Alcohol dependence-induced neuroadaptations in prelimbic K V 7 channels contribute to working memory deficits in mice

Alcohol dependence-induced neuroadaptations in prelimbic KV7 channels contribute to working memory deficits in mice | 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 Alcohol dependence-induced neuroadaptations in prelimbic K V 7 channels contribute to working memory deficits in mice Kathy L. Lindquist , View ORCID Profile Jennifer A. Rinker , View ORCID Profile Patrick J. Mulholland doi: https://doi.org/10.1101/2025.11.07.687180 Kathy L. Lindquist Department of Neuroscience, Charleston Alcohol Research Center, Medical University of South Carolina , 70 President Street, Drug Discovery Building, Charleston, SC 29425 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jennifer A. Rinker Department of Neuroscience, Charleston Alcohol Research Center, Medical University of South Carolina , 70 President Street, Drug Discovery Building, Charleston, SC 29425 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jennifer A. Rinker Patrick J. Mulholland Department of Neuroscience, Charleston Alcohol Research Center, Medical University of South Carolina , 70 President Street, Drug Discovery Building, Charleston, SC 29425 Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Patrick J. Mulholland For correspondence: mulholl{at}musc.edu Abstract Full Text Info/History Metrics Preview PDF Abstract Chronic alcohol use can cause executive function deficits, such as diminished working memory, which can facilitate excessive alcohol intake and elevate relapse probability even in prolonged withdrawal. Unfortunately, current FDA-approved medications for alcohol use disorder (AUD) are not designed to also treat alcohol-induced cognitive dysfunction. K V 7 channels are a class of voltage-gated potassium channels that regulate neuronal excitability and are implicated in cognitive function. K V 7 channels are sensitive to acute and chronic alcohol exposure, and positive K V 7 channel modulation reduces alcohol intake in high-drinking rodents. The impact of chronic alcohol exposure on K V 7 physiology in the prelimbic cortex (PL), a region essential for cognitive function, remains unexplored. To address this, cognitive deficits were measured in male and female mice following the chronic intermittent ethanol (CIE) model of alcohol dependence using a delayed alternation working memory task. After establishing that CIE elicits cognitive deficits regardless of sex, whole-cell patch clamp electrophysiology recordings were performed on distinct PL pyramidal neurons; intratelencephalic (IT neurons) and extratelencephalic (ET neurons) projection neurons to measure K V 7 channel mediated M-currents, along with evoked action potential firing following chronic intermittent ethanol (CIE) exposure in male and female mice. CIE exposure led to an increase in intrinsic excitability and a decrease in M-current regardless of sex, which was largely driven by CIE-induced adaptations in IT neurons. These neural adaptations in dependent mice coincided with the emergence of working memory deficits. Microinjections of the K V 7 positive modulator retigabine restored working memory function in male and female CIE-exposed mice. Together, these results indicate that chronic alcohol downregulates K V 7 function within distinct cortical cell types, and that K V 7 modulation may be a promising pharmacological treatment for alcohol induced cognitive impairment. Introduction Alcohol use disorder (AUD) is an escalating public health issue that is associated with a general decline in overall health and the emergence of cognitive dysfunction 1 - 4 . Executive function deficits, such as diminished working memory capacity, are common following alcohol cessation and can lead to an increased probability of relapse 3 , 5 - 7 . Current treatments for AUD are not designed to treat AUD-induced cognitive dysfunction, so the development of novel therapeutics that can specifically target cognitive impairments remains necessary to reduce relapse in AUD individuals. Alcohol dependence decreases activation of the dorsolateral prefrontal cortex in humans during spatial working memory 1 , 8 , alters its coherence during cognitive performance 6 , and is associated with volumetric decrease within the prefrontal cortex 1 , 9 - 11 . The prelimbic cortex (PL), a brain region heavily implicated in cognitive function, is susceptible to ethanol-induced damage 1 , 12 - 14 and is the rodent equivalent to the dorsolateral prefrontal cortex 1 , 15 . Further interrogation of PL mechanisms underlying alcohol dependence induced cognitive impairment are required to identify new therapeutic options. K V 7 channels regulate cognitive function and are highly expressed within the cortex 16 - 20 . K V 7 channels are a class of voltage-gated potassium channels that are essential for regulating neuronal excitability 16 , 21 , 22 . Previous work has indicated that positive modulation of K V 7 channels may serve as a potential AUD treatment 23 - 25 . Our lab has previously demonstrated that positive modulation of K V 7 channels within the nucleus accumbens, ventral tegmental area, and systemically can reduce alcohol intake in rodents that exhibit a high drinking phenotype 23 - 25 . Others have also demonstrated that lateral habenula K V 7 channels could participate in regulating alcohol withdrawal-induced negative affect and hyperalgesia 26 , 27 . They have been proposed as a potential therapeutic target for AUD, but it is currently unknown if targeting them could also improve alcohol withdrawal-induced cognitive decline. Traditionally, K V 7 inhibition, such as via the antagonist XE-991, is considered cognitive-enhancing when administered systemically or directly into the cortex by enhancing excitability 18 , 20 , 28 , 29 . However, our lab has demonstrated that administration of XE-991 to rats with a history of chronic alcohol exposure induces seizure activity 23 . This suggests that chronic alcohol exposure decreases K V 7 channel function, leading to increased neuronal excitability, which is further enhanced in the presence of XE-991. There is some evidence that positive modulation of K V 7 channels systemically may improve cognition following alcohol exposure 30 , but that study administered the positive modulator retigabine concurrently with alcohol exposure via oral gavage, so it remains to be determined if positive modulation of K V 7 channels following chronic alcohol exposure can alleviate cognitive deficits, as well as possible brain regions that could be responsible. Although K V 7 channels are known to be expressed in all neuronal subtypes within the cortex 31 , 32 , it is currently unknown how alcohol exposure alters K V 7 channel function within prefrontal cortical projection subtypes. Deep-layer PL pyramidal neurons have diverse projection targets that can be subdivided into extratelencephalic neurons (ET) and intratelencephalic (IT) neurons, distinguished by the presence of I h ‘sag’ current 33 - 36 . IT neurons project contralaterally within the cortex, as well as to other telencephalic regions, such as the striatum and amygdala 33 , 36 . In contrast, ET neurons project to regions outside of the telencephalon, such as the thalamus, hypothalamus, etc 33 , 36 . ET and IT cortical neurons have demonstrated roles in cognitive domains, such as working memory and