Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity

Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity View ORCID Profile Gregory J. Ordemann , Polina Lyuboslavsky , Alena Kizimenko , View ORCID Profile Audrey C. Brumback doi: https://doi.org/10.1101/2025.02.02.636122 Gregory J. Ordemann 1 Department of Neurology, Dell Medical School at The University of Texas at Austin 3 Center for Learning and Memory at The University of Texas at Austin Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gregory J. Ordemann Polina Lyuboslavsky 1 Department of Neurology, Dell Medical School at The University of Texas at Austin 3 Center for Learning and Memory at The University of Texas at Austin Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alena Kizimenko 1 Department of Neurology, Dell Medical School at The University of Texas at Austin 3 Center for Learning and Memory at The University of Texas at Austin Find this author on Google Scholar Find this author on PubMed Search for this author on this site Audrey C. Brumback 1 Department of Neurology, Dell Medical School at The University of Texas at Austin 2 Department of Pediatrics, Dell Medical School at The University of Texas at Austin 3 Center for Learning and Memory at The University of Texas at Austin Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Audrey C. Brumback For correspondence: audrey.brumback{at}austin.utexas.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Background The neurodevelopmental disorder Fragile X syndrome (FXS) results from hypermethylation of the FMR1 gene which prevents FMRP production. FMRP modulates the expression and function of a wide variety of proteins, including voltage-gated ion channels such as Hyperpolarization-Activated Cyclic Nucleotide gated (HCN) channels, which are integral to rhythmic activity in thalamic structures. Thalamocortical pathology, particularly involving the mediodorsal thalamus (MD), has been implicated in neurodevelopmental disorders. MD connectivity with mPFC is integral to executive functions like working memory and social behaviors that are disrupted in FXS. Methods We used a combination of retrograde labeling and ex vivo brain slice whole cell electrophysiology in 40 wild type and 42 Fmr1 KO male mice to investigate how a lack of Fmr1 affects intrinsic cellular properties in lateral (MD-L) and medial (MD-M) MD neurons that project to the medial prefrontal cortex (MD→mPFC neurons). Results In MD-L neurons, Fmr1 knockout caused a decrease in HCN-mediated membrane properties such as voltage sag and membrane afterhyperpolarization. These changes in subthreshold properties were accompanied by changes in suprathreshold neuron properties such as the variability of action potential burst timing. Conclusions In Fmr1 knockout mice, reduced HCN channel activity in MD→mPFC neurons impairs both the timing and magnitude of HCN-mediated membrane potential regulation. Changes in response timing may adversely affect rhythm propagation in Fmr1 KO thalamocortical circuitry. MD thalamic neurons are critical for maintaining rhythmic activity involved in cognitive and affective functions. Understanding specific mechanisms of thalamocortical circuit activity may lead to therapeutic interventions for individuals with FXS. Introduction Fragile X syndrome (FXS) is a neurodevelopmental condition characterized by cognitive and social / emotional challenges that partially localize to the prefrontal brain network, which includes mediodorsal thalamus (MD) and prefrontal cortex (PFC). MD contributes to executive functioning and social motivation including attention, working memory, behavioral flexibility, cognitive flexibility, and social motivation ( 1 – 10 ). Changes in thalamocortical connectivity are implicated in autism spectrum disorders ( 11 , 12 ). Studies of executive dysfunction in FXS have identified deficits in working memory in human subjects ( 13 ) and cognitive flexibility in a mouse model of FXS ( 14 – 16 ). Previous work has demonstrated that the intrinsic excitability of the neurons that project from the prefrontal cortex to thalamus is disrupted in a mouse model of FXS ( 17 , 18 ). However, the the physiology of the neurons in the mediodorsal (MD) thalamus that provide ascending input to the prefrontal cortex are altered by a lack of Fmr1 remains unknown. The two major projections from MD to medial PFC (MD→mPFC) arise from the medial (MD-M) and lateral (MD-L) subnuclei ( 10 , 19 ). Previously, we found that MD-L→mPFC neurons have more Hyperpolarization-Activated Cyclic Nucleotide-Gated (HCN) channel activity, shorter membrane time constants, lower membrane resistance, and require stronger current injections to generate action potentials compared