Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice

RESEARCH ARTICLE Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice M. Angeles Luque, Pablo Beltran-Matas, M. Carmen Marin, Blas Torres, Luis Herrero* Department of Physiology. University of Seville, Seville, Spain *[email protected] Abstract Fragile X syndrome (FXS) is caused by a failure of neuronal cells to express the gene encoding the fragile mental retardation protein (FMRP). Clinical features of the syndrome include intellectual disability, learning impairment, hyperactivity, seizures and anxiety. Fmr1 knockout (KO) mice do not express FMRP and, as a result, reproduce some FXS behavioral abnormalities. While intrinsic and synaptic properties of excitatory cells in various part of the brain have been studied in Fmr1 KO mice, a thorough analysis of action potential characteristics and input-output function of CA1 pyramidal cells in this model is lacking. With a view to determining the effects of the absence of FMRP on cell excitability, we studied rheobase, action potential duration, firing frequency–current intensity relationship and action potential after-hyperpolarization (AHP) in CA1 pyramidal cells of the hippocampus of wild type (WT) and Fmr1 KO male mice. Brain slices were prepared from 8- to 12-week-old mice and the electrophysiological properties of cells recorded. Cells from both groups had similar resting membrane potentials. In the absence of FMRP expression, cells had a significantly higher input resistance, while voltage threshold and depolarization voltage were similar in WT and Fmr1 KO cell groups. No changes were observed in rheobase. The action potential duration was longer in the Fmr1 KO cell group, and the action potential firing frequency evoked by current steps of the same intensity was higher. Moreover, the gain (slope) of the relationship between firing frequency and injected current was 1.25-fold higher in the Fmr1 KO cell group. Finally, AHP amplitude was significantly reduced in the Fmr1 KO cell group. According to these data, FMRP absence increases excitability in hippocampal CA1 pyramidal cells. Introduction Fragile X syndrome (FXS) is the most common form of inherited human intellectual disability. Many FXS patients display learning impairment, hyperactivity, hypersensitivity to sensory stimuli, seizures and anxiety. Thirty percent of children with FXS are diagnosed with autism [1]. FXS is caused by transcriptional silencing of the FMR1 gene which encodes the fragile mental retardation protein (FMRP). Fmr1 knockout (KO) mice do not express FMRP, and reproduce some of the behavioral abnormalities seen in FXS; these animals are commonly used as a model to understand the molecular-, synaptic-, cellular-, and neural network-bases of the syndrome [2–7]. PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 1 / 18 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Luque MA, Beltran-Matas P, Marin MC, Torres B, Herrero L (2017) Excitability is increased in hippocampal CA1 pyramidal cells of Fmr1 knockout mice. PLoS ONE 12(9): e0185067. https://doi.org/10.1371/journal.pone.0185067 Editor: Ste ´phane Charpier, University Paris 6, FRANCE Received: May 11, 2017 Accepted: September 6, 2017 Published: September 20, 2017 Copyright: ©2017 Luque et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The authors received no specific funding for this work. Competing interests: The authors have declared that no competing interests exist. Electrophysiological research carried out on brain tissue from Fmr1 KO mice has identified impairment of long- and short-term synaptic plasticity [8–10], abnormal dendritic excitability associated with alterations in the expression and/or function of several types of voltage-gated ion channels [11–15], and presynaptic dysfunction dependent on N-type voltage-gated calcium channels [16]. The abnormal dendritic excitability attributed to ion channels appears to be specific both to the brain region and to the cell type under investigation [13,15,17]. Studies of intrinsic excitability using somatic patch-clamp recordings have also been carried out. Some reports suggested unaltered membrane properties of layer 5 pyramidal neurons in the somatosensory cortex [15,18]. On the other hand, an increased input resistance probably underlies a decrease of the minimum current step required to evoke an action potential and the increased firing frequency seen in response to a given suprathreshold current injection in layer 4 excitatory neurons in the barrel sensory cortex [19]. This neuronal population, under an epileptiform condition, switch from a regular spiking pattern to a seizure-like activity [20]. An absence of FMRP increased the persistent sodium current which diminished action potential threshold and caused pyramidal cell hyperexcitability in the entorhinal cortex [21]. Layer 2/3 neurons of the prefrontal cortex in Fmr1 KO mice display a higher excitability as measured at the soma, which could result from a larger transient Na + current and a depolarizing shift in the activation of A-type K + conductance [22]. Action potential broadening, via a reduction in the activity of BK channels, has been reported in layer 5 pyramidal cells of the entorhinal cortex and in CA3 pyramidal neurons of the hippocampus in Fmr1 KO mice [23]. In this way, a primary objective of the current work was to increase our present understanding of cell excitability by studying the hippocampal CA1 pyramidal neurons in wild-type (WT) and Fmr1 KO mice. The hippocampus is widely recognized as a critical structure for learning and memory; cell hyperexcitability could, at least in part, underlie behavioral deficits associated with the absence of FMRP [for review see 5,6,7]. FMRP is highly expressed in the somatodendritic domains of neurons in all hippocampal areas [24] and acts through multiple mechanisms, including as a translational regulator of its mRNA targets, some of which, encode voltage-gated ion channels, and interaction with ion channel-associated proteins [11,16,23,25–29]. In physiological conditions, cell bodies integrate incoming signals from synaptic transmission. Abnormal channel expression and/or function can have substantial effects on the processes of cell body signal integration and the generation of neuronal outputs [30]. The manner in which the absence of FMRP can affect cell computations can be captured by the input–output function. The input– output function is characterized by the threshold and the gain [31]. Here we define the threshold from rheobase and the gain from the slope of the relationship between firing frequency and injected current. Modifications in resting membrane potential, input resistance, and /or action potential voltage threshold could shift rheobase, and therefore increase or decrease cell excitability. A change in the slope (gain) can lead to the amplification or scaling-down of the sensitivity of the neuron to stimuli [31,32]. On the other hand, FMRP-dependent alterations in action potential duration can modify neurotransmitter release [23]. Based on this knowledge, we investigated whether the absence of FMRP by genetic modification alters rheobase, the firing frequency–current injection relationship, and/or the shape of the action potential, including after-hyperpolarization (AHP). Materials and methods Slice preparation Fmr1 KO mice (FVB.129P2-Pde6b + Tyr c-ch Fmr1 tm1Cgr /J; stock# 4624, Jackson Laboratory) and wild-type (WT) mice (FVB.129P2-Pde6b + Tyr c-ch /AntJ; stock# 4828, Jackson Laboratory) were used in these studies. All experimental procedures were approved by the Ethics Committee on Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 2 / 18 Animal Experimentation at the University of Seville (Permit number 12-06-15.260) 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). Male mice (8–12 weeks old) were anesthetized deeply with sodium pentobarbital (50 mg kg-1) and all efforts were made to minimize suffering. Mice were then 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 (300-μm thickness) including the hippocampi were cut using a vibrating microtome (Leica 1200S). Slices were first incubated in a chamber containing ACSF for 30–45 minutes at 32˚C, and then maintained at room temperature. Single slices were transferred to the recording chamber and superfused at 2 ml min -1 with ACSF bubbled with 95% O2–5% CO2 (pH 7.4; 32 ±1˚C). The composition of ACSF was as follows (in mM): 126 NaCl, 2 KCl, 1.25 