Kv7/M Channel Dysfunction Produces Hyperexcitability in Hippocampal CA1 Pyramidal Cells of Fmr1 Knockout Mice

J Physiol 602.15 (2024) pp 3769–3791 3769 The Journal of Physiology Kv7/M channel dysfunction produces hyperexcitability in hippocampal CA1 pyramidal cells of Fmr1 knockout mice M. Angeles Luque, Sara Morcuende, Blas Torres and Luis Herrero Departamento Fisiología, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain Handling Editors: David Wyllie & Gareth Morris The peer review history is available in the Supporting Information section of this article (https://doi.org/10.1113/JP285244#support-information-section). Abstract Fragile X syndrome (FXS), the most frequent monogenic form of intellectual disability, is caused by transcriptional silencing of the FMR1 gene that could render neuronal hyperexcitability. Here we show that pyramidal cells (PCs) in the dorsal CA1 region of the hippocampus elicited a larger action potential (AP) number in response to suprathreshold stimulation in juvenile Fmr1 knockout (KO) than wild-type (WT) mice. Because Kv7/M channels modulate CA1 PC excitability in rats, we investigated if their dysfunction produces neuronal hyperexcitability in Fmr1 KO mice. Immunohistochemical and western blot analyses showed no differences in the expression of Kv7.2 and Kv7.3 MªAngeles Luque is currently Associate Professor in the Department of Physiology at the University of Seville, Spain. In May 2005 she completed her PhD in Pharmacy at the University of Seville. Subsequently, she worked as a Postdoctoral Research Fellow at the University of Malaga, Spain. Her research combines electrophysiological, immunohistochemical, molecular and behavioural methods to investigate the bases of the cellular hyperexcitability of hippocampal neurons in the mouse model of fragile X syndrome, trying to revert it to a normal phenotype. © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. DOI: 10.1113/JP285244 This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 3770 M. A. Luque and others J Physiol 602.15 channel subunits between genotypes; however, the current mediated by Kv7/M channels was reduced in Fmr1 KO mice. In both genotypes, bath application of XE991 (10 μM), a blocker of Kv7/M channels: produced an increased AP number, produced an increased input resistance, produced a decreased AP voltage threshold and shaped AP medium afterhyperpolarization by increasing mean velocities. Retigabine (10 μM),anopenerofKv7/Mchannels,producedoppositeeffectsto XE991. Both XE991 and retigabine abolished differences in all these parameters found in control conditions between genotypes. Furthermore, a low concentration of retigabine (2.5 μM) normalized CA1PCexcitabilityofFmr1 KO mice. Finally, ex vivo seizure-like events evoked by 4-aminopyiridine (200 μM) in the dorsal CA1 region were more frequent in Fmr1 KO mice, and were abolished by retigabine (5–10 μM). We conclude that CA1 PCs of Fmr1 KO mice exhibit hyperexcitability, caused by Kv7/M channel dysfunction, and increased epileptiform activity, which were abolished by retigabine. (Received 4 July 2023; accepted after revision 18 June 2024; first published online 8 July 2024) Corresponding author L. Herrero: Departamento Fisiologa, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes, 6, 41012 Sevilla, Spain. Email: lherrer[email protected] Abstract figure legend Fmr1 knockout (KO) mice are commonly used as a model to understand the molecular, synaptic, cellular and neural network bases of fragile X syndrome as they reproduce some of the behavioural abnormalities of the syndrome. Dorsal pyramidal cells (PCs) of the hippocampal CA1 region of Fmr1 KO mice exhibit hyperexcitability, which is due to a reduced Kv7/M channel activity. Indeed, Kv7/M channel blockage with XE991 or activation with retigabine increases or decreases, respectively, excitability in both wild-type and Fmr1 KO mice, and abolishes differences in excitability between genotypes. Finally, low doses of retigabine, a Kv7/M channel opener, normalize neuronal excitability in CA1 PCs of the Fmr1 KO mice. Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome. Key points rDorsal pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice exhibit hyperexcitability. rKv7/M channel activity, but not expression, is reduced in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. rKv7/M channel dysfunction causes hyperexcitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice by increasing input resistance, decreasing AP voltage threshold and shaping medium afterhyperpolarization. rA Kv7/M channel opener normalizes neuronal excitability in pyramidal cells of the hippocampal CA1 region of Fmr1 knockout mice. rEx vivo seizure-like events evoked in the dorsal CA1 region were more frequent in Fmr1 KO mice, and such an epileptiform activity was abolished by a Kv7/M channel opener depending on drug concentration. rKv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in fragile X syndrome. Introduction Fragile X syndrome (FXS) is the most frequent monogenic form of intellectual disability and autism. FXS is usually caused by transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein 1 (FMRP) (Penagarikano et al., 2007; Sitzmann et al., 2018). Fmr1 knockout (KO) mice do not express FMRP and reproduce many of the behavioural symptoms displayed by FXS patients, including learning deficits, hyperactivity, hypersensitivity to sensory stimuli and anxiety (Melancia & Trezza, 2018). Channelopathies (i.e. abnormal ion channel expression and/or function) contribute to many FXS symptoms (Brager & Johnston, 2014; Contractor et al., 2015; Deng & Klyachko, 2021; Ferron, 2016). In recent years, channelopathies for potassium (Kv 4.2, Kv7, BK, SK), sodium and HCN ionchannelshavebeenreportedinthehippocampus © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3771 of Fmr1 KO mice (Brager et al., 2012; Brandalise et al., 2020; Deng & Klyachko, 2016; Deng et al., 2013, 2019, 2024; Dwivedi et al., 2019; Gross et al., 2011; Lee et al., 2011; Routh et al., 2013). Some of these channel dysfunctions yield an augmentation of the persistent sodium current or a reduction of the voltage-independent small calcium-activated potassium current and, consequently, increase action potential (AP) frequency in response to membrane depolarization of pyramidal cells (PCs) in the entorhinal cortex and in the CA3 hippocampal region of Fmr1 KO mice (Deng & Klyachko, 2016; Deng et al., 2019). We recently reported that the AP number evoked by current steps of the same intensity was larger in hippocampal CA1 PCs of adult Fmr1 KO mice than in wild-type (WT) (Luque et al., 2017). Several types of ion channels (Kv1, SK, HCN and Kv7) have been studied in relation to the firing properties of CA1 PCs in rodents (Chen et al., 2014; Gu et al., 2005, 2008; Hönigsperger et al., 2015; Ordemann et al., 2019). In mice, the AP threshold in PCs lying in the ventral CA1 region is influenced by a greater functional expression of D-type potassium (Kv1-like) channels, resulting in a depolarized AP threshold compared with the dorsal hippocampus. However, AP firing was not significantly different between ventral and dorsal CA1 PCs of the hippocampus because ventral neurons also had a more depolarized resting membrane potential and higher input resistance than dorsal CA1 neurons in the mouse hippocampus (Ordemann et al., 2019). In rats, HCN channels have little modulation of AP frequency when the membrane potential is close to rest (−60 mV), whereas the contribution of SK channels to neuronal excitability is little or none (Chen et al., 2014; Gu et al., 2005, 2008). By contrast, Kv7/M (KCNQ/M) channels play a major role in modulating AP frequency in dorsal CA1 PCs (Hönigsperger et al., 2015). According to these findings, we hypothesized that hyperexcitability of dorsal CA1 PCs of Fmr1 KO mice arises from abnormal expression and/or function of Kv7/M channels. Kv7/M channels are composed of tetrameric assemblies of Kv7 αsubunits (Kv7.1−7.5). In the hippocampus, Kv7 channels are composed principally of Kv7.2/ Kv7.3 subunits, but with a possible contribution of Kv7.3/7.5 subunits (Jentsch, 2000; Shah et al., 2002). Kv7 channels generate the M current (Wang et al., 1998). M current was first identified in frog sympathetic ganglion cells, and it was suppressed by muscarinic acetylcholine receptor activation (hence its name), and later in CA1 PCs (Brown & Adams, 1980; Halliwell & Adams, 1982). Many other neurotransmitters converge to up- or downregulate M current by a variety of second-messenger cascades (Marrion, 1997). M current is a low threshold, slowly activating, deactivating and non-inactivating voltage-dependent potassium current (Brown & Adams, 1980). Kv7/M channels modulate overall cell excitability in CA1 PCs of rats, including input resistance, AP voltage threshold and AP medium afterhyperpolarization (mAHP), which, in