decision making. In an auditory-based decision task, ET neurons displayed robust activity surrounding choices, whereas IT activity did not seem tied to decision choice 37 . In contrast, a separate study found that inhibition of cortical IT neurons caused working memory deficits, while inhibiting ET neurons had no impact 38 . So, further research is required to fully elucidate IT and ET function within cognition. IT and ET function following alcohol exposure have begun to be assessed by others 39 , 40 . While Joffe et al. found increased glutamatergic postsynaptic strength in IT neurons without impacting neuronal excitability following the Intermittent access to ethanol voluntary alcohol intake model 39 in mice, Hughes et al. found increased neuronal excitability and glutamatergic release of ET neurons following 2 weeks of chronic intermittent ethanol (CIE) exposure in rats 40 . The impact of a chronic alcohol model on IT and ET neurons has not previously been studied in mice. Although it is hypothesized that ET neurons may have inhibited baseline activity of K V 7 channels due to concurrent expression of muscarinic receptors 41 , little is known about K V 7 physiology differences between IT and ET neurons. To determine the role of K V 7 channels in regulating alcohol-induced cognitive impairment, a series of behavioral and electrophysiological studies using the chronic intermittent ethanol (CIE) model of alcohol dependence in male and female C57BL/6J mice were performed. First, four cycles of CIE exposure caused spatial working memory deficits in male and female mice. Then, whole-cell patch clamp electrophysiology recordings were performed to measure K V 7-mediated physiological measures in the PL following CIE. To determine circuit specificity of electrophysiological measures, recordings were classified as IT or ET neuron recordings based on their calculated physiological index, a measure able to predict projection subtype with over 90% accuracy 41 , 42 . Retigabine was microinfused into the PL to determine if K V 7 modulation could reverse CIE-induced cognitive deficits. Methods Mice All procedures were performed in male and female C57BL/6J (Jackson Laboratory, ME; strain # 000664) mice that were ∼8-10 weeks old at experimental onset. All mice were individually housed within a temperature and humidity-controlled vivarium. Mice were given libitum access to food and water (Harlan Teklad Diet 2918) and kept on a 12-hr. reverse light/dark cycle (Lights off at 11 am). Animal care was approved by the Medical University of South Carolina Institutional Animal Care and Use Committee, which was in accordance with NIH guidelines for humane care of laboratory animals. Alcohol dependence model To establish alcohol dependence, mice were exposed to the standard chronic intermittent ethanol (CIE) model 43 , 44 . Mice were injected with the alcohol dehydrogenase inhibitor pyrazole (1 mmol/kg; IP; Sigma–Aldrich, St. Louis, MO), dissolved in 1.6 g/kg ethanol (8% w/v in 0.9% v/v sterile saline; IP; 20 ml/kg dose volume), before placement in plexiglass ethanol inhalation chambers (Plas Labs, Inc, Lansing, MI) for a 16-hour exposure period, followed by an 8-hour withdrawal period. Ethanol concentration within the chambers was monitored so that blood ethanol levels were over 200 mg/dl. BECs were measured via retro-orbital blood samples (40 μL) collected with heparinized capillary tubes (Drummond Scientific Company, Broomall, PA) after each session. After samples were centrifuged at 10000×g for 10 minutes, plasma was separated for BEC measurement using an Analox Instrument (Lunenburg, MA). Control mice were exposed to air and injected with pyrazole dissolved in saline, such that all mice received the same number of injections and handling. CIE vapor was administered Monday through Thursday, followed by a 72-hour withdrawal period. This cycle was repeated four times, as this amount of CIE exposure has been shown to cause cognitive deficits when tested 72 hours after the final CIE session 13 , 45 . Whole-cell patch-clamp slice electrophysiology Mice ( n = 7-8/sex/treatment) were anesthetized via isoflurane before rapid decapitation. Slices including the PL were collected using a Leica VT 1200S vibratome before placement into oxygenated artificial cerebral spinal fluid (ACSF) and incubation at 34°C for 30 minutes, followed by 30 minutes at room temperature. Slices were transferred to the recording chamber and continually perfused with oxygenated ACSF. Cutting ACSF solution during slice preparation was composed of the following: 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 0.4 mM C 6 H 8 O 6 , 10 mM C 6 H 12 O 6 , 200 mM sucrose, 25 mM CHNaO 3 , 6 mM MgCl 2 , 0.5 mM CaCl 2 . Recording ACSF solution was composed of the following: 125 mM NaCl, 2.5 mM KCl, 1.25 mM NaH 2 PO 4 , 0.4 mM C 6 H 8 O 6 , 10 mM C 6 H 12 O 6 , 25 mM CHNaO 3 , 1.3 mM MgCl 2 , 2 mM CaCl 2 , 2.4 mM sodium pyruvate. Reagents for ACSF and internal solutions were purchased from Sigma Aldrich. Recordings were conducted using Axon Multiclamp 700B amplifier and the Instrutech ITC 18 analog digital converters, which were controlled by Axograph software. Thin wall borosilicate glass electrodes (Sutter instruments) were pulled on a Sutter Instrument P97 micropipette puller and had tip resistances ranging from 2-3.5 MΩ. Internal solution was composed of the following: 10 mM Hepes, 0.1 mM EGTA, 4 mM ATP-Mg, 2 mM GTP-Na, 10 mM Na-phosphocreatine salt hydrate, 110 mM KCl, 10 mM tetrapotassium pyrophosphate. Only neurons with a series resistance under 10 mΩ were recorded, and neurons whose resistance changed by over 20% were excluded. Deep-layer pyramidal neurons were identified within the PL based on location and neuronal morphology. To identify IT vs ET neurons, a 200-pA hyperpolarizing current was applied to generate I H sag current. This allowed for the calculation of the physiological index (PI) using the following equation 41 , 42 : Recordings were then taken in current clamp mode to measure evoked action potential firing, which was induced with a series of direct current injections in increments of 20 pA for 1 s from 0-300 pA. All recordings were analyzed for total action potentials generated at each current step, rheobase, resting membrane potential, action potential half-width, amplitude, rise time, and afterhyperpolarization using the Easy Electrophysiology Software. M-current was recorded in voltage clamp in the presence of 200 nM of tetrodotoxin (Alomone Labs, Jerusalem, Israel) following established protocols 21 , 46 . In whole cell mode, neurons were initially held at −70 mV, and series resistance was compensated at 80%. A depolarizing protocol consisting of five voltage steps incrementing in 10 mV was applied at −70mV. Following this baseline recording, the K V 7 modulator retigabine (30 μM, Alomone Labs, Jerusalem, Israel) was bath applied before repeating the depolarizing protocol. Lastly, the K V 7 antagonist XE-991 (20 μM, Tocris Bioscience, Bristol, UK) was bath applied to abolish M-current. Using Axograph software offline, the final recording was subtracted from the initial recording give the K V 7-specific current. Delayed Alternation Spatial Working Memory Task The T-maze protocol was adapted studies showing working memory deficits in mice exposed to a chronic ethanol liquid diet model 47 - 49 . For two 10-minute sessions on consecutive