DOI: 10.5061/dryad.5x69p8dh7
title: Data from: Cntnap2 loss drives striatal neuron hyperexcitability and behavioral inflexibility

description: 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.
description: <strong>Mice</strong> 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).
 <em>Cntnap2^-/-^</em> mice and littermate
 <em>Cntnap2^+/+^</em> 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 <em>Drd1a</em>-tdTomato,
 <em>Thy1</em>-ChR2-YFP, PV-Cre, Ai32, or Ai9 transgenes to
 avoid potential physiological or behavioral alterations.
 <strong>Electrophysiology</strong>
 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% O<sub>2</sub>
 and 5% CO<sub>2</sub>). 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 &gt; 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. <strong>Current-clamp
 recordings</strong> 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.
 <strong>Voltage-clamp recordings</strong>
 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.
 <strong>Dendritic imaging and spine
 analysis</strong> Neonatal (P1-3)
 <em>Cntnap2^-/-^</em>;D1-tdT and
 <em>Cntnap2^+/+^</em>;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.
 <strong>Brain sectioning and
 immunohistochemistry</strong> 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). <strong>PV
 cell counting</strong> 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.
 <strong>Western Blot</strong>
 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). <strong>In situ
 hybridization</strong> Fluorescent in situ
 hybridization was performed to quantify <em>Pvalb</em> mRNA
 expression in the striatum of <em>Cntnap2^+/+^</em> and
 <em>Cntnap2^-/-^</em> 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). <em>Drd1a</em> mRNA was visualized with a
 probe in channel 2 (ACD, #406491-C2) and <em>Pvalb</em> 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.
 <em>Pvalb</em>-expressing cells were quantified across the
 entire striatum using the ROI manager tool in ImageJ. A cell was
 considered <em>Pvalb</em> positive if over 50% of the cell
 contained fluorescent puncta when compared to the DAPI channel.
 Experimenter was blind to genotype.
 <strong>Behavioral analysis</strong>
 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.
 <strong>Open field assay</strong>
 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. <strong>Open field assay with DeepLabCut
 Keypoint-MoSeq analysis</strong> 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
 .: <strong>Marble burying
 assay</strong> 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 (&gt;50% exposed) was recorded
 after the session.  <strong>Holeboard
 assay</strong> 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.
 <strong>Accelerating rotarod assay</strong>
 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. <strong>Four choice
 odor-based reversal learning test</strong> 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:
 <em>Cntnap2^+/+^</em> mice:  60% thyme, 25% anise, 12.5%
 clove, and 2.5% litsea. <em>Cntnap2^-/-^</em> mice:  47.5%
 thyme, 45% anise, 7.5% clove, 0% litsea. We note that both
 <em>Cntnap2^+/+^</em> and <em>Cntnap2^-/-^</em>
 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.
 <strong>Quantification and statistical
 analysis</strong> 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
 &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001.
