson GP: FM1-43 dye behaves as a permeant blocker of the hair-cell mechan- nels where it is inactive (up to 30 µM) [3]. However, as in otransducer channel. J Neurosci 2001, 21:7013-7025. hair cells, FM3-25 was not taken up by the cell in response 4. Meyers JR, MacDonald RB, Duggan A, Lenzi D, Standaert DG, Corwin to mechanical stimulation. These data suggest that the JT, Corey DP: Lighting up the senses: FM1-43 loading of sen- sory cells through nonselective ion channels. J Neurosci 2003, polar head group of FM3-25 is able to block the DRG 23:4054-4065. channel but the larger size of FM3-25 makes it imper- 5. Drew LJ, Wood JN, Cesare P: Distinct mechanosensitive prop- meant. erties of capsaicin-sensitive and -insensitive sensory neurons. J Neurosci 2002, 22:RC228. 6. Drew LJ, Rohrer DK, Price MP, Blaver KE, Cockayne DA, Cesare P, Local application of FM1-43 and FM3-25 blocked behav- Wood JN: ASIC2 and ASIC3 do not contribute to mechani- cally activated currents in mammalian sensory neurons. J ioural responses to noxious mechanical stimuli as Physiol 2004, 556:691-710. assessed by von Frey hairs and the Randall-Selitto device. 7. Di Castro A, Drew LJ, Wood JN, Cesare P: Modulation of sensory Both compounds increased paw withdrawal thresholds in neuron mechanotransduction by PKC- and nerve growth factor-dependent pathways. Proc Natl Acad Sci USA 2006, response to punctate stimulation with von Frey hairs and 103:4699-4704. it is notable that the degree of behavioural inhibition 8. Dixon WJ: Efficient analysis of experimental observations. appeared directly related to the compounds' efficiency in Annu Rev Pharmacol Toxicol 1980, 20:441-462. 9. Chaplan SR, Bach FW, Pogrel JW, Chung JM, Yaksh TL: Quantita- blocking MA currents. Whereas this test measures the tive assessment of tactile allodynia in the rat paw. J Neurosci acute withdrawal from a punctate stimulus the Randall- Methods 1994, 53:55-63. 10. Ricci AJ, Fettiplace R: Calcium permeation of the turtle hair cell Selitto device measures a response to high levels of pres- mechanotransducer channel and its relation to the composi- sure related to C-fibre activation. FM1-43 did not affect tion of endolymph. J Physiol 1998, 506:159-173. action potential generation so the effect of the dye is likely 11. McCarter GC, Levine JD: Ionic basis of a mechanotransduction current in adult rat dorsal root ganglion neurons. Mol Pain dependent on inhibition of transduction. This is the first 2006, 2:28. demonstration of a mechanosensitive ion channel antag- 12. Lumpkin EA, Marquis RE, Hudspeth AJ: The selectivity of the hair cell's mechanoelectrical-transduction channel promotes onist inhibiting a mammalian behavioural response to 2+ 2+ Ca flux at low Ca concentrations. Proc Natl Acad Sci USA mechanical stimulation. The results are consistent with 1997, 94:10997-11002. the ion channels we have characterised on the somata of 13. Sidi S, Friedrich RW, Nicolson T: NompC TRP channel required for vertebrate sensory hair cell mechanotransduction. Sci- cultured neurons being expressed at the peripheral termi- ence 2003, 301:96-99. Page 7 of 8 (page number not for citation purposes) Molecular Pain 2007, 3:1 http://www.molecularpain.com/content/3/1/1 14. Corey DP, Garcia-Anoveros J, Holt JR, Kwan KY, Lin SY, Vollrath MA, Amalfitano A, Cheung EL, Derfler BH, Duggan A, Geleoc GS, Gray PA, Hoffman MP, Rehm HL, Tamasauskas D, Zhang DS: TRPA1 is a candidate for the mechanosensitive transduction channel of vertebrate hair cells. Nature 2004, 432:723-730. 15. Geleoc GS, Holt JR: Developmental acquisition of sensory transduction in hair cells of the mouse inner ear. Nat Neurosci 2003, 6:1019-1020. 16. Sage C, Huang M, Karimi K, Gutierrez G, Vollrath MA, Zhang DS, Garcia-Anoveros J, Hinds PW, Corwin JT, Corey DP, Chen ZY: Pro- liferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 2005, 307:1114-1118. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 8 of 8 (page number not for citation purposes)

Journal

Molecular Pain – Springer Journals

Published: Jan 6, 2007

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