days, mice were allowed to freely explore the T-Maze. On the following day, mice were tested to determine if they successfully choose between alternate arms on a T-maze following a 30-second inter-trial interval (ITI). For each trial, they were confined in their choice arm for 30 seconds before being placed back in the stem arm for 30 seconds to begin the next trial. On the test day, mice underwent seven of these trials, followed by an 8 th trial with a 5-second ITI to ensure that a potential cognitive deficit isn’t a loss of motivation to alternate. Any urine or fecal matter produced during ongoing trials will be cleaned out with water to avoid becoming a visual or odor cue. Mice underwent this 3-day process before beginning CIE to establish a baseline working memory level. Based on their initial performance, mice were pseudo-randomly assigned to control or CIE treatment groups. The task was then repeated 72-96 hours following the final CIE session to model cognitive deficits. Performance on the working memory task was reported as percent correct alternations. Stereotaxic surgery and microinjections Mice ( n = 7-10/sex/treatment) were anesthetized using isoflurane and head-fixed into a stereotaxic instrument (David Kopf Instruments, Tujunga, CA) for the duration of the surgical procedure. An incision was performed to expose the skull, the following coordinates were used to target the PL: A/P + 1.78 mm, M/L ± 0.4 mm, and D/V −2.25 mm (relative to Bregma). Microinjection cannulas (Protech International, Boerne, TX) targeting both hemispheres were chronically implanted into the PL. Mice underwent postoperative care for a minimum of 5 days following surgery. Retigabine (10 ng/hemisphere; Alomone Labs, Jerusalem, Israel) was dissolved in sterile 0.9% saline and microinfused 30 minutes before T-maze testing at a dose volume of 0.15 μl/min over 2 minutes. Cannula placements were verified via alcian blue staining and cannula tracks. Mice were excluded if the cannula tip was outside of the PL. Statistics Multi-factor ANOVAs were used for analysis with Bonferroni post-hoc tests for multiple comparisons, when appropriate (GraphPad Prism 10.2.3). An α of 0.05 was used to determine statistical significance. All data are reported mean ± SEM. Results Chronic alcohol-induced cognitive deficits Previous reports indicated that CIE exposure model could induce cognitive deficits during early withdrawal 13 , 45 , 50 - 53 , so a delayed alternation task was employed following CIE exposure to determine if working memory deficits would emerge. Male and female mice ( n = 10-13 mice/group) were assessed for baseline working memory function using a delayed alternation task ( Fig. 1A, B ) before initiating the CIE exposure paradigm ( Fig. 1B ). Following 72 hours after the final CIE exposure session, working memory was reassessed using the delayed alternation task. At baseline performance, both male and female mice performed at 70.129 ± 1.185 % ( Fig. 1C ). Following CIE exposure, mice performed significantly worse on the task regardless of sex ( Fig. 1C , 3-way ANOVA, F(1,41) = 7.832, p < 0.01). To determine whether or not this deficit emerged due to locomotor effects, as some have reported that C57BL/6J mice can be hypoactive during early withdrawal 54 , 55 , the total time required to complete the delayed alternation task was quantified. While CIE did not impact task completion time, females performed the task significantly quicker than males, regardless of exposure treatment ( Fig. 1D , 3-way ANOVA, F(1,43) = 14.72, p < 0.001), suggesting that the a mice may be employing sex-dependent strategies to perform the task, as sex hormones can influence spatial-based task strategies 56 , 57 . Download figure Open in new tab Fig 1. (A) Schematic of delayed alternation task. (B) Timeline for testing working memory performance along with the CIE model. (C) CIE reduced the number of correct alternations on the task, p<.001. (D) Time to complete the delayed alternation task varied based on sex, p < 0.001. Prelimbic physiological adaptations in alcohol dependent mice To determine a possible mechanism underlying the identified CIE-induced cognitive impairment, M-current was measured to assess if K V 7 channel function, an ion channel implicated in cognition 20 , 28 , 58 - 64 , was diminished. During withdrawal from CIE, whole cell patch clamp electrophysiology was performed in layer V PL cortex pyramidal neurons ( Fig. 2A ). Analysis of evoked firing revealed a significant treatment by current step interaction with post-hoc comparisons showing that CIE exposure significantly elevated firing across 20-180pA current steps in male and female mice ( Fig. 2B ; 3-way ANOVA, F(15,1515) = 3.117, p < .001). CIE exposure also significantly increased the total amount of evoked action potentials fired ( Fig. 2C , 2-way ANOVA, F(1,106) = 12.75, p < 0.001). Other measures of intrinsic excitability that were significantly altered by CIE included a decreased rheobase ( Fig. 2D , 2-way ANOVA, F(1,100) = 14.40, p < 0.001) and a depolarized resting membrane potential ( Fig. 2E , 2-way ANOVA, F(1,102) = 7.482, p < .01), but we found no changes to action potential kinetics ( Table 1 ). View this table: View inline View popup Download powerpoint Table 1. Action potential kinetics of deep layer prelimbic pyramidal neurons from figure 1 . Download figure Open in new tab Fig 2. ( A ) Experimental timeline of alcohol exposure and whole cell patch clamp electrophysiology of layer 5 prelimbic pyramidal neurons. ( B ): CIE increased evoked firing regardless of sex. ( C ) The total summation of evoked action potentials was elevated in male and female mice exposed to CIE (p < 0.001), along with decreased rheobase ( D , p < .001) and depolarized resting membrane potential ( E , p < 0.01). To directly determine if the CIE-induced neuronal excitability adaptations were partially related to K V 7 channel dysfunction, the K V 7-specific current, M-current, was recorded using a standard depolarizing voltage step protocol 16 , 21 , 46 . Following baseline recording, the current was modulated using retigabine (30 μM) and blocked using the K V 7 blocker XE-991 (20μM) ( Fig. 3A ). Using a repeated measure 3-way ANOVA with voltage step as the repeated measure, there was a significant interaction of voltage step and sex (F(4,264) = 3.401, p < .01) as well as voltage step and CIE (F(4,264) = 4.981, p < 0.001), but only a significant effect of CIE at −30 and −20 voltage steps was found to be significant following Bonferroni post hoc analysis ( Fig. 3B , F(4,272) = 5.588, p < 0.001, Bonferroni adjusted p-value −30--20pA: p < .05). Because M-current is maximized at the most depolarized current, the −20mV M-current step was separately analyzed. At the −20 voltage step, M-current significantly differed based on sex, while CIE significantly reduced M-current in both sexes, despite no significant interaction of CIE and sex ( Fig. 3C , F sex (1,62) = 9.010, p<.01; F CIE (1,62) = 13.13, p<.001). A 3-way repeated measure ANOVA of Retigabine-modulated M-current found significant interactions of voltage step and sex (F(4,228) = 3.517, p<.01) as well as voltage step and CIE (F(4,228) = 3.291, p<.01) but they were not significant following Bonferroni posthoc ( Fig. 4D , F(4,236) = 3.334, p <.05; F(4,236) = 3.098 p <.05). Only at the most depolarized current step did CIE produce a significant deficit