turn, modulate AP frequency (Gu et al., 2005, 2008; Hönigsperger et al., 2015; Shah et al., 2008; Yue & Yaari, 2004). Here we aimed to study if dysfunction of Kv7/M channels produces hyperexcitability of CA1 PCs of Fmr1 KO mice and to determine if firing properties of these cells in Fmr1 KO mice can be normalized by pharmacological modulation of Kv7/M channel activity. Methods Ethical approval Procedures were approved by the Ethics Committee on Animal Experimentation at the University of Seville (Permit number 24-06-18-100) in accordance with the guidelines of the European Community Directive 2010/63/EU, as well as with the Spanish legislation (R.D. 53/2013 BOE 34/11370-421). Fmr1 KO (FVB.129P2-Pde6b+Tyrc−chFmr1tm1Cgr/J; stock 004624) and WT mice (FVB.129P2-Pde6b+Tyrc−ch/AntJ; stock 004828) were purchased from The Jackson Laboratory (BarHarbor,M,USA;donatedbyDrA.Ozaita,Univ. Pompeu Fabra, Spain), and cohoused in standard caging (three–four per cage) on wood shavings with a 12 h light–dark cycle, controlled room temperature and ad libitum access to food and water. Slice preparation Mice (3–5 weeks old) were deeply anaesthetized with sodium pentobarbital (50 mg kg−1i.p.) and all efforts were made to minimize suffering. Then, mice were perfused intracardially with ice-cold sucrose-artificial cerebrospinal fluid (ACSF) and decapitated; brains were removed quickly and placed in a dissection medium containing cold sucrose–ACSF. Coronal slices (thickness 300 μm) including the hippocampi were cut using a vibrating microtome (Leica 1200S, Wetzlar, Germany). Slices were first incubated in a chamber containing ACSF for 30–45 min at 30°C, and then transferred to a second chamber containing ACSF at room temperature. Single slices were transferred to the recording chamber and superfused at 2 ml min−1with a solution of ACSF, bubbled with 95% O2/5% CO2(pH 7.4; 30 ±1°C). The composition of ACSF was as follows (in mM): 126 NaCl, 2KCl,1.25Na 2HPO4,26NaHCO 3,10glucose,2MgSO 4 and 2 CaCl2.Forthesucrose–ACSFsolution,theNaClwas substituted by sucrose (240 mM), and the concentrations of MgSO4and CaCl2were modified to 4 mM and 0.1 mM, respectively. © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3772 M. A. Luque and others J Physiol 602.15 Intracellular recordings Whole-cell patch clamping was performed on visually identified somata of CA1 PCs, using a Nikon Eclipse FN1 equipped with infrared-differential interference contrast optics, a 40×water immersion objective and a Hamamatsu C-7500 camera. Recordings were carried out on neurons located approximately between Bregma −1.3 and −1.9 mm in the dorsal region of the hippocampus. Patch pipettes (4–6 MΩ) contained (in mM): 120 KMeSO4, 10 KCl, 10 phosphocreatine disodium salt, 2 MgATP, 0.3 NaGTP and 10 Hepes, adjusted to pH 7.4 with KOH. The osmolality of intracellular solutions was 280–290 mosmol l−1adjusted with sucrose. Voltage- and current-clamp recordings were carried out using a MultiClamp 700B Amplifier (Molecular Devices, Sunnyvale, CA, USA). Gigaseals (>1G) were obtained beforetheruptureofthepatch,andpipettecapacitance and bridge-balance were compensated. Series resistance was 20 Mor less during recording. Recordings were low-pass Bessel-filtered at 10 kHz; data were digitized at 20 kHz with a Digidata 1440A analog-to-digital converter and acquired using pCLAMP 10 software (Molecular Devices). Data were stored on a computer disk andanalysedofflinebyusingClampfit10.2(Molecular Devices) software. Voltages were not corrected for the liquid-junction potential (estimated as ∼8mV). Voltage clamp recordings and analysis Voltage clamp experiments were carried out to determine whether the Kv7/M channel current was altered in the Fmr1 KO cell group. To isolate this current, we prepared afreeCa 2+solution of ACSF and added TTX (0.5 μM), ZD7288 (10 μM) and 4-aminopyridine (4-AP, 4.0 mM) to block or reduce several voltage- and calcium-dependent Ca2+,Na +,K +currents (i.e. ICav,INaT,INaP,Ih,IKv1 ,IBK, ISK). Series resistance was compensated by 80%. We used the traditional method to isolate M current by a voltage step protocol (Adams et al., 1982; Brown & Adams, 1980): cells were held at −70 mV, then a 1 s step to −10 mV was applied to activate M current and inactivate other voltage-dependent potassium currents, followed by a step to −50 mV for 1 s to elicit a slow current ‘relaxation’ due to channel deactivation (tail current), and a 500 ms step to −10 mV, before returning to the holding potential. This protocolwasrepeatedevery20sfor7min;1minafter its onset, we added XE991 (10 μM), a Kv7/M channel blocker, to the external solution. We assumed XE991 sensitive current, which is likely to be exclusively due to Kv7/M channels, as the Kv7/M current; and Kv7/M current was quantified by subtracting current in the presence of XE991 from that before drug application. Current measurements were taken during the last 100 ms of the voltage step to −50 mV from a holding potential of −10 mV (Iss, steady state current), and on the tail current (Itail). Tail currents were fitted to a single exponential function and τwas the time constant. Drugs used for experiments (XE991, retigabine, TTX, ZD7288, 4-AP) were purchased from Tocris Bioscience (Bristol, UK). Current clamp recordings and analysis To check if excitability differences between genotypes wereduetoKv7/Mchanneldysfunction,CA1PCswere recorded before (control conditions) and during bath application of XE991 (10 μM) or retigabine (10 μM), respectively a blocker and opener to Kv7/M channels. Resting membrane potential was measured as the difference between the intracellular and extracellular potentials.Membranepotentialwasfittedto−62 mV by injecting direct current just before quantifying any ofthefollowingparameters:inputresistancethatwas determined by −50 pA current steps during 500 ms. We injected depolarizing current ramps (200 pA every 2 s) to assess the AP voltage threshold, which was determined only from the first AP of ramp-evoked AP trains. The rheobase was the current of the ramp that elicited the first AP. Single APs were evoked by 1 ms depolarizing current pulses and current intensity was adjusted to the AP voltage threshold. AP amplitude was the voltage increment between the resting level and spike voltage peak. AP duration was determined as the width of the spike at half-maximal amplitude. Repetitive firing was evoked by depolarizing current steps of 1 s with 50 pA increments until 300 pA and with 20 pA increments until 100 pA. After each current step and before a new one, the cell was allowed to recover resting membrane potential and then fitted to −62 mV. To measure mAHP afteranactionpotentialburst,wesetthemembrane potential at −55 mV and delivered a pulse train (train duration =200 ms and pulse width =1ms)toyield20 APs at 100 Hz. mAHP amplitude corresponded to the difference between −55 mV and the lowest membrane potentialafterthetrain.WealsomeasuredmAHP amplitude and duration during repetitive discharge: amplitude was the difference between the action potential voltage threshold and negative peak of the membrane potential after a fast AHP, while duration was the spent time until the membrane potential reached the voltage threshold. mAHP mean velocity was calculated as amplitude/duration. Extracellular recordings Slices were placed on a mesh within a submerged chamber to be perfused above and below at 2 ml min−1with ACSF (32°C), or with 4-AP (200 μM) dissolved in ACSF to evoke seizure-like activity, or with 4-AP (200 μM) © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3773 plus retigabine at different concentrations (2.5, 5 and 10 μM) in ACSF to determine if the manipulation of Kv7/M channel activity mitigates epileptiform activity. Extracellular recordings were carried out with glass electrodes filled with ACSF (2–3 MΩ, resistance) placed in the dorsal CA1 pyramidal cell layer. Signals were sampled at5kHz,lowpassfilteredat10kHz,digitizedandstored on a computer disk to be analysed offline. After 1 h of continuous perfusion with 4-AP, seizure-like events were quantified (number and duration) for 30 min. As criteria for quantification were used that ictal activity was longer than 10 s and repetitive. Equipment used for extracellular recordings was that previously described for intracellular recordings. Immunohistochemistry against Kv7.2 and Kv7.3 channel subunits Mice (n=3WTandn=3KO)wereperfusedtranscardially under deep anaesthesia with 10 ml of physiological saline, followed by 25 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. Brains were removed and cryoprotected by immersion in a solution of 30% sucrose in sodium phosphate buffer saline (PBS) until sinking. Tissue was then cut coronally into 30 μm thick sections using a cryostat (Leica CM1850). Sections containing the hippocampus were incubated for 10 min in 1% sodium borohydride prepared in PBS for antigen retrieval. After rinsing in PBS, sections