Cntnap2 loss drives striatal neuron hyperexcitability and behavioral inflexibility

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by two major diagnostic criteria – persistent deficits in social communication and interaction, and the presence of restricted, repetitive patterns of behavior (RRBs). Evidence from both human and animal model studies of ASD suggests that alteration of striatal circuits, which mediate motor learning, action selection, and habit formation, may contribute to the manifestation of RRBs. CNTNAP2 is a syndromic ASD risk gene, and loss of function of Cntnap2 in mice is associated with RRBs. How loss of Cntnap2 impacts striatal neuron function is largely unknown. In this study, we utilized Cntnap2^-/-^ mice to test whether altered striatal neuron activity contributes to aberrant motor behaviors relevant to ASD. We assessed excitatory, inhibitory, and intrinsic physiological function of the primary striatal cell type, SPNs, as well as a primary striatal interneuron class, PV-INs, using whole cell patch clamp electrophysiology in Cntnap2^+/+^ and Cntnap2^-/-^ mice. We find that Cntnap2^-/-^ mice exhibit increased cortical drive of direct pathway striatal projection neurons (dSPNs). This enhanced drive is likely due to increased intrinsic excitability of dSPNs, as we find no change in interneuron number or function. We hypothesize that this enhanced excitability of dSPNs underlies their increased responsiveness to cortical inputs. Behaviorally, we find that Cntnap2^-/-^ mice exhibit spontaneous repetitive behaviors in the open field, marble burying and holeboard assays, increased motor routine learning on the accelerating rotarod assay, and increased perseveration and cognitive inflexibility in the four-choice reversal learning assay. We conclude that increased corticostriatal drive of the direct pathway may therefore contribute to the acquisition of repetitive, inflexible behaviors in Cntnap2 mice.

Methods
Mice

All animal procedures were conducted by protocols approved by the University of California, Berkeley Institutional Animal Care and Use Committee (IACUC) and Office of Laboratory Animal Care (OLAC) (AUP-2016-04-8684-3). Cntnap2^-/-^ mice and littermate Cntnap2^+/+^ controls with the following alleles were utilized for each experiment.

Mice were group housed on a 12 h light/dark cycle (dark cycle 9:00 AM – 9:00 PM) and given ad libitum access to standard rodent chow and water. Both male and female animals were used for experimentation. The ages, sexes, and numbers of mice used for each experiment are indicated in the respective method details and figure legends. All mice used for experiments were heterozygous or hemizygous for the Drd1a-tdTomato, Thy1-ChR2-YFP, PV-Cre, Ai32, or Ai9 transgenes to avoid potential physiological or behavioral alterations.

Electrophysiology

Mice (P50-60) were briefly anesthetized with isoflurane and perfused transcardially with ice-cold ACSF (pH = 7.4) containing (in mM): 127 NaCl, 25 NaHCO3, 1.25 NaH2PO4, 2.5 KCl, 1 MgCl2, 2 CaCl2, and 25 glucose, bubbled continuously with carbogen (95% O2 and 5% CO2). Brains were rapidly removed and coronal slices (275 μm) were cut on a VT1000S vibratome (Leica) in oxygenated ice-cold choline-based external solution (pH = 7.8) containing (in mM): 110 choline chloride, 25 NaHCO3, 1.25 NaHPO4, 2.5 KCl, 7 MgCl2, 0.5 CaCl2, 25 glucose, 11.6 sodium ascorbate, and 3.1 sodium pyruvate. Slices were recovered in ACSF at 36°C for 15 min and then kept at room temperature (RT) before recording. Recordings were made with a MultiClamp 700B amplifier (Molecular Devices) at RT using 3-5 MOhm glass patch electrodes (Sutter, #BF150-86-7.5). Data were acquired using ScanImage software, written and maintained by Dr. Bernardo Sabatini (https://github. com/bernardosabatini/ SabalabAcq). Traces were analyzed in Igor Pro (Wavemetrics). Recordings with a series resistance > 25 MOhms or holding current more negative than -200 pA were rejected. Passive properties were calculated using the double exponential curve fit of the average of five -5 mV, 100 ms long pulse steps applied at the beginning of every experiment.