Na2HPO4, 26 NaHCO3, 10 glucose, 2 MgSO4, and 2 CaCl 2 . For the sucrose-ACSF solution, NaCl was substituted by sucrose (240 mM) and the concentrations of MgSO 4 and CaCl 2 were modified to 4 mM and 0.1 mM, respectively. Electrophysiological recordings Whole-cell patch clamping was performed on visually identified somata of CA1 pyramidal cells 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 [33] in the dorsal region of the hippocampus. For current clamp recordings, patch pipettes (4–6 MΩ) contained (in mM): 120 potassium gluconate, 10 KCl, 10 phosphocreatine disodium salt, 2 MgATP, 0.3 NaGTP, 0.1 EGTA and 10 Hepes, adjusted to pH 7.3 with KOH. The osmolality of intracellular solutions was 280–290 mosmol l −1 , adjusted with sucrose. Whole-cell patch clamp recordings were carried out using a MultiClamp 700B Amplifier (Molecular Devices). Gigaseals (>1 GO) were obtained before rupture of the patch, pipette capacitance was compensated before breaking in and the bridge was balanced using the auto-adjust button. Series resistance was 20 MOor less during recording. Current clamp 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 and analyzed offline using Clampfit 10.2 (Molecular Devices) software. Voltages were not corrected for the liquid-junction potential (estimated as ~14 mV). Data acquisition and analysis Current clamp experiments were conducted to determine whether FMRP modulates the cell excitability of hippocampal CA1 pyramidal cells and a number of parameters were quantified. Resting membrane potentials were measured as the difference between the intracellular and extracellular potentials. Input resistance was determined by passing current steps (500 ms, 1 Hz; with 10 pA increments from 20 to—60 pA), and calculated as the slope of the current-volt- age plot. To measure inward rectification, or sag, a current pulse that drifted the membrane potential from rest to -85 ±2 mV was applied; sag amplitude was calculated as the difference between membrane potentials at peak and at steady-state; the percentage of sag was equal to (sag amplitude / voltage difference between rest and peak amplitude) x 100. The membrane time constant was obtained by recording the membrane response to 20 pA hyperpolarizing current pulses (500 ms duration, 1 Hz) and fitting the response to a single exponential curve. We chose 20 pA of hyperpolarizing current because such a current intensity did not produce sag. The rheobase was the minimum injected current that generated an action potential in 50% of cases, and was measured for pulse durations of 500 ms (1Hz; with 2 pA increments). Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 3 / 18 Depolarization voltage was the increase in membrane potential required for the cell to reach the spike voltage threshold. To assess the spike voltage threshold, we used phase-plane plots and the spike onset was taken as the value of the membrane potential at which the first derivative exceeded 10 V s −1 . Single action potentials were evoked by short (100 μs) depolarizing current pulses; the current intensity was progressively increased by 0.5 nA current steps until the spike voltage threshold was reached. Action potential amplitude was the voltage increment between the resting level and spike voltage peak. The value of the duration of the action potential was determined as the width of the spike at half-maximal amplitude. Repetitive firing was evoked by depolarizing current steps (500 ms, 0.5 Hz) with 10–50 pA increments, and the mean firing for each injected current in WT and Fmr1 KO cells was calculated. Next, the relationship between the firing frequency and injected current was plotted for each cell and the slope (gain) determined. Evoked action potentials were followed by an after-depolarization (ADP) and /or AHP. A 100 μs stimulus with current intensity adjusted just above the spike voltage threshold yielded an action potential often followed by an ADP, the amplitude of which was taken as the voltage difference between rest and membrane potential peak during ADP. The duration of the ADP corresponded to the time between ADP onset and rest recovery. The amplitude and /or duration of the fast and medium components of the action potential AHP were