were immersed in a blocking solution with 0.1% triton X-100 in PBS (PBS-T) and 10% normal donkey serum (NDS, Sigma Aldrich, Saint Louis, MO, USA) for 45 min. Subsequently, sections were incubated overnight at room temperature and shaken in a solution containing the primary antibody against NeuN (mouse anti-NeuN, 1:300, Merck MAB377, Darmstadt, Germany), a specific marker for neurons, together with one of the following antibodies: Kv7.2 (rabbit anti-Kv7.2, Abcam ab22897, 1:200, Cambridge, MA, USA) or Kv7.3 (rabbit anti-Kv7.3, Abcam ab66640, 1:200) diluted in PBS-T +5% NDS +0.1% sodium azide. After washing with PBS-T, sections were incubated in the dark with the secondary antibodies coupled to the fluorophore fluorescein-5-isothiocyanate (FITC) (donkey anti-mouse IgG FITC, 1:250, Jackson ImmunoResearch, 715-095-150, West Grove, PA, USA) and tetramethyl-rhodamine 5,6-isothiocyanate (TRITC) (donkey anti-rabbit IgG TRITC, 1:250, Jackson Immuno- Research, 711-025-152) for 2 h with shaking at room temperature. After washing, the serial sections were mounted on gelatinized slides and covered with a mounting medium for immunofluorescence (Fluoromount, Sigma F4680). The immunofluorescent staining was visualized with a Zeiss LSM 7 DUO scanning confocal microscope (Carl Zeiss, Jena, Germany), equipped with an argon laser for visualization of FITC-labelled cells (green, excitation wavelength 488 nm), and a DPSS laser for TRITC (red, excitation wavelength 543 nm). The images were captured using a Plan-Apochromat 40×Oil DIC M27 objective, numerical aperture 1.3, pixel size/sampling size 4.8, and were stored and processed by the Zen Software Light Edition Software (Carl Zeiss). The images taken with the confocal microscope were analysed using ImageJ software (NIH, Bethesda, MD, USA). To quantify Kv7.2 and Kv7.3 immunostaining,thesomatalayerofPCatCA1was outlined in the green channel (NeuN), and measurements of the optical density of the red channel (Kv7.2 or Kv7.3) were performed in the selected area. Western blot analyses Coronalslicesof300μmfromnineWTandnineKOmice were obtained as described above. The dorsal CA1 region was dissected from the slices, transferred to an Eppendorf tube (hippocampi from three mice per tube) with 1 ml of cold ACSF, and allowed to recover on ice for 10 min before starting the biotinylation procedure using the Pierce Cell Surface Biotinylation and Isolation Kit (Pierce, A44390, Rockford, IL, USA). Briefly, slices were incubated in 1 ml of EZ-Link Sulfo-NHS-SS-Biotin for 1 h on ice with gentle stirring every 15 min. Unbound biotin was washed twice for 10 min with ice-cold ACSF, once for 10 min in quenching buffer and twice for 10 min again with ACSF. Then,tissuewashomogenizedin300μlofcoldlysis buffer containing a cocktail of protease and phosphatase inhibitors (Merck; 11836170001) for 30 min at 4°C, vortexing, pipetting tissue up and down several times, and sonicating. The lysate was centrifuged at 12,500 r.p.m. for 30 min at 4°C. The supernatant was isolated, and 50 μl was kept for analysis of total protein. The remaining supernatant was used to isolate biotinylated membrane proteins by transferring it to a NeutrAvidin Agarose column and eluting the labelled proteins following the manufacturer’s instructions. Concentrations of total and superficial proteins were determined by the Bradford method. For western blot analysis, total and surface proteins from both genotypes were diluted in sample buffer, and equal amounts of protein (50 μg) were loaded onto a 10% polyacrylamide-SDS gel and then transferred to a PVDF membrane by electroblotting. The membranes were blocked with 10% bovine serum albumin (BSA) for 1 h and incubated overnight at 4°C in a solution containing anti-Kv7.2 (Abcam; ab22897, 1:1000) or anti-Kv7.3 (Abcam; ab66640, 1:1000) rabbit polyclonal antibodies, diluted in TBS-Tween 0.1% with 5% BSA. After washing, the membranes were incubated with HRP-conjugated anti-rabbit antibody (Vector Labs; PI-1000, 1:2000, Burlingame, CA, USA) for 1 h at room temperature. The immunoreaction was detected usingtheWesternBrightQuantumkit(Advansta,Menlo © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3774 M. A. Luque and others J Physiol 602.15 Park, CA, USA; K-12042). The chemiluminiscence was visualized using a chemiluminiscence imaging system (Vilber, Collégien, France; Fusion-Solo S). After washing the membranes for 10 min with stripping buffer, blots were re-probed with anti-GAPDH mouse monoclonal antibody (Millipore; MAB374, 1:1000), followed with an HRP-conjugated anti-mouse antibody (Vector Labs; PI-2000, 1:3500) 1 h at room temperature to ensure equal loading. Densitometric analysis of the bands corresponding to the predicted molecular weight of Kv7.2 (95 kDa) and Kv7.3 (97 kDa) was carried out using the Multi Gauge software (Fuji Photo Film, Japan). The data were normalized to the GAPDH level for each sample, and protein expression in KO animals was expressed relative to WT level of the same experiment (normalized to 1). The specificity of the antibodies against Kv7.2 and Kv7.3 channel subunits was probed by the omission of the primary or secondary antibodies, and they have already been used for both immunohistochemistry and western blotting (Galvin et al., 2020; Wei et al., 2018; Zhang et al., 2011). Statistical analysis All data supporting the results presented here are included in the figures as well as in the supporting information published with the paper online. Each slice was exposed to only one drug, XE991 or retigabine (see Figs 3–5). When experiments were addressed to normalize AP number in Fmr1 KO mice, slices were sequentially exposed to 2.5, 5 and 10 μM retigabine (see Fig. 6). Therefore, individual comparisons between WT and Fmr1 KO cell groups before and during XE991 or retigabine applications were determined separately by using Student’s ttest for unpaired samples when values were normally distributed within each group with equal variance between groups. When a normal distribution could not be assumed, data were analysed using a Mann–Whitney test. Multiple comparisons were determined by the two-way ANOVA test with Bonferroni correction. Wilcoxon matched-pairs signed rank analysis was also used to detect differences in inmunohistochemistry and western blot data between WT and KO mice. The significance level was established at P<0.05. All data are reported as means ±standard deviation (SD). The number of recorded cells and mice are indicated as nand N,respectively. Results Dorsal CA1 PCs of juvenile Fmr1 KO exhibit hyperexcitability Within the hippocampal formation, increased AP frequency has been reported in juvenile entorhinal and CA3 PCs, and in dorsal CA1 PCs of adult Fmr1 KO mice (Deng & Klyachko, 2016; Deng et al., 2019; Luque et al., 2017). We first investigated if such hyperexcitability was also present in juvenile (3–5 weeks old) PCs located in the dorsal CA1 region of the Fmr1 KO mice. PCs of both genotypes (WT: n=14, N=10; and Fmr1 KO mice: n=16, N=12) produced an AP repetitive discharge in response to 1 s depolarizing current steps, which increased with current intensity. However, there were remarkable differences between genotypes. Thus, most cells from Fmr1 KO mice (13/16, ∼80%) were firing at 40 pA current steps, while ∼40% (6/14) were in WT mice; at 50 pA current steps, all Fmr1 KO PCs, but only ∼65% (9/14) of WT PCs, were active (Fig. 1A,C). In addition, the number of APs yielded by current steps of the same intensity from 50 to 300 pA was significantly larger in PCs of the Fmr1 KO mice (Fig. 1A,C). For instance, in response to 100 pA current steps, CA1 PCs fired about 12 and 20 APs in WT and Fmr1 KO mice, respectively (Fig. 1C); and, in response to 300 pA current steps, they discharged about 32 and 41 APs, respectively (Fig. 1C). Therefore, the absence of FMRP produced an AP number larger by 1.5-fold in CA1 PCs of Fmr1 KO than in WT mice. Similarly, depolarizing ramps (200 pA/2 s) also produced an ∼1.5-fold larger number of APs in Fmr1 KO PCs (WT: 22.57 ±8.36, n=14, N=10; KO: 33.25 ±7.68, n=16, N=12; P=0.0012, Student’s unpaired ttest; Fig. 1B,D). Kv7/M channel activity, but not expression, is reduced in the hippocampal CA1 PCs of Fmr1 KO mice We studied if hyperexcitability in CA1 PCs of Fmr1 KO mice could arise from abnormal expression and/or function of Kv7/M channels. Because axo-somatic and apical dendritic Kv7/M channels differentially regulate the intrinsic excitability of adult rat CA1 PC (Yue & Yaari, 2006), we searched for potential changes in localization between WT and Fmr1 KO mice by means of immunohistochemistry. Kv7.2 and K7.3 immunoreactivity was stronginthesomataofPCofWTandFmr1 KO mice in the dorsal region of CA1, but in the apical dendrites was scarce, if any. Somata quantification of Kv7.2 and Kv7.3 immunostaining showed a similar optical density of Kv7.2 (P=0.75, Wilcoxon matched-pairs signed rank