A, Phaseplane plots of the first action potential in response to a repeated urine stimulation of 10 s duration with increasing IPIs ranging from 2 to 60 s, as indicated. The authors first used paired pulses of diluted urine and found that VSNs exhibited spike adaptation to repeated stimulations of natural stimulants. Voltage-gated Na+ channels undergo rapid activation to initiate the rising phase of action potential, followed by fast and slow inactivation processes. We added this observation in the discussion, page 9 lines 319-323. C, D, Scatter dot plots with the average SD of normalized spike frequencies for each IPI for urine pulses of 2 s (C) or 10 s (D; for C: n = 9, Demsars test after Friedman test (p=0.045 for IPI 2 s; for D: n=6; paired t test with Bonferroni correction after ANOVA for repeated measurements: p=0.018 for IPI 2 s; p=0.008 for IPI 5 s; and p=0.036 for IPI 10 s). Voltage-gated sodium channels open (activate) when the membrane is depolarized and close on repolarization (deactivate) but also on continuing depolarization by a process termed inactivation, which leaves the channel refractory, i.e., unable to open again for a period of time. Revision of the manuscript eN-NWR-0471-21 Slow inactivation of sodium channels contributes to short-term adaptation in vomeronasal sensory neurons. 5. The authors further analyzed the inactivation properties of voltage-gated Na+ channels and demonstrated that voltage-gated Na+ channels in VSNs exhibited slow inactivation. (2017). Before use, male and female urine samples were mixed in a 1:1 ratio, and the mixture was diluted to 1:50 in ACSF, pH 7.4. The 3 subunit can increase persistent current in certain sodium channels. In the "classical" fast inactivation, this time is of the . We believe that the revised manuscript has been improved by taking into account all the comments. Activation of Na* Channels The Na* channels are closed at RMP of the ventricular muscle cells. Lacosamide inhibited sustained repetitive firing during a 10-s burst but not within the . No eLetters have been published for this article. Fast inactivation occurs by a 'hinged lid' mechanism in which an . On average, the Na+ current was 832% (n = 8) of the total inward current at 20mV. Moreover, we have shown that bypassing signal transduction activation with repetitive current injections still produced spike frequency adaptation and that slow inactivation of Na+ channels is one of the relevant molecular mechanisms involved in short-term adaptation in VSNs. Mice were handled according to the guidelines of the Italian Animal Welfare Act (Decreto legislativo 26/2014) and European Union guidelines on animal research (2010/63) under a protocol approved by the Italian Ministry of Health. The ball is at the N-terminus of the subunit and consists of a disordered part (residues 110) and a loop-helix motif formed by a block of amino acids spanning from serine at position 11 to aspartate at position 16. Owing to a neurotransmitter release, there is depolarization of the plasma membrane around the channel. We found that slow inactivation of Na+ channels in mouse VSNs contributes to spike frequency adaptation to repeated stimuli. The following reviewer(s) agreed to reveal their identity: Roberto Tirindelli, Haiqing Zhao. The resistance unit is missing. For example, it would be nice to know whether the parameters of the Boltzmann equations for fast inactivation are in agreement with those found by others. They show that VSNs display short-term spike adaptation using whole-cell patch-clamp recording and paired-pulse stimulation protocol. To further investigate the dependence of short-term adaptation on stimulus duration, we applied urine pulses of shorter (2 s) and longer (10 s) durations than 5 s (Fig. The channel closure is a. A. 2A,B). Ligand binding also causes Ca2+ release from intracellular stores that may also directly activate Ca2+-activated Cl channels (Kim et al., 2011). Fast inactivation occurs by a 'hinged lid' mechanism in which an inactivating . This means the cell loses positively charged ions, and returns back toward its . 7C). Rapid inactivation of sodium channels is crucial for the normal electrical activity of excitable cells. [14] When voltage-gated sodium channels open, the S4 segment moves outwards from the channel and into the extracellular side. Structurally, Na V channels are composed of one pore-forming -subunit, which may be associated with either one . The inactivated state is mainly achieved through fast inactivation, by which a channel transitions rapidly from . We first stimulated VSNs with a current step lasting 5 s, followed by an identical stimulus at time intervals increasing from 2 to 60 s (Fig. To measure the voltage dependence of steady-state fast and slow inactivation of Na+ channels, we used typical voltage protocols composed of prepulses and a test pulse to evaluate the noninactivated fraction of Na+ channels (all recordings were performed in the presence of 100 m Cd2+ in the bath). In the classical E, Phaseplane plots of the first action potential in response to a repeated 5pA current step of 10 s duration with increasing IPIs ranging from 2 to 60 s, as indicated. To test cell viability, we used a high-K+ solution (25 mm KCl) by replacing equimolar amounts of NaCl with KCl in ACSF. We hypothesized that lacosamide modulates voltage-gated sodium channels (VGSCs) at clinical concentrations (32-100 M). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license, which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. The blockage is caused by a "ball" of amino acids connected to the main protein by a string of residues on the cytoplasmic side of the membrane. Voltage-gated ion channels open upon depolarization of the cell membrane. [10] Structural studies have shown that the inner pore of the potassium channel is accessible only through side slits between the cytoplasmic domains of the four -subunits, rather than from a central route as previously thought. microO . Rufinamide is a structurally novel, antiepileptic drug approved for the treatment of Lennox-Gastaut syndrome. TTX (2 m; LATOXAN) was used to block Na+ channels. For instance, inherited or acquired . On the other hand, paired-pulse protocols with longer urine stimulations of 10 s resulted in spike frequency adaptation when IPIs of 2, 5, and 10 s were used (average frequency ratios: 0.680.21 for 2 s; 0.790.14 for 5 s; and 0.760.2 for 10 s; n=9; Fig. A, Representative whole-cell current-clamp recordings of a VSN repetitively stimulated with a 5pA current step for 5 s with increasing intervals between steps of 2, 5, 10, 20, or 60 s, as indicated. (2018) used in situ Ca2+ imaging and extracellular electrophysiological recordings to measure responses to various chemical cues (diluted urine and bile acid ligands) and found evidence for short- and long-term adaptation in VSNs stimulated with repetitive pulses of natural stimuli. The recovery time between each paired pulse was at least 2min. Shortly after opening, the channel is blocked by the peptide ball. We changed the Figure 7D and F with the means and SD values. The ball and chain domains are on the cytoplasmic side of the channel. Voltage gated sodium (Na+) channels are critically important for a variety of neurobiological phenomena, most notably for the Na+ spike action potentials of . As soon as the potential reaches a fixed threshold value, there is a change in the conformation of the sodium channel. Many types of pain reflect neuronal hyperexcitability, so the use-dependent block of Na + channels may contribute to the efficacy of menthol as analgesic compound. 4. 2 legend n = 9. Inactivation occurs in the presence of an activating stimulus, e.g. They showed that the inactivation properties of the voltage-gated sodium current (INa) might play a role in the reduced response to repeated stimulation. However, the parameter of the Boltzmann equation we found for fast inactivation are indeed in good agreement with the values reported by Ukhanov et al. However, the voltage-dependent inactivation curves were shifted to more negative potentials and the recovery from the inactivation was decelerated. A, B, Representative whole-cell voltage-clamp recordings of Na+ currents in a VSN in the presence of 100 m Cd2+. Lacosamide reduced spiking evoked in cultured rat cortical neurons by 30-s depolarizing ramps but not by 1-s ramps. 6G), while for current pulses of 2 s we did not measure any significant difference in maximal dV/dt (Fig. The initial residues have a sequence motif of phenylalanine, isoleucine and tryptophan without which inactivation does not occur. With the curve obtained at 0 mV, how could the authors know that the channel underwent slow inactivation? Fast channel inactivation, which is required for proper physiological function, is mediated by a cytoplasmic loop proposed to occlude the ion pore via a hinged lid mechanism with the triad IFM serving as a hydrophobic "latch". Electrophysiological recordings were made using an Axopatch 200B amplifier controlled by Clampex 10 via a Digidata 1440A digitizer (Molecular Devices). The reviewers and I agree that this manuscript expands our understanding of sensory adaptation and makes an important contribution to the field. Channels containing the NIP domain behave as mutated non-inactivating channels, as they have no inactivation activity. B, Currentvoltage relations for the peak inward currents from the recordings in A. A, Families of whole-cell voltage-gated inward currents recorded from a VSN elicited by voltage steps from 80 to +60mV with 10mV increments from a holding potential of 100mV. The peptide was built based on the sequence of a 20 amino acid residue from the Drosophila melanogaster's Shaker ShB protein and applied on the intracellular side of a non-inactivating channel in Xenopus