Postnatal reduction of eIF4E overexpression in D1-SPNs ameliorates KCNQ channel dysfunction, hyperexcitability and ASD-like behaviours

Alina Aaltonen; Ayu Tamaki; Andrés Peris Ramón; Anders Borgkvist; Emanuela Santini

doi:10.1007/s00018-026-06115-2

. 2026 Feb 18;83(1):117. doi: 10.1007/s00018-026-06115-2

Postnatal reduction of eIF4E overexpression in D1-SPNs ameliorates KCNQ channel dysfunction, hyperexcitability and ASD-like behaviours

Alina Aaltonen ¹, Ayu Tamaki ¹, Andrés Peris Ramón ¹, Anders Borgkvist ^1,^✉, Emanuela Santini ^1,^✉

PMCID: PMC12932781 PMID: 41703231

Abstract

An imbalance between the direct and indirect pathways of the striatum has been linked to the pathophysiology of autism spectrum disorder (ASD), manifesting as repetitive behaviours and hyperactivity. We have investigated cell-specific dysfunctions in spiny projection neurons (SPNs) in a mouse model of ASD characterised by elevated expression of the eukaryotic initiation factor 4E (eIF4E), a key regulator of cap-dependent translation. eIF4E-TG mice, which exhibit ASD-like motor behaviours, were examined using a combination of fibre photometry, electrophysiology, conditional gene silencing, and behavioural assays. Direct pathway SPNs showed elevated activity during exploratory behaviour, along with hyperexcitability and reduced KCNQ potassium channel function in striatal slices. Conditional reduction of eIF4E in direct pathway SPNs of adult mice ameliorated KCNQ channel function, reduced excitability, and attenuated repetitive and hyperactive behaviours. These findings provide novel evidence that eIF4E-dependent translational dysregulation is associated with altered potassium channel function in direct pathway SPNs, and that postnatal reduction of eIF4E can mitigate motor phenotypes relevant to ASD in a cell-type-specific manner.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00018-026-06115-2.

Keywords: Autism spectrum disorder; Striatum; D1 spiny projection neurons; EIF4E,; Translational control; KCNQ potassium channels; Neuronal excitability; Mmouse model

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental condition defined by core symptoms such as stereotyped, repetitive behaviours and deficits in social interactions and communication [1]. These behaviours are key for clinical diagnosis, yet the heterogeneity of ASD symptomatology and significant overlap with other neurodevelopmental and psychiatric conditions complicate diagnosis. This complexity underscores the need to better ascertain the neuronal circuits involved in ASD to develop effective biomarkers and targeted therapies. Recent MRI and fMRI studies have implicated specific brain regions in ASD pathology, including the striatum, the main input nucleus of the basal ganglia. Individuals with ASD show atypical striatal development [2–4], altered striatal volume [5–7], and disrupted corticostriatal connectivity [8, 9]. In addition, preclinical investigations have further established that striatal dysfunctions contribute to ASD-like motor behaviours, such as stereotypies and perseverative behaviours [10–18]. Together, these findings suggest that striatal abnormalities play a critical role in the expression of ASD-related motor symptoms and repetitive behaviours [19–22].

The striatum mainly consists of GABAergic spiny projection neurons (SPNs), which form two anatomically and functionally distinct striatal output pathways: the direct striatonigral pathway, primarily consisting of Drd1a-dopamine receptor-expressing SPNs (D1-SPNs), and indirect striatopallidal pathways consisting of Drd2-dopamine receptor-expressing SPNs (D2-SPNs). These two pathways are central to movement regulation [23–25]. Although initially conceptualised as operating in an antagonistic balance [26–28], recent evidence instead suggests that both pathways are coactivated during movement [25, 29]. These findings support models where either direct-pathway SPNs drive specific motor programs and indirect-pathway SPNs suppress competing actions [25, 29, 30], or coordinated activity between D1- and D2-SPNs within specific ensembles determines the motor program executed [31]. It has also been proposed that motor actions may result from a combination of these mechanisms [32]. Dysfunction in either pathway can disrupt motor control, resulting in disorders characterised by hyperactivity or stereotyped behaviours. Hyperactivity in neurodevelopmental and psychiatric disorders may result from inadequate suppression of actions by the indirect pathway [10, 16, 33] or excessive activity in the direct pathway [13, 15, 34, 35]. Therefore, identifying specific disruptions in D1- or D2-SPNs in neurodevelopmental disorders could reveal mechanisms underlying motor impairments and hyperactivity.

Aberrant protein synthesis has been implicated as a potential convergent mechanism across both genetic and idiopathic ASD-associated disorders [36–38]. Several ASD-implicated genes converge on the mammalian target of rapamycin (mTOR) signalling pathway, which is also implicated in idiopathic autism [36, 38]. The mTOR pathway plays a key role in regulating activity-dependent protein synthesis by releasing eIF4E-mRNA complexes through inhibition of eIF4E-binding protein 2 (4E-BP2) [39–41]. The cap-binding protein eIF4E drives translational initiation [39, 40, 42], critical for neurodevelopment [43], as well as synaptic and structural plasticity [42]. Notably, disruption of eIF4E-dependent translation is frequently implicated in neurological and psychiatric disorders, including ASD [36, 44–46]. Supporting this connection, the gene encoding eIF4E has also been identified as a candidate ASD gene in humans [47, 48]. Moreover, genetic variations on chromosome 4q, which contains the gene encoding eIF4E, have been described in ASD patients [49, 50]. Altered eIF4E-dependent translation has also been described in postmortem brains of individuals with ASD [51]. Further evidence comes from animal models, where global [52, 53] or microglia-specific overexpression of eIF4E [54] or knockout of 4E-BP2, which encodes the eIF4E repressor protein [55], results in aberrant synaptic function and neuronal excitability. These alterations lead to ASD-like behaviours, including increased grooming, marble burying and novelty-induced hyperlocomotion [52–55]. Thus, examining the effect of eIF4E overexpression may provide insight into the mechanism by which specific disruptions in SPNs contribute to ASD-related behaviours.

In this study, we utilised the eIF4E-transgenic (eIF4E-TG) mice [52, 53] to test the hypothesis that altered excitability in the D1- or D2-SPNs causes an imbalance in the striatonigral and striatopallidal pathways that drives hyperactivity and repetitive behaviours. By integrating in vivo neuronal activity measurements, ex vivo patch clamp electrophysiology, and conditional gene silencing, we demonstrate that eIF4E overexpression leads to elevated Ca²⁺ activity in D1-SPNs during exploratory activity in freely behaving mice. Furthermore, we found that eIF4E-TG mice exhibit D1-SPN hyperexcitability after postnatal maturation and reduced conductance through voltage-gated Kv7 (KCNQ) potassium channels, which play a pivotal role in the control of membrane excitability. Importantly, reducing eIF4E overexpression in D1-SPNs in adult eIF4E-TG mice increased the KCNQ current, reduced hyperexcitability, and ameliorated novelty-induced motor behaviours. These findings underscore the importance of understanding cell-specific mechanisms, as targeting D1-SPNs may offer new therapeutic approaches for motor dysfunctions in ASD and related disorders.

Materials and methods

Mice

Male mice were sex-separated and group-housed (up to five mice per cage) in a temperature (23 °C) and humidity (55%) controlled environment, on a 12-h light/dark cycle with water and food available ad libitum. In all cases, eIF4E^wt/wt mice are referred to as wild type (WT), and eIF4E^wt/βtEif4e mice are referred to as eIF4E-transgenic (eIF4E-TG or simply TG) [52, 56]. Unless otherwise specified, eIF4E refers to the eIF4E protein throughout the manuscript. Each of the transgenic lines described below was maintained in hemizygosis by crossing with C57BL/6J mice (Janvier) and subsequently crossed with the eIF4E-TG line to generate the indicated double-transgenic mice. All mice were maintained on the C57BL/6J genetic background. In these experiments, we primarily used male mice, because the current work expands on our previous studies where phenotypes were observed in male mice only [52, 53], and as our study aimed to establish the cell-type specificity of the previously published repetitive and hyperactive behaviours, the presence of the behavioural phenotypes was imperative. For early postnatal (P10) experiments, all available pups were used irrespective of sex (see below).

For the fibre photometry experiment, we used three-to-four-month-old double-transgenic mice generated by crossing Tg(Drd1a-cre)FK150Gsat^+/− (D1-Cre) or Tg(Adora2a-cre)KG139Gsat^+/− (A2a-Cre) mice [57] with eIF4E^wt/βtEif4e, producing D1-Cre/WT, D1-Cre/TG, A2a-Cre/WT and A2a-Cre/TG to target D1- and D2-SPNs, respectively. For immunostaining, and intrinsic and K+ channel recordings, we used postnatal day 60–90 double-transgenic mice generated by crossing Drd1a-TdTomato^+/− (D1-Tomato) mice [58] (Jackson Laboratory, #016204) with eIF4E^wt/βtEif4e producing D1-Tomato/WT and D1-Tomato/TG. For developmental recordings, we used P10 (postnatal day 10–12) and P28 (postnatal day 28–32) mice of the same strain. For the P10 experiment, we used all available mice from each litter without assessing sex. For immunostaining and neuronal morphology experiments, we used P60-90 double-transgenic mice generated by crossing Tg(Drd2-EGFP)S118Gsat^+/−(D2-EGFP) mice [59] with eIF4E^wt/βtEif4e yielding D2-EGFP/WT and D2-EGFP/TG. For eIF4E reduction experiments (RNAi), we used three-to-four-month-old double-transgenic mice generated by crossing TRE-GFP.shmiR-4E mice [60, 61] with eIF4E^wt/βtEif4e, generating TRE-GFP.shmiR-4E/WT and TRE-GFP.shmiR-4E/TG mice. In all experiments involving surgical procedures, surgeries were performed at two-to-three months of age, and experiments were performed one month later. Mice were randomly assigned to experimental groups, and experimenters were blinded to the genotype during data acquisition and analysis.