Current-clamp recordings

Current clamp recordings were made using a potassium-based internal solution (pH = 7.4) containing (in mM): 135 KMeSO4, 5 KCl, 5 HEPES, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine, and 1 EGTA. For corticostriatal excitability experiments, optogenetic stimulation consisted of a full-field pulse of blue light (470 nm, 0.5 ms pulse width, CoolLED) through a 63x objective (Olympus, LUMPLFLN60XW). Light power was linear over the range of intensities tested. No synaptic blockers were included. For intrinsic excitability experiments (SPN, PV interneuron, and SPN + a-DTX experiments), NBQX (10 μM, Tocris, #1044), CPP (10 μM, Tocris, #0247) and picrotoxin (50 μM, Abcam, #120315) were added to the external solution to block synaptic transmission. For Kv1.2 inhibition experiments, a-DTX (100nM, Alomone Labs, #D-350) was added to the external solution. Control recordings in the absence of a-DTX were performed on slices prior to drug application or on fresh slices after drug washout in alternating order across recording days. Bovine serum albumin (BSA, 0.005%, Sigma, #A7030) was included in both control and a-DTX-containing external solutions to minimize nonspecific binding. One second depolarizing current steps were applied to induce action potentials. No holding current was applied to the membrane.

Voltage-clamp recordings

Voltage-clamp recordings were made using a cesium-based internal solution (pH = 7.4) containing (in mM): 120 CsMeSO4, 15 CsCl, 10 TEA-Cl, 8 NaCl, 10 HEPES, 1 EGTA, 5 QX-314, 4 Mg-ATP, and 0.3 Na-GTP. Recordings were acquired with the amplifier Bessel filter set at 3 kHz. Corticostriatal synaptic stimulation experiments to measure evoked EPSCs were performed in picrotoxin (50 μM), and optogenetic stimulation consisted of a full-field pulse of blue light (470 nm, 0.15 ms pulse width) through a 63x objective. To record AMPAR-mediated EPSCs, cells were held at -70mV; to record NMDAR-mediated EPSCs, cells were held at +40mV. Synaptic stimulation experiments to measure evoked IPSCs were performed in NBQX (10 μM) and CPP (10 μM). For electrically evoked IPSCs, a concentric bipolar stimulating electrode (FHC, #30214) was placed in dorsal striatum, roughly 200 μm medial to the recording site in dorsolateral striatum, and a 0.15 ms stimulus was applied. For PV-interneuron optically evoked IPSCs, a full-field pulse of blue light (470 nm, 0.15 ms pulse width) was applied through a 63x objective at the recording site. All evoked IPSC experiments were recorded with cells held at -70mV.

Dendritic imaging and spine analysis

Neonatal (P1-3) Cntnap2^-/-^;D1-tdT and Cntnap2^+/+^;D1-tdT mice were cryoanesthetized and injected bilaterally with 200 nL AAV1.hSyn.eGFP.WPRE.bGH (Penn Vector Core, #p1696 (Keaveney et al., 2018)), diluted 1:75 in saline to achieve sparse transduction. Injections were targeted to the dorsal striatum, with coordinates approximately 1.3 mm lateral to midline, 2.0 mm posterior to bregma, and 1.5 mm ventral to the head surface. At P50-60, mice were anesthetized with isoflurane and transcardial perfusion was performed with 10 mL of 1x PBS followed by 10 mL of ice cold 4% PFA (EMS, #15710-S) in 1x PBS. Brains were post-fixed in 4% PFA in 1x PBS overnight at 4° C. 80 μm coronal sections were made using a freezing microtome (American Optical, AO 860) and stored in 1x PBS at 4° C. Sections were blocked for 1 hour at RT in BlockAid (ThermoFisher, #B10710) and incubated for 48 hours with gentle shaking at 4° C with antibodies against GFP (1:2500, Abcam, #13970) and RFP (1:1000, Rockland (VWR, #600-401-379) diluted in PBS-Tx (1x PBS with 0.25% Triton X-100 (Sigma, #T8787). Sections were washed 3 x 10 min in PBS-Tx and incubated with gentle shaking for 1 hour at RT with Alexa Fluor 488 and 546 secondary antibodies (1:500, Invitrogen, #A11039, #A11035). Sections were washed 3 x 10 min in 1x PBS and mounted onto SuperFrost slides (VWR, #48311- 703) using VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, #H-1400-10). Z stack images of individual dendrites were taken on a confocal microscope (Olympus FLUOVIEW FV3000) with a 60x oil immersion objective (Olympus #1-U2B832) at 2.5x zoom with a step size of 0.4 μm and deconvoluted using Olympus CellSens software. To quantify spine density, dendrites and spines were reconstructed using the FilamentTracer module in Imaris software (Oxford Instruments). The spine density of each dendrite was calculated using Imaris. Dendrites analyzed varied in total length, but excluded the most proximal and distal portions of the dendrite.