to MD-M→mPFC neurons ( 19 ). Animal models of FXS show that intrinsic ion channelopathies can result in changes to synaptic processing and plasticity ( 20 – 23 ). Additionally, HCN channelopathies have been identified in multiple brain regions in mouse models of FXS ( 18 , 20 , 24 ). The importance of the prefrontal thalamocortical network to executive and social / emotional function suggests MD involvement in cognitive and behavioral symptoms observed in patients with FXS. We hypothesized that Fmr1 KO MD→mPFC neurons would have intrinsic and circuit dysfunction, in part, due to ion channel dysfunction. To test this hypothesis, we used a retrograde tracer to fluorescently label MD neurons that project to prelimbic and infralimbic cortices in mouse (MD→mPFC neurons). In ex vivo thalamic slices from adult mice, we used whole cell current-clamp recordings to measure subthreshold and suprathreshold physiological properties of labeled MD-M→mPFC and MD-L→mPFC neurons. We compared Fmr1 knockout (KO) animals to their wildtype (WT) littermates. Loss of Fmr1 caused MD-L→mPFC neurons to display less HCN channel activity, which caused a mild increase in input resistance ( R N ). Loss of Fmr1 did not cause major changes in action potential generation in response to direct current injections. However, MD-L→mPFC neurons in Fmr1 KO mice showed dampening of HCN-dependent membrane resonator dynamics. First, in Fmr1 KO mice, MD-L→mPFC neurons had lower amplitude post-depolarization afterhyperpolarizations (AHP). Second, these same neurons showed a slowing of rebound spiking following release of hyperpolarization. These differences in cellular physiology related to membrane resonance have implications for slow wave oscillations in thalamic neurons which may influence prefrontal network activity in FXS with broad implications for sleep and cognitive function. Methods and Materials Animals All experiments were conducted in accordance with procedures established by the Institutional Animal Care and Use Committee (IACUC) at The University of Texas. Male mice (8-12 weeks) were used due to the significantly greater prevalence of FXS in males. Fmr1 het females (gift of Kimberly Huber, UT Southwestern) were crossed with wildtype C57Bl/6J males from Jackson Labs (stock #000664) to yield Fmr1 +/y (wildtype) and Fmr1 KO mice that were group-housed with same sex littermates in open-top cages in reverse lighting conditions (9am-9pm dark) and fed ad libitum . Fluorescent labeling of specific neuron populations Fluorescent labeling of wildtype and Fmr1 KO MD-L→mPFC and MD-M→mPFC neurons with cholera toxin subunit B (CTB, 500 µg / 100 µL, Molecular Probes, Thermo Fisher Scientific), was performed as in ( 19 ) . mPFC injection (450 nL at 100 nL/minute) was delivered to coordinates (in mm relative to Bregma): –1.7 anterior– posterior (AP), +0.3 mediolateral (ML) and –2.75 dorsoventral (DV). After 3-6 days brain tissue was processed for histology or electrophysiological recording. Experiments were performed after 3-6 days on neurons located in MD from bregma = -1.00 to bregma = -1.50. Neurons recorded anterior to this range were considered part of a transition area in MD and excluded from analysis ( 25 ). In this manuscript WT and Fmr1 KO refers to “WT MD-L” and “ Fmr1 KO MD-L” unless otherwise indicated. Histology Injected animals used for histological confirmation of injection location were transcardially perfused with paraformaldehyde (PFA, Sigma-Aldrich) 4% in phosphate-buffered saline (1x PBS). Tissue was sectioned in 50 µm thick coronal slices with DAPI-containing mounting medium (VectaShield HardSet with DAPI, Vector laboratories) and slices containing mPFC and MD were imaged at 5x using Zeiss Axio Imager 2. Slice preparation 250 μm thick slices (Leica VT1200) were prepared from mice 8 to 12 weeks old after intraperitoneal injection of ketamine / xylazine (90 / 10 mg/kg; Acor/Dechra). Mice were perfused with cutting solution containing (in mM): 205 sucrose, 25 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 7 MgCl 2 , 7 dextrose, 3 Na pyruvate, 1.3 sodium ascorbate, and 0.5 CaCl 2 bubbled with 95% O 2 / 5% CO 2 . Slices were incubated in holding solution containing (in mM): 125 NaCl, 25 NaHCO 3 , 2.5 KCl, 1.25 NaH 2 PO 4 , 25 dextrose, 2 CaCl 2 , 2 MgCl 2 , 1.3 sodium ascorbate, and 3 Na pyruvate at 37±1°C for 30 minutes then kept for at least 30 minutes at room temperature before recording began. Intracellular recordings Artificial cerebrospinal fluid (ACSF) contained (in mM): 125 NaCl, 25 NaHCO 3 , 12.5 dextrose, 2.5 KCl, 1.25 NaH 2 PO 4 2 CaCl 2 and 1 MgCl 2 . Slices were continuously perfused with ACSF in an immersion chamber (Warner Instruments) with temperature maintained at 