also measured [34]. AHP parameters were quantified for three conditions: (1) with current depolarizing pulses of 500 ms that shifted the membrane potential to the spike threshold value and evoked spikes at a low firing frequency (2 action potential s -1 ); (2) with suprathreshold current steps lasting 500 ms and producing 8–12 action potentials; and (3) at the end of the repetitive discharge evoked by the previous current injections. For low firing frequency, fast AHP amplitude was the difference between resting membrane potential and the lowest value of membrane potential during this AHP phase. The amplitude of the medium AHP was measured as the difference between rest and membrane potential in a time window of 5–15 ms after the lowest value of membrane potential during fast AHP. To measure the medium AHP component after repetitive discharge, the amplitude and duration were assessed relative to the resting membrane potential. For spike trains, AHP amplitudes were assessed relative to the action potential threshold. To study spike-frequency adaptation, the instantaneous frequency was calculated as the reciprocal of the inter-spike interval duration. The spike-frequency adaptation was then quantified by the adaptation index, which is equal to [1—(frequency in the steady-state ⁄instantaneous frequency in the first inter-spike interval)]. The steady-state firing frequency was taken as the average of the instantaneous frequency during the last 250 ms of the repetitive discharge. Adaptation index was measured from trains of 8–12 action potentials evoked by 500 ms current injections. Statistical analysis Significant differences between WT and Fmr1 KO cell groups were determined by using the Student’s t test for unpaired samples as values were normally distributed within each group with equal variance between groups. The correlation between variables was measured by Pearson’s correlation coefficient (r). The significance level was established at P <0.05. All data are reported as mean ±standard error of the mean. The percentage change in each parameter was normalized relative to the WT cell group and calculated as: (Fmr1 KO mean value—WT mean value / WT mean value) x 100. Results Rheobase in CA1 pyramidal cells is not modified in Fmr1 KO mice Whole-cell patch clamp recordings were performed on the somas of 33 WT and 32 Fmr1 KO pyramidal cells located within the hippocampal CA1 region. These cells were from slices Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 4 / 18 prepared from 14 WT and 16 Fmr1 KO mice (details in S1 and S2 Tables). Resting membrane potential, input resistance and action potential voltage threshold were measured to assess if the absence of FMRP leads to changes in rheobase. All cells included for analysis showed a stable resting membrane potential, and the mean value of the resting membrane potential was close to—63 mV in both cell groups (Fig 1D;Table 1). The Fmr1 KO cell group exhibited a significantly higher input resistance (~15%; Fig 1A and 1B;Table 1). The amplitude of sag was measured, as the altered functional expression of h-channels in dendrites has been linked to input resistance modifications in Fmr1 KO mice [13,15], and there were no differences between WT and Fmr1 KO cell groups (Table 1). The Fmr1 KO cell group showed a significantly higher membrane time constant (~15%) than the WT cell group (Table 1). Next, we measured whether spike voltage threshold and depolarization voltage (i.e. the difference between spike voltage threshold and resting membrane potential) were different between WT and Fmr1 KO cell groups and found non-significant differences (Fig 1C and 1D;Table 1). Higher values of input resistance in the Fmr1 KO cell group were accompanied by a reduction of rheobase (in the Fmr1 KO group was ~13% lower than in the WT group); however, differences in rheobase were not statistically significant (Fig 1D;Table 1). Action potential duration is longer in CA1 pyramidal cells from Fmr1 KO mice Action potential broadening associated with the absence of FMRP was recently reported for pyramidal cells within CA3 region of the hippocampus [23]. When this parameter was assessed in relation to excitatory neurons located in layer 5 of the