test; N=3) and Kv7.3 (P=0.75, Wilcoxon matched-pairs signed rank test; N=3)channelsubunitsinWTandFmr1 KO mice (Fig. 2A). Western blot analyses, carried out from thedorsalregionofCA1,showedsimilartotal(P>0.999 for Kv7.2; P=0.75 for Kv7.3; Wilcoxon matched-pairs signed rank test; N=3) and surface (P>0.999 for Kv7.2; P>0.999 for Kv7.3; Wilcoxon matched-pairs signed rank test; N=3) protein expression of Kv7.2 and Kv7.3 channel subunits in WT and Fmr1 KO mice (Fig. 2B). © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3775 B 20 mV 200 ms 200 pA 200 pA – 62 mV – 62 mV – 62 mV – 62 mV WT KO A 200 ms 20 mV 100 pA 50 pA 40 pA 100 pA 50 pA 40 pA –62 mV –62 mV –62 mV –62 mV C WT * * * * KO 50 100 200 300 Current (pA) 60 40 20 AP number AP number 50 40 30 20 10 WT KO * D Figure 1. Pyramidal cells of the CA1 hippocampal region are hyperexcitable in the Fmr1 KO mice Aand B, current steps and ramps elicited action potential (AP) repetitive firing in both wildtype (WT) and Fmr1 KO (KO) mice, but AP number was larger in Fmr1 KO mice. Cand D, histograms showing AP numbers in response to different intensities of current steps (C)orramp(D). Asterisks indicate significant differences (two-way ANOVA test with Bonferroni correction, P<0.05 in C; Student’s unpaired ttest, P<0.05 in D). Data bars are shown as means ±SD for WT (n= 14 cells, N= 10 animals) and Fmr1 KO (n= 16 cells, N= 12 animals) mice. [Colour figure can be viewed at wileyonlinelibrary.com] © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3776 M. A. Luque and others J Physiol 602.15 A B C Figure 2. Kv7/M channel activity, but not expression, is reduced in the hippocampal CA1 pyramidal cells (PC) of Fmr1 KO mice A, immunohistochemical localizations of Kv7.2 and Kv7.3 channel subunits in the somata of pyramidal cells lying dorsal to the CA1 region of WT and Fmr1 KO mice. Arrowheads point to apical dendrites. Scale bar = 20 μm. Histograms showing Kv7.2 and Kv7.3 channel subunit labelling were not different between WT and Fmr1 KO mice. Data were relativized to that of wildtype (WT) mice of the same experiment (normalized to 1). n.s., not significant (Wilcoxon matched-pairs signed rank test). Data bars are shown as means ±SD for WT (N=3)andFmr1 KO (N=3) mice. B, western blot analyses show unaltered total and surface expression of Kv7.2 and Kv7.3 channel subunits © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3777 in Fmr1 KO mice. GAPDH immunoblotting was used as a loading control. Kv7 channel densitometry data were relativized to that of wildtype (WT) mice of the same experiment (normalized to 1). n.s., not significant (Wilcoxon matched-pairs signed rank test). Data bars shown as means ±SD for WT (N=3)andFmr1 KO (N=3)mice. C, representative whole-cell voltage clamp recordings in response to the voltage command protocol indicated by the upper trace in WT and Fmr1 KO mice. The lower traces show the tail current evoked by a repolarizing step to −50 mV in control (blue) and after the application of 10 μM XE991 (red). The bottom trace shows the XE991-sensitive current obtained by digital subtraction of the red trace (XE991) from the blue trace (control). Dashed lines indicate state-state current (Iss) and tail current (Itail) mediated by Kv7/M channels. Histograms showing Iss and Itail current densities in both WT and Fmr1 KO mice. Asterisks indicate significant differences (Student’s unpaired ttest, P<0.05). n.s., not significant. Data bars are shown as means ±SD for WT (n= 5 cells, N=4 animals) and Fmr1 KO (n= 6 cells, N= 4 animals) mice. [Colour figure can be viewed at wileyonlinelibrary.com] Next,wetestediftheKv7/Mchannelactivitywas altered in the Fmr1 KO cell group using a classical voltage step protocol (Brown & Adams, 1980), blocking or reducing several voltage- and calcium-dependent Ca2+,Na +and K+currents, and quantifying steady-state and tail Kv7/M currents as XE991-sensitive current (see Methods). The time constants of the tail currents were similar (P=0.6573, Student’s unpaired ttest) in CA1 PCs of both WT (τ=24 ±3.69 ms, n=5, N=4) and Fmr1 KO (τ=25.3 ±2.82 ms, n=6, N=4) mice, and consistent with those previously reported for deactivation kinetics of the Kv7/M channel (Battefeld et al., 2014; Nigro et al., 2014; Safiulina et al., 2008). As illustrated in Fig. 2C,steady-stateandtail Kv7/M current densities evoked by stepping voltage from −10 to −50 mV were significantly lower (steady-state P=0.0175; tail P=0.0040; Student’s unpaired ttest) in Fmr1 KO cells (steady-state =12.43 ±4.56 pA pF−1; tail =2.28 ±0.62 pA pF−1;n=6, N=4) than in WT mice (steady-state =24.44 ±8.88 pA pF−1; tail =4.54 ±1.27 pA pF−1;n=5, N=4). These results indicate that the expression of Kv7/M channels is normal in the dorsal CA1 region of Fmr1 KO mice, but their function is reduced in CA1 PCs. Kv7/M channel blockage or activation abolished excitability differences between genotypes To determine if the Kv7/M channel dysfunction produces CA1 PC hyperexcitability in Fmr1 KO mice, we superfused brain slices with the Kv7/M channel blocker XE991 (10 μM). In response to 1 s current steps, the AP number increasedwithbathapplicationofXE991inbothWTand Fmr1 KO mice. Thus, in response to 100 pA current steps, AP number increased from 13.37 ±3.85 to 24 ±3.42 in the WT cell group (n=8, N=5), while it increased from 18.87 ±5.38 to 27.13 ±10.25 in the Fmr1 KO cell group (n=8, N=6; Fig 3A,B). More importantly, excitability differences between genotypes were abolished in the presence of the Kv7/M channel blocker (Fig. 3B;Table1). These results indicate that a lower activity of the Kv7/M channel underlies CA1 PC hyperexcitability in Fmr1 KO mice.Toalsocheckthisresult,wesuperfusedbrainslices with the Kv7/M channel opener retigabine (10 μM). Under this experimental condition, CA1 PCs of both genotypes decreased AP number in response to current stimuli. Retigabine produced a cessation of repetitive discharge before finishing stimulation for current steps lower than 200 pA (Fig. 3C). Thus, in response to current steps of 200 pA, AP number decreased from 25 ±3.46 to 12.33 ±7.98 in CA1 PCs of WT mice (n=6, N=5), while it decreased from 34.25 ±4.33 to 14.50 ±9.05 in Fmr1 KO mice (n=8, N=6). Furthermore, in response to the same intensity current step, retigabine abolished differences in AP number between genotypes found in control conditions (Fig. 3D; Table 2). These data also support the proposal that hyperexcitability in CA1 PCs of Fmr1 KO mice is due to a Kv7/M channel dysfunction. Kv7/M channel dysfunction produces hyperexcitability of CA1 PCs in Fmr1 KO mice by increasing input resistance and reducing AP voltage threshold and interspike interval Kv7/M channels are active at subthreshold membrane potential and their voltage-dependent characteristics have been associated with various roles controlling neuronal excitability (Greene & Hoshi, 2017). Resting membrane potential was similar in CA1 PCs of both genotypes (WT =−62.5 ±3.67 mV, n=14, N=10; KO =−63.6 ±4.81 mV, n=16, N=12; P=0.4829, Student’s unpaired ttest). XE991 (10 μM) and retigabine (10 μM) yielded slight changes (<2 mV) in resting membranepotential,buttheydidnotproducesignificant differences between CA1 PCs of WT and Fmr1 KO mice (Tables 1 and 2). Input resistance was significantly higher in PCs of Fmr1 KO mice (WT =140.29 ±16.7 MΩ, n=14, N=10; KO =189.8 ±27.89 MΩ,n=16, N=12; P=0.0001, Student’s unpaired ttest). Bath application of XE991 (10 μM) and retigabine (10 μM) increased and decreased, respectively, input resistance in both genotypes (Fig. 4A), and abolished input resistance differences found in control conditions (Fig. 4B; Tables 1 and 2). StudiescarriedoutinCA1PCsofratshave demonstrated that Kv7/M current significantly contributes to determining the AP voltage threshold (Hönigsperger et al., 2015; Schwarz et al., 2006; Shah et al., © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3784 M. A. Luque and others J Physiol 602.15 A B C Figure 6. Low concentrations of the Kv7/M channel opener retigabine normalize action potential (AP) firing in the CA1 pyramidal cells (PCs) of Fmr1KO mice A, AP firing of an Fmr1 KO cell in response to 100 pA current steps in control conditions and during bath application of retigabine at 2.5, 5 and 10 μM. B, histograms showing the effect of retigabine concentration on AP number evoked by 100, 200 or 300 pA current steps in the Fmr1 KO cell group. Asterisks indicate significant differences between PCs in control conditions and during bath application of different retigabine concentrations (P<0.05). Asterisks indicate significant differences (repeated measures two-way ANOVA, P<0.05). Data bars are shown as means ±SD for Fmr1 KO (n= 6 cells, N= 4 animals) mice. C, histograms comparing AP number elicited by 100, 200 and 300 pA current steps in wildtype (WT) PCs in control conditions and Fmr1 KO (KO) PCs exposed to 2.5 μM retigabine. n.s., not significant (two-way ANOVA test with