Brain sectioning and immunohistochemistry

Adult mice were perfused as above and brains were post-fixed with 4% paraformaldehyde overnight, then sectioned coronally at 30 μm. For immunohistochemistry, individual wells of sections were washed for 3 x 5 min with 1x PBS, then blocked for 1 hour at RT with BlockAid blocking solution. Primary antibodies diluted in PBS-Tx were added and tissue was incubated for 48 hours with gentle shaking at 4° C. Sections were then washed 3 x 10 min with PBS-Tx. Secondary antibodies diluted 1:500 in PBS-Tx were added and incubated with gentle shaking for 1 hour at RT. Sections were washed 3 x 10 min in 1x PBS. Sections were mounted onto SuperFrost slides (VWR, #48311- 703) and coverslipped with VECTASHIELD HardSet with DAPI (Vector Laboratories, #H-1500-10) or VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories, #H-1400-10). The following antibodies were used: mouse anti-PV (1:1000, Sigma, #P3088), rabbit anti-PV (1:1000, Abcam, #11427), anti-RFP (1:500, Rockland, #600-401-379), Alexa Fluor 405, 488 and 546 conjugated secondary antibodies (1:500, Invitrogen, #A-31553, #A-11001, #A-11003, and #A-11035).

PV cell counting

To count PV+ interneurons, Z-stack images of immunostained striatal sections were taken on a confocal microscope (Olympus FLUOVIEW FV3000) with a 10x or 20x objective (Olympus # 1-U2B824 or Olympus # 1-U2B825) and step size of 2 μm. For quantification, image stacks were Z-projected to maximum intensity using Fiji (ImageJ) and cropped to a 400 μm x 400 μm image in anatomically matched sections of the DLS. All PV-expressing cells within this region were counted using the ROI manager tool in ImageJ. Designation of a cell as PV positive was determined by the experimenter and consistently maintained across animals. Experimenter was blind to genotype and ROIs were made on the DAPI channel to avoid selecting regions based on PV expression. To quantify bulk PV fluorescence, ROIs were manually defined in ImageJ using the Freehand tool to cover as much of the DLS as possible, and mean fluorescence intensity was measured.  To quantify individual cell PV fluorescence, ROIs were manually defined around every PV positive cell in the previously drawn DLS ROI using the Freehand tool, and mean fluorescence intensity was measured.

Western Blot

Adult mice (P48-55) were deeply anesthetized with isoflurane and decapitated. Brains were rapidly dissected and 1.5 mm dorsal striatum punches (Biopunch, Ted Pella, #15111-15) were collected from both hemispheres, flash-frozen in liquid nitrogen, and stored at −80° C. On the day of analysis, frozen samples were sonicated (QSonica Q55) until homogenized in 200 μl lysis buffer containing 1% SDS in 1x PBS with Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific, #PI78420) and Complete mini EDTA-free protease inhibitor cocktail (Roche, #4693159001). Sample homogenates were then boiled on a heat block at 95° C for 5 min and allowed to cool to RT. Total protein content was determined using a BCA assay (Thermo Fisher Scientific, #23227). Following the BCA assay, protein homogenates were mixed with 4x Laemmli sample buffer (BioRad, #161-0747). 12.5μg of total protein per sample were then loaded onto 12% Criterion TGX gels (BioRad, #5671044) and run at 65 V. Proteins were transferred to a PVDF membrane (BioRad, #1620177) at 11 V for 14 hours at 4° C using the BioRad Criterion Blotter (BioRad, #1704070). Membranes (BioRad, #1620177) were briefly reactivated in methanol and rinsed in water 3x. After rinsing, membranes were blocked in 5% milk in 1x TBS with 1% Tween (TBS-Tween) for 1 hour at RT before being incubated with primary antibodies diluted in 5% milk in TBS-Tween overnight at 4° C. The following day, after 3 x 10 min washes with TBS-Tween, membranes were incubated with secondary antibodies for 1 hour at RT. Following 6 × 10 min washes, membranes were incubated with chemiluminescence substrate (PerkinElmer #NEL105001EA) for 1 min and exposed to Amersham Hyperfilm ECL (VWR, #95017-661).