in Retigabine modulated M-current ( Fig. 3E , 2-way ANOVA, F(1,57) = 4.042, p < .05). Download figure Open in new tab Fig 3. (A) M-current recording drug wash schedule, where the K V 7 positive modulator retigabine (RTG) and the K V 7 blocker XE-991 are applied following baseline recording, which allowed for the subtraction of the K V 7-mediated current, M-current. (B-D) CIE diminished M-current regardless of sex. (−30mV and −20mV: p < 0.05, Bonferroni posthoc; −20mV sex: p < 0.01, treatment: p < 0.001) (E-G) Positively modulated M-current is significantly larger from air exposed mice at −20mV (p < 0.05). Download figure Open in new tab Fig 4. Method of cell type classification. A hyperpolarizing current was applied to induce sag current (A) which is an identifier of IT and ET projection neurons. ET neurons display a larger sag ratio and higher membrane resistance (B) , and larger physiological index (C , p < 0.0001 ) in comparison to IT neurons. Differentiating Extratelencephalic and Intratelencephalic projection neurons using physiological measures Previous work has demonstrated that alcohol exposure can elicit distinct electrophysiological alterations based on prefrontal projection subtype 39 , 40 , so previous recordings from nonexposed animals were categorized into type A extratelencephalic (ET) and type B intratelencephalic (IT) neurons based on known physiological differences such as sag ratio, membrane resistance, and physiology index to determine if this method could be used to extrapolate to the remaining findings ( Fig. 4A ). When graphed based on their sag ratio and membrane resistance, IT and ET neurons largely form their own distinct clusters ( Fig. 4B ). Upon applying this data to calculate the physiology index, ET neurons have a significantly larger index size regardless of sex ( Fig. 4C , F(1,61) = 74.48, p < .0001). These results indicated that the rest of the data could be further classified into IT vs ET cell types based on sag ratio and physiology index. Prelimbic Extratelencephalic neurons do not exhibit CIE induced hyperexcitability and reduced K V 7 function Within the ET population, although there was a significant 3-way interaction of CIE, current step, and sex ( Fig. 5A , F(15,735) = 3.969, p < .0001) there were no significant comparisons following Bonferroni post hoc. The summation of total evoked action potential firing was not significantly altered due to CIE, but a sex trend was detected ( Fig. 5B , 2-way ANOVA, F(1,46) = 3.770, p = .0583). No significant effects were observed with regards to rheobase ( Fig. 5C ), resting membrane potential ( Fig. 5D ), or other action potential kinetic properties ( Table 2 ). View this table: View inline View popup Download powerpoint Table 2. Action potential kinetics of deep layer ET prelimbic pyramidal neurons from Figure 5 . Download figure Open in new tab Fig 5. (A) ET neurons were not hyperexcitable following CIE. (B ) The summation of action potentials are potentially sex dependent. (B) CIE does not alter rheobase (C) or resting membrane potential (D) . ET M current is not impacted by CIE, but females displayed significantly higher evoked M-current at −20mV (E) . The RTG exposed M-current is significantly higher in female neurons due to CIE exposure only at −20mV (p < 0.05) (F) . M-current within the ET population was then identified. A 3-way repeated measures ANOVA measured a significant interaction of voltage step and sex ( Fig. 5E , F(4,88) = 7.331, p < .001), but nothing was significant following post-hoc comparison. At the maximized −20 current step, females exhibited a significantly larger M-current regardless of exposure treatment ( Fig. 5F , 2-way ANOVA, F(1,23) = 8.211, p < .01). Retigabine modulated M-current elicited a significant interaction of voltage step x CIE x sex ( Fig. 5G , F(4,84) = 4.668, p<.01), but post hoc analysis found no significant comparisons. A significant CIE x sex interaction was measured for the maximal retigabine modulated M-current, which post hoc comparison found to be a significant increase in M-current in female CIE-exposed neurons in comparison to female air neurons ( Fig. 5H , F(1,21) = 11.50, p < .01, Bonferroni post hoc p < .05). Based on ET evoked action potential firing and M-current results, ET neurons were deemed less sensitive to CIE and are not driving the physiological findings from the collapsed PL data. CIE reduced K V 7 function predominantly in Intratelencephalic projecting Prelimbic neurons Within the IT population, a 3-way repeated measured ANOVA determined a significant interaction of CIE and current step across multiple current steps ( Fig. 6A , F(15,795) = 4.277, p < 0.0001; 2-way consolidated data F(15,825) = 4.429, p < 0.0001, Bonferroni adjusted p-value 40pA: p < .05, 60-200pA: p < .01, 220pA: p < .05). Total evoked action potential firing was significantly elevated due to CIE in both sexes ( Fig. 6B , F(1,52) = 10.14, p < 0.01). CIE-exposed IT neurons had a significantly lower rheobase ( Fig. 6C , F(1,55) = 13.51, p < 0.001) and depolarized resting membrane potential ( Fig 6D , F(1,52) = 4.175, p < 0.05). Download figure Open in new tab Fig 6. (A) IT neurons displayed elevated evoked firing following CIE. (40pA: p < 0.05, 60-200pA: p < 0.01, 220pA: p < .05, Bonferroni posthoc). The summation of action potentials was larger in CIE exposed neurons (B) along with a decreased rheobase (p < .01) (C) and depolarized resting membrane potential (p < 0.001 ) (D) . IT M current is depressed in male and female CIE exposed neurons (−20mV: p < 0.01, Bonferroni posthoc) (E) . At −20mV, CIE reduced M-current and was larger in females (sex: p < .05; CIE: p < 0.001). The RTG exposed M-current is significantly lower due to CIE exposure at −20 mV (p < 0.05) (F) . Upon analyzing IT neuron M-current, a 3-way repeated measure ANOVA revealed a significant interaction of voltage and sex, as well as voltage and treatment, which following post hoc comparison yielded an effect of decreased M-current at −20mV ( Fig. 6E , F(4,152) = 9.771 p<0.0001; F(4,152) = 2.734, p < 0.05; F(4,160) = 10.36, p < 0.0001, −20 adjusted p-value < 0.01). A 2-way ANOVA also found a main effect of CIE and sex, but no interaction, at the maximal m-current step ( Fig. 6F , F(1,35) = 4.155 p < .05; F(1,35) = 13.02, p < .01). There were no significant differences in retigabine modulated M current following post-hoc comparisons ( Fig. 6G ), but there was a main effect of CIE diminishing the current at the maximal current step ( Fig. 6H , F(1,32) = 5.973 p < 0.05). Along with the elevated neuronal excitability results, these results lead to the conclusion that the global findings of decreased K V 7 function were largely driven by IT projection neurons. Positive modulation of Prelimbic K V 7 channels rescued cognitive function Because the physiology data indicated an overall decrease in K V 7 function when data from all cell types were collapsed, global modulation of PL K V 7 was performed following CIE to determine whether enhancement of K V 7 function within the PL would be sufficient to restore cognitive function following alcohol dependence. Mice (n=7-10/treatment/sex) first had a guide cannula directed towards the PL cortex surgically implanted before undergoing baseline working memory testing and CIE exposure ( Fig. 7A ). CIE exposure significantly reduced working memory performance in mice administered vehicle, and