32.5 ± 1°C (Warner Instruments TC-324C). We did not add synaptic blockers to the ACSF unless otherwise specified. In a subset of experiments ZD7288 (20 μM; Tocris Cat#1000) or TTX (0.5 μM; Abcam Cat# were added to extracellular ACSF. Somatic whole-cell patch recordings were obtained from retrogradely-labeled neurons in the medial (MD-M) or lateral (MD-L) subnuclei of WT and Fmr1 KO mice, using DODT (Zen 2.5 blue addition, Zen pro) contrast microscopy and epifluorescence on an upright microscope (Zeiss Examiner D1). Patch electrodes (tip resistance = 3–6 MΩ) were filled with the following (in mM): 118 K-gluconate, 10 KCl, 10 HEPES, 4 MgATP, 1 EGTA, 0.3 Na3GTP and 0.3% biocytin (pH adjusted to 7.2 with KOH; 282 mOsM). For some cells, 16 µM Alexa 488 or Alexa 594 was added to the internal solution to visualize the dendritic arbor under epifluorescence. Recordings were made with Clampex 10.7 software running a Multiclamp 700B (Molecular Devices). Signals were digitized at 20 kHz and lowpass filtered at 4 kHz. Data were collected at resting membrane potential (RMP) and -65 ± 3 mV. Unless stated otherwise, all data reported here were taken from recordings performed at -65 mV. Experiments were discontinued if series resistance rose above 30 MΩ or action potentials failed to overshoot 0 mV. Liquid junction potential was estimated to be 14.3 mV using Patchers Power Tools (IGORpro 7, Wavemetrics). Liquid junction potential was not corrected. The data sets were sampled from 40 WT and 42 Fmr1 KO mice. Electrophysiological properties The analysis of electrophysiological data was performed as in ( 19 ) . Briefly, RMP, τ, and R N . were measured from voltage responses to subthreshold current injections. Analyses were performed on a series of current injections ranging from -60 to 60 pA in 5 pA intervals for 1000 ms or from -250 to 350 in 25 pA intervals. Voltage sag is the difference between peak hyperpolarization and steady state voltage deflection in the trace with a peak hyperpolarization of -100 mV. Afterhyperpolarzation is the difference between peak hyperpolarization and baseline membrane potential following the offset of the first current stimulus to have 12 or more action potentials. In Figs. 3F & I and Fig. 5C , neurons in the wildtype ZD wash-on experiments were previously analyzed in ( 19 ). Burst and tonic action potentials We measured action potential threshold as the point at which the third derivative of the membrane potential exceeded 0.3 V/s 3 . Firing frequency measurements for tonic firing included sweeps in which no action potentials were fired, however, frequency measurements for burst action potentials excluded instances in which no action potentials were present. Accommodation was measured as the slope of the linear relationship between the interspike intervals for each successive action potential during the first current step to elicit ≥12 action potentials. Low threshold calcium spikes (LTS) were isolated with 0.5 μM TTX. LTS analysis was performed on the first depolarizing step that elicited an LTS or on the hyperpolarizing step that reached -100 mV for rebound LTSs. α EPSPs were generate using the equation , where 𝐼 t is the current 𝐼 at elapsed time 𝑡. 𝐼 max is the maximum current. 𝛼 is a constant representing the decay rate. Statistics We used the ‘sampsizepwr’ function in MATLAB to calculate sample sizes based on preliminary data. To detect a difference in membrane time constant of 25% with a standard deviation of 20 ms, given α of 0.05 and power of 0.8, we estimated at least 16 cells per group. All quantifications are presented in Tables 1 - 2 . Data compared with a two-way ANOVA or Fixed effects (type III) are reported as mean ± 95% CI. Data compared with Mann-Whitney or Wilcoxon tests are presented with all data points and median with 95% confidence intervals. We defined ɑ as p < 0.05. Effect size is expressed as η 2 and only calculated only when p<0.05. η 2 effect sizes are defined as small – 0.01, medium – 0.06, and large – 0.14 ( 26 ). Quantifications were performed using custom-written code in MATLAB 2022b (Mathworks). Statistical analyses were performed using Prism version 8.0.0 (GraphPad). Graphs were made using GraphPad Prism and figures were made using Adobe Illustrator version 24.3. View this table: View inline View popup Table 1: Descriptive statistics and test statistics of pairwise comparisons. View this table: View inline View popup Table 2: ANOVA and Fixed effects statistics tables for experiments with multiple measurement points. Results To investigate the intrinsic effects of Fmr1 KO on MD neurons projecting to mPFC (“MD→mPFC neurons” but for simplicity referred to as “MD neurons” in this manuscript) we made whole cell