Bonferroni correction). Data bars are shown as means ±SD for WT (n= 14 cells, N= 10 animals) and Fmr1 KO (n= 6 cells, N=4 animals) mice. [Colour figure can be viewed at wileyonlinelibrary.com] Fig. 7B). As a consequence, the percentage of time with seizure-like events was 1.5-fold larger in Fmr1 KO mice (20.99 ±3.14%, n=6, N=4; KO: 31.42 ±4.50%, n=6, N=4; P=0.0021, Student’s unpaired ttest; Fig. 7B). These resultsshowthatepileptiformactivityisincreasedinFmr1 KO mice. Next, we studied the effects of retigabine on ex vivo evoked epileptiform activity in the dorsal CA1 region. We first exposed two slices from WT mice and two slices from Fmr1 KO mice that exhibited seizure-like events to 10 μM retigabine and found that the drug abolished ictal activity. We also assessed the effects of retigabine at 2.5 and 5 μMonfourslicesfromFmr1 KO mice showing seizure-like events. Retigabine at 2.5 μMproduceda significant diminution in the number and duration of ictal activity, while retigabine at 5 μMabolishedictalactivity. Therefore, a Kv7/M channel opener such as retigabine can mitigate and/or abolish seizure-like activity in the CA1 dorsal region depending on drug concentration. Discussion Here, we report that the AP number elicited by suprathreshold current stimulation was larger in dorsal CA1 PCs of juvenile Fmr1 KO mice than in WT mice. Because AP firing has been closely related to Kv7/M channel activity in this neuronal pool of rats, we investigated whetherexpressionand/orfunctiontheKv7/Mchannel was altered in Fmr1 KO mice. We found that Kv7/M channelactivityisreducedinthehippocampalCA1PCs of Fmr1 KO mice, while expression of Kv7.2 and 7.3 channel subunits was normal. Superfusion of brain slices with XE991 (10 μM), a blocker of Kv7/M channels, increased input resistance and decreased AP voltage threshold in both genotypes and abolished differences found for these parameters in control conditions. In addition, XE991 shaped mAHP, increasing interspike interval mean velocities, and abolished differences in the interspike interval features found in control conditions. Consistent with these findings, retigabine, an opener of © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3785 A B Figure 7. Epileptiform activity is greater in the dorsal CA1 region of Fmr1 KO than WT mice and is abolished by retigabine in a concentration-dependent manner A, representative field potential recordings obtained in slices from WT and Fmr1 KO mice showing seizure-like events evoked by 4-AP (200 μM). Retigabine at 2.5 or 5 μM diminished or abolished ictal activity, respectively. Retigabine washout led to a recovery of epileptiform activity. Recordings were bandpassed (2–2500 Hz) for illustrations. Arrowheads point the ictal events shown at the right of each panel with an expanded time scale. Note, however, the cease of activity caused by retigabine 5 μM (arrow) as illustrated at an expanded time scale. B, © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3786 M. A. Luque and others J Physiol 602.15 histograms showing number and duration of seizure-like events (SLE) recorded for 30 min; % SLE activity indicates the percentage of time exhibiting ictal activity for 30 min. cand 2.5 indicate control conditions and exposure to 2.5 μM retigabine, respectively. Data corresponding to bath application of 5 μM retigabine are not shown because ictal activity was abolished at this concentration. Asterisks indicate significant differences (Student’s unpaired ttest, P<0.05). n.s., not significant. Data bars are shown as means ±SD for WT (n= 6 cells, N= 4 animals) and Fmr1 KO (n= 6 cells, N= 4 animals) mice. [Colour figure can be viewed at wileyonlinelibrary.com] theKv7/Mchannel,producedtheoppositeeffectsoninput resistance, AP voltage threshold and mAHP features. Therefore, Kv7/M channel dysfunction produces hyperexcitability in CA1 PCs of Fmr1 KO mice by increasing input resistance, decreasing AP voltage threshold and shaping mAHP. In addition, a low external concentration (2.5 μM) of retigabine normalizes neuronal excitability in the CA1 PCs of Fmr1 KO mice. Kv7/channel activity, but not expression, is reduced in the CA1 PCs of Fmr1 KO mice This work has tested the hypothesis that hyperexcitability of hippocampal CA1 PCs of Fmr1 KO mice can arise from abnormal expression and/or function of Kv7/M channels. FMRP is highly expressed in cell bodies of the CA1 PCs of mice (Zorio et al., 2017) and, among other functions, FMRP binds to hundreds of mRNAs, some of which code for voltage-gated channels, and regulates its translation (Darnell et al., 2011). Western blot analyses have shown that FMRP can negatively regulate channel synthesis (Lee et al., 2011), promote translation of certain mRNAs (Gross et al., 2011; Yang et al., 2020), and not affect channel expression (Brown et al., 2010; Strumbos et al., 2010) or affect expression depending on channel subunit (Brager et al., 2012; Zhang et al., 2014). Kv7/M channels are composed principally of Kv7.2/7.3 subunits in the CA1 region of the hippocampus (Marguet et al., 2015; Shah et al., 2002; Tzingounis et al., 2010). The mRNAs coding for Kv7.2 and Kv7.3 channel subunits have been identified as FMRP targets (Darnell et al., 2011), but immunohistochemical and western blot analyses carried out in the present study revealed normal expression in thehippocampalCA1regionoftheFmr1 KO mice. In addition, FMRP modulates ion channel trafficking and surface expression (Gross et al., 2011). Because, in response to suprathreshold current stimulation, AP number produced by bath application of the Kv7/M channel-blocker (XE991) or opener (retigabine) increased or decreased in both genotypes and were not significantly different,weconcludethatexpressioninthemembrane was similar in CA1 PCs of both WT and Fmr1 KO mice. Besides FMRP roles in channel expression, the absence of FMRP could modulate channel activity via second messenger signalling pathways and/or by interacting with channels. Thus, exaggerated glutamatergic metabotropic signalling pathways (mGLuR5) reduce the AP threshold and cause hyperexcitability in the PCs of the entorhinal cortex of Fmr1 KO mice (Deng & Klyachko, 2016). Various neurotransmitters activating Gq-protein-coupled receptors induce suppression of the Kv7/M current inthehippocampus,includingcholinergic,glutamate, serotonin and opioid receptors (Charpak et al., 1990; Colino & Halliwell, 1987; Halliwell & Adams, 1982; Moore et al., 1994). Their contribution, acting alone or in an orchestrated manner, to reduce the Kv7/M current reported here in CA1 of Fmr1 KO mice is not yet known. FMRPcanalsomodulatecellexcitabilitybydirectinteraction with the pore-forming subunit and/or with the regulatory subunits of channels (Brandalise et al., 2020; Brown et al., 2010; Deng et al., 2013, 2019; Ferron et al., 2014; Yang et al., 2020; Zhan et al., 2020). Finally, FXS a neurodevelopmental disorder, and the impact of FMRP absence on the biophysical membrane properties is age-dependent (Dwivedi et al., 2019; He et al., 2014; Martin et al., 2015). Dorsal CA1 PCs of juvenile Fmr1 KO mice exhibit hyperexcitability A larger AP number evoked by current steps has been reported in neurons of the prefrontal (Routh et al., 2017) the somatosensory cortex (Gibson et al., 2008; Zhang et al., 2014, 2016), and the dorsal root ganglion (Deng et al., 2021) of Fmr1 KO mice. Similarly, PCs in the entorhinal cortex and in the CA3 hippocampal region exhibit hyperexcitability by setting the resting membrane potential close to −50 mV (Deng & Klyachko, 2016; Deng et al., 2019). In agreement with these findings, CA1 PCs fired significantly more APs in Fmr1 KO mice than in WT mice in response to suprathreshold stimulation (Luque et al., 2017; and present data). These latter results are inconsistent with those showing no significant change in AP number in CA1 PCs of juvenile Fmr1 KO mice and lower in adults (Dwivedi et al., 2019). Several possible explanations could, in part, underlie such a discrepancy. The present study was carried out in mice with an FVB genetic background, instead of the C57BL/6 strain, and the opposite results have been explained by strain-dependent differences (Paradee et al. 1999; Routh et al., 2013). Besides, it has been reported that potassium methylsulphate internal solution is more effective in recording cell excitability than potassium gluconate because gluconate has also been shown to bind Ca2+ions with low affinity (Woehler et al., 2014; Zhang et al., 1994). AP number differences between WT © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3787 and Fmr1 KOmicecoulddependonstimulusduration (Dwivedi et al., 2019; Routh et al., 2017); for instance, the neocortical PCs of Fmr1 KO mice fired significantly more APs than WT neurons at 1 s duration or longer (Routh et al., 2017). Although the above-mentioned studies reported a larger AP number in Fmr1 KO mice, it should be borne in mind that