retigabine increased working memory performance in CIE exposed mice ( Fig. 7B , 3-Way ANOVA, F(1,31) = 14.24, p < 0.001; subsequent sex collapsed 2-way ANOVA, F(1,33) = 13.38, p < 0.001) Download figure Open in new tab Fig 7. (A) Timeline for microinjection study. (B) Retigabine administration to the PL restored CIE induced working memory deficits. (Air vs CIE; p < 0.01, RTG vs saline; p < 0.001, Bonferroni posthoc) (C) locations of cannula tracks. Discussion Consistent with known literature 13 , 50 , 52 , 65 , 66 , chronic intermittent ethanol model consistently produced cognitive deficits in male and female C57BL/6J mice as measured using a spatial working memory task. In parallel, CIE increased the neuronal excitability of layer V PL projection neurons from male and female mice, while concurrently reducing K V 7-mediated M-current. Using electrophysiological parameters, these findings were largely driven by IT projecting neurons, as ET neurons were not sensitive to CIE-induced dysregulation. Lastly, positive modulation of PL K V 7 channels restored cognitive function in both sexes of alcohol dependent mice. Together, this set of experiments is the first to demonstrate a role for cortical K V 7 channels in alcohol induced cognitive impairment. Chronic alcohol exposure decreased the function of K V 7 channels within the PL cortex. Expression of K V 7 channels is altered following alcohol exposure in the nucleus accumbens 23 , 24 , and others have shown diminished M-current in the lateral habenula 26 , 27 , ventral tegmental area 67 , and hippocampus 68 . Interestingly, K V 7 current appeared most prominent in IT neurons, with ET neurons having smaller current in comparison. ET neurons have preferential muscarinic activation in comparison to IT neurons, and this mechanism may suppress K V 7 current 41 , so it is possible that there may not be a difference in total K V 7 current across these cell types if one were to block muscarinic activation. A caveat of this study is that the total amount of K V 7 channels within each cell type was not quantified. Future studies should explore if the amount of K V 7 channels is altered, and if that is the reason K V 7 channel function was diminished. K V 7 channels require PI(4,5)P 2 for stabilization of the ion channel pore to allow potassium ions to flow through and close when there is not enough 16 , 21 , 22 . Cell culture studies have identified that ethanol can diminish the amount of PI(4,5)P 2 available, alter sensitivity of K V 7 channels to PI(4,5)P 2 , and ultimately inhibit K V 7 channels 19 , 69 , 70 . Thus, the reported effects may in part be due to altered PI(4,5)P 2 levels, though additional work would be required in the future to confirm the levels of PI(4,5)P 2 following CIE. Chronic alcohol induced cognitive deficits were reversed following direct modulation of K V 7 channels within the prefrontal cortex. Although the working memory task used previously demonstrated ethanol induced behavioral deficits and is prefrontal cortex dependent 47 , 48 , this was following long term voluntary liquid diet, and did not reverse deficits via direct cortical modulation. Ethanol can disrupt memory in drosophila unless KCNQ is mutated, suggesting a role for K V 7 channels in ethanol induced memory impairment 71 , 72 . Prevention of alcohol induced deficits via systemic administration of the K V 7 modulator retigabine has previously been established 30 , but this was not brain region specific, and it is unknown if their cognitive deficit would have been reversed with sole administration of retigabine following alcohol exposure instead of concurrently. Typically, blocking K V 7 channels has been shown to enhance cognitive performance 18 , 20 , 28 , 29 , 59 , 73 . In contrast to this notion, genetic disruption of K V 7 channels have demonstrated that limiting K V 7 channel function can also cause cognitive deficits 64 , 71 , 72 , 74 . Inhibiting K V 7 channels in alcohol exposed rodents leads to seizures 23 , so K V 7 inhibition may operate on an inverted U like fashion in its role in regulating cognition. Indeed, Galvin et al 59 demonstrated an inverted U like pattern when modulating working memory in nonhuman primates with a muscarinic agonist, which was directly linked to modulation of K V 7 channels. Although global modulation of PL K V 7 channels modulated IT neurons, it also modulated ET neurons, so the conclusion that IT K V 7 neurons are responsible for the behavioral results cannot be made. Chemogenetic inhibition of IT neurons in the prefrontal cortex can inhibit working memory performance 38 , lending to the argument of IT controlling working memory in the cortex. Currently, validation of a CRISPR/Cas9 construct (Courtesy of the Zweifel Lab) to knock down Kcnq3 , the K V 7.3 subunit is underway. Once validated, this construct will be used to knock down K V 7 function within an IT circuit, with the intent to mimic CIE induced cognitive deficits. This would further lend credence to the role of IT K V 7 channels in alcohol induced cognitive impairment. The escalation in neuronal excitability following alcohol exposure parallels similar findings in other brain regions, such as dopaminergic VTA neurons 67 , nucleus accumbens 75 , lateral habenula 26 , 27 and orbitofrontal cortex 76 . Although some groups have identified hyperexcitable phenotypes within the prefrontal cortex 40 , 77 , other studies have reported no ethanol induced intrinsic excitability adaptations 12 , 14 , 50 . One reason for this could be related to the choice of internal solution for performing evoked action potential firing recordings. While the cited papers used a k-gluconate based internal solution, these recordings were performed using a KCl-based internal solution. This was done specifically to allow for the additional recording of the K V 7 mediated m-current from the same neurons, as the KCl internal facilitates the recording M-current 78 . Additionally, while many of these studies recorded evoked activity at −70mV, the current recordings were performed at the neuron’s natural resting membrane potential. K V 7 channels are known to regulate the resting membrane potential 79 - 81 , so the possibility that adaptations to excitability, like the overall enhancement identified, may in part be K V 7-mediated. The results for IT and ET neuron excitability differ from Joffe et al and Hughes et al. Hughes et al demonstrated an increase in excitability in male ET neurons, and no difference in female ET neurons or any IT neurons. It is important to note that despite including both sexes, they did not use sex as a variable in their analysis. Although no difference in evoked firing was found in the ET neurons, there is a trend of increased excitability from male ET neurons. It is possible if male ET data was analyzed separately from the females that the current findings would appear more like the effects identified in Hughes et al. It is possible that the IT effects identified are different due to animal differences, as they employed rats while the current study used mice. It is also important to note that, despite them also using CIE for alcohol exposure, CIE in rats is a continuous exposure method across 2 weeks instead of the 4-week exposure period used in mice, which could also account for physiological differences. Although Joffe et al saw no changes in intrinsic excitability, this could