Bands were quantified by densitometry using ImageJ software. GAPDH was used to normalize protein content and data are expressed as a percentage of control within a given experiment. The following antibodies were used: anti-Caspr2 (1:5000, Abcam, #153856), anti-PV (1:2500, Abcam, #11427), anti-GAPDH (1:5000, Cell Signaling, #51745S), and anti-rabbit goat HRP conjugate (1:5000, BioRad, #1705046).

In situ hybridization

Fluorescent in situ hybridization was performed to quantify Pvalb mRNA expression in the striatum of Cntnap2^+/+^ and Cntnap2^-/-^ mice. Mice were briefly anesthetized with isoflurane and brains were harvested, flash-frozen in OCT mounting medium (Thermo Fisher Scientific, #23-730-571) on dry ice and stored at -80° C for up to 6 months. 16 µm sections were collected using a cryostat (Thermo Fisher Scientific, Microm HM 550), mounted directly onto Superfrost Plus glass slides (VWR, #48311-703) and stored at -80° C for up to 6 months. In situ hybridization was performed according to the protocols provided with the RNAscope Multiplex Fluorescent Reagent Kit (ACD, #323100). Drd1a mRNA was visualized with a probe in channel 2 (ACD, #406491-C2) and Pvalb mRNA in channel 3 (ACD, #421931-C3). After incubation, sections were secured on slides using ProLong Gold Antifade Mountant with DAPI (Invitrogen, P36935) and 60 x 24 mm rectangular glass coverslips (VWR, #16004-096). Sections were imaged on an Olympus FluoView 3000 confocal microscope using a 10x objective with 1.5x zoom and a step size of 2 µm. Pvalb-expressing cells were quantified across the entire striatum using the ROI manager tool in ImageJ. A cell was considered Pvalb positive if over 50% of the cell contained fluorescent puncta when compared to the DAPI channel. Experimenter was blind to genotype.

Behavioral analysis

All behavior studies were carried out in the dark phase of the light cycle under red lights (open field) or white lights (marble burying, holeboard, rotarod, and four choice reversal learning). Mice were habituated to the behavior testing room for at least 30 min prior to testing. Mice were given at least one day between different tests. All behavior equipment was cleaned between each trial and mouse with 70% ethanol and rinsed in diluted soap followed by water at the end of the day. If male and female mice were to be tested on the same day, male mice were run first then returned to the housing room, after which all equipment was thoroughly cleaned prior to bringing in female mice for habituation. Behavioral tests were performed with young adult male and female mice (7-11 weeks old). The experimenter was blind to genotype throughout the testing and scoring procedures.

Open field assay

Exploratory behavior in a novel environment and general locomotor activity were assessed by a 60 min session in an open field chamber (40 cm L x 40 cm W x 34 cm H) made of transparent plexiglass. Horizontal infrared photobeams (Stoelting, 60001-02A) were positioned to detect rearing. The mouse was placed in the bottom right-hand corner of the arena and behavior was recorded using an overhead camera and analyzed using ANY-maze software (Stoelting). An observer manually scored self-grooming behavior during the first 20 minutes of the test. A grooming bout was defined as an unbroken series of grooming movements, including licking of body, paws, or tail, as well as licking of forepaws followed by rubbing of face with paws.