entorhinal cortex, the magnitude of the effect of FMRP absence on the action potential duration appeared to fall into two different cell populations: one of these populations showed action potential broadening similar to CA3 pyramidal cells, whereas the other population exhibited highly exaggerated broadening. According to these reports, the absence of FMRP produces an increased action potential duration, the magnitude of which depends on the cell population under investigation. To determine if such a finding can be extended to CA1 pyramidal cells, the action potential duration was assessed following short- and long-duration current steps. Short current steps (100 μs) evoked single action potentials. These spikes were lower in amplitude (WT = 118.56 ±1.51 mV from 28 cells of 11 mice versus Fmr1 KO = 105.9 ±2.22 from 29 cells of 14 mice) and longer in duration (~7%) in cells from the Fmr1 KO group compared to cells from WT mice (Fig 2A;Table 1). Differences between groups were statistically significant for both parameters. Because action potential duration increases during burst firing [23], 500 ms current steps were injected to evoke trains of 8–12 action potentials and the durations of the first, middle (250 ms after stimulus onset) and last spike of the train were measured in both cell populations. Action potentials were longer in duration (~6%) in the Fmr1 KO cell population compared with the WT cell group (Fig 2B and 2C;Table 1). In conclusion, the absence of FMRP results in an increased action potential duration. The slope (gain) of the firing frequency–current intensity relationship is higher in CA1 pyramidal cells from Fmr1 KO mice CA1 pyramidal cells showed a repetitive discharge of action potentials in response to suprathreshold depolarizing current steps. Current steps of equal intensity produced a higher number of spikes in Fmr1 KO cells than in WT cells (Fig 3A and 3B). The increased firing frequency of Fmr1 KO cells was statistically significant for current steps of 100, 200, 300 and 400 pA (Fig 3B;Table 1). Fig 3C shows the firing frequency–current intensity relationships of representative WT and Fmr1 KO cells. As shown, firing frequency increases with the stimulus Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 5 / 18 Fig 1. Rheobase is not modified in CA1 pyramidal cells of Fmr1 KO mice. (A) Voltage responses of two representative WT and Fmr1 KO cells to depolarizing and hyperpolarizing current steps. (B) Histogram showing input resistance in WT and Fmr1 KO cell groups. (C) Representative recordings of the rheobase that was 70 pA for both WT and Fmr1 KO cells (left panel). Phase-plane plots to determine the action potential voltage threshold (right panel). (D) Histograms showing resting membrane potential, depolarization voltage, action potential voltage threshold and rheobase in WT and Fmr1 KO cell groups. Calibrations as indicated. In this and following figures, histograms show the mean value and standard error of the mean. Asterisks (*) indicate statistically significant differences (P<0.05). n.s. indicates non-significant statistical differences. https://doi.org/10.1371/journal.pone.0185067.g001 Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 6 / 18 Table 1. Membrane properties of the CA1 pyramidal cells in wild type and Fmr1 KO mice. Parameter Wild type mice (n) 1 Fmr1 KO mice (n) 1 Statistics 2 (P)*% Change 3 Resting membrane potential (mV) -63.33 ±0.49 (33/14) -63.02 ±0.87 (32/16) 0.760 -0.50 Input resistance (MΩ) 151.8 ±6.90 (32/13) 174.1 ±8.16 (32/16) 0.040*14.69 Time constant (ms) 20.41 ±0.78 (32/13) 23.39 ±0.93 (32/16) 0.017*14.60 Sag (%) 16.42 ±0.72 (13/9) 13.91 ±0.75 (21/11) 0.030 -9.19 Rheobase (pA) 76.64 ±11.68 (22/10) 66.94 ±9.02 (17/11) 0.538 -12.65 Voltage threshold (mV) -47.62 ±1.06 (22/10) -49.03 ±0.89 (17/11) 0.336 2.96 Depolarization voltage (mV) 15.24 ±1.29 (22/10) 15.15 ±1.23 (17/11) 0.963 -0.59 Action potential duration 4 (ms) Single action potential 0.99 ±0.01 (28/11) 1.06 ±0.02 (29/14) 0.026*7.07 Firing train (8–12 AP during 500 ms) First 0.96 ±0.02 (12/9) 1.02 ±0.01 (11/8) 0.046*6.25 Middle 0.98 ±0.01 (12/9) 1.04 ±0.01 (11/8) 0.035*6.12 Last 0.99 ±0.01 (12/9) 1.03 ±0.01 (11/8) 0.041*4.04 Firing frequency 5 (AP • s -1 ) 