Fibre photometry

Fibre photometry measurements were conducted using custom-built optical components and custom LabVIEW software [62]. The setup included two fibre-coupled LEDs: one for exciting Ca²⁺-bound GCaMP7s (M470F3, Thorlabs) modulated at 573 Hz, and the other for exciting the isosbestic signal (M405F1, Thorlabs) modulated at 211 Hz. The LED beams passed through emission filters (FF02-472/30 − 25, Semrock; FB405-10, Thorlabs) and a long pass dichroic mirror (DMLP425R, Thorlabs). They were then reflected via a dichroic beamsplitter (FF495-Di03-25 × 36, Semrock) and directed through a fibre-optic patch cord (400 μm core, 0.5 NA, R-FC-P-N5-400-L1, RWD Life Science) connected to the mouse using a ceramic mating sleeve (ADAF1-5, Thorlabs). Fluorescence emitted from the sample was collected through the same patch cord, filtered by a bandpass filter (FF01-535/50 − 25, Semrock), and focused onto the sensor of a battery-powered femtowatt photoreceiver (Newton, Model 2151) using a plano-convex lens (62–561, Edmund Optics). The excitation LED modulation and lock-in amplification of the photoreceiver signal were managed by a Field-Programmable Gate Array board (National Instruments, sbRIO-9637). The light power was set to 50 µW for both LEDs.

Novel cage experiment during fibre photometry

Mice were habituated to an empty cage (Tecniplast GM500, 500 cm²) across two days prior to recording. On the day of fibre photometry recording, mice were introduced to the same cage for at least 30 min, to allow for collection of baseline GCaMP7s fluorescence. After this baseline period, mice were placed in a novel cage with minimal bedding covering the bottom and allowed to explore, and the GCaMP7s activity was monitored for 10 min. In a separate experiment, mice were intraperitoneally (i.p.) injected with D1-receptor blocker SCH39166 (0.25 mg/kg, Tocris) during recording to assess the effect of D1-receptor blockade on GCaMP7s activity. Mice were excluded if they did not exhibit sufficient baseline GCaMP7s fluorescence or if the implant location was inappropriate, which was determined post hoc upon collecting and examining brain slices. During fibre photometry recordings, the novel cage exposure was conducted under dim light conditions (~ 15 lx) to avoid behavioural freezing or stress induced by bright light.

Fibre photometry analysis

Recordings were analysed using a custom-made MATLAB script. For the pre-processing we used steps similar to those previously described [63]. In brief, we applied a low-pass Butterworth filter of 25 Hz and performed exponential curve fitting to both the GCaMP data signal (470 nm) to the isosbestic (405 nm) control. We then fitted the GCaMP signal to the isosbestic control using scaled linear regression as described [63–65]. We performed Z-score normalisation of the fitted signal prior to analysis.

We then quantified signal peak frequency and size from one-minute recording sections of baseline and novelty-induced responses, to detect small scale changes in GCaMP signal – similar to parameters measured previously during longitudinal fibre photometry recordings [66, 67]. For these, we used the Find Peaks function in MATLAB to quantify the frequency of peaks in Hz in the given time frame, and we used the envelope function to measure the amplitude of the signal by quantifying the difference between the average upper and lower limits of the Z-score fluctuations during the same time frame. We also analysed the area under the curve of the one-minute recordings of the signal to detect bulk changes in GCaMP activity. These three parameters (frequency, size and area under curve) were normalised to their respective baseline values and then combined to create an aggregate ‘composite’ score, which provides an overall, multi-parameter change in GCaMP7s activity, expressed as a percentage from baseline (0%).

Stereotaxic surgeries

Mice were injected with buprenorphine (i.p. 0.1 mg/kg) and anaesthetised with 2% isoflurane. Adeno-associated virus (AAV) injection was administered via a Quintessential Stereotaxic Injector (Stoelting) with a 30 nl/min flow rate. Post-operative treatment was given in the form of carprofen and buprenorphine i.p. injection (5 mg/kg and 0.1 mg/kg, respectively). Mice were left to recover for at least three weeks prior to experimentation, to allow sufficient viral expression. For the fibre photometry experiment, unilateral injection of 250 nl pGP-AAV-syn-FLEX-jGCaMP7s-WPRE (Addgene #104491) and implant of fibre optic cannula (RWD Life Sciences: 0.5 NA, 400 μm core, 2.5 mm) were performed in the dorsal striatum (AP + 0.8, ML −1.8, DV −2.4 taken from dura). In separate control experiments, mice were injected with 250 nl pAAV-hSyn-EGFP (Addgene #50465) to confirm the absence of non-GCaMP mediated events.

Selective eIF4E reduction (RNAi) was performed as previously described [60, 68]. Briefly, for the initial characterisation of eIF4E reduction in SPNs, we performed bilateral injections of 300 nl AAV5-hSyn-mCherry-Cre (UNC Vector Core) and 300 nl pAAV-ihSyn1-DIO-tTA (Addgene #99121) or 300 nl pAAV-FLEX-tdTomato (Addgene #28306) in the dorsal striatum. To specifically target D1-SPNs, bilateral injections of 150 nl pAAV-EF1a-Cre (Addgene #55636) in the substantia nigra pars reticulata (SNr) (AP −3.28, ML +/−1.5, DV −4.3) were performed to retrogradely target direct pathway neurons, i.e., D1-SPNs, with bilateral striatal injection of 300 nl pAAV-ihSyn1-DIO-tTA (Addgene #99121) or 300 nl pAAV-FLEX-tdTomato (Addgene #28306). For the intrinsic property recordings, we bilaterally injected 150 nl pAAV-EF1a-Cre retrograde (Addgene #55636) in the SNr and unilaterally injected 300 nl pAAV-ihSyn1-DIO-tTA (Addgene #99121) in the left dorsal striatum (AP + 1 and 0, ML + 1.8, DL −2.4) and 300 nl pAAV-FLEX-tdTomato (Addgene #28306) in the right striatum (AP + 1 and 0, ML −1.8, DL −2.4). For the KCNQ and behavioural experiments, we bilaterally injected either the pAAV-FLEX-tdTomato (Addgene #28306) or pAAV-ihSyn1-DIO-tTA (Addgene #99121) in the dorsal striatum.

Electrophysiology

Recordings were obtained using a patch clamp rig fitted with SciCam pro camera (Scientifica, United Kingdom) equipped with a 40 × 0.8 NA water-immersion objective (LUMPlanFLN, Olympus, United States) and Dodt contrast tube optics. Cell fluorescence was visualised using a CoolLED PE-300 Ultra LED. Recordings were obtained using either a Multiclamp 700B or 200B amplifier (Molecular Devices) and Axon Digidata 1550B digitizer (Molecular Devices, United States), using either pCLAMP 11 software (Molecular Devices, United States) or WinWCP software (University of Strathclyde). Data were low pass filtered at 10 kHz. Recordings were not corrected for liquid junction potentials. Series resistance compensation was not applied during any of the recordings; however, for all recordings, it was monitored by applying hyperpolarizing 10 mV voltage steps at regular intervals. Recordings were excluded if series resistance exceeded 30 MΩ or changed by over 10%, or in the event of declining cell health.

Slice Preparation

Acute brain slices were prepared and maintained using the two-step N-methyl-D-glucamine (NMDG) based protective recovery method [69]. Slices were initially prepared using NMDG-based solution containing (in mM): 92 NMDG, 30 NaHCO3, 2.5 KCl, 20 HEPES, 2 thiourea, 1.25 NaH2PO4, 3 Na-pyruvate, 5 ascorbic acid, 25 glucose, 0.5 CaCl2 and 10 MgCl2 titrated to pH 7.4. During the experiment, the slices were stored in the HEPES-based holding solution containing (in mM): 92 NaCl, 30 NaHCO3, 2.5 KCl, 20 HEPES, 2 thiourea, 1.25 NaH2PO4, 3 Na-pyruvate, 5 ascorbic acid, 25 glucose, 2.5 CaCl2 and 2 MgCl2 titrated to pH 7.4. Briefly, mice underwent cardiac perfusion with 25 ml chilled NMDG solution prior to brain removal and dissection. 250 μm coronal slices were collected containing the dorsal striatum and stored in NMDG-based solution for 10 min at + 32 °C before transferring to HEPES-based holding solution. All solutions were oxygenated with 95% O and 5% C2.

Recording protocols

All recordings were obtained using a potassium-gluconate based internal solution containing (in mM): 120 K-gluconate, 20 KCl, 4 MgATP, 0.3 Na2-GTP, 5 Na2-phosphocreatine, 0.1 EGTA and 10 HEPES at pH 7.25 and osmolarity ~ 300 mOsm, with a pipette resistance of 3–5 MΩ. During the experiments, slices were placed in the submersion recording chamber in the rig with oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) supplied at 2–3 ml/min and maintained at 32–34 °C, containing (in mM): 125 NaCl, 2.5 KCl, 25 NaHCO3, 1.25 NaH2PO4, 10 glucose, 2 CaCl2 and 1 MgCl2. Potentials were not corrected for the liquid junction potential. For recordings of Kir2 channel activity, ACSF was prepared with 300 nM tetrodotoxin citrate (TTX, Tocris) to prevent depolarisation and 1 mM CsCl to block potassium channels. For recordings of Kv7 channel activity, we used 10 µM XE991-dihydrochloride (Tocris) in ACSF.

D1- and D2-SPNs were identified based on presence or absence, respectively, of TdTomato fluorescent marker. Intrinsic properties were determined in current-clamp configuration using somatic current injections ranging from − 300 pA to + 450 pA delivered as a ramp (0.75 nA/s) or stepwise in 50 pA increments. Each current step was held for 500 ms with 500 ms inter-sweep interval. The Kir2 channel recordings were performed in voltage-clamp configuration, as previously described [70]. Cells were held at −60 mV and voltage steps were performed in −10 mV increments until − 140 mV, after which CsCl (1 mM) was bath-applied, and the voltage steps were performed again 10 min after bath application. To activate the M-current, we clamped the membrane potential at 0 mV for four seconds, followed by a series of repolarising, deactivating voltage steps from − 20 mV to −60 mV, before and after application of XE991 to block KCNQ2/3 channels [71].