current clamp recordings of fluorescently labeled thalamocortical neurons in the MD region of thalamus in acute ex vivo mouse brain slices ( Fig. 1A ). MD neurons were visually identified and distinguished as medial (MD-M) or lateral (MD-L) based on the distance from the midline and anatomical landmarks ( Fig. 1B-D ). Both MD-M and MD-L neurons in WT and Fmr1 KO mice were investigated during this study, however, intrinsic properties only differed between WT and Fmr1 KO MD-L neurons. As a result, MD-L data is presented in the main b ody of this manuscript ( Figs. 1 - 8 ) while all MD-M data is presented in the supplement ( Supp. Figs. 2-3). Download figure Open in new tab Figure 1: CTB injections in mPFC label MD-L and MD-M neurons that project to mPFC. A. Adult wildtype and Fmr1 KO mice were stereotaxically injected with the retrograde fluorescent tracer CTB into mPFC to target prelimbic and infralimbic cortex. Fluorescently-labeled MD→mPFC neurons were visually identified for patch clamp electrophysiology experiments. B. Bright field photomicrographs of coronal mouse brain slices with the estimated distance from Bregma (in mm) where MD→mPFC neurons were patched in this investigation. C. Photomicrographs (10x) of the boxed areas from B demonstrating MD→mPFC labeled neurons (red) in each of the representative slices. D. Map of the approximate locations of recorded neurons reported in this manuscript placed on a single representative atlas drawing for each coronal slice. MD-M is dark blue. MD-L is highlighted in cyan. Fmr1 KO MD-L neurons display greater R N compared with WT MD neurons We measured subthreshold, intrinsic neuronal properties of WT and Fmr1 KO fluorescently labeled neurons ( Fig. 2A ). We found no difference in either RMP ( Fig. 2B ; Mann-Whitney test: p = 0.87) or membrane tau ( Fig. 2C ; Mann-Whitney test: p = 0.92) of Fmr1 KO neurons compared with WT. We observed greater input resistance ( R N ) in Fmr1 KO MD neurons compared with WT controls ( Fig. 2D ; Mann-Whitney test: p = 0.037, η 2 = 0.052). Based on previous reports of HCN expression in MD neurons ( 19 , 27 ) and extensive literature identifying modifications in HCN expression in Fmr1 KO mouse models ( 18 , 20 , 22 – 24 , 28 ), we next investigated intrinsic measures of HCN activity to assess potential differences in HCN function between WT and Fmr1 KO MD neurons. Download figure Open in new tab Figure 2: Greater R N in Fmr1 KO MD neurons compared with WT. A. Representative traces showing voltage deflections in response to hyperpolarizing current steps ranging from 0 to -60 pA in 5 pA increments. B . Resting V m in WT and Fmr1 KO neurons. Mann-Whitney test: p = 0.87. C. Membrane tau of WT and Fmr1 KO neurons. Mann-Whitney test: p = 0.92. D. R N in WT and Fmr1 KO MD neurons measured from the linear portion of each neurons IV plot. Mann-Whitney test: p = 0.037. Fmr1 KO MD-L neurons have reduced sag when compared with WT MD neurons We used voltage sag, a well-established measure of HCN activity, to investigate differences in HCN function ( Fig. 3A ). Figure 3B shows V-I plots of peak voltage deflection and steady state voltage deflection for both WT and Fmr1 KO MD-L neurons. While both WT and Fmr1 KO neurons exhibited clear sag in response to a range of current injections, voltage sag was reduced in Fmr1 KO neurons compared with WT controls ( Fig. 3C ; 2-way ANOVA: Effect of genotype - p = 0.009; η 2 = 0.08). The difference reported in R N in Figure 2D can affect comparisons of sag measured only by current injection. To account for differences in R N we compared sag in WT and Fmr1 KO neurons from a peak hyperpolarization of -100 mV. After normalizing for membrane hyperpolarization, the difference in sag persisted ( Fig. 3D ; Mann-Whitney test: p = 0.004, η 2 = 0.08). We used the HCN channel blocker ZD7288 (20 μM) to verify that the differences observed in intrinsic neuronal properties stem from HCN channel activity. R N increased significantly in both WT and Fmr1 KO neurons with the application of ZD7288 ( Fig. 3E-F ; Wilcoxon test – WT: p = 0.002, η 2 = 3.53; Fmr1 KO: p = 0.002, η 2 = 3.14). We additionally measured the effect of ZD7288 on sag. As expected, sag was completely abolished by the wash-on of ZD7288 in both WT and Fmr1 KO MD neurons ( Fig. 3G-H ; Wilcoxon test – WT: p = 0.0005, η 2 = 3.12; Fmr1 KO: p = 0.004, η 2 = 3.16). Download figure Open in new tab Figure 3: Fmr1 KO MD neurons exhibit decreased HCN channel activity compared with WT neurons. A. Representative traces of voltage responses from WT and Fmr1 KO neurons to current stimuli ranging from 0 to -250 pA in 25 pA intervals. B. VI plot of WT and Fmr1 KO neurons measured from both the peak and steady state voltage deflections. C. Sag, measured as the difference between peak and steady state voltage deflections, in response to current stimuli from -25 to -250 pA in 25 pA intervals. 