100 pA 10.64 ±1.71 (22/11) 15.58 ±1.58 (19/13) 0.042*46.42 200 pA 22.55 ±2.04 (22/11) 31.37 ±2.21 (19/13) 0.005*39.11 300 pA 32.57 ±2.16 (22/11) 41.05 ±1.79 (19/12) 0.006*26.03 400 pA 38.80 ±2.44 (20/10) 45.05 ±1.75 (19/12) 0.046*16.10 Gain 6 0.12 ±0.006 (22/11) 0.15 ±0.006 (19/12) 0.013*25 ADP/AHP 7 ADP single action potential Amplitude (mV) 8.01 ±0.66 (24/14) 6.93 ±0.55 (26/13 0.220 -13.48 Duration (ms) 65.96 ±4.08 (24/14) 92.89 ±7.49 (26/13) 0.003*40.82 AHP low frequency (2 AP • s -1 ) fAHP Amplitude (mV) 10.29 ±0.92 (22/11) 7.68 ±0.80 (17/11) 0.047*-25.36 fAHP Duration (ms) 3.33 ±0.41 (22/11) 3.52 ±0.19 (17/11) 0.709 5.70 mAHP Amplitude (mV) 7.57 ±0.76 (22/11) 4.40 ±0.57 (16/11) 0.003*-41.87 mAHP Duration (ms) 167.73 ±9.65 (22/11) 153.5 ±16.63 (15/11) 0.436 -8.84 AHP firing train (8–12 AP during 500 ms) fAHP Amplitude (mV) 10.67 ±1.214 (12/9) 7.72 ±0.59 (11/8) 0.044*-27.64 fAHP Duration (ms) 2.05 ±0.19 (12/9) 3.17 ±0.12 (11/8) 0.001*54.63 mAHP Amplitude (mV) 9.64 ±1.32 (12/9) 6.13 ±0.90 (11/8) 0.043*-36.41 mAHP Duration (ms) 58.28 ±2.9 (12/9) 50.39 ±3.64 (11/8) 0.102 -13.53 mAHP after a firing train (8–12 AP during 500 ms) Amplitude (mV) 2.15 ±0.16 (5/3) 1.39 ±0.24 (4/3) 0.029*-35.34 Duration (ms) 121.2 ±16.29 (5/3) 141.78 ±25.3 (4/3) 0.498 16.98 Adaptation index 8 0.46 ±0.06 (12/9) 0. 43 ±0.06 (11/8) 0.763 -6.52 1 (n) indicates the number of cells / mice quantified for each parameter. 2 Statistical significance level (P value) after comparing mean values using Student’s t test for unpaired samples *indicates significant differences between both cell groups (P<0.05). 3 Data in this column correspond to the percentage of change between the mean values from Fmr1 KO cells compared to WT cells and was calculated as: (Fmr1 KO mean value—WT mean value / WT mean value) x 100. Positive and negative values indicate increments and decrements of Fmr1 KO cell group relative to WT cell group. 4 Action potential (AP) durations were measured after short stimuli (100 μs–single AP) and during 500 ms depolarizing current pulses. 5 Action potential firing frequencies were evoked by 500 ms depolarizing current steps at 100, 200, 300 and 400 pA and measured as action potentials (AP) per second. 6 Gain was the slope of the linear relationship between the firing frequency and injected current. 7 Action potentials after-depolarization (ADP) and after-hyperpolarization (AHP) were measured. Fast (fAHP) and medium (mAHP) were quantified for two conditions: with current steps of 500 ms that shifted membrane potential to the spike voltage threshold and evoked spikes at a low firing frequency (<2 action potential • s -1 ); with suprathreshold current steps that evoked 8–12 spikes. At the end of the repetitive discharge evoked by the previous suprathreshold current, mAHP was also quantified. 8 The adaptation index was calculated as [1—(frequency in the steady-state ⁄instantaneous frequency in the first inter-spike interval)]. The repetitive discharge (8–12 AP) was evoked by 500 ms depolarizing current steps. https://doi.org/10.1371/journal.pone.0185067.t001 Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 7 / 18 intensity, reaching a firing plateau with stimuli 300 pA. For this reason, data were fitted to a linear regression up to 300 pA. Both WT and Fmr1 KO neurons had a good fit (r>0,9), and Fmr1 KO cell group exhibited a significantly higher slope (gain) than WT (Fig 3D and 3E; Table 1). The mean gain in Fmr1 KO cell group was 1.25-fold higher than WT cell group. Next, we studied spike-frequency adaptation and found that these were similar (~ 0.45) in both cell groups (Table 1). In summary, the absence of FMRP resulted in an 1.25-fold increase in the gain of the firing frequency–current intensity relationship; this alteration leads to a heightened sensitivity of Fmr1 KO cells to changes in input. CA1 pyramidal cells showed an action potential ADP and AHP in response to short and long suprathreshold current steps, respectively (Fig 4). Stimuli of 100 μs duration evoked single action potentials that were often followed by an ADP (n = 24 of 28 WT cells and n = 26 of 29 Fmr1 KO cells). The amplitude of this response was similar in Fmr1 KO and WT cells, but the ADP duration was significantly