oocytes. With the curve obtained at 0 mV, how could the authors know that the channel underwent slow inactivation? The results show that a conformational change involving the intracellular segment between . The latter pro-cess is termed inactivation and leaves the channel refrac-tory for some time after repolarization. Several Na+ channel isoforms have been found to be expressed in VSNs either at mRNA level by RT-PCR or by immunohistochemistry, as follows: Nav1.1, Nav1.2, Nav1.3, Nav1.6, and Nav1.7. alanine in domain III's S4-S5 segments and the asparagine in domain IV's S4-S5 segments. In this manuscript, the authors investigate short-term spike adaptation of vomeronasal neurons (VSNs). A pvalue of <0.05 was considered statistically significant. The test potential should not affect the parameters of either slow or fast inactivation. There is no influx of sodium. 8AC). The peptide restored inactivation to the channel, giving further support to the ball and chain model. For each IPI, the peak current measured at the test pulse was normalized to the prepulse peak current and superimposed. The results of this study demonstrate the importance of the intrinsic biophysical properties of ion channels in normalizing axonal function, which depends on relations between the kinetics of slow inactivation and the firing frequency, and which can compensate for uneven channel distributions. In this in vitro/in silico study, we found that long-term inactivation of sodium channels causes adaptation in the firing rate that could potentially skew the firing of CA1 hippocampal pyramidal neurons earlier within a place field. A, Representative whole-cell current-clamp recordings from a VSN repetitively stimulated with diluted urine for 5 s with increasing intervals between pulses of 5, 10, 20, or 60 s, as indicated. D, F, Normalized currents versus membrane potential from the experiments shown in C and E were fitted with a Boltzmann equation with Vhalf = 46.71.6mV and k=4.91.3mV (n= 8) for fast inactivation (D) and Vhalf = 50.62.4mV and k=8.71.3mV (n = 6) and asymptotic value of A=0.18 for slow inactivation (F). . There are many different types of inactivation, including fast, slow and ultra-slow, and each of these can be modulated by cellular factors or accessory subunits. For each experiment, the response to high-K+ stimulation (Fig. Currents were elicited by a paired-pulse protocol consisting of a depolarization prepulse from 80 to 20mV of 1 s (A) or 10 s (B) duration followed by a short (10ms) test pulse at increasing recovery intervals ranging from 1 to 15 s. The holding potential was 80mV. Kinetic properties and inactivation of the gating currents of sodium channels in squid axon. Here, we measured spike frequency adaptation to repeated identical pulses of urine. Figure 8, A and B, shows the superimposition of ratios of the peak currents evoked by the second and first pulses at IPIs from 1 to 15 s for depolarizing prepulses of 1 or 10 s, respectively. Time course of recovery of Na+ channels from inactivation induced by depolarization steps varying from 1 to 10 s duration. neuroscience neurology action-potential. Moreover, the authors did not find any spike frequency adaptation when VSNs responded to paired current steps of 2pA lasting 20 s and separated by an IPI of 30 s, thus excluding a contribution of voltage-gated channels. [25], Inactivation anomalies have also been linked to Brugada syndrome. Such analysis would make this paper much more impactful. It is important to note that time scales are different in our experiments, and therefore the results cannot be directly compared. A voltage-gated ion channel can be in three states: open, closed, or inactivated. Voltage-dependent Na channels are always fast sodium channels. It may also have an intermediate selectivity between calcium and sodium. Due to the poor clamp conditions and to the different slope of the inactivating curves it is difficult to drive conclusions about the significance of the difference of the Vhalf for fast and slow inactivation. Urine pulse durations of 10 s (B), 5 s (C), and 2 s (D; for B: n=6; Demsars test after Friedman test: p=0.0021 for IPI 2 s; p=0.0041 for IPI 5 s; for C: n=6; Friedman test, p=0.89; for D: n=9; Friedman test, p=0.051). First, in the case of the current injection experiments (Figs 4 and 5), I understand it is easier to use a current of 5 pA to generate spikes, but would it be more informative to use a current that would generate a comparable number of spikes (as the diluted urine would (e.g., some 20 spikes/5 sec)? Indeed, the Vhalf of fast inactivation measured by us was 46.7mV, more similar to 53.5mV in V2R1b than to 65.7mV in V1Rb2. The process is also called hinged-lid inactivation or N-type inactivation. We corrected the Figure moving the -100 mV at the right position. Acknowledgment: We thank Professor Michele Giugliano [Scuola Internazionale Superiore di Studi Avanzati (SISSA)] and Professor Andrea Nistri (SISSA) for helpful discussions. The recordings were performed from the soma of neurons located in the basal zone of the VNO; therefore, we expect that most of the recorded neurons expressed receptors of the V2R family (Herrada and Dulac, 1997; Matsunami and Buck, 1997; Ryba and Tirindelli, 1997). D, Recovery from inactivation as a function of IPIs at the indicated prepulse duration. NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. Stimuli activate a transduction cascade in vomeronasal neurons that leads to spiking activity. The 1 subunit aids recovery from inactivation,[22] while 2 accelerates inactivation. Wikipedia currently has a useful table on the sodium channel page showing the different states that a sodium channel goes through during different phases of an action potential. To measure the inactivation properties of Na+ channels in VSNs, we recorded inward currents from individual VSNs in the whole-cell voltage-clamp configuration using a Cs+-based intracellular solution to block outward K+ currents. Slow inactivation of voltage-gated Na+ channels is well known to play a role in controlling membrane excitability, firing properties, and spike frequency adaptation in the nervous system (Fleidervish et al., 1996; Toib et al., 1998). Moreover, as urine contains K+, we also performed a control by applying diluted artificial urine (Fig. The model proposes that the inactivated state, which is stable and non-conducting, is caused by the physical blockage of the pore. As the authors also implied in the manuscripts (Line 186), an obvious question here is the relative contribution of the transduction-originated adaptation and Na channel-originated adaptation to the spike adaption. 5,202. The authors declare no competing financial interests. However the percentage of firing neurons that responded to diluted urine was quite high (>80%). [13] The T and F interact directly with the docking site in the channel pore. Continuous and dashed lines were calculated from the first action potential of the first and second urine pulses, respectively. To evaluate the time course of adaptation, we first applied a 5 s urine pulse followed by a second pulse of the same duration. This process involves a conformational change, which allows the ball and chain blocker to elongate and reach the inner center of the channel.[12]. These gates are called the activation gate (on the extracellular side) and the inactivation gate on the intracellular side. I would not use the term resting potential because any measurement with the patch-clamp technique is inevitably affected by the shunt seal resistance. IPR044564 Voltage-gated sodium channel alpha subunit, inactivation gate. Two alternatively spliced variants, encoding the same protein, have been identified. Voltage-gated sodium channels (VGSC) are multi-molecular protein complexes expressed in both excitable and non-excitable cells. Localization of recorded VSNs inside the slice can be an indication of the type of cell recorded, but without the use of a genetically encoded label we cannot be sure what type of cells we are recording from. 6H). In this study, we have shown that VSNs undergo spike frequency short-term adaptation when stimulated with repetitive pulses of natural stimuli in current-clamp whole-cell recordings. The authors should at least discuss this question in the Discussion. As Na+ channels may enter a state of slow inactivation on long depolarizations from which they recover only after several seconds, we measured the time course of recovery of Na+ channels in VSNs with a typical paired-pulse protocol with depolarizing prepulses varying from 1 to 10 s in duration. Second, in the experiments characterizing the slow inactivation of Na+ channels (Fig 7), Fig 7F curve should contain both the fast inactivation and the slow inactivation components. Indeed, Wong et al. 6B). Statistical significance was determined using the following tests. C, Scatter dot plots with the average SD of the normalized peak currents measured at 1 s after the prepulse of the indicated duration (t test with Bonferroni correction after ANOVA for repeated measurements: p =1.7 * 106 for 1 s; p=1.97 * 106 for 2 s; p=0.22 for 5 s; n=1417). DOI: 10.1161/CIRCULATIONAHA.106.653949. In hippocampus CA1 pyramidal neurons, cumulative inactivation is involved in regulating back-propagating action potential amplitude and can influence dendritic excitation (Jung et al., 1997; Mickus et al., 1999). However also in the response to diluted urine the number of spikes was quite variable, as shown in fig 1B. Together, these data indicate that voltage-gated Na+ channels involved in action potential generation contribute to short-term spike frequency adaptation measured with paired urine pulses. The effects of site-directed antibodies on single sodium channel currents in excised membrane patches from rat brain neurons have