be related to the alcohol exposure method used, the intermittent access model. Although both groups reported IT neurons being more excitable than ET neurons, our data showed the opposite. ET neurons are composed of corticothalamic projection neurons and pyramidal tract neurons 33 , 35 , with the latter being more excitable. Because viral tracers were not employed, it is impossible to know if ET neurons were pyramidal tract or corticothalamic neurons, so it is possible that many were pyramidal tract neurons. Of note, layer V pyramidal tract ET neurons have been demonstrated to be more excitable than IT neurons 36 , 82 , so these findings are still in line with established literature. To conclude, the current findings support the effectiveness of K V 7 channel positive modulation in improving chronic alcohol-induced cognitive impairment. Along with our previous work indicating its efficacy in decreasing alcohol intake in high drinkers 23 , 24 , 83 , these results further highlight the potential for K V 7 modulators as a therapeutic option for those with alcohol use disorder. Funder Information Declared NIAAA , AA023288 , AA007474 , AA031433 REFERENCES 1. ↵ Abernathy , K. , Chandler , L. J. & Woodward , J. J. Alcohol and the prefrontal cortex . Int Rev Neurobiol 91 , 289 – 320 ( 2010 ). doi: 10.1016/s0074-7742(10)91009-x OpenUrl CrossRef PubMed 2. Staples , M. C. & Mandyam , C. D. Thinking after Drinking: Impaired Hippocampal-Dependent Cognition in Human Alcoholics and Animal Models of Alcohol Dependence . Front Psychiatry 7 , 162 ( 2016 ). doi: 10.3389/fpsyt.2016.00162 OpenUrl CrossRef PubMed 3. ↵ Ambrose , M. L. , Bowden , S. C. & Whelan , G. Working memory impairments in alcohol-dependent participants without clinical amnesia . Alcohol Clin Exp Res 25 , 185 – 191 ( 2001 ). OpenUrl CrossRef PubMed Web of Science 4. ↵ Stavro , K. , Pelletier , J. & Potvin , S. Widespread and sustained cognitive deficits in alcoholism: a meta-analysis . Addiction Biology 18 , 203 – 213 ( 2013 ). doi: 10.1111/j.1369-1600.2011.00418.x OpenUrl CrossRef PubMed 5. ↵ Balcells-Oliveró , M. et al. A systematic review of treatments for alcohol-related cognitive impairment: lessons from the past and gaps for future interventions . Psychological Medicine 50 , 2113 – 2127 ( 2020 ). doi: 10.1017/S0033291720002925 OpenUrl CrossRef PubMed 6. ↵ Canessa , N. , Basso , G. , Manera , M. , Poggi , P. & Gianelli , C. Functional Coherence in Intrinsic Frontal Executive Networks Predicts Cognitive Impairments in Alcohol Use Disorder . Brain Sci 13 ( 2022 ). doi: 10.3390/brainsci13010045 OpenUrl CrossRef 7. ↵ Charlet , K. et al. Increased neural activity during high working memory load predicts low relapse risk in alcohol dependence . Addiction Biology 19 , 402 – 414 ( 2014 ). doi: 10.1111/adb.12103 OpenUrl CrossRef PubMed 8. ↵ Pfefferbaum , A. et al. Reorganization of Frontal Systems Used by Alcoholics for Spatial Working Memory: An fMRI Study . NeuroImage 14 , 7 – 20 ( 2001 ). doi: 10.1006/nimg.2001.0785 OpenUrl CrossRef PubMed Web of Science 9. ↵ de la Monte , S.M. Disproportionate atrophy of cerebral white matter in chronic alcoholics . Archives of neurology 45 , 990 – 992 ( 1988 ). OpenUrl CrossRef PubMed Web of Science 10. Hayes , V. , Demirkol , A. , Ridley , N. , Withall , A. & Draper , B. Alcohol-related cognitive impairment: current trends and future perspectives . Neurodegenerative Disease Management 6 , 509 – 523 ( 2016 ). doi: 10.2217/nmt-2016-0030 OpenUrl CrossRef PubMed 11. ↵ Pandey , A. K. et al. Lower Prefrontal and Hippocampal Volume and Diffusion Tensor Imaging Differences Reflect Structural and Functional Abnormalities in Abstinent Individuals with Alcohol Use Disorder . Alcohol Clin Exp Res 42 , 1883 – 1896 ( 2018 ). doi: 10.1111/acer.13854 OpenUrl CrossRef PubMed 12. ↵ Pleil , K. E. et al. Effects of chronic ethanol exposure on neuronal function in the prefrontal cortex and extended amygdala . Neuropharmacology 99 , 735 – 749 ( 2015 ). doi: 10.1016/j.neuropharm.2015.06.017 OpenUrl CrossRef PubMed 13. ↵ Kroener , S. et al. Chronic Alcohol Exposure Alters Behavioral and Synaptic Plasticity of the Rodent Prefrontal Cortex . PLOS ONE 7 , e37541 ( 2012 ). doi: 10.1371/journal.pone.0037541 OpenUrl CrossRef PubMed 14. ↵ Varodayan , F. P. , Sidhu , H. , Kreifeldt , M. , Roberto , M. & Contet , C. Morphological and functional evidence of increased excitatory signaling in the prelimbic cortex during ethanol withdrawal . Neuropharmacology 133 , 470 – 480 ( 2018 ). doi: 10.1016/j.neuropharm.2018.02.014 OpenUrl CrossRef PubMed 15. ↵ Laubach , M. , Amarante , L. M. , Swanson , K. & White , S. R. What, If Anything, Is Rodent Prefrontal Cortex? eNeuro 5 ( 2018 ). doi: 10.1523/eneuro.0315-18.2018 OpenUrl CrossRef 16. ↵ Brown , D. A. & Passmore , G. M. Neural KCNQ (Kv7) channels . Br J Pharmacol 156 , 1185 – 1195 ( 2009 ). doi: 10.1111/j.1476-5381.2009.00111.x OpenUrl CrossRef PubMed Web of Science 17. Lee , S. & Kwag , J. M-channels modulate the intrinsic excitability and synaptic responses of layer 2/3 pyramidal neurons in auditory cortex . Biochem Biophys Res Commun 426 , 448 – 453 ( 2012 ). doi: 10.1016/j.bbrc.2012.08.057 OpenUrl CrossRef PubMed 18. ↵ Wang , J. , Yu , W. , Gao , Q. , Ju , C. & Wang , K. Prefrontal inhibition of neuronal K(v) 7 channels enhances prepulse inhibition of acoustic startle reflex and resistance to hypofrontality . Br J Pharmacol 177 , 4720 – 4733 ( 2020 ). doi: 10.1111/bph.15236 OpenUrl CrossRef PubMed 19. ↵ Kim , K. S. , Duignan , K. M. , Hawryluk , J. M. , Soh , H. & Tzingounis , A. V. The Voltage Activation of Cortical KCNQ Channels Depends on Global PIP2 Levels . Biophys J 110 , 1089 – 1098 ( 2016 ). doi: 10.1016/j.bpj.2016.01.006 OpenUrl CrossRef PubMed 20. ↵ Esaki , H. et al. Nicotine enhances object recognition memory through inhibition of voltage-dependent potassium 7 channels in the medial prefrontal cortex of mice . J Pharmacol Sci 147 , 58 – 61 ( 2021 ). doi: 10.1016/j.jphs.2021.05.009 OpenUrl CrossRef PubMed 21. ↵ Delmas , P. & Brown , D. A. Pathways modulating neural KCNQ/M (Kv7) potassium channels . Nature Reviews Neuroscience 6 , 850 – 862 ( 2005 ). doi: 10.1038/nrn1785 OpenUrl CrossRef PubMed Web of Science 22. ↵ Soldovieri , M. V. , Miceli , F. & Taglialatela , M. Driving with no brakes: molecular pathophysiology of Kv7 potassium channels . Physiology (Bethesda) 26 , 365 – 376 ( 2011 ). doi: 10.1152/physiol.00009.2011 OpenUrl CrossRef PubMed 23. ↵ McGuier , N. S. et al. Kv7 channels in the nucleus accumbens are altered by chronic drinking and are targets for reducing alcohol consumption . Addict Biol 21 , 1097 – 1112 ( 2016 ). doi: 10.1111/adb.12279 OpenUrl CrossRef PubMed 24. ↵ McGuier , N. S. et al. Identification and validation of midbrain Kcnq4 regulation of heavy alcohol consumption in rodents . Neuropharmacology 138 , 10 – 19 ( 2018 ). doi: 10.1016/j.neuropharm.2018.05.020 OpenUrl CrossRef PubMed 25. ↵ Rinker , J. A. et al. Differential potassium channel gene regulation in BXD mice reveals novel targets for pharmacogenetic therapies to reduce heavy alcohol drinking . Alcohol 58 , 33 – 45 ( 2017 ). doi: 10.1016/j.alcohol.2016.05.007 OpenUrl CrossRef PubMed 26. ↵ Kang , S. et al. Downregulation of M-channels in lateral habenula mediates hyperalgesia during alcohol withdrawal in rats . Scientific Reports 9 , 2714 ( 2019 ). doi: 10.1038/s41598-018-38393-7 OpenUrl CrossRef PubMed 27. ↵ Kang , S. et al. Ethanol Withdrawal Drives Anxiety-Related Behaviors by Reducing M-type Potassium Channel Activity in the Lateral Habenula . Neuropsychopharmacology 42 , 1813 – 1824 ( 2017 ). doi: 10.1038/npp.2017.68 OpenUrl CrossRef PubMed 28. ↵ Fontán-Lozano , A. , Suárez-Pereira , I. , Delgado-García , J. M. & Carrión , A. M. 