2-Way ANOVA – effect of genotype: p = 0.009. D. Sag in WT and Fmr1 KO neurons when peak hyperpolarization reached -100 mV. Mann-Whitney test: p =0.004. E. Representative traces showing WT and Fmr1 KO neuron voltage responses to current stimuli from 0 to -60 pA in 5 pA intervals before and after the application of 20 μM ZD7288. The linear portion of neuron VI curves were used to calculate R N . F. Before and after plots of the effect of ZD7288 on WT and Fmr1 KO R N . WT: Wilcoxon test, p = 0.002. Fmr1 KO: Wilcoxon test, p = 0.002. G. Representative traces showing sag before and after ZD7288 application in WT and Fmr1 KO neurons. H. Before and after plots showing the effect of ZD7288 on sag measurements in WT and Fmr1 KO neurons. WT: Wilcoxon test: p = 0.0005. Fmr1 KO: Wilcoxon test, p = 0.004. αEPSP summation is not different between WT and Fmr1 KO MD-L neurons To test if differences in HCN function between WT and Fmr1 KO neurons affect signal summation, we injected trains of alpha wave EPSPs ( α EPSP) at frequencies of 50, 100, and 200 Hz. Stimulus amplitudes were adjusted to approximately 1-2 mV. We found no difference in the amplitude of the first α EPSP injected between WT and Fmr1 KO neurons (Mann-Whitney test: p = 0.62). There was no difference in summation between WT and Fmr1 KO neurons at 50 Hz ( Fig 4A ; 2-way ANOVA – effect of genotype: p = 0.23), 100 Hz ( Fig 4B ; 2-way ANOVA – effect of genotype: p = 0.54), or 200 Hz ( Fig. 4C ; 2-way ANOVA – effect of genotype: p = 0.49). The summation ratio (10 th α EPSP / 1 st α EPSP) revealed no difference between WT and Fmr1 KO neurons ( Fig. 4D ; 2-way ANOVA – effect of genotype: p = 0.46). These results suggest that the differences in HCN channel function between WT and Fmr1 KO neurons do not affect subthreshold summation of α EPSP inputs. Download figure Open in new tab Figure 4: α EPSP summation is not different between WT and Fmr1 KO MD neurons. A. Normalized voltage response to α EPSPs delivered at 50 Hz in WT and Fmr1 KO neurons. 2-Way ANOVA – effect of genotype: p = 0.23. B. Normalized voltage response to α EPSPs delivered at 100 Hz in WT and Fmr1 KO neurons. 2-Way ANOVA – effect of genotype: p = 0.54. C. Normalized voltage response to α EPSPs delivered at 200 Hz in WT and Fmr1 KO neurons. 2-Way ANOVA – effect of genotype: p = 0.49 D. Summation ratio at 50, 100, and 200 Hz α EPSP rate in WT and Fmr1 KO neurons. 2-Way ANOVA – effect of genotype: p = 0.46. Fmr1 KO MD neurons have greater AHP and delayed RST compared with WT MD neurons To investigate differences in AHP after a train of action potentials we measured the minimum voltage amplitude reached within 500 ms of the offset of current injection. These measurements were performed both before and after the application of 20 μM ZD7288 ( Fig. 5A ). Measurements of AHP showed a consistent difference in amplitude between WT and Fmr1 KO neurons ( Fig. 5B ; Mann-Whitney test: p = 0.036, η 2 = 0.05). AHP was significantly reduced by the application of ZD7288 in both WT and Fmr1 KO neurons ( Fig. 5C ; Wilcoxon test – WT: p = 0.001, η 2 = 3.13; Fmr1 KO: p = 0.004, η 2 = 3.16). These results suggest that the AHP is strongly influenced by HCN activity and has a reduced amplitude in Fmr1 KO compared with WT neurons. Download figure Open in new tab Figure 5: Decreased HCN activity results in decreased AHP amplitude in Fmr1 KO neurons compared with WT controls. A. Representative traces showing AHP amplitude in WT and Fmr1 KO neurons before and after the application of 20 μM ZD7288. B. AHP measured in WT and Fmr1 KO neurons. Mann-Whitney test: p = 0.036. C. Before and after plots of the effect of ZD7288 on AHP in WT and Fmr1 KO neurons. WT: Wilcoxon test, p = 0.001. Fmr1 KO: Wilcoxon test: p = 0.002. We next evaluated RST, defined as the time between the offset of the hyperpolarizing current step and the peak of the first action potential in a rebound burst ( Fig. 6A ). RST measured against hyperpolarizing current step amplitude revealed a delay in RST in Fmr1 KO neurons compared with WT controls ( Fig. 6B ; Fixed effects (type III) analysis – Effect of genotype: p = 0.015, η 2 = 0.07). To account for the peak level of depolarization we plotted RST against peak hyperpolarization for WT and Fmr1 KO MD neurons ( Fig. 6C ). To further account for level of peak hyperpolarization due to differences in R N we compared RST when peak hyperpolarization reached -100 mV in WT and Fmr1 KO neurons and found that Fmr1 KO neurons show a greater delay in RST compared with WT controls ( Fig. 6D ; Mann-Whitney test: p = 