Electrophysiological data analysis

All analysis of electrophysiological data was performed using the open-access Python-based software Stimfit [72]. The current at which the first action potential was fired was used to determine the rheobase current. The first action potential with step current injection was used to calculate the action potential threshold, height, half-height duration, rise time, decay time and fast afterhyperpolarisation amplitude. AP kinetics were calculated from threshold. Current-frequency (IF) plots were generated from the action potential frequency (Hz) at each current step. Membrane capacitance was calculated using 𝞃/Ri, where Ri is the input resistance calculated from the voltage response to −100 pA current injection, and 𝞃 is the membrane time constant, calculated using an exponential fit from the initial voltage change with − 100 pA current. The membrane resistance (Rm) was determined using Ohm’s law (V = IR) from the current-voltage slope across the range of current steps performed. For Kir2 and KCNQ channel recordings, the drug-sensitive current was calculated as the difference between the current responses before and after CsCl or XE991 application.

Behaviour

All behavioural testing was performed in a randomised order with respect to genotype and experimental condition, and experimenters were blinded during both data acquisition and analysis.

Novel cage experiment

Mice were transferred from their homecage and individually placed in clean cages (Tecniplast GM500, 501 cm²) with minimal bedding material covering the bottom and allowed to explore for two hours. The novelty-induced locomotor activity test was conducted in complete darkness using infrared illumination between 9 AM and 5 PM. During the test, mice were video recorded, and their distance travelled was analysed using Ethovision XT16 video tracking software (Noldus, The Netherlands).

Marble burying

For the marble burying test, 20 marbles were evenly arranged in a large cage (Tecniplast GR900, 904 cm²) with at least 5 cm of fine wood shavings. Mice were introduced to the cage and allowed to move freely for 30 min. After this period, the number of marbles buried was scored, where a marble was considered buried if less than half of it was visible above the surface. No mice were excluded during analysis from the behavioural experiments.

Western blotting

Mice were sacrificed via cervical dislocation and striata from the left and right hemispheres were dissected and flash-frozen in liquid nitrogen. The samples were sonicated in 1% SDS and boiled for 10 min [73]. Protein concentration was determined using the Pierce™ BCA Protein Assay Kit. A standard curve was generated from a colorimetric assay of bovine serum albumin (BSA) with known concentrations to quantify the protein levels in our samples. Samples were diluted in Laemmli buffer (4X) and boiled for one minute. Equal quantities of protein were loaded (5–30 µg) into 10% plyacrylamide gels. Proteins were transferred to Immobilon FL PVDF membranes (pore size 0.2 μm). Membranes were blocked for one hour in 5% mlk in TBS with 0.5% Teen-20 (TBS-T) before incubation with primary antibody for two hours at room temperature (R&D Systems MAB3228 mouse anti-eIF4E, Abcam ab13970 chicken anti-GFP, and Cell Signalling 2306 S rabbit anti-DARPP32; diluted 1:20,000, 1:1,000 and 1:50,000, respectively). GCaMP7s protein levels were assessed using an anti-GFP antibody, as GCaMP7s is a GFP-based genetically encoded Ca²⁺ indicator [74]. Membranes were washed in TBS-T and incubated with HRP-conjugated secondary antibody (Invitrogen A16096 anti-rabbit, A16066 anti-mouse or Thermo Scientific anti-chicken; diluted 1:50,000, 1:25,000, or 1:10,000, respectively) for 30 min. Following further washes, blots were developed using Pierce ECL Plus Western Blotting Substrate and visualised with a Protec OPTIMAX X-Ray Film Processor. Films were scanned and analysed using ImageJ to quantify protein expression levels.

In vivo sunset assay

To assess the rate of nascent protein synthesis in vivo, we performed a surface sensing of translation (SUnSET) assay [75] through stereotaxic delivery of puromycin into the striatum. Mice were injected with buprenorphine (i.p. 0.1 mg/kg) and anaesthetised with 2% ioflurane, as described above in the stereotaxic surgeries section. A total volume of 2 µl puromycin (8 µM in sterile dH₂O) was delivered bilaterally into the striatum (AP + 0.8, ML ± 1.8, DV − 3.0 from dura) using a Quintessential Stereotaxic Injector (Stoelting) at a flow rate of 0.5 µl/min. The injection needle was kept in place for 5 min before withdrawal to minimise reflux. Following the procedure, mice were transferred to a clean home cage and allowed to recover for approximately one hour to permit incorporation of puromycin into elongating polypeptide chains, after which they were perfused and brains processed as described in the subsequent immunofluorescence section.

Immunofluorescence

Mice were sacrificed and transcardial perfusion with 4% paraformaldehyde was performed. Whole brains were extracted and 30–40 μm coronal slices were prepared with a vibratome in PBS. Slices containing striatum were selected for immunostaining. Slices were washed in TBS before permeabilization for five minutes in solution containing 1:10 methanol and 1:3 hydrogen peroxide in TBS. A second permeabilization step was performed using TBS with 0.2% Triton X-100 (TBS-Triton) and incubated for 15 min. Slices were washed in TBS and then incubated in primary antibody overnight at 4 °C (1:200 R&D system MAB3228 mouse anti-eIF4E, 1:1000 EMD MABE343 anti-puromycin, 1:500 Synaptic System 382004 guinea pig anti-DARPP32, 1:400 ImmunoStar 20065 rabbit anti-enkephalin, 1:2000 Abcam ab13970 chicken anti-GFP, 1:1000 Rockland 600-40−379 rabbit anti-RFP, 1:500 Rockland 200–301−379 mouse anti-RFP). Slices were washed and then incubated in secondary antibody for 45–60 min at room temperature (Invitrogen Alexa fluor: A-11001 anti-mouse 488, A-11004 anti-mouse 548, A-21450 anti-guinea pig 647, A-11039 anti-chicken 488, A-11008 anti-rabbit 488, A-11011 anti-rabbit 568; all diluted 1:500). Slices were mounted onto glass slides with Fluromount-G with DAPI (Invitrogen). Sections were imaged at 10x or 20x with a Zeiss LSM800 confocal microscope, and images were processed with ImageJ for manual counting.

Neuronal morphology

The process of microinjection was followed as previously described [76]. Briefly, mice underwent cardiac perfusion, and brains were removed and fixed for four hours in 4% praformaldehyde (PFA) in 0.1 M PBS (pH 7.4) at 4 °C. Brains were sliced at 200 μm and stored in PBS with 0.1% N-azide. During microinjection, D2-SPNs were identified via expression of GFP and D1-SPNs via absence of GFP, and cells were filled with 8% Lcifer Yellow CH lithium salt (Invitrogen) in PBS and Tris-HCl via current injection of 2–10 nA over five-10 min. Cells were filled until distal dendrites were visible, and post-fixed in PFA before imaging. To amplify the fluorescent signal from Lucifer Yellow via the conjugated biotin, following a series of washes in tris-buffered saline (TBS), slices were incubated with Streptavidin Alexa Fluor™−488 (Invitrogen, 1:200) in 0.6% Titon X-100 in TBS for 48 h. Slices were then incubated overnight in 2% NS/0.6% TS-triton with goat anti-mouse Alexa Fluor™ 488 (Invitrogen, 1:500). Cell morphology was analysed using the Sholl analysis function of the Neuroanatomy plug-in for ImageJ.

Statistical analysis

All statistical analyses were performed using Graphpad Prism. For behavioural, fibre photometry and western blot experiments, the n-values indicate the number of mice, and data are presented as mean ± SEM. For electrophysiology and neuronal morphology experiments, the N values indicate number of mice and n values indicate the number of cells and data are presented as median ± quartiles, whereas for immunofluorescence experiments the n-values indicate the number of slices. One-sample t-tests were used to make comparisons between baseline and experimental effect within one data group. Either two-tailed student’s t-tests or paired t-tests were used for two-group comparisons, whereas two-way ANOVAs were used for grouped variable comparisons and repeated-measures (RM) two-way ANOVA for matched variable comparisons, with Tukey’s multiple comparisons test for post-hoc comparisons between groups. Ordinary one-way ANOVA was used for datasets with a single parameter with multiple groups. When comparing three parameters, a three-way ANOVA was used. Multiple comparisons tests were performed only in the case of an interaction effect, and comparisons were made only between genotypes in the same condition, or between conditions in the same genotype. In all cases, * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, ns p > 0.05. Key statistical parameters, including N and n values, mean ± SEM, median ± quartiles, t- and f-values, degrees of freedom, p-values and significance levels are indicated for each statistical test in Supplementary Tables 1–12.

Results

Increased novelty-induced D1-SPN activity in the eIF4E-TG mice

The eIF4E-transgenic (eIF4E-TG) mice exhibit hyperactivity when introduced to a novel, unfamiliar environment [52]. Novel environment exposure stimulates concurrent direct and indirect pathway activity associated with locomotion [29, 77]. To explore whether dysfunctions in the striatal pathways are responsible for the hyperlocomotion observed in the eIF4E-TG mice, we assessed novelty-induced changes in fluorescence of the genetically encoded Ca²⁺ indicator GCaMP7s used as a proxy for spiking activity in direct and indirect SPNs using fibre photometry. We injected adult 3–4-month-old mice with pGP-AAV-syn-FLEX-jGCaMP7s-WPRE containing Cre-inducible GCaMP7s into the dorsal striatum (Fig. 1a) of mice generated by crossing the eIF4E-TG line with D1- and A2a-Cre mice (D1-Cre/WT and -/TG and A2a-Cre/WT and -/TG; see materials and methods). Mice were left to recover for three weeks, after which we recorded GCaMP7s activity.

We recorded baseline GCaMP7s fluorescence first in a familiar home cage and then in a novel environment (Fig. 1b). To assess the novelty response, GCaMP7s fluorescence after novel cage exposure in each mouse was normalised to its baseline, yielding a percentage change in fluorescence. Exposure to the novel environment enhanced the Ca²⁺ activity in both direct and indirect SPNs in WT mice (Fig. 1c, e) (D1: 31.33% WT, one-sample T-test: t(5) = 4.057, p = 0.0098; A2a: 83.5% WT, one-sample T-test: t(7) = 4.986, p = 0.0016). Importantly, the eIF4E-TG mice exhibited an enhanced novelty-induced GCaMP7s fluorescence (57.17%) in direct SPNs compared to WT (unpaired t-test, t(10) = 2.27, *p = 0.0465; Fig. 1c, d), but not in indirect SPNs (Fig. 1e, f). Further analysis of individual parameters revealed increases in peak frequency (Supplementary Fig. 1a) and area under the curve (Supplementary Fig. 1e) in direct SPNs of eIF4E-TG mice, with no change in signal amplitude (Supplementary Fig. 1c). In indirect SPNs, no difference between eIF4E-TG mice and WT was observed in any parameter (Supplementary Fig. 1b, d, f).