description: # Data from: Cntnap2 loss drives striatal neuron hyperexcitability and
 behavioral inflexibility Dataset DOI:
 [10.5061/dryad.5x69p8dh7](10.5061/dryad.5x69p8dh7) ## Description of the
 data and file structure The following data repository contains the raw and
 pre-processed data for the experiments contained within the eLife
 publication "*Cntnap2* loss drives striatal neuron hyperexcitability
 and behavioral inflexibility". This includes patch-clamp slice
 electrophysiology data, confocal images, western blot gels, and behavior
 data ### Files and variables **File: Figure_1.zip**
 **Description:** Contains subfolders Electrophysiology data and Sample
 images and traces. Electrophysiology data includes the pre-processed
 electrophysiology data contained within Figure 1 (Corticostriatal
 excitability - oAPs, Corticostriatal Synapses - oEPSCs, Corticostriatal
 Synapses - AMPA:NMDA). Sample images and traces contains raw files for the
 sample images and sample traces seen in Figure 1.  For all
 electrophysiology Excel files, tab names are formatted "date of
 experiment - animal number - cell number - tdt+ or -", and the final
 tab of the sheet contains an animal number genotype key. Missing values
 are indicated with "NA". Units for baseline and peak amplitudes
 is pA. Other units are abbreviated as follows: * Vm = resting membrane
 potential * AP = action potential * ISI = inter-spike interval * Ih =
 injected current * Rs = series resistance * Rm = membrane resistance * Cm
 = membrane capacitance * tdt+ = D1-tdTomato positive * tdt- = D1-tdTomato
 negative **File: Figure_1_-_Figure_Supplement_1.zip**
 **Description:** Contains subfolders Mut dSPN dendritic spine images, Mut
 iSPN dendritic spine images, WT dSPN dendritic spine images, WT iSPN
 dendritic spine images, and Sample images. The dendritic spine image
 folders contain the raw .tif files for all of the dendrite images used to
 quantify spine density for the corresponding genotypes and cell types in
 Figure 1 - Figure Supplement 1. *Sample images* contains raw files for the
 sample images seen in Figure 1 - Figure Supplement 1.  **File:
 Figure_2.zip** **Description:** Contains subfolders Electrophysiology data
 and Sample images and traces. Electrophysiology data includes the
 pre-processed electrophysiology data contained within Figure 2 (Synaptic
 Inhibition - eIPSCs, PV-interneuron Specific Inhibition - oIPSCs). Sample
 images and traces contains raw files for the sample images and sample
 traces seen in Figure 2.  For all electrophysiology Excel files, tab names
 are formatted "date of experiment - animal number - cell number -
 tdt+ or -", and the final tab of the sheet contains an animal number
 genotype key. Units for baseline and peak amplitudes is pA. Units are
 abbreviated as follows: * Ih = injected current * Rs = series resistance *
 Rm = membrane resistance * Cm = membrane capacitance * tdt+ = D1-tdTomato
 positive * tdt- = D1-tdTomato negative **File:
 Figure_2_-_Figure_Supplement_1.zip** **Description:** Contains subfolders
 Electrophysiology data and Sample images and traces. Electrophysiology
 data includes the pre-processed electrophysiology data contained within
 Figure 2 - Figure Supplement 1 (PV-interneuron Intrinsic Excitability).
 Sample images and traces contains raw files for the sample images and
 sample traces seen in Figure 2 - Figure Supplement 1. For the
 electrophysiology Excel file, tab names are formatted "date of
 experiment - animal number - cell number", and the final tab of the
 sheet contains an animal number genotype key. Missing values are indicated
 with "NA". Other units are abbreviated as follows: * Vm =
 resting membrane potential * AP = action potential * ISI = inter-spike
 interval * Ih = injected current * Rs = series resistance * Rm = membrane
 resistance * Cm = membrane capacitance **File:
 Figure_2_-_Figure_Supplement_2.zip** **Description:** Contains subfolders
 DLS IHC - p21 PV images, DLS IHC - p50 PV images, Dorsal striatum - p50 PV
 images, Dorsal striatum - PV western blot images, and Pvalb mRNA - PV in
 situ images for all IHC, western blot and in situ images quantified in