longer in Fmr1 KO cells (Fig 4A;Table 1). The injection of 500 ms depolarizing current steps shifted the membrane potential to values close to the spike threshold, yielding action potentials at low firing frequencies (2 action potentials s -1 ). Under this condition, AHP recordings were noticeably different in cells from the two groups. WT cells exhibited a fast and a medium AHP, as already reported in rat CA1 pyramidal cells [33]. In Fmr1 KO cells, fast AHPs were observed, whereas medium AHPs were absent or weak (Fig 4B). Consistent with previously reported data for CA3 pyramidal cells [23], an absence of FMRP causes a significant reduction of the amplitude of fast AHPs in CA1 pyramidal cells (Table 1). Fast and medium AHP characteristics were also studied during repetitive discharge. Because firing frequency produces different amounts of calcium influx which can shape AHP [35,36], trains of 8–12 spikes were generated by 500 ms current injections and the fast and medium AHPs were studied. Amplitude and duration of these AHP phases were quantified for the action potential closest to 250 ms after stimulus onset. In the Fmr1 KO cell group, fast AHP amplitude (~28%) and duration (~55%) were reduced (Table 1); medium AHP amplitude decreased (~36%), while duration did not (Fig 4C, 4E and 4F;Table 1). Medium AHPs after similar repetitive firing were also studied. Some recorded cells of both groups showed an AHP following the action potential train. When present, the AHP amplitude was lower in the Fmr1 KO cell group (Fig 4D;Table 1). These data support the notion that the absence of FMRP has a considerable impact on conductances underlying AHP. Discussion The present study demonstrates an increase in excitability in hippocampal CA1 pyramidal cells from Fmr1 KO mice compared with WT mice. The origin of this increase was not due to modifications in rheobase. The action potential duration was longer in cells from the Fmr1 KO group, and the firing frequency evoked by current steps of the same intensity (100, 200, 300 and 400 pA) and duration (500 ms) were higher for this group. Moreover, the relationship between firing frequency and injected currents showed an almost 1.25-fold increase in the slope (gain) for cells from the Fmr1 KO cell group in comparison with WT cells. Firing frequency increase of Fmr1 KO cells was accompanied by a decrease in AHP amplitude. Modifications in input resistance, firing frequency gain and action potential duration reveal that the cell body input–output function is altered by the absence of FMRP in CA1 pyramidal neurons. Rheobase is not modified in CA1 pyramidal cells of Fmr1 KO mice The function of FMRP in altering membrane properties–excitability–from somatic recordings has previously been studied, and published reports suggest that FMRP-dependent intrinsic properties appear to be cell type-, brain region- and mouse strain-specific, as already proposed Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 8 / 18 Cell excitability in Fmr1 KO mice PLOS ONE | https://doi.org/10.1371/journal.pone.0185067 September 20, 2017 9 / 18 S2 Table. Membrane properties of the CA1 pyramidal cells in Fmr1 KO mice. (PDF) Author Contributions Conceptualization: M. Angeles Luque, Blas Torres, Luis Herrero. Formal analysis: M. Angeles Luque, Luis Herrero. Investigation: M. Angeles Luque, Pablo Beltran-Matas, M. Carmen Marin, Luis Herrero. Methodology: M. Angeles Luque, Blas Torres, Luis Herrero. Supervision: M. Angeles Luque, Blas Torres, Luis Herrero. Validation: M. Angeles Luque, Luis Herrero. Visualization: M. Angeles Luque, Blas Torres, Luis Herrero. Writing – original draft: M. Angeles Luque, Blas Torres, Luis Herrero. Writing – review & editing: M. Angeles Luque, Blas Torres, Luis Herrero. References 1. Pfeiffer BE, Huber KM. The state of synapses in fragile X syndrome. Neuroscientist 2009; 15: 549–567. https://doi.org/10.1177/1073858409333075 PMID: 19325170 2. Bear MF, Huber KM, Warren ST. The mGluR theory of fragile X mental retardation. Trends Neurosci 2004; 27: 370–377. https://doi.org/10.1016/j.tins.2004.04.009 PMID: 15219735 3. Penagarikano O, Mulle JG, Warren ST. The pathophysiology of fragile x syndrome. 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