Given the selective enhancement of D1-SPN Ca²⁺ activity, we wanted to confirm that eIF4E overexpression is comparable in D1- and D2-SPNs, and that GCaMP7s expression does not differ between genotypes. We performed immunostaining in striatal slices from Drd1a-TdTomato mice [58] crossed with the eIF4E-TG line (D1-Tomato/WT and -/TG mice) and Drd2-eGFP [59] mice crossed with eIF4E-TG (D2-eGFP/WT and -/TG mice) to assess whether eIF4E overexpression is comparable between the two neuronal types in the eIF4E-TG mice. We confirmed that eIF4E overexpression occurs in both cell populations (Supplementary Fig. 2a-c). We also observed increased puromycin incorporation in both cell types following an in vivo SUnSET assay [75], indicating elevated protein synthesis rates (Supplementary Fig. 2d-e). Together, these results suggest that eIF4E expression, and protein synthesis rates, are similarly impacted in D1- and D2-SPNs.

We then assessed whether differences in GCaMP7s expression between WT and TG mice could contribute to the observed signal. Western blot analysis of GCaMP7s-expressing striatal extracts, quantified using an anti-GFP antibody (see Materials and Methods), revealed no significant difference between WT and TG mice (Supplementary Fig. 2f), suggesting that differences in GCaMP7s reporter expression do not account for the increased activity observed in the D1-SPNs of the eIF4E-TG mice. To ensure that our in vivo measurements reported the correct cell-type specific changes in Ca²⁺ signal, we performed post hoc tissue analysis with immunostaining using antibodies detecting GCaMP7s and DARPP-32, a marker for SPNs. We confirmed a 50% co-expression of GCaMP7s and DARPP-32 in the respective experimental groups (Supplementary Fig. 2g-h), in agreement with the equinumerous distribution of D1- and D2-SPNs [78]. In addition, treatment of D1-Cre/WT mice with the D1-receptor selective antagonist SCH39166 (0.25 mg/kg, i.p.) reduced D1-SPN GCaMP7s fluorescence (Supplementary Fig. 2i-l). Fibre photometry recordings performed on mice expressing GFP via injection of pAAV-hSyn-EGFP did not reveal any movement-related changes in the parameters we used to quantify fluorescence signals [29] (Supplementary Fig. 2 m), confirming that movement does not artefactually drive signals measured with GCaMP7s. Altogether, our results show that the eIF4E-TG mice exhibit a direct pathway-specific hyperactivity in response to novelty.

Overexpression of eIF4E results in cell-type specific hyperexcitability in SPNs

We investigated whether alterations in membrane conductances regulating excitability might underlie the hyperactivity observed in D1-SPNs in vivo. We performed whole-cell patch-clamp electrophysiology in striatal slices obtained from 2–3-month-old D1-Tomato/WT and -/TG mice to isolate TdTomato-fluorescent D1-positive or D1-negative SPNs (used as a proxy for D2-SPNs). In D1-SPNs, we found a significant reduction in membrane capacitance in TG mice (Fig. 2a) and a depolarised resting membrane potential (RMP, Fig. 2b). In addition, membrane resistance (Rm) of D1-SPNs was significantly increased in eIF4E-TG mice compared to WT mice (Fig. 2c). Together, these findings indicate genotype-related differences in passive membrane properties and membrane conductances that regulate excitability.

We did not observe differences in action potential (AP) kinetics including AP half-height duration (Fig. 2d), AP peak height (Fig. 2e), rise time (Fig. 2f), decay time (Fig. 2g) or fast afterhyperpolarisation amplitude (Fig. 2h). In addition, no difference was observed in AP threshold (measured from the first rheobase-evoked action potential; Fig. 2i). However, the rheobase current in D1-SPNs was reduced (Fig. 2j), accompanied by an increase in maximum firing frequency (Fig. 2k-l) and enhanced current-frequency responses (Fig. 2m), indicating a lower current threshold for firing, and heightened intrinsic excitability.

Since differences in membrane properties could reflect morphological changes [79], we performed Sholl analysis on D1-SPNs microinjected with the fluorescent dye Lucifer Yellow. However, no difference was found in the distribution of dendritic intersections or cell size (Fig. 2n-o). These findings indicate that D1-SPNs of the eIF4E-TG mice exhibit preserved dendritic architecture despite altered passive membrane properties and increased excitability, which could contribute to the hyperactivity phenotype observed in vivo.

We also analysed the intrinsic properties of D2-SPNs (Supplementary Fig. 3) and found a reduction in membrane capacitance in eIF4E-TG mice (Supplementary Fig. 3a), but no change in RMP or Rm (Supplementary Fig. 3b-c). Furthermore, while we identified no changes in AP duration, AP height or rise time (Supplementary Fig. 3d-f), we did observe a significant increase in AP decay time (Supplementary Fig. 3 g) and a reduction in fast afterhyperpolarisation amplitude (Supplementary Fig. 3 h). Despite these kinetic changes, we did not observe any differences in AP threshold, rheobase or firing activity (Supplementary Fig. 3i-m). As in D1-SPNs, we found no change in the dendritic morphology of D2-SPNs (Supplementary Fig. 3n-o).

In summary, while we observed subtype-specific alterations in passive membrane properties and action potential kinetics, these changes did not translate into increased firing output in D2-SPNs. In contrast, we identified increased excitability specifically in D1-SPNs of eIF4E-TG mice, as evidenced by a reduced rheobase and enhanced firing response to depolarising current injection. In the absence of morphological changes, these findings indicate that altered passive membrane properties and increased intrinsic excitability are selective features of D1-SPNs in eIF4E-TG mice and likely reflect changes in ion channel-mediated membrane conductance.

D1-SPN hyperexcitability emerges late during postnatal SPN maturation

SPNs undergo extensive maturation of their electrophysiological and structural properties between the first and fourth postnatal weeks, ultimately acquiring adult-like phenotypes [73, 80, 81]. Because eIF4E is overexpressed prenatally in eIF4E-TG mice, we sought to determine whether the aberrant firing properties of D1-SPNs emerge during a specific phase of postnatal development. To test this, we recorded the intrinsic properties and depolarisation-induced membrane responses of D1 and D2-SPNs around postnatal day P10 (P10-12), early in the developmental period, and around P28 (P28-32), towards the end of this period [70], comparing D1-Tomato/WT and D1-Tomato/TG mice.

While the capacitance of D1-SPNs did not change during this interval (Fig. 3a), we found a significant age-associated decrease in RMP (Fig. 3b) and Rm in WT mice (Fig. 3c), as previously reported [70]. Interestingly, RMP and Rm in eIF4E-TG mice did not change between P10 and P28 (Fig. 3b-c), indicating an altered developmental trajectory of passive membrane property maturation. No maturation-dependent differences were found in AP kinetics, rheobase or maximum firing frequency across genotypes (Fig. 3d-h). However, D1-SPNs of eIF4E-TG mice generated more action potentials in response to depolarising current injection at P28, but not at P10 (Fig. 3g, i). To determine whether these developmental alterations were specific to D1-SPNs, we also analysed the corresponding intrinsic membrane properties, AP peak and duration, and depolarisation-evoked firing output in D2-SPNs. Across postnatal development, these parameters were comparable between WT and eIF4E-TG mice (Supplementary Fig. 4).

In conclusion, we observed dysregulation in the maturation of passive membrane properties in D1-SPNs but little alteration in the developmental trajectory of excitability during postnatal development in eIF4E-TG mice. Together, these findings suggest that while alterations in passive membrane property maturation are already evident during early postnatal development, increased D1-SPNs firing emerges later, toward the end of the main postnatal maturation period when SPNs acquire adult-like membrane properties.

Conditional Silencing of eIF4E reduces firing frequency of D1-SPNs

Given the cell-type specificity and postnatal establishment of the firing abnormalities observed in eIF4E-TG mice, we hypothesised that this dysregulation occurs in a cell-autonomous manner and that reducing eIF4E levels in D1-SPNs in adulthood could ameliorate the observed impairments. To reduce eIF4E expression in adult mice, we utilised an inducible RNAi approach based on a genetically encoded, germline knock-in shRNA mouse line (TRE-GFP.shmiR-4E), previously established to target Eif4e [61, 68, 82]. In this model, the shmiR-4E cassette is integrated into the mouse genome and is not virally delivered. The shmiR-4E sequence is embedded within a GFP reporter and placed under the control of tet-responsive elements (TRE), enabling conditional, cell-specific silencing of eIF4E upon activation by tTA [61, 68].

We assessed the efficacy of the double-conditional gene expression system for silencing eIF4E in SPNs in the striatum of TRE-GFP.shmiR-4E mice. We co-infused AAV5-hSyn-mCherry-Cre together with pAAV-ihSyn1-DIO-tTA, which transcribes a neuron-specific Cre-dependent tet-transactivator (tTA) into the striatum. Cre-dependent activation of tTA drives expression of shmiR-4E, resulting in eIF4E reduction and concomitant GFP expression [61, 68]. As a control, we co-injected AAV5-hSyn-mCherry-Cre with pAAV-FLEX-tdTomato, which does not induce shmiR-4E expression but instead labels Cre-expressing neurons with TdTomato. Importantly, in all experiments the RNAi cassette itself was provided by the TRE-GFP.shmiR-4E mouse line, while viral vectors were used exclusively to deliver Cre and/or tTA to activate the endogenous shmiR-4E allele. To quantify eIF4E reduction, we performed Western Blot analysis on striatal extracts and confirmed a robust overall reduction in eIF4E levels in samples from double transgenic TRE-GFP.shmiR-4E/WT and -/TG littermates with AAV5-hSyn-mCherry-Cre/pAAV-ihSyn1-DIO-tTA co-injected into the striatum, compared to those co-infected with AAV5-hSyn-mCherry-Cre/pAAV-FLEX-tdTomato (Supplementary Fig. 5a), suggesting effective eIF4E silencing.