the impact of FMRP absence on electrophysiological properties is region- and cell-type dependent (Brandalise et al., 2020; Kalmbach et al., 2015). Indeed, it was recently reported that an increased Kv7 channel function causes mossy cell hypo-excitability of the dentate gyrus in Fmr1 KO mice (Deng et al., 2024). Alterations on input resistance, AP voltage threshold and mAHP shape converge to produce hyperexcitability in CA1 PCs of Fmr1 KO mice We found increased input resistance and decreased AP voltage threshold which, in turn, produced lower rheobase in CA1 PCs of Fmr1 KO mice. These cells also exhibited a larger mAHP mean velocity. Differences in these parameters between genotypes were abolished with bath application of XE991 (10 μM) and retigabine (10 μM). Modifications in these three parameters have been reported as underlying hyperexcitability in different neuronal populations of Fmr1 KO mice. Thus, layer 4 excitatory neurons of the somatosensory cortex mice fired more APs and exhibited a higher input resistance and lower rheobase in Fmr1 KO than in WT mice (Gibson et al., 2008). Small/medium diameter neurons of the dorsal root ganglia also fire more APs in Fmr1 KO; and associated with such a finding was reported an increased input resistance, reduced AP voltage threshold and reduced rheobase (Deng et al., 2021). In Fmr1 KO mice, a decreased AP threshold causes cell hyperexcitabilityinthePCsoftheentorhinalcortex,whilea reduced AP threshold and mAHP produce an elevated AP firing rate in the hippocampal CA3 region (Deng & Klyachko, 2016). Dorsal CA1 hippocampal region of Fmr1 KO mice exhibit an increased epileptiform activity and can be abolished by a Kv7/M channel opener Seizures are often diagnosed in FXS patients, they are generally well controlled with anti-epileptic drugs and disappear during childhood following a benign course (Berry-Kravis et al., 2021). However, sometimes they are severe and unresponsive to treatments (Incorpora et al., 2002; Kenmuir et al., 2015; Sabaratnam et al., 2001). In addition, FXS patients with seizures are more to likely have autism than those without seizures (Berry-Kravis et al., 2021). Therefore, a better understanding of epileptic activity is pivotal to improve the quality of life of FXS patients. Fmr1 KO mice also exhibit a significant increased susceptibility to seizures (Armstrong et al., 2022; Musumeci et al., 2000). Consistent with these latter findings,andeventhoughthedorsalCA1hippocampal region is less prone to seizures (Isaeva et al., 2015; Papatheodoropoulos et al., 2005), here we show that the pro-convulsant 4-AP elicited seizure-like events with a higher frequency in the dorsal CA1 region of Fmr1 KO than WT mice. Ictal discharge differences between both genotypes reported here can arise, in part, from circuit dysfunction (i.e. excitation/inhibition imbalance). In agreement with this suggestion, excitatory inputs to theCA1regionareheightenedbecausereducedactivity of calcium-activated potassium channels (BK and SK) in CA3 PCs of Fmr1 KO mice causes increased firing rate, excessive action potential broadening, elevated presynaptic Ca2+influx and increased glutamate release (Deng et al., 2013, 2019). In addition, excitatory transmission to CA1 inhibitory interneurons of Fmr1 KO mice is diminished, because expression of AMPA receptor subunits for glutamate is altered (Hwang et al., 2022), and could result in inhibition deficits from CA1 inhibitory interneurons to CA1 PCs. We propose that an increased excitatory input to CA1 PCs together with their hyperexcitability (reported here), by a reduced Kv7/M channel activity, contribute to increase epileptiform activity in the dorsal CA1 hippocampal region of Fmr1 KO mice. Mutations in Kv7/M channel subunits (Kv 7.2 and 7.3) are responsible for epilepsy diseases: from benign familial neonatal seizures to severe epileptic encephalopathy (Biervert et al., 1998; Borgatti et al., 2004; Singh et al., 1998). The relationship between reduced Kv7/M channel activity and seizure susceptibility has also been shown in transgenic mice (Otto et al., 2004; Peters et al., 2005; Singh et al., 1998). Given these antecedents, efforts to develop Kv7/M channel openers have been devoted for therapy of epilepsy (Liu et al., 2021). Because retigabine is an anti-seizure drug whose primary mechanism is to diminish neuronal excitability by promoting the open state of Kv7/M channels (Kv7.2–7.5) (Gunthorpe et al., 2012), we assessed its effect on ictal activity in the dorsal CA1 hippocampal region of Fmr1 KO mice. We found that retigabine mitigated or abolished seizure-like events in a dose-dependent manner. Nevertheless, retigabine was approved in 2011 by the FDA as an anti-convulsant but withdrawn in 2017 due to its side effects. Thus, further novel Kv7 channel openers may be used as effective treatment of FXS and other neurological disorders linked to with epilepsy and autism. Functional consequences Knowledge of alterations in expression and/or function of ion channels produced by FMRP absence is relevant © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3788 M. A. Luque and others J Physiol 602.15 to better understanding the neurophysiological bases of FXS and it could provide a potentially rich source of pharmacological and genetic therapies to treat this neurological disorder. Kv7/M channels have been involved in controlling neuronal excitability and learning in the hippocampus (Fontán-Lozano et al., 2011; Gu et al., 2005, 2008; Hönigsperger et al., 2015; Petrovic et al., 2012), and its dysfunction has been linked to epilepsy (Peters et al., 2005; Marguet et al., 2015) and to CA1 PC hyperexcitability in FXS (present data). Thus, Kv7/M channels may represent a therapeutic target for treating symptoms associated with hippocampal alterations in FXS. However, as already pointed out by Brandalise et al. (2020), targets need to be specific because disparate effects on a single ion channeldependonboththecelltypeandbrainregion. Alternatively, broad interventions would need to target the regulation (or restoration) of the FMR1 geneoracellular process upstream from the effector. References Adams, P. R., Brown, D. A., & Constanti, A. (1982). PharmacologicalinhibitionoftheM-current.The Journal of Physiology,332(1), 223–262. Armstrong, J. L., Saraf, T. S., Bhatavdekar, O., & Canal, C. E. (2022). Spontaneous seizures in adult Fmr1 knockout mice: FVB.129P2-Pde6b+Tyrc-chFmr1tm1Cgr/J. Epilepsy Research,182, 106891. Battefeld,A.,Tran,B.T.,Gavrilis,J.,Cooper,E.C.,&Kole,M. H. (2014). Heteromeric Kv7.2/7.3 channels differentially regulate action potential initiation and conduction in neocortical myelinated axons. Journal of Neuroscience., 34(10), 3719–3732. Berry-Kravis,E.,Filipink,R.A.,Frye,R.E.,Golla,S.,Morris, S.M.,Andrews,H.,Choo,T.H.,Kaufmann,W.E.,& FORWARD Consortium. (2021). Seizures in fragile X syndrome: Associations and longitudinal analysis of a large clinic-based cohort. Frontiers in Pediatrics,9, 736255. Biervert,C.,Schroeder,B.C.,Kubisch,C.,Berkovic,S.F., Propping, P., Jentsch, T. J., & Steinlein, O. K. (1998). A potassium channel mutation in neonatal human epilepsy. Science,279(5349), 403–406. Borgatti, R., Zucca, C., Cavallini, A., Ferrario, M., Panzeri, C., Castaldo, P., Soldovieri, M. V., Baschirotto, C., Bresolin, N., Dalla Bernardina, B., Taglialatela, M., & Bassi, M. T. (2004). A novel mutation in KCNQ2 associated with BFNC, drug resistant epilepsy, and mental retardation. Neurology,63(1), 57–65. Brager, D. H., Akhavan, A. R., & Johnston, D. (2012). Impaired dendritic expression and plasticity of h-channels in the Fmr1(-/y) mouse model of fragile X syndrome. Cell Reports,1(3), 225–233. Brager, D. H., & Johnston, D. (2014). Channelopathies and dendritic dysfunction in fragile X syndrome. Brain Research Bulletin,103, 11–17. Brandalise, F., Kalmbach, B. E., Mehta, P., Thornton, O., Johnston, D., Zemelman, B. V., & Brager, D. H. (2020). Fragile X Mental Retardation Protein bidirectionally controls dendritic Ih in a cell-type specific manner between mouse hippocampus and prefrontal cortex. Journal of Neuroscience,40(27), 5327–5340. Brown, D. A., & Adams, P. R. (1980). Muscarinic suppression of a novel voltage-sensitive K+current in a vertebrate neurone. Nature,283(5748), 673–676. Brown,M.R.,Kronengold,J.,Gazula,V.R.,Chen,Y., Strumbos, J. G., Sigworth, F. J., Navaratman, D., & Kaczmarek, L. K. (2010). Fragile X mental retardation proteincontrolsgatingofthesodium-activatedpotassium channel Slack. Nature Neuroscience,13(7), 819–821. Charpak, S., Gähwiler, B. H., Do, K. Q., & Knöpfel, T. (1990). Potassium conductances in hippocampal neurons blocked by excitatory amino-acid transmitters. Nature,347(6295), 765–767 Chen, S., Benninger, F., & Yaari, Y. (2014). Role of small conductance Ca2+-activated K+channels in controlling CA1 pyramidal cell excitability. Journal of Neuroscience, 34(24), 