been examined. We compared both responses to paired stimuli to diluted urine and to current steps, to bypass the transduction cascade. In the "classical" fast inactivation, this time is of the millisecond range, but it can last much longer (up to . Thank you for sharing this eNeuro article. The opening and closing of voltage-gated sodium and potassium channels at different threshold voltages and inactivation of sodium channels occur because gates in the proteins move to open and close the pore region in the centre of the channel that allows ions . The patch pipette contained a Cs+-based intracellular solution. The voltage-gated sodium channel Na v 1.4 of the skeletal muscle is a heterodimer consisting of a pore-forming and a regulatory 1-4 subunit [6,7]. This blockage causes inactivation of the channel by stopping the flow of ions. . Scorpion analgesic peptide N58A is a -type toxin that can play a role by regulating the activation and inactivation of voltage-gated sodium channels. Furthermore, we analyzed the adaptation properties of voltage-gated channels independent of stimuli. a change in membrane voltage. In epilepsy, mutations in sodium channels genes delay inactivation. 8D,E). B, Raster plot of urine responses of 10 different neurons stimulated for 5 s. For voltage-clamp recordings, the intracellular solution filling the patch pipette contained the following (in mm): 135 CsCl, 5 NaCl, 10 HEPES, and 10 EGTA, adjusted to pH 7.2 with NaOH. The slice preparation maintained the VNO cross-sectional structure, and VSNs could be distinguished by their morphology. Inactivation of Na+ currents in VSNs. Lipopolysaccharide can exacerbate loss-of-function of sodium channels in Brugada syndrome through ROS/PKC signaling: . The results point to [Na(+)] overload in DRG neurons expressing mutant G856D Nav1.7, which triggers reverse mode of NCX and contributes to Ca(2+) toxicity, and suggest subtype-specific blockade of Nav 1.7 or inhibition of reverse NCX as strategies that might slow or prevent axon degeneration in small-fiber neuropathy. This study was supported by Grant 2010599KBR from the Italian Ministry of Education, Universities, and Research. 6A, continuous line), while increasing IPIs narrowed the differences between the maximal values of dV/dt between the first action potentials of the repeated urine pulses. The results showed that compared to the sham group, the steady-state . These data show that voltage-gated Na+ channels in VSNs present slow inactivation and that recovery from slow inactivation requires several seconds and depends on the duration of the inactivation step. Our study confirms and significantly extends a previous study that revealed adaptation of VSNs when stimulated with paired-pulse protocols. The suggestion of a physical basis for non-conductance came from experiments in squid giant axons, showing that internal treatment with pronase disrupted the inactivation phenomenon. Diluted urine elicits spike activity in VSNs. A rapid, nearly . Primer pair 4. The authors should at least discuss this question in the Discussion. The voltage-gated inactivation curves were fitted to the following Boltzmann equation: I/Imax = A + (1 A)/(1+ exp((V Vhalf)/k), where I is the peak sodium current, Imax the maximal peak sodium current, V is the membrane potential, Vhalf is the membrane potential at which I is half of Imax, k is the slope constant, and A is the asymptotic value. Moreover, to allow a complete recovery of the Na+ current, the time between each consecutive paired pulse was at least 1min. Gating current (Ig) has been studied in relation to inactivation of Na channels. A depolarizing prepulse of a selected duration was given from a holding potential of 80 to 20mV to induce inactivation, followed by a second pulse (test) with IPIs varying from 1 to 15 s (Fig. Ligand binding activates a phospholipase C signaling cascade that leads to activation of transient receptor potential canonical 2 (TRPC2) channels allowing the influx of Na+ and Ca2+ and the subsequent activation of the Ca2+-activated Cl channels TMEM16A and TMEM16B (Liman and Buck, 1994; Lucas et al., 2003; Dibattista et al., 2012; Amjad et al., 2015; Leinders-Zufall et al., 2018; Hernandez-Clavijo et al., 2021). Negative regulation of sodium channel conductance occurs through a process known as inactivation, which can proceed from either the open or closed states, termed "fast" or "steady-state" inactivation (SSI), respectively. These data indicate that, at least partially, ion channels involved in the generation of action potentials, independent of signal transduction cascade components, might contribute to the development of short-term spike frequency adaptation in VSNs. Analysis of action potential machinery indicates that slow inactivation of Na+ channels contributes to short-term spike frequency adaptation to repeated stimuli. 