Having established the efficacy of this approach, we next sought to silence eIF4E expression selectively in D1-SPNs. Because the Cre-dependent tTA construct (pAAV-ihSyn1-DIO-tTA) restricts shmiR-4E expression to Cre-positive neurons, selective targeting of Cre allows cell-type-specific eIF4E reduction. To this end, we injected a retrograde Cre virus (pAAV-EF1a-Cre) bilaterally into the substantia nigra pars reticulata (SNr) of TRE-GFP.shmiR-4E/WT mice, thereby selectively infecting direct pathway SPNs. In the same animals, pAAV-ihSyn1-DIO-tTA was injected into the striatum of the left hemisphere (tTA^L), while pAAV-FLEX-tdTomato was injected into the contralateral (right) striatum (TdTomato^R) as an internal control (Fig. 4a). This bilateral, within-animal design enabled direct comparison of eIF4E-reduced and control D1-SPNs under identical experimental conditions. We confirmed widespread GFP expression, reflecting shmiR-4E activation, throughout the dorsal striatum (Supplementary Fig. 5b).

Fig. 4 — Conditional reduction of eIF4E in adult D1-SPNs decreases firing frequency. a) Schematic of the experimental design to selectively reduce eIF4E in D1-SPNs. A retrograde pAAV-EF1a-*Cre* was injected into the substantia nigra pars reticulata (SNr) of TRE-GFP.shmiR-4E/WT and -/TG mice to target D1-SPNs. An AAV-ihSyn1-DIO-tTA was injected into the left striatum (tTA^L), activating RNAi for eIF4E and driving GFP expression and activation of the endogenous shmiR-4E cassette in D1-SPNs. In parallel, an AAV-FLEX-tdTomato was injected into the contralateral right striatum (TdTomato^R), driving the expression of a control fluorescent marker in D1-SPNs. b) Schematic depicting the timeline of viral infection and subsequent measurement of intrinsic neuronal properties. c) Capacitance in D1-SPNs from TRE-GFP.shmiR-4E/WT and -/TG mice in tTA^L or TdTomato^R hemispheres. d) Resting membrane potential in D1-SPNs from tTA^L or TdTomato^R hemispheres. e) Membrane resistance in D1-SPNs from tTA^L or TdTomato^R hemispheres. f) Action potential half-height duration in D1-SPNs from tTA^L or TdTomato^R hemispheres. g) Action potential peak amplitude in D1-SPNs from tTA^L or TdTomato^R hemispheres. h) Action potential rise time in D1-SPNs from tTA^L or TdTomato^R hemispheres. i) Action potential decay time in D1-SPNs from tTA^L or TdTomato^R hemispheres. j) Fast afterhyperpolarisation amplitude in D1-SPNs from tTA^L or TdTomato^R hemispheres. k) Action potential threshold voltage in D1-SPNs from tTA^L or TdTomato^R hemispheres (genotypeXRNAi interaction, 2-way ANOVA: F(1, 53) = 5.331, # p = 0.0249). l) Rheobase current in D1-SPNs from tTA^L or TdTomato^R hemispheres (genotypeXRNAi interaction, 2-way ANOVA: F(1, 53) = 8.028, ## p = 0.0065). m) Representative traces of action potentials at + 450 pA current injection for 500 ms (scale bar: 200 ms, 50 mV). n) Maximum firing frequency D1-SPNs from tTA^L or TdTomato^R hemispheres (genotypeXRNAi interaction, 2-way ANOVA: F(1, 54) = 10.10, ## p = 0.0024). o) Current-frequency (IF) plots for D1-SPNs from tTA^L or TdTomato^R hemispheres (currentXgenotypeXRNAi interaction, 3-way ANOVA: F(9, 486) = 6.46, #### p < 0.0001). Data are presented as median ± quartiles within the box plots, with whiskers indicating minimum and maximum values (a-n), or as mean ± SEM, with the mean represented by a connecting line and SEM by error lines with shading (o). Dots represent individual values for each neuron. All recordings were collected from 4 TRE-GFP.shmiR-4E/WT and 4 TRE-GFP.shmiR-4E/TG mice (n = 14 WT TdTomato^R, n = 17 WT tTA^L, n = 12 TG TdTomato^R, n = 15 TG tTA^L). Significance is denoted as * p < 0.05, *** p < 0.001, ^# p < 0.05 and ^## p < 0.01 calculated using two-way or RM-2way ANOVA followed by Tukey’s post-hoc multiple comparisons test. Unless otherwise indicated, panels c–j did not show genotype-dependent differences at either the TdTomato^R or tTA^L hemispheres. For full details of statistical analysis including negative results, refer to Supplementary Table 4

To verify cell-type specificity of the conditional silencing strategy, we performed immunohistochemical analyses in mice that received bilateral but hemisphere-specific viral injections (tTA in the left striatum, tTA^L; TdTomato control in the right striatum, TdTomato^R), with retrograde Cre delivered to the SNr (Fig. 4a). Analysis was performed across slices within the region of the dorsal striatum that received the strongest viral expression (Supplementary Fig. 5b). We confirmed that the total count of DARPP-32-positive neurons was similar between the two hemispheres (see Supplementary Table 10), indicating that viral expression did not result in overt changes in striatal neuronal density at the injection sites. Both GFP (tTA-dependent reporter) and TdTomato expression were predominantly observed in DARPP-32-positive neurons, with 81% of fluorophore-positive cells (i.e. tTA^L or TdTomato^R) expressing DARPP-32 (Supplementary Fig. 5c-d). Given that approximately 95% of striatal neurons are SPNs, with roughly equal proportions of D1- and D2-SPNs [78], and that D2-SPNs selectively express enkephalin [83, 84], we further assessed enkephalin immunoreactivity. In striatal sections from double-injected mice, 93% of fluorophore-positive neurons were enkephalin-negative (Supplementary Fig. 5e-f), confirming selective targeting of D1-SPNs. Consistent with effective knockdown, we also observed a significant reduction in the density of eIF4E-positive neurons in tTA^L striatal sections compared to TdTomato^R sections (Supplementary Fig. 5g-h).

We then recorded intrinsic electrophysiological properties of D1-SPNs in striatal slices from TRE-GFP.shmiR-4E/WT and -/TG mice, comparing tTA^L neurons (in the left hemisphere) with contralateral TdTomato^R control neurons (in the right hemisphere) from the same animal (Fig. 4a). This experimental design enabled within-animal comparisons, minimising inter-animal variability. Mice were left to recover for at least four weeks prior to recording (Fig. 4b). Control TdTomato^R neurons exhibit passive membrane properties consistent with previously reported values for D1-SPNs [83, 84] (Fig. 2), suggesting that viral expression alone does not substantially perturb neuronal physiology. Reducing eIF4E expression did not alter membrane capacitance, RMP or membrane resistance in D1-SPNs (Fig. 4c-e), nor did it affect AP kinetics (Fig. 4f-j). However, we observed a selective increase in AP threshold in TG neurons following eIF4E reduction (Fig. 4k). Importantly, we detected a significant genotype X treatment interaction in rheobase current (Fig. 4l), and a marked reduction of the elevated firing frequency previously observed in eIF4E-TG mice (Fig. 4m-o). Thus, reducing eIF4E levels in adult D1-SPNs of eIF4E-TG mice attenuated the aberrant firing activity associated with global eIF4E overexpression.

Reduction of eIF4E in D1-SPNs ameliorates hyperactivity and repetitive behaviours

As the targeted reduction of eIF4E in D1-SPNs reduced their hyperexcitability in adult mice, we hypothesised that interfering with eIF4E expression may also decrease novelty-induced hyperlocomotion and ameliorate repetitive behaviours. To test this, we used TRE-GFP.shmiR-4E/WT and -TG littermates, injected with AAVrg-Cre in the SNr and either DIO-tTA (tTA^L+R) or AAV-FLEXtdTomato (TdTomato^L+R) bilaterally in the striatum, to achieve D1-SPN-specific reduction of eIF4E or expression of a control fluorophore (Fig. 5a). During the fourth week post-surgery (Fig. 5b), we first assessed novelty-induced locomotion using a behavioural paradigm similar to that employed for the fibre photometry experiments (see Materials and Methods). Subsequently, we performed a marble burying test, a paradigm previously used to quantify repetitive behaviour in ASD models [15, 85], including eIF4E-TG mice [52].

Fig. 5 — Conditional reduction of eIF4E in adult D1-SPNs ameliorates hyperactivity, repetitive behaviours and KCNQ hypofunction in eIF4E-TG mice. a) Schematic of the experimental design to selectively reduce eIF4E in D1-SPNs. A retrograde pAAV-EF1a-*Cre* was injected into the substantia nigra pars reticulata (SNr) of TRE-GFP.shmiR-4E/WT and -/TG mice to target D1-SPNs. An AAV-ihSyn1-DIO-tTA was injected into both the left and right striata (tTA^L+R), activating RNAi for eIF4E and driving GFP expression in D1-SPNs. In control mice, AAV-FLEX-tdTomato was injected into both hemispheres (TdTomato^L+R), driving the expression of a control fluorescent marker in D1-SPNs. b) Schematic depicting the timeline of viral infection, behavioural and electrophysiology experiments. **c-e**) Behavioural experiments were performed using 9 TRE-GFP.shmiR-4E/WT TdTomato^L+R and 10 TRE-GFP.shmiR-4E/TG TdTomato^L+R mice, and 8 TRE-GFP.shmiR-4E/WT tTA^L+R and 10 TRE-GFP.shmiR-4E/TG tTA^L+R mice. c) Time-course of the distance travelled during the novel cage experiment, with data binned in five-minute intervals (shown for illustrative purposes; no statistical comparisons were performed across time bins). d) Total distance travelled over the two-hour experimental period (genotypeXRNAi interaction, 2-way ANOVA: F(1, 33) = 7.607, ## p = 0.0094). e) Total number of marbles buried during the 30-minute marble burying experiment (genotypeXRNAi interaction, 2-way ANOVA, F(1, 33) = 13.13, # p = 0.018). Data are presented as mean ± SEM, with mean indicated by bar height or connecting line and SEM by error bars or lines with shading. f-g) Recordings with XE991 in ACSF were collected from D1-positive cells (n = 18 D1-Tomato/WT, n = 11 D1-Tomato/TG) from 5 D1-Tomato/WT and 5 D1-Tomato/TG mice, and from D1-positive cells (n = 10 tTA^L+R WT and n = 16 tTA^L+R TG) from 4 TRE-GFP.shmiR-4E/WT tTA^L+R and 6 TRE-GFP.shmiR-4E/TG tTA^L+R mice. f) Representative traces from D1-SPNs from D1-Tomato/WT, -/TG, TRE-GFP.shmiR-4E/WT tTA^L+R (WT tTA^L+R) and -/TG tTA^L+R (TG tTA^L+R) before (ACSF) and after (XE991) bath application of XE991, with one-second current steps from a brief (four second) holding potential of 0mV and from − 20 to −50 mV in 10 mV increments (scale bar: 600 ms and 300 pA). g) Percentage current reduction by XE991 −20 mV, comparing D1-Tomato/WT, -/TG, GFP.shmiR-4E/WT tTA^L+R and -/TG tTA^L+R D1-SPNs (genotypeXRNAi interaction, 2-way ANOVA: F(1, 51) = 5.218, ^# p = 0.0266; * p < 0.05). Data are presented as median ± quartiles within the box plot, and whiskers indicate minimum and maximum values. Dots represent individual values for each neuron. Significance is denoted as * p < 0.05, ** p < 0.01, **** p < 0.0001, ^# p < 0.05 and ^## p < 0.01 calculated with 2-way ANOVA followed by Tukey’s post-hoc multiple comparisons test. For full details of statistical analysis including negative results, refer to Supplementary Table 5