8219–8230. Colino, A., & Halliwell, J. V. (1987). Differential modulation of three separate K-conductances in hippocampal CA1 neurons by serotonin. Nature,328(6125), 73–77. Contractor, A., Klyachko, V. A., & Portera-Cailliau, C. (2015). Altered neuronal and circuit excitability in fragile X syndrome. Neuron,87(4), 699–715. Darnell,J.C.,VanDriesche,S.J.,Zhang,C.,Hung,K.Y.S., Mele,A.,Fraser,C.E.,Stone,E.F.,Chen,C.,Fak,J.J., Chi, S. W., Licatalosi, D. D., Richter, J. D., & Darnell, R. B. (2011). FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell,146(2), 247–261. Delmas, P., & Brown, D. A. (2005). Pathways modulating neural KCNQ/M (Kv7) potassium channels. Nature Reviews Neuroscience,6(11), 850–862. Deng, P. Y., Avraham, O., Cavalli, V., & Klyachko, V. A. (2021). Hyperexcitability of sensory neurons in fragile X mouse model. Frontiers in Molecular Neuroscience,14, 796053. Deng,P.Y.,Carlin,D.,Oh,Y.M.,Myrick,L.K.,Warren,S.T., Cavalli, V., & Klyachko, V. A. (2019). Voltage-independent SK-channel dysfunction causes neuronal hyperexcitability in the hippocampus of Fmr1 knock-out mice. Journal of Neuroscience,39(1), 28–43. Deng, P. Y., & Klyachko, V. A. (2016). Increased persistent sodium current causes neuronal hyperexcitability in the entorhinal cortex of Fmr1 knockout mice. Cell Reports, 16(12), 3157–3166. Deng, P. Y., & Klyachko, V. A. (2021). Channelopathies in fragile X syndrome. Nature Reviews Neuroscience,22(5), 275–289. Deng, P. Y., Kumar, A., Cavalli, V., & Klyachko, V. A. (2024). Circuit-based intervention corrects excessive dentate gyrus output in the fragile X mouse model. Elife,12, RP92563. Deng, P. Y., Rotman, Z., Blundon, J. A., Cho, Y., Cui, J., Cavalli, V., Zakharenko, S. S., & Klyachko, V. A. (2013). FMRP regulates neurotransmitter release and synaptic information transmission by modulating action potential duration via BK channels. Neuron,77(4), 696–711. © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3789 Dulla,C.G.,Janigro,D.,Jiruska,P.,Raimondo,J.V.,Ikeda,A., Lin, C. K., Goodkin, H. P., Galanopoulou, A. S., Bernard, C., & de Curtis, M. (2018). How do we use in vitro models to understand epileptiform and ictal activity? A report of the TASK1-WG4 group of the ILAE/AES Joint Translational Task Force. Epilepsia Open,3(4), 460–473. Dwivedi, D., Chattarji, S., & Bhalla, U. S. (2019). Impaired reliability and precision of spiking in adults but not juvenilesinamousemodeloffragileXsyndrome.eNeuro, 6(6), ENEURO.0217–19.2019. Ferron, L. (2016). Fragile X mental retardation protein controls ion channel expression and activity. The Journal of Physiology,594(20), 5861–5867. Ferron, L., Nieto-Rostro, M., Cassidy, J. S., & Dolphin, A. C. (2014). Fragile X mental retardation protein controls synaptic vesicle exocytosis by modulating N-type calcium channel density. Nature Communications,5, 3628. Fontán-Lozano, A., Suárez-Pereira, I., Delgado-García, J. M., & Carrión, A. M. (2011). The M-current inhibitor XE991 decreases the stimulation threshold for long-term synaptic plasticity in healthy mice and in models of cognitive disease. Hippocampus,21(1), 22–32. Galvin, V. C., Yang, S. T., Paspalas, C. D., Yang, Y., Jin, L. E., Datta,D.,Morozov,Y.M.,Lightbourne,T.C.,Lowet,A.S., Rakic, P., Arnsten, A. F. T., & Wang, M. (2020). Muscarinic M1 receptors modulate working memory performance and activity via KCNQ potassium channels in the primate prefrontal cortex. Neuron,106(4), 649–661.e4. Gibson,J.R.,Bartley,A.F.,Hays,S.A.,&Huber,K.M. (2008). Imbalance of neocortical excitation and inhibition and altered UP states reflect network hyperexcitability in the mouse model of fragile X syndrome. Journal of Neurophysiology,100(5), 2615–2626. Greene, D. L., & Hoshi, N. (2017). Modulation of Kv7 channels and excitability in the brain. Cellular and Molecular Life Sciences,74(3), 495–508. Gross, C., Yao, X., Pong, D. L., Jeromin, A., & Bassell, G. J. (2011). Fragile X mental retardation protein regulates protein expression and mRNA translation of the potassium channel Kv4.2. Journal of Neuroscience,31(15), 5693–5698. Gu, N., Hu, H., Vervaeke, K., & Storm, J. F. (2008). SK (KCa2) channels do not control somatic excitability in CA1pyramidalneuronsbutcanbeactivatedbydendritic excitatory synapses and regulate their impact. Journal of Neurophysiology,100(5), 2589–2604. Gu, N., Vervaeke, K., Hu, H., & Storm, J. F. (2005). Kv7/KCNQ/M and HCN/h, but not KCa2/SK channels, contribute to the somatic medium after-hyperpolarization and excitability control in CA1 hippocampal pyramidal cells. The Journal of Physiology,566(Pt 3), 689–715. Gunthorpe, M. J., Large, C. H., & Sankar, R. (2012). The mechanism of action of retigabine (ezogabine), a first-in-class K+channel opener for the treatment of epilepsy. Epilepsia,53(3), 412–424. Halliwell, J. V., & Adams, P. R. (1982). Voltage-clamp analysis of muscarinic excitation in hippocampal neurons. Brain Research,250(1), 71–92. He, Q., Nomura, T., Xu, J., & Contractor, A. (2014). The developmentalswitchinGABApolarityisdelayedinfragile X mice. Journal of Neuroscience,34(2), 446–450. Heuzeroth, H., Wawra, M., Fidzinski, P., Dag, R., & Holtkamp, M. (2019). The 4-aminopyridine model of acute seizures in vitro elucidates efficacy of new antiepileptic drugs. Frontiers in Neuroscience,13, 677. Hönigsperger,C.,Marosi,M.,Murphy,R.,&Storm,J.F. (2015). Dorsoventral differences in Kv7/M-current and its impact on resonance, temporal summation and excitability in rat hippocampal pyramidal cells. The Journal of Physiology,593(7), 1551–1580. Hwang,J.Y.,Monday,H.R.,Yan,J.,Gompers,A.,Buxbaum, A.R.,Sawicka,K.J.,Singer,R.H.,Castillo,P.E.,&Zukin, R. S. (2022). CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X. Cell Reports,39(10), 110853. Incorpora, G., Sorge, G., Sorge, A., & Pavone, L. (2002). Epilepsy in fragile X syndrome. Brain & Development, 24(8), 766–769. Isaeva, E., Romanov, A., Holmes, G. L., & Isaev, D. (2015). Status epilepticus results in region-specific alterations in seizure susceptibility along the hippocampal longitudinal axis. Epilepsy Research,110, 166–170. Jentsch, T. J. (2000). Neuronal KCNQ potassium channels: Physiology and role in disease. Nature Reviews Neuroscience,1(1), 21–30. Kalmbach, B. E., Johnston, D., & Brager, D. H. (2015). Cell-type specific channelopathies in the prefrontal cortex of the Fmr1−/y mouse model of fragile X syndrome. eNeuro, 2(6), ENEURO.0114–15.2015. Kenmuir, C., Richardson, M., & Ghearing, G. (2015). Surgical treatment for medically refractory focal epilepsy in a patient with fragile X syndrome. Brain & Development,37(9), 916–918. Lee,H.Y.,Ge,W.P.,Huang,W.,He,Y.,Wang,G.X., Rowson-Baldwin, A., Smith, S. J., Jan, Y. N., & Jan, L. Y. (2011). Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein. Neuron,72(4), 630–642. Liu, Y., Bian, X., & Wang, K. (2021). Pharmacological activation of neuronal voltage-gated Kv7/KCNQ/M-channels for potential therapy of epilepsy and pain. Handbook of Experimental Pharmacology, 267, 231–251. Luque, M. A., Beltran-Matas, P., Marin, M. C., Torres, B., & Herrero, L. (2017). Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice. PLoS ONE, 12(9), e0185067. Marguet, S. L., Le-Schulte, V. T. Q., Merseburg, A., Neu, A., Eichler, R., Jakovcevski, I., Ivanov, A., Hanganu-Opatz, I. L., Bernard, C., Morellini, F., & Isbrandt, D. (2015). Treatment during a vulnerable developmental period rescues a genetic epilepsy. Nature Medicine,21(12), 1436–1444. Marrion, N. V. (1997). Control of M-current. Annual Review of Physiology,59, 483–504. © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3790 M. A. Luque and others J Physiol 602.15 Martin, H. G. S., Lassalle, O., Brown, J. T., & Manzoni, O. J. (2015). Age-dependent long-term potentiation deficits in the prefrontal cortex of the Fmr1 knockout mouse model of fragile X syndrome. Cerebral Cortex,26(5), 2084–2092. Mateos-Aparicio, P., Murphy, R., & Storm, J. F. (2014). Complementary functions of SK and Kv7/M potassium channels in excitability control and synaptic integration in rat hippocampal dentate granule cells. The Journal of Physiology,592(4), 669–693. Melancia, F., & Trezza, V. (2018). Modelling fragile X syndrome in the laboratory setting, A behavioral perspective. Behavioural Brain Research,350, 149–163. Moore, S. D., Madamba, S. G., Schweitzer, P., & Siggins, G. R. (1994). Voltage-dependent effects of opioid peptides on hipocampal CA3 pyramidal neurons in vitro. Journal of Neuroscience,14(2), 809–820. Musumeci,S.A.,Bosco,P.,Calabrese,G.,Bakker,C.,De Sarro, G. B., Elia, M., Ferri, R., & Oostra, B. A. (2000). Audiogenic seizures susceptibility in transgenic mice with fragile X syndrome. Epilepsia,41(1), 19–23. Myers, T. L., Gonzalez, O. C., Stein, J. B., & Bazhenov, M. (2018). Characterizing concentration-dependent neural dynamics of 4-aminopyridine-induced epileptiform activity. Journal of Epilepsy,4(2), 128. Nigro, M. J., Mateos-Aparicio, P., & Storm, J. F. (2014). Expression and functional roles of Kv7/KCNQ/M-channels in rat medial entorhinal cortex layer II stellate cells. Journal of Neuroscience,34(20), 6807–6812. Ordemann, G. J., Apgar, C. J., & Brager, D. H. (2019). D-type potassium channels normalize action potential firing between dorsal and ventral CA1 neurons of the mouse hippocampus. Journal of Neurophysiology,121(3), 983–995. Otto, J. F., Yang, Y., Frankel, W. N., Wilcox, K. S., & White, H. S. (2004). Mice carrying the szt1 mutation exhibit increased seizure susceptibility and altered sensitivity to compounds acting at the m-channel. Epilepsia,45(9), 1009–1016. Papatheodoropoulos, C., Moschovos, C., & Kostopoulos, G. (2005). Greater contribution of N-methyl-D-aspartic acid receptors in ventral compared to dorsal hippocampal slices in the expression and long-term maintenance of epileptiform activity. Neuroscience,135(3), 765–779. Paradee, W., Melikian, H. E., Rasmussen, D. L., Kenneson, A., & Conn, P. J. (1999). Fragile X mouse: Strain effects of knockout phenotype and evidence suggesting deficient amygdala function. Neuroscience,94(1), 185–192. Penagarikano, O., Mulle, J. G., & Warren, S. T. (2007). The pathophysiology of fragile x syndrome. Annual Review of Genomics and Human Genetics,8, 109–129. Peters, H. C., Hu, H., Pongs, O., Storm, J. F., & Isbrandt, D. (2005). Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nature Neuroscience,8(1), 51–60. Petrovic, M. M., Nowacki, J., Olivo, V., Tsaneva-Atanasova, K., Randall, A. D., & Mellor, J. R. (2012). Inhibition of post-synaptic Kv7/KCNQ/M channels facilitates long-term potentiation in the hippocampus. PLoS ONE,7(2), e30402. Routh, B. N., Johnston, D., & Brager, D. H. (2013). Loss of functional A-type potassium channels in the dendrites of CA1 pyramidal neurons from a mouse model of fragile X syndrome. Journal of Neuroscience,33(50), 19442–19450. Routh,B.N.,Rathour,R.K.,Baumgardner,M.E.,Kalmbach, B. E., Johnston, D., & Brager, D. H. (2017). Increased transient Na+conductance and action potential output in layer 2/3 prefrontal cortex neurons of the Fmr1−/y mouse. The Journal of Physiology,595(13), 4431–4448. Sabaratnam, M., Vroegop, P. G., & Gangadharan, S. K. (2001). Epilepsy and EEG findings in 18 males with fragile X syndrome. Seizure: The Journal of the British Epilepsy Association,10(1), 60–63. Safiulina, V. F., Zacchi, P., Tagliatela, M., Yaari, Y., & Cherubini, E. (2008). Low expression of Kv7/M channels facilitates intrinsic and network bursting in the developing rat hippocampus. The Journal of Physiology,586(22), 5437–5453. Schwarz, J. R., Glassmeier, G., Cooper, E. C., Kao, T. C., Nodera, H., Tabuena, D., Kaji, R., & Bostock, H. (2006). KCNQ channels mediate IKs, a slow K+current regulating excitability in the rat node of Ranvier. The Journal of Physiology,573(Pt 1), 17–34. Shah, M. M., Migliore, M., Valencia, I., Cooper, E. C., & Brown, D. A. (2008). Functional significance of axonal Kv7 channels in hippocampal pyramidal neurons. Proceedings National Academy of Science USA,105(22), 7869– 7874. Shah,M.M.,Mistry,M.,Marsh,S.J.,Brown,D.A.,&Delmas, P. (2002). Molecular correlates of the M-current in cultured rat hippocampal neurons. The Journal of Physiology,544(Pt 1), 29–37. Singh,N.A.,Charlier,C.,Stauffer,D.,DuPont,B.R.,Leach, R.J.,Melis,R.,Ronen,G.M.,Bjerre,I.,Quattlebaum,T., Murphy, J. V., McHarg, M. L., Gagnon, D., Rosales, T. O., Peiffer, A., Anderson, V. E., & Leppert, M. (1998). A novel potassium channel gene, KCNQ2, is mutated in an inherited epilepsy of newborns. Nature Genetics,18(1), 25–29. Sitzmann,A.F.,Hagelstrom,R.T.,Tassone,F.,Hagerman, R. J., & Butler, M. G. (2018). Rare FMR1 gene mutations causing fragile X syndrome: A review. American Journal of Medical Genetics. Part A,176(1), 11–18. Strumbos,J.G.,Brown,M.R.,Kronengold,J.,Polley,D.B., & Kaczmarek, L. K. (2010). Fragile X mental retardation protein is required for rapid experience-dependent regulation of the potassium channel Kv3.1b. Journal of Neuroscience,30(31), 10263–10271. Tzingounis, A. V., Heidenreich, M., Kharkovets, T., Spitzmaul, G., Jensen, H. S., Nicoll, R. A., & Jentsch, T. J. (2010). The KCNQ5 potassium channel mediates a component of the afterhyperpolarization current in mouse hippocampus. Proceedings National Academy of Science USA,107(22), 10232–10237. Wang,H.S.,Pan,Z.,Shi,W.,Brown,B.S.,Wymore,R.S., Cohen, I. S., Dixon, J. E., & McKinnon, D. (1998). KCNQ2 and KCNQ3 potassium channel subunits: Molecular correlates of the M-channel. Science,282(5395), 1890–1893. Wei, X., Zhang, Y., Yin, B., Wen, J., Cheng, J., & Fu, X. (2018). The expression and function of KCNQ potassium channels in human chorionic plate arteries from women with normal pregnancies and pre-eclampsia. PLoS ONE,13(3), e0192122 © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License J Physiol 602.15 M current and cell excitability in FXS 3791 Woehler, A., Lin, K. H., & Neher, E. (2014). Calcium-buffering effects of gluconate and nucleotides, as determined by a novel fluorimetric titration method. The Journal of Physiology,592(22), 4863–4875. Yang, Y. M., Arsenault, J., Bah, A., Krzeminski, M., Fekete, A.,Chao,O.Y.,Pacey,L.K.,Wang,A.,Forman-Kay,J., Hampson, D. R., & Wang, L. Y. (2020). Identification of a molecular locus for normalizing dysregulated GABA release from interneurons in the Fragile X brain. Molecular Psychiatry,25(9), 2017–2035. Yue, C., & Yaari, Y. (2004). KCNQ/M channels control spike afterdepolarization and burst generation in hippocampal neurons. Journal of Neuroscience,24(19), 4614–4624. Yue, C., & Yaari, Y. (2006). Axo-somatic and apical dendritic Kv7/M channels differentially regulate the intrinsic excitability of adult rat CA1 pyramidal cells. Journal of Neurophysiology,95(6), 3480–3495. Zhan, X., Asmara, H., Cheng, N., Sahu, G., Sanchez, E., Zhang, F. X., Zamponi, G. W., Rho, J. M., & Turner, R. W. (2020). FMRP(1–297)-tat restores ion channel and synaptic function in a model of Fragile X syndrome. Nature Communications,11(1), 2755. Zhang, L., Liang, Z., Zhu, P., Li, M., Yi, Y. H., Liao, W. P., & Su, T. (2016). Altered intrinsic properties and bursting activities of neurons in layer IV of somatosensory cortex from Fmr-1 knockout mice. Experimental Neurology,280, 60–69. Zhang, L., Weiner, J. L., Valiante, T. A., Velumian, A. A., Watson, P. L., Jahromi, S. S., Schertzer, S., Pennefather, P., & Carlen, P. L. (1994). Whole-cell recording of the Ca(2+)-dependent slow afterhyperpolarization in hippocampal neurones: Effects of internally applied anions. Pflugers Archiv: European Journal of Physiology,426(3–4), 247–253. Zhang, X. F., Han, P., Neelands, T. R., McGaraughty, S., Honore, P., Surowy, C. S., & Zhang, D. (2011). Coexpression and activation of TRPV1 suppress the activity of the KCNQ2/3 channel. Journal of General Physiology,138(3), 341–352 Zhang,Y.,Bonnan,A.,Bony,G.,Ferezou,I.,Pietropaolo,S., Ginger, M., Sans, N., Rossier, J., Oostra, B., LeMasson, G., & Frick, A. (2014). Dendritic channelopathies contribute to neocortical and sensory hyperexcitability in Fmr1(−/y) mice. Nature Neuroscience,17(12), 1701–1709. Zorio,D.A.,Jackson,C.M.,Liu,Y.,Rubel,E.W.,&Wang, Y. (2017). Cellular distribution of the fragile X mental retardation protein in the mouse brain. Journal of Comparative Neurology,525(4), 818–849. Additional information Data availability statement Thedatathatsupportthefindingsofthisstudyareavailablefrom the corresponding author upon reasonable request. Competing interests None declared. Author contributions All experiments were performed in the Department of Physiology, Faculty of Biology, University of Seville, Spain. M.A.L., B.T. and L.H. were responsible for the conception and design of the work. M.A.L, S.M. and L.H. performed the experiments and the analysis. M.A.L., B.T. and L.H. interpreted the results. B.T. and L.H. drafted the manuscript. All authors edited and revised the manuscript and approved the final version for publication. L.H. is responsible for all aspects of the reported work, including the integrity of the data collected and the accuracy of the data analysis. Funding ThisworkwassupportedbytwograntsfromtheVIIPlan Propio de Investigación of the University of Seville (Grant numbers 2022/00000326 and 2024/00000601), and a grant from the Asociacion Sindrome X Fragil Pais Vasco (3607/0980). Keywords action potential threshold, firing properties, FMRP, FXS, KCNQ channels Supporting information Additional supporting information can be found online in the Supporting Information section at the end of the HTML view of the article. Supporting information files available: Statistical Summary Document Peer Review History © 2024 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society. 14697793, 2024, 15, Downloaded from https://physoc.onlinelibrary.wiley.com/doi/10.1113/JP285244 by Readcube (Labtiva Inc.), Wiley Online Library on [13/12/2024]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License