0.0014, η 2 = 0.13). Download figure Open in new tab Figure 6: RST is decreased by HCN activity. Fmr1 KO neurons show a greater delay in RST compared with WT controls. A. Representative traces of RST measured as the time from the offset of the hyperpolarizing current to the peak of the first action potential in the burst. Boxed region is expanded in the inset at the top of the panel. B. RST measured at current stimuli ranging from 0 to -250 pA in 25 pA intervals. Mixed effects analysis – effect of genotype: p = 0.015. C. Plot of RST by the peak hyperpolarization reached in response to current stimuli. D. RST measured when peak hyperpolarization reached -100 mV. Mann-Whitney test: p = 0.0014. E. Representative traces showing rebound bursts before and after the application of 20 μM ZD7288 in WT and Fmr1 KO neurons. Insets represent expanded views of the boxed regions. F. RST measurements in response to hyperpolarizing current stimuli from 0 to -250 pA in 25 pA intervals in WT MD neurons before and after wash-on of 20 μM ZD7288. WT Fixed effects (type III) analysis – effect of genotype: p = 0.015. G. RST measurements in response to hyperpolarizing current stimuli from 0 to -250 pA in 25 pA intervals in Fmr1 KO MD neurons before and after wash-on of 20 μM ZD7288. Fmr1 KO Fixed effects (type III) analysis – effect of genotype: p = 0.032. H. Before and after plots of RST showing the effects of 20 μM ZD7288 wash-on in WT and Fmr1 KO neurons measured when peak hyperpolarization reached -100 mV. WT: Wilcoxon test, p = 0.004. Fmr1 KO: Wilcoxon test, p = 0.56. In the absence of rebound burst firing, the release of hyperpolarization causes an HCN channel-mediated depolarization before returning to rest ( 29 ) . We hypothesized that this effect contributes to the difference in RST observed here. To test HCN involvement, we measured RST before and after wash-on of 20 μM ZD7288 ( Fig. 6E ). When compared against current amplitude both WT ( Fig. 6F ; WT Fixed effects (type III) analysis – effect of genotype: p = 0.015, η 2 = 0.42) and Fmr1 KO ( Fig 6G ; Fmr1 KO Fixed effects (type III) analysis – effect of genotype: p = 0.032, η 2 = 0.23) neurons showed a significant delay in RST with blockade of HCN channels. When normalized to steps in which peak hyperpolarization reached -100 mV, only WT neurons showed a significant difference in RST after ZD7288 wash-on ( Fig. 6H ; Wilcoxon test – WT: p = 0.016, η 2 = 3.2; Fmr1 KO: p = 0.56), suggesting that decreased HCN channel activity results in delayed RST in Fmr1 KO neurons. Burst firing and calcium spike properties are not different between WT and Fmr1 KO MD neurons Thalamic neurons display state-dependent firing properties based on the availability of Ca v 3 channels: neurons fire tonic spikes when resting at depolarized potentials (when Ca V 3 channels are inactivated) and fire bursts when hyperpolarized (when Ca V 3 channels are available) ( 30 ). Figs. 7 - 8 investigate differences in action potential firing based on neuron state and stimulus type. Tonic firing in WT and Fmr1 KO neurons was investigated and no differences were identified ( Supp. Fig. 1 ). Download figure Open in new tab Figure 7: Bursting evoked by depolarizing current steps is not different between WT and Fmr1 KO MD neurons. A. Representative traces showing burst firing in response to 5, 15, 25, and 35 pA steps. Slashes on current steps show where voltage traces were truncated to better show bursts. B. Number of action potentials per burst for current steps from 0 to 60 pA in 5 pA intervals. Fixed effects (type III) analysis – effect of genotype: p = 0.51. C. Burst threshold measured from the first step to elicit a burst. Mann-Whitney test: p = 0.81. D. Representative traces of LTS isolated by the application of 0.5 μM TTX. E. LTS threshold measured from the first current step to evoke a spike. Mann-Whitney test: p = 0.47. F. LTS time to threshold measured from the first current step to evoke a spike. Mann-Whitney test: p = 0.31. We recorded bursts of action potentials in response to depolarizing current steps ranging from 0 to 60 pA in 5 pA intervals ( Fig. 7A ). There were no differences in either the number of action potentials fired in each burst ( Fig. 7B ; Fixed effects (type III) analysis – effect of genotype: p = 0.51) or the threshold for the first action potential in the first step to elicit a burst ( Fig. 7C ; Mann-Whitney test: p = 0.81). To compare calcium spikes evoked by depolarization we applied the sodium channel blocker TTX (0.5 μM) ( Fig. 7D ). We found no difference in LTS threshold ( Fig. 7E ; Mann-Whitney test: p = 0.47), or the time from the offset of the current injection and the peak of the LTS (Mann-Whitney test: p = 0.47) between