Mice in all experimental groups habituated to the novel environment, showing a gradual decrease in the distance travelled over the course of two hours (Fig. 5c). However, TRE-GFP.shmiR-4E/TG mice expressing TdTomato^L+R, which maintain elevated eIF4E levels in D1-SPNs, travelled a significantly greater cumulative distance compared to the tTA^L+R group, in which eIF4E levels were reduced (Fig. 5c-d). This difference could primarily be driven by an increased locomotor velocity (measured in cm/s averaged over five-minute time intervals; Supplementary Fig. 6a-b), as the total movement duration (Supplementary Fig. 6c-d) and immobile time (Supplementary Fig. 6e-f) were unaffected by eIF4E reduction. Furthermore, D1-SPN-targeted reduction of eIF4E in TRE-GFP.shmiR-4E/TG mice decreased the number of marbles buried to a level indistinguishable from WT mice injected with control TdTomato-expressing AAVs (Fig. 5e).

Together, these results indicate that elevated eIF4E expression in D1-SPNs contributes to increased locomotor activity and repetitive behaviours in vivo. Importantly, reducing eIF4E levels selectively in adult D1-SPNs mitigated these behavioural phenotypes, demonstrating that ASD-related hyperactivity and repetitive behaviours in eIF4E-TG mice can be ameliorated by postnatal intervention in adulthood.

Reduced KCNQ potassium channel function in D1-SPNs

We found alterations in passive membrane properties in D1-SPNs of eIF4E-TG mice, consistent with changes in membrane conductances regulating excitability, rather than gross structural remodelling (Fig. 2). An increasing body of evidence links various channelopathies to ASDs, including those involving K+ channels [86]. SPNs highly express Kir2 channels, which contribute to the hyperpolarised resting membrane potential of mature SPNs [70, 83, 87]. Alterations in Kir2 channel activity have been shown to influence excitability, including in D1-SPNs [70]. Given the increased membrane resistance and depolarised RMP observed in D1-SPNs of eIF4E-TG mice, we first tested whether Kir2 channel activity was altered. To this end, we subjected D1-SPNs from D1-Tomato/WT and -/TG mice to hyperpolarising voltage steps within the voltage-range of Kir2 channel activation and then applied the K-channel blocker Cs⁺ to isolate the Kir2-mediated current [70]. Under these recording conditions, we did not detect a significant difference in the amplitude of the Cs⁺-sensitive current between eIF4E-TG and WT mice (Supplementary Fig. 7), suggesting that large changes in Kir2-mediated current are unlikely to account for the observed excitability phenotype.

While Kir2 channels are important regulators of resting membrane properties in SPNs, these neurons also express additional K+ channel subtypes that contribute to excitability regulation [87]. Among these, voltage-gated KCNQ channels mediate a non-inactivating K⁺ current (M-current) and have been shown to modulate D1-SPN excitability [71, 88]. Therefore, we next examined whether KCNQ channel function is altered in association with D1-SPN hyperexcitability observed in eIF4E TG mice. Patch-clamp recordings were performed in striatal slices from D1-Tomato/WT and -/TG mice, as well as from TRE-GFP.shmiR-4E/WT and -/TG mice treated with tTA^L+R (Fig. 5a). Recordings were conducted nine weeks after viral manipulation (Fig. 5b).

To isolate the M-current, D1-SPNs were subjected to a KCNQ-activating voltage-step protocol initially performed in standard ACSF, followed by application of the KCNQ2/3 blocker XE991 [71, 89]. Consistent with altered KCNQ function, we observed a significantly reduced XE991-sensitive current in D1-TdTomato/TG mice compared to D1-TdTomato/WT (Fig. 5f-g). Strikingly, reducing eIF4E expression in D1-SPN eliminated the genotype-dependent difference, as XE991-sensitive currents recorded from D1-SPNs of TRE-GFP.shmiR-4E/TG mice treated with tTA^L+R were no longer significantly different from those measured in WT mice (Fig. 5f-g). Together, these data indicate that altered KCNQ channel-mediated currents are associated with increased D1-SPN excitability in eIF4E-TG mice. Furthermore, targeted reduction of eIF4E expression in adulthood mitigates these channel-associated differences, paralleling the amelioration of D1-SPN firing and behavioural phenotypes.

Discussion

Using whole-cell ex vivo patch clamp electrophysiology and in vivo fibre photometry, we have demonstrated that eIF4E overexpression, which alters protein synthesis, results in cell type-specific functional dysregulation in the striatum. Our results show that D1-SPNs are more active during novelty-induced locomotion in the eIF4E-TG mice, and the D1-SPNs selectively exhibit altered intrinsic properties suggestive of hyperexcitability and hyperactivity, which emerge progressively across postnatal maturation and are robust in adulthood. Additionally, selective reduction of eIF4E in adult D1-SPNs is sufficient to reduce enhanced firing frequency, attenuate the associated KCNQ channel dysfunction and ameliorate ASD-like behaviours such as novelty-induced hyperlocomotion and marble burying. These findings support a model in which altered D1-SPN activity associated with eIF4E overexpression contributes to ASD-related cellular and behavioural phenotypes.

Enhanced D1-SPN activity in eIF4E-TG mice

The striatum plays a central role in exploratory behaviour and eventual habituation to a novel environment, with movement initiation and exploration correlated with an increased in GCaMP fluorescence in both D1- and D2-SPNs [29, 77]. Our findings align with these studies, as we observed increased GCaMP7s fluorescence in both D1- and D2-SPNs in WT mice during locomotor responses to a novel environment (Fig. 1c, e). Recent work has further shown that while both D1- and D2-SPNs are active, they participate in distinct neuronal ensembles to select and suppress behaviours via D1-SPN activation and D2-SPN silencing [32, 90]. As we measured Ca²⁺ activity across a large dorsal striatal population of direct and indirect SPNs, subpopulation-specific dynamics may be undetected, limiting the degree to which our data align with ensemble-specific models.

Interestingly, in the eIF4E-TG mice, we observed a significant increase in Ca²⁺ activity in D1-SPNs during exploration (Fig. 1c-d), correlating with a heightened novelty response and suggesting cell-type-specific mechanisms underlying hyperactivity and other ASD-related behaviours in the eIF4E-TG mice. Whole-cell recordings further revealed hyperexcitability in D1-SPNs of the eIF4E-TG mice, characterised by a reduction in rheobase (Fig. 2j) and an elevated firing frequency (Fig. 2k-m). These changes indicate that less current is required to initiate action potentials, and once activated, D1-SPNs sustain higher firing rates. This hyperexcitability is consistent with the increased Ca²⁺ activity observed in vivo during exploration, as reduced rheobase and increased membrane resistance (Fig. 2c) enhance the neuronal responsiveness to novel stimuli. Together, these data indicate that elevated D1-SPN activity during novelty reflects enhanced recruitment of the direct pathway rather than global striatal hyperactivity.

Progressive emergence of D1-SPN hyperexcitability during postnatal maturation

Our developmental analyses indicate that D1-SPN hyperexcitability emerges progressively across postnatal maturation rather than appearing abruptly in adulthood. At P10, we did not detect genotype-dependent differences in depolarisation-evoked firing in D1-SPNs (Fig. 3i). However, by P28, D1-SPNs from eIF4E-TG mice generated more action potentials in response to current injection (Fig. 3j), indicating that increased firing output is already evident by the end of the main maturation period. In parallel, passive membrane properties followed an altered developmental trajectory in TG mice, with differences in resting membrane potential and membrane resistance becoming apparent earlier (Fig. 3b-c). Together, these findings suggest that alteration in passive membrane properties emerge earlier during postnatal development and precede the later increase in depolarisation-evoked firing, consistent with a gradual divergence in intrinsic excitability rather than an abrupt change arising in adulthood.

In contrast, D2-SPNs did not exhibit genotype-dependent changes in intrinsic properties or firing output during early postnatal maturation, indicating that developmental trajectories of excitability diverge between SPN subtypes (Supplementary Fig. 4).

Cell-type specificity of eIF4E-dependent excitability changes

In contrast to D1-SPNs, although eIF4E expression and rate of protein synthesis are similarly enhanced in D2-SPNs of eIF4E-TG mice (Supplementary Fig. 2a-c), Ca²⁺ activity in A2a-expressing indirect SPNs during novelty exploration was comparable between eIF4E-TG and WT littermates (Fig. 1e-f). Consistently, whole-cell recordings revealed no increase in depolarisation-evoked firing in D2-SPNs (Supplementary Fig. 3), despite detectable changes in intrinsic properties, including reduced membrane capacitance (Supplementary Fig. 3a), and changes in action potential kinetics such as prolonged decay and decreased fAHP (Supplementary Fig. 3g-h). Although changes in AP kinetics such as fAHP can influence firing frequency [91], these alterations were insufficient to increase firing output in D2-SPNs under our recording conditions. These D2-SPN alterations may nonetheless influence striatal circuit balance or state-dependent processing, without manifesting as increased intrinsic firing under basal conditions. Overall, these results indicate that elevated eIF4E expression does not uniformly drive hyperexcitability across SPN subtypes but instead engages distinct intrinsic mechanisms in D1- and D2-SPNs, resulting in divergent functional outcomes.