Open field assay with DeepLabCut Keypoint-MoSeq analysis

Mice were placed in the open field arena and video recorded with a monochrome camera (FLIR Grasshopper 3, GS3-U3-41C6NIR-C) and a 16 mm wide angle lens (Kowa, LM16HC) placed above the arena from a height of 50 cm. To extract the body part (keypoint) coordinates from the video recordings, DeepLabCut (DLC) 2.3.4 (Mathis, et al. 2018; Nath, et al. 2019) was used. Fourteen body parts including nose, head, left ear, right ear, left forelimb, right forelimb, spine 1, spine 2, spine 3, left hindlimb, right hindlimb, tail 1, tail 2, and tail 3 were manually labeled on a small subset of the video frames. A DLC model was then trained using the annotated frames to label those 14 body parts for all videos recorded. The total distance traveled, and number of center entries were calculated using the coordinate of bodypart tail 1. Discrete behavior syllables were extracted using Keypoint-MoSeq 0.4.4 (Weinreb, et al. 2023). Syllable usage and transition data were obtained using built-in functions of the Keypoint-MoSeq package. Decoding and entropy analysis were performed using customized Python 3.9 script. Code available upon request in Github. Entropy was calculated using the following equation, where  denotes the frequency of the syllable  and denotes the transition probability from syllable  to syllable .:

Marble burying assay

The marble burying assay was used to test for repetitive behavior. 20 black marbles were arranged in an orderly 4 x 5 grid on top of 5 cm of clean corn cob bedding in a standard mouse cage. Overhead room lights were on and white noise was played to induce mild stress. Mice were placed in the cage with the marbles for 30 minutes. The number of unburied marbles (>50% exposed) was recorded after the session. 

Holeboard assay

The holeboard assay was used to measure exploratory and repetitive behavior. The holeboard apparatus consisted of a smooth, flat, opaque gray plastic platform, suspended 10 cm from the base by four plastic pegs in each corner. The board contained 16 evenly spaced 2 cm diameter holes and was surrounded by a 30 cm high clear plastic square encasing. During testing, mice were placed into the center of the holeboard. Mice explored the board for 10 minutes while video was recorded from both an above and side-view camera. Videos were used post-hoc to manually count and map the number of nose pokes made during the task. Nose pokes were defined as the mouse’s nose passing through the board barrier when viewed through the side-view camera.

Accelerating rotarod assay

The accelerating rotarod test was used to examine motor coordination and learning. Mice were trained on a rotarod apparatus (Ugo Basile, #47650) for four consecutive days. Three trials were completed per day with a 5 min break between trials. The rotarod was accelerated from 5-40 revolutions per minute (rpm) over 300 s for trials 1-6 (days 1 and 2), and from 10-80 rpm over 300 s for trials 7-12 (days 3 and 4). On the first testing day, mice were first acclimated to the apparatus by being placed on the rotarod rotating at a constant 5 rpm for 60 s and returned to their home cage for 5 min prior to starting trial 1. Latency to fall, or to rotate off the top of the rotarod barrel, was measured by the rotarod stop-trigger timer.

Four choice odor-based reversal learning test

The four-choice odor-based reversal learning test was used to assess learning and cognitive flexibility. Animals were food restricted for 6 days in total, with unrestricted access to drinking water, and maintained at 90-95% of ad lib feeding body weight. Food was given at the end of the day once testing was completed. Food restriction and introduction to Froot Loop cereal piece (Kellogg’s, Battle Creek, MI) began 48 hours before pre-training. The four-choice test was performed in a custom-made square box (30.5 cm L × 30.5 cm W × 23 cm H) constructed of clear acrylic. Four internal walls 7.6 cm wide partially divided the arena into four quadrants. A 15.2 cm diameter removable cylinder fit in the center of the maze and was lowered between trials (after a digging response) to isolate the mouse from the rest of the maze. Odor stimuli were presented mixed with wood shavings in white ceramic pots measuring 7.3 cm in diameter and 4.5 cm deep. All pots were sham baited with a piece of Froot Loop cereal secured underneath a mesh screen at the bottom of the pot. This was to prevent mice from using the odor of the Froot Loop to guide their choice. The apparatus was cleaned with 2.5% acetic acid followed by water and the pots were cleaned with 70% ethanol followed by water between mice. The apparatus was cleaned with diluted soap and water at the end of each testing day.