WT and Fmr1 KO neurons. Added variability from the slower activation time of Ca v 3 channels compared with Na v channels may obscure differences in LTS onset. To address this confound we measured the latency from the onset of current to LTS threshold. We found no difference in spike timing using this measure ( Fig. 7F ; Mann-Whitney: p = 0.31). We then recorded the properties of rebound bursts evoked in response to hyperpolarizing current steps from 0 to -60 pA in 5 pA intervals ( Fig. 8A ). There was no difference between WT and Fmr1 KO neurons in the number of action potentials fired in each rebound burst ( Fig. 8B ; Fixed effects (type III) analysis – effect of genotype: p = 0.9) or the rebound burst threshold ( Fig. 8C ; Mann-Whitney test: p = 0.37). With the addition of TTX to isolate calcium spikes ( Fig. 8D ) we found no difference in spike threshold ( Fig. 8E ; Mann-Whitney test: p = 0.87) or the timing between current offset and spike peak (Mann-Whitney test: p = 0.2) between WT and Fmr1 KO neurons. As in Fig 7F , we measured the time to LTS threshold to assess LTS latency using more concise measure. We found that the latency to LTS threshold was greater in Fmr1 KO compared with WT MD neurons after the offset of hyperpolarizing current ( Fig. 8F ; Mann-Whitney: p = 0.028, η 2 = 0.22). Our results show waveform properties of rebound bursts and LTS associated with Ca v 3 channels are not different between Fmr1 KO and WT MD neurons. Consistent with measurements of RST in Fig. 6 , we identified greater latency to hyperpolarization-evoked rebound LTS in Fmr1 KO compared with WT MD neurons. Download figure Open in new tab Figure 8: Bursting evoked by hyperpolarizing current steps is not different between WT and Fmr1 KO MD neurons. A. Representative traces showing burst firing in response to -5, - 15, -25, and -35 pA steps. Slashes on current steps show where voltage traces were truncated to better show bursts. B. Number of action potentials per burst for current steps from 0 to -60 pA in 5 pA intervals. Fixed effects (type III) analysis – effect of genotype: p = 0.9. C. Burst threshold measured from the first hyperpolarizing step to elicit a burst. Mann-Whitney test: p = 0.37. D. Representative traces of LTSs isolated by the application of 0.5 μM TTX. E. LTS threshold measured from the first hyperpolarizing current step to evoke a spike. Mann-Whitney test: p = 0.87. Black dots represent threshold measurements for LTS. F. LTS time to threshold measured from the first hyperpolarizing current step to evoke a spike. Mann-Whitney test: p = 0.028. Discussion Reciprocal connectivity between MD and mPFC is required for higher order operations like executive function and social behavior ( 2 , 3 , 6 , 31 ) . The symptomatology of FXS displays a high degree of overlap with functions controlled by thalamocortical circuitry ( 32 , 33 ), providing strong implications of prefrontal circuit pathology in FXS. Research has revealed disruptions to prefrontal circuitry through changes in both intrinsic and synaptic properties as well as in mPFC-mediated learning ( 18 , 34 , 35 ). The importance of MD in executive and affective functions led to our hypothesis that disruptions in MD function may contribute to pathology associated with FXS. We found that Fmr1 KO MD-L neurons that project to mPFC displayed reduced HCN activity compared with WT MD-L neurons. Further investigation revealed HCN-channel-dependent differences in membrane voltage dynamics after the offset of depolarizing and hyperpolarizing current injections manifesting as decreased AHP amplitude and delayed RST. We identified differences in events following the offset of both hyperpolarizing (RST) and depolarizing (AHP) current stimuli when comparing Fmr1 KO with WT MD-L neurons. AHP amplitude was decreased, and RST was delayed in bursts and LTS in Fmr1 KO neurons when compared to WT controls. These findings suggest that Fmr1 KO MD-L neurons may have difficulty maintaining phase locked rhythmic firing, which is critical for normal thalamocortical circuit function. The thalamus is important for the generation and maintenance of rhythmic activity in the brain ( 30 ). Furthermore, rhythmic activity in MD is an integral component to normal higher order brain function, particularly working memory ( 36 , 37 ) and normal sleep function ( 38 , 39 ). Because Fmr1 KO MD-L neurons had increased input resistance, we predicted that modeled synaptic currents would show greater summation in Fmr1 KO compared with WT neurons. However, injected α EPSPs revealed no difference in summation properties at any recorded frequency. This contrasts with observed effects of HCN channel activity on EPSP summation in hippocampal pyramidal neurons ( 40 ). It remains unknown how HCN channels localize within the membrane of MD neurons. Dendritic HCN channel localization may impact α EPSPs differences observed in somatic recordings ( 41 ). Although this study focuses on differences in HCN channel activity, it is likely that other channelopathies are present in MD neurons of Fmr1 KO animals ( 42 ). For instance, the lack of difference in α EPSP summation between Fmr1 KO neurons could be explained by increased resting K + conductance in Fmr1 KO MD-L neurons. MD neurons express 2-pore K + channels (K 2P ) which contribute to the control over state-dependent firing within thalamic neurons ( 43 ). Increased activity of these channels could account for the lack of difference in α EPSP summation between WT and Fmr1 KO MD-L neurons despite the difference in input resistance reported here. Recordings from human MD reveal the presence of both sleep spindles and ripples, establishing a role for MD in normal sleep function ( 44 ). Spindle activity in thalamic neurons is thought to stem from rebound bursting in response to hyperpolarizing GABAergic inputs ( 45 ). Reduced HCN activity in Fmr1 KO MD-L neurons contributes to delayed spike timing and reduced AHP amplitude, changes that will functionally impair a neuron’s ability to generate rhythmic bursting activity. Rhythmic bursting in thalamic neurons depends on HCN channel activity as is shown by disrupted burst generation in an Hcn4 conditional KO mouse ( 46 ). Sleep is disrupted in patients with FXS ( 47 , 48 ). Fmr1 KO mice have reduced sleep spindle density and sleep spindle discoordination in cortical areas including the prefrontal network ( 49 ). Activation of G-protein receptor coupled inward rectifying K + (GIRK) channels restored normal cortical sleep spindle activity in Fmr1 KO mice ( 50 ). Our results implicate disruption of rhythmic bursting activity in MD neurons in Fmr1 KO mice that are likely involved in sleep disruptions observed in both mice models of FXS and humans with FXS. In this study we performed whole cell current clamp recordings from CTB-labeled MD neurons that project to mPFC in WT and Fmr1 KO MD-M and MD-L neurons. This study focused on identifying the effects of Fmr1 KO on intrinsic neuronal properties in MD, however, synaptic dysfunction is widely reported in FXS ( 51 ) . HCN plays a prominent role in the integration, filtering, and coordination of synaptic inputs in neuronal dendrites ( 40 , 52 – 54 ). Although somatically-generated α EPSPs did not reveal differences in subthreshold summation between WT and Fmr1 KO MD-L neurons, the localization and dendritic effects of HCN channel expression remain unexplored. Thalamic neurons, through their unique ion channel expression profile, generate and maintain rhythmic activity. Coupled with the widespread interconnectivity between cortical and subcortical brain structures, MD is a promising target for therapeutic intervention for the treatment of symptoms associated with FXS. DISCLOSURES The authors disclose biomedical financial interests or potential conflicts of interest. FUNDING This work was supported by NINDS K08 NS094643, NIMH R56 MH122810, NIMH R01 MH131857, the PERF Elterman research grant, the Phillip R. Dodge Young Investigator Award, the STARS award from The University of Texas System, startup funds from Dell Medical School, laboratory space from the College of Natural Sciences at UT Austin, The University of Texas System STARS award, and the Mulva Clinic for the Neurosciences at Dell Medical School at the University of Texas at Austin. CONTRIBUTIONS G.J.O. wrote the paper and interpreted and analyzed data. P.L. performed electrophysiological recordings. A.K. performed imaging and projection tracing. A.B. conceived of the project, designed all experiments, wrote all code, analyzed data, and wrote portions of the paper. All authors approved the final manuscript prior to publication. DATA AVAILABILITY/ SUPPLEMENTAL MATERIALS Source data and supplemental materials are available through github. ACKNOWLEDGEMENTS We thank Meredith McCarty, Aurora Weiden, Madelynn Campbell, Joy Adler, and Mendee Geist for their technical assistance. 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Share Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity Gregory J. Ordemann , Polina Lyuboslavsky , Alena Kizimenko , Audrey C. Brumback bioRxiv 2025.02.02.636122; doi: https://doi.org/10.1101/2025.02.02.636122 Share This Article: Copy Citation Tools Fmr1 KO causes delayed rebound spike timing in mediodorsal thalamocortical neurons through regulation of HCN channel activity Gregory J. Ordemann , Polina Lyuboslavsky , Alena Kizimenko , Audrey C. 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