Importantly, the cell-type–specific electrophysiological outcomes of translational dysregulation likely arise from the interaction between intrinsic molecular identity and extrinsic circuit-level modulation [92]. D1- and D2-SPNs are defined by divergent transcriptional programs, encompassing differences in receptor expression, neuropeptides, signalling scaffolds, and ion channel complements [93, 94]. As a result, changes in eIF4E-dependent translational control are expected to reshape neuronal function in a manner that reflects each cell type’s underlying transcriptomic landscape [95], rather than producing uniform physiological effects. Moreover, the intrinsic molecular identity of striatal SPNs is continuously shaped by the local neuromodulatory environment. For instance, dopamine and acetylcholine exert powerful and cell-type–specific control over both synaptic integration and intrinsic excitability, differentially tuning D1- and D2-SPNs through distinct receptor signalling pathways [83, 96]. Within this framework, translational dysregulation is unlikely to act in isolation, but instead biases how each SPN subtype responds to ongoing extrinsic striatal neurotransmission, including dopaminergic and cholinergic modulation. Consequently, increased eIF4E-dependent translation may preferentially amplify spike-generating mechanisms in D1-SPNs, where neuromodulatory signalling strongly promotes excitability, whereas in D2-SPNs it may manifest as alterations in membrane and action potential dynamics without producing a net increase in firing under basal conditions. This interaction between intrinsic translational state and extrinsic circuit context provides a principled explanation for the divergent functional outcomes observed across SPN subtypes and motivates our subsequent focus on ion channel effectors that tightly couple neuromodulation to firing output, such as KCNQ channels.

Importantly, this framework does not contradict the cell-autonomous nature of the eIF4E-dependent phenotype demonstrated here. Rather, it indicates that translational dysregulation within D1-SPNs biases how these neurons integrate and respond to an altered neuromodulatory context, including the dysregulation of striatal DA release previously reported in eIF4E-TG mice [53].

Impaired KCNQ channel function

To investigate mechanisms that may underlie D1-SPN hyperexcitability, we focused on ion channel function, as increased neuronal excitability was associated with coordinated changes in passive membrane properties, including membrane resistance, resting membrane potential, and capacitance, as well as alterations in active firing properties, in the absence of overt changes in neuronal morphology. Specifically, we examined the function of Kir2 and KCNQ potassium channels, both of which are critical regulators of neuronal excitability [97] and have been implicated in neurodevelopmental disorders, including ASD [98].

We did not detect large differences in Cs⁺-sensitive inwardly rectifying currents in D1-SPNs under our experimental conditions, as assessed using hyperpolarising voltage steps and Cs⁺-based current isolation (Supplementary Fig. 7). However, Kir channels are one of the key determinants of resting membrane potential and input resistance in mature SPNs [99, 100], and subtle or developmentally regulated alterations may not be fully resolved with this approach. In particular, Ba²⁺-based isolation, which more effectively blocks inwardly rectifying potassium channels [101], was not employed in the present study and may provide greater sensitivity for detecting changes in Kir-mediated conductances. Thus, while our data do not support large alterations in Kir2 currents, they do not exclude more modest or developmentally dynamic contributions of Kir2 or other leak potassium channels to the observed changes in resting membrane properties [70].

In contrast, voltage-clamp recordings of D1-SPNs from eIF4E-TG mice revealed a significant reduction in XE991-sensitive currents (Fig. 5f-g), consistent with impaired KCNQ channel-mediated conductance. KCNQ channels mediate the M-current, a non-inactivating potassium current activated by depolarisation that plays a key role in regulating neuronal excitability [97]. Notably, KCNQ channels are preferentially activated at more depolarised membrane potentials [97] and are therefore thought to contribute primarily to subthreshold excitability, spike initiation, and firing gain, rather than serving as dominant determinants of the highly hyperpolarised resting membrane potential characteristic of mature SPNs. Mutations in KCNQ2, such as frameshift insertions [102] and deletions [103], have been identified in ASD patients. Moreover, conditional deletion of Kcnq2 in mouse cortical neurons reduces potassium currents and induces hyperexcitability [104], while heterozygous deletion leads to ASD-like behaviours such as altered exploratory and repetitive behaviours [105]. Conversely, pharmacological activation of KCNQ channels reduces spontaneous locomotor activity and attenuates dopamine-driven hyperactivity caused by methylphenidate or cocaine [106]. Importantly, our data indicate that reduced XE991-sensitive currents are associated with D1-SPNs hyperexcitability in eIF4E-TG mice. Targeted reduction of eIF4E expression in adult D1-SPNs (Supplementary Fig. 5) was sufficient to restore KCNQ-mediated currents and attenuate elevated firing (Fig. 4k-o), supporting a cell-autonomous link between translational dysregulation and ion channel function, while not excluding contributions from additional conductances or signalling pathways.

As eIF4E regulates the expression of a broad set of proteins [107, 108], it is likely that multiple ion channels contribute to the full electrophysiological phenotype observed in eIF4E-TG mice. In particular, channels that regulate resting membrane properties may contribute to the unresolved changes in resting membrane potential and input resistance, whereas channels that shape subthreshold and suprathreshold dynamics are likely to influence firing output. For example, K+ conductances involved in dendritic integration and excitability control, such as A-type channels (Kv4) [109], or channels shaping fAHP and firing patterns, including BK channels [91, 110], as well as non-K+ conductances such as voltage-gated Na+ channels [111], may also contribute to intrinsic excitability. Our data do not identify KCNQ channels as the sole ion channel affected by translational dysregulation; rather, they establish KCNQ-mediated currents as a functionally relevant effector associated with altered firing gain in D1-SPNs. Importantly, targeted manipulation of eIF4E levels (Supplementary Fig. 5) was sufficient to ameliorate aberrant firing and behavioural phenotypes (Figs. 4 and 5), supporting its relevance even in the context of additional, unresolved conductance changes.

Possible molecular mechanisms linking eIF4E overexpression to KCNQ dysfunction

The molecular mechanisms underlying the cell-autonomous dysregulation of KCNQ function remain to be determined. KCNQ2 and KCNQ3 subunits can form both homo- and heterodimers, which exhibit distinct biophysical properties, with heteromeric channels conveying a larger M-current [112]. One possibility is that chronic eIF4E overexpression selectively alters the translation of Kcnq2 or Kcnq3 mRNAs, or of auxiliary or channel-interacting proteins, thereby changing KCNQ subunit stoichiometry, trafficking, or surface stability and leading to a net reduction in functional M-current. This idea is consistent with our finding that KCNQ function can be rescued in a cell-autonomous manner by reducing eIF4E in D1-SPNs.

A second, not mutually exclusive, possibility is that eIF4E overexpression increases the translation of D1 receptors (D1R) or downstream signalling components, thereby amplifying D1R signalling in response to dopaminergic stimulation. This mechanism would align with our recent findings that eIF4E-TG mice display reduced striatal dopamine release [53] which could drive compensatory increases in D1R translation and signalling sensitivity. KCNQ channels are inhibited by MAP-kinase (ERK)-dependent phosphorylation downstream of D1R activation, which increases D1-SPN excitability [71], and eIF4E itself is phosphorylated via ERK-Mnk1/2 signalling, further promoting cap-dependent translation [113, 114]. Thus, aberrant eIF4E activity could couple altered translational control to both the molecular composition and phosphorylation state of KCNQ channels within D1-SPNs, potentially resulting in KCNQ hypofunction and increased excitability.

A third possibility is that eIF4E overexpression alters development of striatal circuits, leading to changes in Ca²⁺ signalling that secondarily impact KCNQ function. Dysregulation of eIF4E or its binding proteins have been shown to disrupt circuit function, including corticostriatal connectivity [115, 116], and corticostriatal abnormalities are also observed in other ASD models [117–119]. Activity of SPNs can modulate circuit function in a cell-type-specific manner, as striatal synaptogenesis and corticostriatal connectivity correlate positively with D1-SPN activity and inversely with D2-SPN activity [120]. Because KCNQ activity is downregulated by elevated intracellular Ca²⁺ signaling [121], enhanced corticostriatal drive and increased D1-SPN Ca²⁺ activity could both contribute to reduced KCNQ channel function.

Our observations of increased D1-SPN Ca²⁺ activity in vivo and altered D1-SPN firing properties during development are consistent with this possibility and align with our previous findings of altered striatal function in eIF4E-TG mice [52]. Further studies will be required to define how synaptic and circuit-level changes interact with intrinsic conductances to shape excitability in this model.

Broader circuit-level consequences of eIF4E overexpression

Although the present study focuses on cell-type–specific intrinsic excitability changes and their rescue in adulthood, it is important to consider that eIF4E overexpression is likely to exert broader effects at the circuit level. Because eIF4E acts as a key regulator of cap-dependent translation [107, 108], sustained dysregulation during development could influence multiple aspects of neuronal maturation, synaptic connectivity, and circuit assembly. In the striatum, prior work has demonstrated that alterations in corticostriatal synapse development, spine maturation, and activity-dependent wiring can profoundly shape striatal output and behavioural flexibility [115, 118, 120].

This principle is well illustrated in other neurodevelopmental disorders linked to dysregulated translation. For example, loss of the Fragile X Mental Retardation Protein (FMRP) or hyperactivation of mTOR signalling broadly alter protein synthesis, including cap-dependent translation processes that converge on eIF4E function [108], yet result in highly selective alterations in synaptic function, circuit organisation, and neuronal excitability across specific brain regions and cell types [122–124]. These observations underscore that global changes in translational control can yield restricted and context-dependent phenotypes, shaped by cell-intrinsic programs and circuit-level interactions rather than uniform physiological effects.