On the first habituation day of pre-training (day 1), animals were allowed to freely explore the testing arena for 30 min and consume small pieces of Froot Loops placed inside empty pots positioned in each of the four corners. On the second shaping day of pre-training (day 2), mice learned to dig to find cereal pieces buried in unscented coarse pine wood shavings (Harts Mountain Corporation, Secaucus, NJ). A single pot was used and increasing amounts of unscented wood shavings were used to cover each subsequent cereal reward. The quadrant containing the pot was alternated on each trial and all quadrants were rewarded equally. Trials were untimed and consisted of (in order): two trials with no shavings, two trials with a dusting of shavings, two trials with the pot a quarter full, two trials with the pot half full, and four trials with the cereal piece completely buried by shavings. The mouse was manually returned to the center cylinder between trials.

On the days for odor discrimination (day 3, acquisition) and reversal (day 4), wood shavings were freshly scented on the day of testing. Anise extract (McCormick, Hunt Valley, MD) was used undiluted at 0.02 ml/g of shavings. Clove, litsea, and eucalyptus oils (San Francisco Massage Supply Co., San Francisco, CA) were diluted 1:10 in mineral oil and mixed at 0.02 ml/g of shavings. Thymol (thyme; Alfa Aesar, A14563) was diluted 1:20 in 50% ethanol and mixed at 0.01 ml/g of shavings. During the discrimination phase (day 3), mice had to discriminate between four pots with four different odors and learn which one contained a buried food reward. Each trial began with the mouse confined to the start cylinder. Once the cylinder was lifted, timing began, and the mouse could freely explore the arena until it chose to dig in a pot. Digging was defined as purposefully moving the shavings with both front paws. A trial was terminated if no choice was made within 3 min and recorded as omission. If a mouse had three pairs of omissions, they were removed to their homecage for a 15-20 minute break. After the break, if the mouse had three additional pairs of omissions then the task was terminated and the mouse excluded from the dataset. Similarly, if the mouse took longer than 3 hours on the reversal without varying its response behavior, then it was also excluded from the dataset. Criterion was met when the animal completed eight out of ten consecutive trials correctly. The spatial location of the odors was shuffled on each trial. The rewarded odor during acquisition was anise.

The first four odor choices made during acquisition were analyzed to determine innate odor preference by the percentage of choices for a given odor: Cntnap2^+/+^ mice:  60% thyme, 25% anise, 12.5% clove, and 2.5% litsea. Cntnap2^-/-^ mice:  47.5% thyme, 45% anise, 7.5% clove, 0% litsea. We note that both Cntnap2^+/+^ and Cntnap2^-/-^ mice exhibited the strongest innate preference for thyme, an unrewarded odor. There were no significant differences in innate odor preference.

The reversal phase of the task was carried out on day 4. Mice first performed the task with the same rewarded odor as the discrimination day to ensure they learned and remembered the task. After reaching criterion on recall (eight out of ten consecutive trials correct), the rewarded odor was switched, and mice underwent a reversal learning test in which a previously unrewarded odor (clove) was rewarded. A novel odor (eucalyptus) was also introduced, which replaced thyme. Perseverative errors were choices to dig in the previously rewarded odor that was no longer rewarded. Regressive errors were choosing the previously rewarded odor after the first correct choice of the newly rewarded odor. Novel errors were choices to dig in the pot with the newly introduced odor (eucalyptus). Irrelevant errors were choices to dig in the pot that had never been rewarded (litsea). Omissions were trials in which the mouse failed to make a digging choice within 3 min from the start of the trial. Total errors were the sum of perseverative, regressive, irrelevant, novel, and omission errors. Criterion was met when the mouse completed eight out of ten consecutive trials correctly. The spatial location of the odors was shuffled on each trial.

Quantification and statistical analysis

Experiments were designed to compare the main effect of genotype. The sample sizes were based on prior studies and are indicated in the figure legend for each experiment. Whenever possible, quantification and analyses were performed blind to genotype. GraphPad Prism version 10 was used to perform statistical analyses. The statistical tests and outcomes for each experiment are indicated in the respective figure legend. Two-tailed unpaired t tests were used for comparisons between two groups. For data that did not pass the D’Agostino & Pearson normality test, a Mann-Whitney test was used. Two-way ANOVAs or mixed effects models were used to compare differences between groups for experiments with two independent variables. Statistical significance was defined in the figure panels as follows: *p < 0.05, **p < 0.01, ***p < 0.001.