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 , 22 – 32 ( 2011 ). doi: 10.1002/hipo.20717 OpenUrl CrossRef PubMed Web of Science 29. ↵ Arnsten , A. F. T. et al. Role of KCNQ potassium channels in stress-induced deficit of working memory . Neurobiol Stress 11 , 100187 ( 2019 ). doi: 10.1016/j.ynstr.2019.100187 OpenUrl CrossRef PubMed 30. ↵ Zwierzyńska , E. , Krupa-Burtnik , A. & Pietrzak , B. Beneficial effect of retigabine on memory in rats receiving ethanol . Pharmacol Rep 73 , 480 – 489 ( 2021 ). doi: 10.1007/s43440-020-00205-z OpenUrl CrossRef PubMed 31. ↵ Cooper , E. C. , Harrington , E. , Jan , Y. N. & Jan , L. Y. M Channel KCNQ2 Subunits Are Localized to Key Sites for Control of Neuronal Network Oscillations and Synchronization in Mouse Brain . The Journal of Neuroscience 21 , 9529 ( 2001 ). doi: 10.1523/JNEUROSCI.21-24-09529.2001 OpenUrl Abstract / FREE Full Text 32. ↵ Geiger , J. , Weber , Y. G. , Landwehrmeyer , B. , Sommer , C. & Lerche , H. Immunohistochemical analysis of KCNQ3 potassium channels in mouse brain . Neurosci Lett 400 , 101 – 104 ( 2006 ). doi: 10.1016/j.neulet.2006.02.017 OpenUrl CrossRef PubMed Web of Science 33. ↵ Anastasiades , P. G. & Carter , A. G. Circuit organization of the rodent medial prefrontal cortex . Trends in Neurosciences 44 , 550 – 563 ( 2021 ). doi: 10.1016/j.tins.2021.03.006 OpenUrl CrossRef PubMed 34. Anastasiades , P. G. , Boada , C. & Carter , A. G. Cell-Type-Specific D1 Dopamine Receptor Modulation of Projection Neurons and Interneurons in the Prefrontal Cortex . Cerebral Cortex 29 , 3224 – 3242 ( 2018 ). doi: 10.1093/cercor/bhy299 OpenUrl CrossRef PubMed 35. ↵ Anastasiades , P. G. , Marlin , J. J. & Carter , A. G. Cell-Type Specificity of Callosally Evoked Excitation and Feedforward Inhibition in the Prefrontal Cortex . Cell Reports 22 , 679 – 692 ( 2018 ). doi: 10.1016/j.celrep.2017.12.073 OpenUrl CrossRef PubMed 36. ↵ Baker , A. et al. Specialized Subpopulations of Deep-Layer Pyramidal Neurons in the Neocortex: Bridging Cellular Properties to Functional Consequences . The Journal of Neuroscience 38 , 5441 – 5455 ( 2018 ). doi: 10.1523/jneurosci.0150-18.2018 OpenUrl Abstract / FREE Full Text 37. ↵ Musall , S. et al. Pyramidal cell types drive functionally distinct cortical activity patterns during decision-making . Nature Neuroscience 26 , 495 – 505 ( 2023 ). doi: 10.1038/s41593-022-01245-9 OpenUrl CrossRef 38. ↵ Bae , J. W. et al. Parallel processing of working memory and temporal information by distinct types of cortical projection neurons . Nature Communications 12 , 4352 ( 2021 ). doi: 10.1038/s41467-021-24565-z OpenUrl CrossRef PubMed 39. ↵ Joffe , M. E. , Winder , D. G. & Conn , P. J. Increased Synaptic Strength and mGlu2/3 Receptor Plasticity on Mouse Prefrontal Cortex Intratelencephalic Pyramidal Cells Following Intermittent Access to Ethanol . Alcoholism: Clinical and Experimental Research 45 , 518 – 529 ( 2021 ). doi: 10.1111/acer.14546 OpenUrl CrossRef PubMed 40. ↵ Hughes , B. A. , O’Buckley , T. K. , Boero , G. , Herman , M. A. & Morrow , A. L. Sex-and subtype-specific adaptations in excitatory signaling onto deep-layer prelimbic cortical pyramidal neurons after chronic alcohol exposure . Neuropsychopharmacology 46 , 1927 – 1936 ( 2021 ). doi: 10.1038/s41386-021-01034-1 OpenUrl CrossRef PubMed 41. ↵ Baker , A. L. , O’Toole , R. J. & Gulledge , A. T. Preferential cholinergic excitation of corticopontine neurons . The Journal of Physiology 596 , 1659 – 1679 ( 2018 ). doi: 10.1113/JP275194 OpenUrl CrossRef PubMed 42. ↵ Gulledge , A. T. Cholinergic Activation of Corticofugal Circuits in the Adult Mouse Prefrontal Cortex . The Journal of Neuroscience 44 , e1388232023 ( 2024 ). doi: 10.1523/jneurosci.1388-23.2023 OpenUrl Abstract / FREE Full Text 43. ↵ Becker , H. C. & Lopez , M. F. Increased Ethanol Drinking After Repeated Chronic Ethanol Exposure and Withdrawal Experience in C57BL/6 Mice . Alcoholism: Clinical and Experimental Research 28 , 1829 – 1838 ( 2004 ). doi: 10.1097/01.ALC.0000149977.95306.3A OpenUrl CrossRef PubMed Web of Science 44. ↵ Lopez , M. F. & Becker , H. C. Effect of pattern and number of chronic ethanol exposures on subsequent voluntary ethanol intake in C57BL/6J mice . Psychopharmacology (Berl) 181 , 688 – 696 ( 2005 ). doi: 10.1007/s00213-005-0026-3 OpenUrl CrossRef PubMed 45. ↵ Pradhan , G. et al. Calcium chloride mimics the effects of acamprosate on cognitive deficits in chronic alcohol-exposed mice . Psychopharmacology (Berl) 235 , 2027 – 2040 ( 2018 ). doi: 10.1007/s00213-018-4900-1 OpenUrl CrossRef 46. ↵ Parrilla-Carrero , J. et al. Restoration of Kv7 Channel-Mediated Inhibition Reduces Cued-Reinstatement of Cocaine Seeking . J Neurosci 38 , 4212 – 4229 ( 2018 ). doi: 10.1523/JNEUROSCI.2767-17.2018 OpenUrl Abstract / FREE Full Text 47. ↵ Dominguez , G. et al. Alcohol withdrawal induces long-lasting spatial working memory impairments: relationship with changes in corticosterone response in the prefrontal cortex . Addict Biol 22 , 898 – 910 ( 2017 ). doi: 10.1111/adb.12371 OpenUrl CrossRef PubMed 48. ↵ Dominguez , G. et al. Repeated diazepam administration reversed working memory impairments and glucocorticoid alterations in the prefrontal cortex after short but not long alcohol-withdrawal periods . Cogn Affect Behav Neurosci 18 , 665 – 679 ( 2018 ). doi: 10.3758/s13415-018-0595-3 OpenUrl CrossRef PubMed 49. ↵ Dominguez , G. et al. Sustained corticosterone rise in the prefrontal cortex is a key factor for chronic stress-induced working memory deficits in mice . Neurobiology of Stress 10 , 100161 ( 2019 ). doi: 10.1016/j.ynstr.2019.100161 OpenUrl CrossRef PubMed 50. ↵ Hu , W. , Morris , B. , Carrasco , A. & Kroener , S. Effects of acamprosate on attentional set-shifting and cellular function in the prefrontal cortex of chronic alcohol-exposed mice . Alcohol Clin Exp Res 39 , 953 – 961 ( 2015 ). doi: 10.1111/acer.12722 OpenUrl CrossRef PubMed 51. Badanich , K. A. , Becker , H. C. & Woodward , J. J. Effects of chronic intermittent ethanol exposure on orbitofrontal and medial prefrontal cortex-dependent behaviors in mice . Behav Neurosci 125 , 879 – 891 ( 2011 ). doi: 10.1037/a0025922 OpenUrl CrossRef PubMed 52. ↵ Athanason , A. C. et al. Chronic ethanol alters adrenergic receptor gene expression and produces cognitive deficits in male mice . Neurobiol Stress 24 , 100542 ( 2023 ). doi: 10.1016/j.ynstr.2023.100542 OpenUrl CrossRef 53. ↵ Nippert , K. E. , Rowland , C. P. , Vazey , E. M. & Moorman , D. E. Alcohol, flexible behavior, and the prefrontal cortex: Functional changes underlying impaired cognitive flexibility . Neuropharmacology 260 , 110114 ( 2024 ). doi: 10.1016/j.neuropharm.2024.110114 OpenUrl CrossRef PubMed 54. ↵ Logan , R. W. , McCulley , W. D. , 3rd . , Seggio , J.A. & Rosenwasser , A. M. Effects of withdrawal from chronic intermittent ethanol vapor on the level and circadian periodicity of running-wheel activity in C57BL/6J and C3H/HeJ mice . Alcohol Clin Exp Res 36 , 467 – 476 ( 2012 ). doi: 10.1111/j.1530-0277.2011.01634.x OpenUrl CrossRef PubMed 55. ↵ Logan , R. W. , Seggio , J. A. , Robinson , S. L. , Richard , G. R. & Rosenwasser , A. M. Circadian wheel-running activity during withdrawal from chronic intermittent ethanol exposure in mice . Alcohol 44 , 239 – 244 ( 2010 ). doi: 10.1016/j.alcohol.2010.02.011 OpenUrl CrossRef PubMed 56. ↵ Fleischer , A. W. & Frick , K. M. New perspectives on sex differences in learning and memory . Trends Endocrinol Metab 34 , 526 – 538 ( 2023 ). doi: 10.1016/j.tem.2023.06.003 OpenUrl CrossRef PubMed 57. ↵ Frick , K. M. & Berger-Sweeney , J. Spatial reference memory and neocortical neurochemistry vary with the estrous cycle in C57BL/6 mice . Behav Neurosci 115 , 229 – 237 ( 2001 ). doi: 10.1037/0735-7044.115.1.229 OpenUrl CrossRef PubMed Web of Science 58. ↵ Baculis , B. C. , Zhang , J. & Chung , H. J. The Role of K(v)7 Channels in Neural Plasticity and Behavior . Front Physiol 11 , 568667 ( 2020 ). doi: 10.3389/fphys.2020.568667 OpenUrl CrossRef PubMed 59. ↵ Galvin , V. C. et al. Muscarinic M1 Receptors Modulate Working Memory Performance and Activity via KCNQ Potassium Channels in the Primate Prefrontal Cortex . Neuron 106 , 649 - 661 .e644 ( 2020 ). doi: 10.1016/j.neuron.2020.02.030 OpenUrl CrossRef PubMed 60. Kim , E. C. et al. Spontaneous seizure and memory loss in mice expressing an epileptic encephalopathy variant in the calmodulin-binding domain of K v 7.2 . Proceedings of the National Academy of Sciences 118 , e2021265118 ( 2021 ). doi: 10.1073/pnas.2021265118 OpenUrl Abstract / FREE Full Text 61. Li , C. et al. KCNQ/Kv7 channel activator flupirtine protects against acute stress-induced impairments of spatial memory retrieval and hippocampal LTP in rats . Neuroscience 280 , 19 – 30 ( 2014 ). doi: 10.1016/j.neuroscience.2014.09.009 OpenUrl CrossRef PubMed 62. Li , C. et al. Retigabine ameliorates acute stress-induced impairment of spatial memory retrieval through regulating USP2 signaling pathways in hippocampal CA1 area . Neuropharmacology 135 , 151 – 162 ( 2018 ). doi: 10.1016/j.neuropharm.2018.02.034 OpenUrl CrossRef PubMed 63. Wei , Y. et al. A novel dual serotonin transporter and M-channel inhibitor D01 for antidepression and cognitive improvement . Acta Pharmaceutica Sinica B ( 2023 ). doi: 10.1016/j.apsb.2023.11.024 OpenUrl CrossRef 64. ↵ Peters , H. C. , Hu , H. , Pongs , O. , Storm , J. F. & Isbrandt , D. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior . Nat Neurosci 8 , 51 – 60 ( 2005 ). doi: 10.1038/nn1375 OpenUrl CrossRef PubMed Web of Science 65. ↵ Phensy , A. et al. Deletion of the Mitochondrial Matrix Protein CyclophilinD Prevents Parvalbumin Interneuron Dysfunctionand Cognitive Deficits in a Mouse Model of NMDA Hypofunction . J Neurosci 40 , 6121 – 6132 ( 2020 ). doi: 10.1523/jneurosci.0880-20.2020 OpenUrl Abstract / FREE Full Text 66. ↵ Lyu , Z. et al. Benefits of exercise on cognitive impairment in alcohol use disorder following alcohol withdrawal . FEBS Open Bio 14 , 1540 – 1558 ( 2024 ). doi: 10.1002/2211-5463.13865 OpenUrl CrossRef PubMed 67. ↵ Koyama , S. , Brodie , M. S. & Appel , S. B. Ethanol Inhibition of M-Current and Ethanol-Induced Direct Excitation of Ventral Tegmental Area Dopamine Neurons . Journal of Neurophysiology 97 , 1977 – 1985 ( 2007 ). doi: 10.1152/jn.00270.2006 OpenUrl CrossRef PubMed Web of Science 68. ↵ Moore , S. D. , Madamba , S. G. & Siggins , G. R. Ethanol diminishes a voltage-dependent K+ current the M-current, in CA1 hippocampal pyramidal neurons in vitro . Brain Research 516 , 222 – 228 ( 1990 ). doi: 10.1016/0006-8993(90)90922-X OpenUrl CrossRef PubMed Web of Science 69. ↵ Kim , K. W. , Kim , K. , Lee , H. & Suh , B. C. Ethanol Elevates Excitability of Superior Cervical Ganglion Neurons by Inhibiting Kv7 Channels in a Cell Type-Specific and PI(4,5)P(2)-Dependent Manner . Int J Mol Sci 20 ( 2019 ). doi: 10.3390/ijms20184419 OpenUrl CrossRef 70. ↵ Kim , K. W. & Suh , B. C. Ethanol inhibits Kv7.2/7.3 channel open probability by reducing the PI(4,5)P2 sensitivity of Kv7.2 subunit . BMB Rep 54 , 311 – 316 ( 2021 ). doi: 10.5483/BMBRep.2021.54.6.231 OpenUrl CrossRef PubMed 71. ↵ Cavaliere , S. , Gillespie , J. M. & Hodge , J. J. L. KCNQ Channels Show Conserved Ethanol Block and Function in Ethanol Behaviour . PLOS ONE 7 , e50279 ( 2012 ). doi: 10.1371/journal.pone.0050279 OpenUrl CrossRef PubMed 72. ↵ Cavaliere , S. , Malik , B. R. & Hodge , J. J. KCNQ channels regulate age-related memory impairment . PLoS One 8 , e62445 ( 2013 ). doi: 10.1371/journal.pone.0062445 OpenUrl CrossRef PubMed 73. ↵ Petrovic , M. M. et al. Inhibition of post-synaptic Kv7/KCNQ/M channels facilitates long-term potentiation in the hippocampus . PLoS One 7 , e30402 ( 2012 ). doi: 10.1371/journal.pone.0030402 OpenUrl CrossRef PubMed 74. ↵ Milh , M. et al. A knock-in mouse model for KCNQ2-related epileptic encephalopathy displays spontaneous generalized seizures and cognitive impairment . Epilepsia 61 , 868 – 878 ( 2020 ). doi: 10.1111/epi.16494 OpenUrl CrossRef PubMed 75. ↵ Padula , A. E. et al. KCNN Genes that Encode Small-Conductance Ca2+-Activated K+ Channels Influence Alcohol and Drug Addiction . Neuropsychopharmacology 40 , 1928 – 1939 ( 2015 ). doi: 10.1038/npp.2015.42 OpenUrl CrossRef PubMed 76. ↵ Nimitvilai , S. , Lopez , M. F. , Mulholland , P. J. & Woodward , J. J. Chronic Intermittent Ethanol Exposure Enhances the Excitability and Synaptic Plasticity of Lateral Orbitofrontal Cortex Neurons and Induces a Tolerance to the Acute Inhibitory Actions of Ethanol . Neuropsychopharmacology 41 , 1112 – 1127 ( 2016 ). doi: 10.1038/npp.2015.250 OpenUrl CrossRef PubMed 77. ↵ Avchalumov , Y. et al. Chronic ethanol exposure differentially alters neuronal function in the medial prefrontal cortex and dentate gyrus . Neuropharmacology 185 , 108438 ( 2021 ). doi: 10.1016/j.neuropharm.2020.108438 OpenUrl CrossRef PubMed 78. ↵ Carver , C. M. & Shapiro , M. S. Gq-Coupled Muscarinic Receptor Enhancement of KCNQ2/3 Channels and Activation of TRPC Channels in Multimodal Control of Excitability in Dentate Gyrus Granule Cells . The Journal of Neuroscience 39 , 1566 – 1587 ( 2019 ). doi: 10.1523/jneurosci.1781-18.2018 OpenUrl Abstract / FREE Full Text 79. ↵ Miceli , F. , Cilio , M. R. , Taglialatela , M. & Bezanilla , F. Gating currents from neuronal K(V)7.4 channels: general features and correlation with the ionic conductance . Channels (Austin) 3 , 274 – 283 ( 2009 ). OpenUrl CrossRef PubMed 80. Gu , N. , Vervaeke , K. , Hu , H. & Storm , J. F. 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 , 689 – 715 ( 2005 ). doi: 10.1113/jphysiol.2005.086835 OpenUrl CrossRef PubMed Web of Science 81. ↵ Peng , H. , Bian , X.-l. , Ma , F.-c. & Wang , K.-W. Pharmacological modulation of the voltage-gated neuronal Kv7/KCNQ/M-channel alters the intrinsic excitability and synaptic responses of pyramidal neurons in rat prefrontal cortex slices . Acta Pharmacologica Sinica 38 , 1248 – 1256 ( 2017 ). doi: 10.1038/aps.2017.72 OpenUrl CrossRef PubMed 82. ↵ Moberg , S. & Takahashi , N. Neocortical layer 5 subclasses: From cellular properties to roles in behavior . Frontiers in Synaptic Neuroscience 14 ( 2022 ). 83. ↵ Rinker , J. A. & Mulholland , P. J. Promising pharmacogenetic targets for treating alcohol use disorder: evidence from preclinical models . Pharmacogenomics 18 , 555 – 570 ( 2017 ). doi: 10.2217/pgs-2016-0193 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 10, 2025. 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