In this context, the selective intrinsic phenotypes we observe in D1-SPNs may reflect the interaction between cell-autonomous translational dysregulation and broader developmental or neuromodulatory influences, including altered dopaminergic transmission [53] and circuit-level homeostatic adaptations. Such mechanisms are not mutually exclusive with the intrinsic changes described here and may bias how excitability phenotypes emerge, stabilise, or are expressed across behavioural states. Importantly, the ability to rescue firing and behavioural abnormalities by reducing eIF4E selectively in adult D1-SPNs indicates that, even in the presence of potential developmental circuit alterations, ongoing translational dysregulation remains a key determinant of pathological output.

Translational implications of KCNQ dysregulation in ASD models

Our results identify KCNQ channel dysfunction as a key downstream effector associated with eIF4E-dependent translation dysregulation in D1-SPNs, thereby linking altered protein synthesis to a well-defined and extensively characterised regulator of neuronal excitability [125]. KCNQ channels are established determinants of firing gain and action potential output across multiple neuronal systems and have a long-standing association with disorders of neuronal hyperexcitability [98, 125]. Drugs that promote KCNQ channel function, such as retigabine (ezogabine), reduce epilepsy and are in clinical trials for various neurological disorders [126–128]. Moreover, genetic studies linking KCNQ2 mutations to ASD [98, 102, 103] further highlight the relevance of this pathway in neurodevelopmental disorders. Together, these observations suggest that eIF4E-dependent translational dysregulation may converge on a functionally tractable excitability control mechanism, rather than producing diffuse or nonspecific effects on neuronal physiology. Importantly, our data do not imply that KCNQ channels represent the sole downstream consequence of altered translation; instead, they establish KCNQ dysfunction as a mechanistically informative and experimentally accessible effector that couples translational control to pathological firing output. This framework motivates future studies to test whether modulation of KCNQ channel function can ameliorate hyperexcitability and behavioural phenotypes in the eIF4E-TG model, while maintaining a cautious interpretation of therapeutic relevance.

Consistent with this framework, modulation of eIF4E in WT D1-SPNs does not significantly alter KCNQ-mediated currents, intrinsic firing properties, or behaviour, despite mild trends in some measures. This suggests that KCNQ dysfunction and hyperexcitability emerge selectively under conditions of pathological translational excess, rather than reflecting a simple bidirectional relationship between eIF4E levels and neuronal output. Such selectivity provides a parsimonious explanation for why targeted reduction of eIF4E restores normal excitability in the eIF4E-TG model while producing minimal effects in WT mice.

One possible interpretation is that physiological levels of eIF4E-dependent translation are sufficient to support normal neuronal function, whereas chronic overexpression drives neurons into a regime where excitability control mechanisms, including KCNQ-mediated currents, become destabilised. In contrast, WT neurons may retain homeostatic mechanisms that buffer moderate postnatal reductions in eIF4E without overt disruption of intrinsic excitability or behaviour. In parallel, alternative strategies to modulate direct pathway excitability may also be relevant. M4 muscarinic receptors are preferentially expressed by D1-SPNs [129] and act as powerful regulators of striatal output [130]. Recent findings demonstrate that M4 receptor activation rescues repetitive behaviours and striatal circuit dysfunction in Fragile X Syndrome models [15]. Given that eIF4E-TG mice exhibit both increased D1-SPN excitability and a reduced M-current, M4 receptor agonism may represent a complementary approach to suppress excessive direct-pathway firing and stabilise membrane excitability [130]. Whether such effects involve modulation of KCNQ channel function or broader changes in intrinsic or synaptic excitability remains an important question for future investigation.

Beyond ion channel-targeted strategies, our findings also inform broader efforts to modulate translational control pathways in neurodevelopmental disorders. Several studies highlight the potential for postnatal mTOR inhibition in reducing ASD pathology. For instance, in VPA-treated rodents, postnatal administration of rapamycin, an mTOR inhibitor, successfully reduced SPN hyperexcitability and aberrant firing patterns [131]. Furthermore, studies in the Tsc1 and Tsc2 models of tuberous sclerosis have demonstrated the efficacy of postnatal mTOR inhibition in ameliorating diverse ASD-like phenotypes [132], ranging from cognitive and social impairments in heterozygote Tsc2^+/− mice [133, 134] to morphological abnormalities and survival deficits in Tsc1 deletion models [135], and epilepsy in mice with glia-specific Tsc1 deletion [136]. However, mTOR regulates numerous cellular processes beyond protein synthesis, including autophagy [137, 138], a process increasingly implicated in neurodevelopmental disorders [73, 139], raising concerns regarding specificity and long-term safety. In this context, targeting eIF4E, a downstream effector of mTOR-dependent translational control, may offer a more selective means of modulating pathological translation while minimising broader disruption of cellular homeostasis.

Overall, our study demonstrates that dysregulated translational control can drive intrinsic excitability changes in a cell-type-specific manner and identifies KCNQ channel dysfunction as a functionally relevant downstream consequence. By showing that conditional reduction of eIF4E in adult D1 SPNs ameliorates intrinsic excitability, KCNQ channel function and behavioural phenotypes, with effects persisting for at least nine weeks, these findings provide a proof-of-concept that cell-specific modulation of translational control can reverse established pathological circuit states. Importantly, this should not be interpreted as evidence for an immediately translatable therapeutic strategy, as the long-term consequences of sustained eIF4E modulation remain unknown. Although the absence of major physiological or behavioural effects in WT mice over this period suggests a degree of tolerance to moderate eIF4E reduction, further work will be required to assess longer-term adaptations, circuit-level consequences, and the impact of developmental timing.

In conclusion, our findings broaden the understanding of translational dysregulation in ASD [36, 45, 140, 141] by demonstrating that its impact extends beyond synaptic dysfunction [52–55] to include cell-type-specific alterations in intrinsic excitability within striatal circuits. By identifying KCNQ channel dysfunction as a downstream consequence of dysregulated translation in D1-SPNs, this work establishes a mechanistic link between mRNA translational control and neuronal firing regulation, highlighting translational pathways as central determinants of circuit function in ASD-relevant systems.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary Material 1 (DOCX 8.42 MB)^{(8.4MB, docx)}

Acknowledgements

We thank the members of Borgkvist and Santini labs for their invaluable methodological support and insightful discussions. We extend special thanks to Eric Klann for generously providing the founders of the eIF4E^wt/βtEif4e and TRE-GFP.shmiR-4E mice and Robert Fetcho for designing the fibre photometry system. We are also grateful to Qian Yu and the KI Animal Behavioral Core Facility for their support during the behavioural experiments, as well as to the staff and veterinarians of the Comparative Medicine Biomedicum (KM-B) for their continuous assistance in maintaining the mouse colonies.

Author contributions

Conceptualisation, A.B. and E.S.; Methodology, A.A., A.B. and E.S.; Validation, A.A., A.B. and E.S.; Formal Analysis, A.A., A.B. and E.S.; Investigation, A.A., A.T., A.P.R., A.B. and E.S.; Resources, A.B. and E.S.; Writing – Original Draft, A.A., A.B. and E.S.; Writing – Review & Editing, A.A., A.B. and E.S.; Visualisation, A.A, A.B. and E.S.; Supervision, A.B. and E.S.; Project Administration, A.B. and E.S.; Funding Acquisition, A.B. and E.S.

Funding

Open access funding provided by Karolinska Institute. This work was supported by the Knut and Alice Wallenberg Foundation (Wallenberg Academy Fellow Grant KAW 2017 − 0169 and project grant 2020-0054 to E.S.), the Swedish Research Council (2016–02758, 2023–02943 to E.S.; 2016–03129, 2024–03684 to A.B.), Olle Engkvist Stiftelse (E.S. and A.B.), Åhlén’s foundation (A.B.), Magn. Bergvall’s foundation (A.B.), The Strategic Research Program in Neuroscience (StratNeuro) starting (E.S. and A.B.) and bridging (E.S.) grants, Karolinska Institute starting grant (E.S.).

Data Availability

The datasets generated during and/or analysed during the current study are not publicly available because they are part of ongoing analyses and follow-up studies but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were compliant with the ethical permit issued by the Swedish Board of Agriculture (Ethical number: 18194 − 2018 and 19345 − 2023) and were performed in accordance with the European Parliament and Council Directive 210&63/EU, 22nd September 2010 for experimentation animals’ protection.

Consent for publication

Not applicable.

Competing interests

The authors report no biomedical financial interests or potential conflicts of interest.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Anders Borgkvist, Email: anders.borgkvist@ki.se.

Emanuela Santini, Email: emanuela.santini@ki.se.

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Supplementary Materials

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Data Availability Statement

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PERMALINK

Postnatal reduction of eIF4E overexpression in D1-SPNs ameliorates KCNQ channel dysfunction, hyperexcitability and ASD-like behaviours

Alina Aaltonen

Ayu Tamaki

Andrés Peris Ramón

Anders Borgkvist

Emanuela Santini

Abstract

Supplementary Information

Introduction

Materials and methods

Mice

Fibre photometry

Novel cage experiment during fibre photometry

Fibre photometry analysis

Stereotaxic surgeries

Electrophysiology

Slice Preparation

Recording protocols

Electrophysiological data analysis

Behaviour

Novel cage experiment

Marble burying

Western blotting

In vivo sunset assay

Immunofluorescence

Neuronal morphology

Statistical analysis

Results

Increased novelty-induced D1-SPN activity in the eIF4E-TG mice

Fig. 1.

Overexpression of eIF4E results in cell-type specific hyperexcitability in SPNs

Fig. 2.

D1-SPN hyperexcitability emerges late during postnatal SPN maturation

Fig. 3.

Conditional Silencing of eIF4E reduces firing frequency of D1-SPNs

Fig. 4.

Reduction of eIF4E in D1-SPNs ameliorates hyperactivity and repetitive behaviours

Fig. 5.

Reduced KCNQ potassium channel function in D1-SPNs

Discussion

Enhanced D1-SPN activity in eIF4E-TG mice

Progressive emergence of D1-SPN hyperexcitability during postnatal maturation

Cell-type specificity of eIF4E-dependent excitability changes

Impaired KCNQ channel function

Possible molecular mechanisms linking eIF4E overexpression to KCNQ dysfunction

Broader circuit-level consequences of eIF4E overexpression

Translational implications of KCNQ dysregulation in ASD models

Supplementary Information

Acknowledgements

Author contributions

Funding

Data Availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

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