 Figure 2 - Figure Supplement 2. DLS IHC - p21 PV images contains the raw
 .tif files used to quantify PV IHC at p21 in panel B, DLS IHC - p50 PV
 images contains the raw .tif files used to quantify PV IHC at p50 in panel
 D, Dorsal striatum - p50 PV images contains the raw .tif files used to
 quantify PV IHC, single-cell and bulk fluorescence at p50 in panels F, G,
 and H, Dorsal striatum - PV western blot images contains the raw and
 annotated western blot gel images used to quantify PV protein expression
 in panel J, and Pvalb mRNA - PV in situ images contains the raw .tif files
 used to quantify Pvalb mRNA-positive cells in panels L, M, and N. All
 folders are separated into subfolders by WT and Mut genotype.  **File:
 Figure_3.zip** **Description:** Contains subfolders Electrophysiology data
 and Sample traces. Electrophysiology data includes the pre-processed
 electrophysiology data contained within Figure 3 (Intrinsic Excitability -
 dSPNs and iSPNs). Sample traces contains raw files for the sample traces
 seen in Figure 3. For the electrophysiology Excel file, tab names are
 formatted "date of experiment - animal number - cell number - tdt+ or
 -", and the final tab of the sheet contains an animal number genotype
 key. Missing values are indicated with "NA". Other units are
 abbreviated as follows: * Vm = resting membrane potential * AP = action
 potential * ISI = inter-spike interval * Ih = injected current * Rs =
 series resistance * Rm = membrane resistance * Cm = membrane capacitance *
 tdt+ = D1-tdTomato positive * tdt- = D1-tdTomato negative **File:
 Figure_3_-_Figure_Supplement_1.zip** **Description:** Contains subfolders
 Electrophysiology data and Sample traces. Electrophysiology data includes
 the pre-processed electrophysiology data contained within Figure 3 -
 Figure Supplement 1 (fAHP - dSPNs and iSPNs, Spike Latency - dSPNs and
 iSPNs, Intrinsic Excitability - dSPNs and iSPNs). Sample traces contains
 raw files for the sample traces seen in Figure 3 - Figure Supplement 1.
 For the *fAHP - dSPNs and iSPNs *and *Spike Latency - dSPNs and iSPNs*
 electrophysiology Excel files, tab names denote and genotype and/or cell
 type of the data in that tab. For the *Intrinsic Excitability - dSPNs and
 iSPNs* electrophysiology Excel file, tab names are formatted "date of
 experiment - animal number - cell number - tdt+ or -" and the final
 tab of the sheet contains an animal number genotype key. Missing values
 are indicated with "NA". Units for baseline and peak amplitudes
 is pA. Other units are abbreviated as follows: * fAHP = fast
 after-hyperpolarization * Vm = resting membrane potential * AP = action
 potential * ISI = inter-spike interval * Ih = injected current * Rs =
 series resistance * Rm = membrane resistance * Cm = membrane capacitance *
 tdt+ = D1-tdTomato positive * tdt- = D1-tdTomato negative **File:
 Figure_4.zip** **Description:** Contains subfolders Electrophysiology data
 and Sample traces. Electrophysiology data includes the pre-processed
 electrophysiology data contained within Figure 4 (Intrinsic Excitability
 with Dendrotoxin - dSPNs and iSPNs). Sample traces contains raw files for
 the sample traces seen in Figure 4. For the electrophysiology Excel file,
 tab names are formatted "date of experiment - animal number - cell
 number - tdt+ or - - pre or post drug application", and the final tab
 of the sheet contains an animal number genotype key. Missing values are
 indicated with "NA". Other units are abbreviated as follows: *
 Vm = resting membrane potential * AP = action potential * ISI =
 inter-spike interval * Ih = injected current * Rs = series resistance * Rm
 = membrane resistance * Cm = membrane capacitance * tdt+ = D1-tdTomato
 positive * tdt- = D1-tdTomato negative **File:
 Figure_4_-_Figure_Supplement_1.zip** **Description:** Contains subfolders
 Electrophysiology data and Sample traces. Electrophysiology data includes
 the pre-processed electrophysiology data contained within Figure 4 -
 Figure Supplement 1 (*f*AHP - dSPNs, Spike Latency - dSPNs, Intrinsic
 Excitability with Dendrotoxin - dSPNs and iSPNs). Sample traces contains
 raw files for the sample traces seen in Figure 4 - Figure Supplement 1.