2. The recording chamber was continuously perfused by gravity flow with oxygenated (95% O2 and 5% CO2) ACSF containing the following (in mm): 120 NaCl, 20 NaHCO3, 5 KCl, 2 CaCl2, 1 MgSO4, 10 HEPES, and 10 glucose, pH 7.4. IgorPro 6.7/8 software (WaveMetrics) was used for data analysis and figure preparation. Lacosamide, developed as an anti-epileptic drug, has been used for the treatment of pain. What is the inactivation of sodium channel? Inactivation of Na* Channels If the membrane potential becomes positive, these . I do have a couple of questions regarding the experimental design that I would like to hear the authors thoughts on. Its mechanism of action involves inhibition of voltage-gated Na+ channels (VGSCs) with possible membrane-stabilizing effects. Here, we measured short-term adaptation performing current-clamp whole-cell recordings by using diluted urine as a stimulus, as it contains many pheromones. As in this type of analysis the maximal value of dV/dt is directly proportional to the availability of voltage-gated Na+ channels (Bean, 2007), these results suggested a contribution of Na+ channels to short-term adaptation. (2009), and we found that spike frequency adaptation was not present at the time interval of 30 s. Thus, our results are not in contrast with those of Spehr et al. By contrast, an enhanced persistent sodium current (59, 60) or an anomalous gating pore current (61, 62) typical of mutations associated with either hyperkalemic or hypokalemic periodic paralysis, respectively, causes sustained depolarization of the resting potential and inexcitability owing to inactivation of WT sodium channels. In sensory systems, the process of adaptation plays an important physiological role as it reduces the responsiveness to a stimulus, allowing the detection of additional variations in stimulus intensity. As reported for point 1 and 2, we compared our results with those reported by Ukhanov et al. [13], Differences in persistent and resurgent currents have been implicated in certain human neurological and neuromuscular disorders. Currents induced by the transduction cascades in response to chemical stimuli generate membrane depolarization and action potentials that are sent to the AOB (Mohrhardt et al., 2018). In Figure 6A, we superimposed phaseplane plots calculated from the first action potential evoked by 10 s urine pulses at different IPIs. (2018), allowed us to bypass the transduction cascade to measure the contribution of voltage-gated channels to spike frequency adaptation. The first 19 amino acids of the N-terminus constitute the ball domain. [24] Higher levels of persistent current are observed in epilepsy. 8, data). Responses to paired current steps, bypassing the signal transduction cascade, also showed spike frequency adaptation. CdCl2 (100 m) was added to extracellular ACSF in all recordings, unless otherwise stated, to block Ca2+ channels. We thank the Reviewing Editor and the two Reviewers for their careful reading and constructive comments on our manuscript. Artificial urine contained the following (in mm): 100 NaCl, 40 KCl, 20 NH4OH, 4 CaCl2, 2.5 MgCl2, 15 NaH2PO4, 20 NaHSO4, and 333 urea, at pH 7.4 adjusted with NaOH (Holy et al., 2000; Wong et al., 2018; Hernandez-Clavijo et al., 2021). Action potentials (AP) were also recorded, Vmax of AP reduced after treatment . 7E) at voltages from 90 to 0mV, followed by a short (20ms) hyperpolarizing step (to separate fast from slow inactivation) and by a test potential (30ms) at 0mV (Fig. Introducing tetraethylammonium (TEA) on the intracellular side of the channel was found to mimic inactivation in non-inactivating channels. voltage-gated sodium channel Introduction The voltage-gated NaC channel (VGSC) is a type of microporous transmembrane protein that is widely distributed on the membranes of excitable cells such as neurons, and it is mainly responsible for the trans-membrane transport of NaC. B, The number of spikes during the first and the second stimulation at the corresponding IPI is shown in the same row in A. In the resting state, the activation gate is closed and the inactivation gate is open. C, Scatter dot plot with the average SD of the normalized spike frequency of the second with respect to the first stimulation for each IPI (n=15; Demsars test after Friedman test: p=7.5 * 105 for 2 s; p=0.006 for 5 s). For example, Ukhanov et al. The ShapiroWilk test was used to verify data normality. Whether and how these neurons adapt to stimuli is still debated and largely unknown. We performed the same analysis when VSNs were stimulated with paired current steps as in Figures 4 and 5. First, in the case of the current injection experiments (Figs 4 and 5), I understand it is easier to use a current of 5 pA to generate spikes, but would it be more informative to use a current that would generate a comparable number of spikes (as the diluted urine would (e.g., some 20 spikes/5 sec)?

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