 For the *fAHP - dSPNs *and Spike Latency - dSPNs electrophysiology Excel
 files, tab names denote and genotype, drug condition, and/or cell type of
 the data in that tab. For the Intrinsic Excitability with Dendrotoxin -
 dSPNs and iSPNs electrophysiology Excel file, tab names are formatted
 "date of experiment - animal number - cell number - tdt+ or - - pre
 or post drug application" and the final tab of the sheet contains an
 animal number genotype key. Missing values are indicated with
 "NA". Units for baseline and peak amplitudes is pA. Other units
 are abbreviated as follows: * fAHP = fast after-hyperpolarization * Vm =
 resting membrane potential * AP = action potential * ISI = inter-spike
 interval * Ih = injected current * Rs = series resistance * Rm = membrane
 resistance * Cm = membrane capacitance * tdt+ = D1-tdTomato positive *
 tdt- = D1-tdTomato negative **File: Figure_4_-_Figure_Supplement_2.zip**
 **Description:** Contains subfolders Electrophysiology data and Sample
 traces. Electrophysiology data includes the pre-processed
 electrophysiology data contained within Figure 4 - Figure Supplement 2
 (fAHP - iSPNs, Spike Latency - iSPNs, Intrinsic Excitability with
 Dendrotoxin - dSPNs and iSPNs). Sample traces contains raw files for the
 sample traces seen in Figure 4 - Figure Supplement 2. For the *fAHP -
 iSPNs *and *Spike Latency - iSPNs* electrophysiology Excel files, tab
 names denote and genotype, drug condition, and/or cell type of the data in
 that tab. For the Intrinsic Excitability with Dendrotoxin - dSPNs and
 iSPNs electrophysiology Excel file, tab names are formatted "date of
 experiment - animal number - cell number - tdt+ or - - pre or post drug
 application" and the final tab of the sheet contains an animal number
 genotype key. Missing values are indicated with "NA". Units for
 baseline and peak amplitudes is pA. Other units are abbreviated as
 follows: * fAHP = fast after-hyperpolarization * Vm = resting membrane
 potential * AP = action potential * ISI = inter-spike interval * Ih =
 injected current * Rs = series resistance * Rm = membrane resistance * Cm
 = membrane capacitance * tdt+ = D1-tdTomato positive * tdt- = D1-tdTomato
 negative **File: Figure_5.zip** **Description:** Contains subfolder
 Repetitive Behavior Data which contains an Excel sheet containing the
 accumulated data for the open field (automatically processed by AnyMaze),
 open field - grooming (processed live by scorer manually), marble
 burying (processed live by scorer manually), and holeboard (processed by
 scorer manually from recorded videos) assays in Figure 5. Note that
 automatically live scored data do not have corresponding video or image
 files. Videos recorded and utilized for holeboard and Deep Lab Cut/MoSeq
 open field were too large to deposit to Dryad - requests for these video
 files can be made to the corresponding author
 ([bateup@berkeley.edu](mailto:bateup@berkeley.edu)). In addition, code
 utilized to process Deep Lab Cut/MoSeq open field videos and create
 Figures 5N-P and all Figure 5 - Figure Supplement 1 figure panels can be
 found
 at [https://github.com/BateupLab/Cording2025/tree/main](https://github.com/BateupLab/Cording2025/tree/main). **File: Figure_6.zip** **Description:** Contains subfolder Accelerating Rotarod Data which contains an Excel sheet containing the accumulated data for the accelerating rotarod assay (automatically outputted by the rotarod) in Figure 6. Note that automatically live scored data do not have corresponding video or image files. Units are abbreviated as follows: * Term = terminal * RPM = rotations per minute #### File: Figure_7.zip **Description:** Contains subfolder *Four Choice Task Data* which contains an Excel sheet containing the accumulated data for the four choice reversal learning assay (automatically outputted by the rotarod machine) in Figure 7. Note that manually live scored data do not have corresponding video or image files. Units are abbreviated as follows: * Term = terminal * RPM = rotations per minute **Code/software** Code (and corresponding README) needed to recreate the Deep Lab Cut/MoSeq analysis performed on our open field videos, and recreate Figures 5N - P and all panels in Figure 5 - Figure Supplement 1 can be found at [https://github.com/BateupLab/Cording2025/tree/main](https://github.com/BateupLab/Cording2025/tree/main).