DOI: 10.6084/m9.figshare.26335684
title: <b>Disrupted stimulus encoding shapes tactile perception in autism.</b> (data)

description: Experimental designWe back-translated a task used in human studies??<sup>4</sup>? and developed a novel Go/No-go detection task for vibrotactile stimuli which we combined with two-photon calcium imaging of excitatory and inhibitory neurons in the forepaw-related primary somatosensory cortex (FP-S1) to study altered tactile perception in autism and define its neural correlates. MiceSecond-generation <i>Fmr1</i> knockout (<i>Fmr1</i><sup>−/y</sup>) and wild-type littermate mice 5-16 weeks old were used in our study. Mice were maintained in a mixed 129/Sv/C57Bl/6 J/FVB background (backcrossed 6 generations into C57Bl/6J) as described in ref.??<sup>28,34</sup>?. Male wild-type and <i>Fmr1</i><sup><em>–/y</em></sup> littermates were generated by crossing <i>Fmr1</i><sup>+/−</sup> females with <i>Fmr1</i><sup>+/y</sup> male mice from the same production, and the resulting progeny used for our experiments was either <i>Fmr1</i><sup>+/y</sup> (wild type) or <i>Fmr1</i><sup><em>–/y</em></sup> (KO). Mice were maintained in collective cages following weaning (2-4 litter males per cage). Cages were balanced for genotype and supplemented with cotton nestlets and carton tubes. Number of mice are given in the figure captions. The genotype of experimental animals was re-confirmed post hoc by tail-PCR.<br>SurgeryMice (P33-42) were anaesthetized with isoflurane (4.5% induction, 1.5-2% maintenance). Proper depth of anesthesia was monitored by testing the absence of a foot-pinch reflex and whisker movement. Mice were head-fixed using non-puncture ear-bars and a nose-clamp, and optic gel (Ocry-gel) was applied to both eyes. Body temperature was maintained at 37 °C with a heating pad and rectal probe. Prior to the beginning of the surgery, saline (0.1 ml/ 10 g) and 0.1 ml of a 1:10 Buprenorphine to saline solution were administered subcutaneously. Following hair trimming and application of Betadine scrub and Betadine dermal solution at the scalp, 0.1 ml of a 1:4 Lidocaine to saline solution was administered subcutaneously and waited for 2–5 min to induce local analgesia. The scalp was carefully removed and a 3 mm diameter craniotomy was made above the S1 forepaw region (0.75 mm anterior and -2.5 mm lateral from Bregma, confirmed with intrinsic imaging coupled with forepaw stimulation) using a dental drill (World Precision Instruments). The cortical surface was rinsed with saline throughout the surgery. Stereotactic injections of 100 nl of AAV1/2-syn-jGCaMP8m (titre: 2.6E+12 gcp/ml, diluted 1:4 in Hank’s Balanced Salt Solution) and AAV1/2-mDlx-HBB-chI-mRuby3-SV40p(A) (titre: 8.6E+13 gcp/ml, diluted 1:2 in Hank’s Balanced Salt Solution) were delivered (50 nl/ min) in layers 2/3 (z depth 0.3mm). A double coverslip, composed of a 3 mm diameter lower glass and a 4 mm diameter upper glass glued together with optical adhesive (NOA 61, Norland Products), was implanted on top of the craniotomy and secured in place with cyanoacrylate glue. A head-post was attached to the skull with cyanoacrylate glue and secured with dental cement. An optical glue (OptiBond Universal, Kerr) and dental filling material (Charisma, Kulzer) were applied on the skull and curated with LED blue light. Dental cement was applied on the top. After the surgery the mice were placed on a warm blanket for 1 h after they awoke from anesthesia.Go/No-Go vibrotactile decision-making taskSetupThe vibrotactile decision-making setup was positioned in an isolation cubicle to minimize interference during the experiment. Mice were placed in a body tube and were head-fixed with their forepaws resting on two steel bars (6 mm diameter, Thorlabs). The right bar was mounted to a Preloaded Piezo Actuator (P-841.6, Physik Instrumente) equipped with a strain gauge feedback sensor and controlled(E-501, Physik Instrumente) in a closed loop, as described before??<sup>50</sup>?. Water reward was delivered through a metal feeding needle (20G, 1,9mm tip<b>, </b><b>Agntho's AB</b>) connected to a lickport interface with a solenoid valve (Sanworks) equipped with a capacitive sensor . The behavioral setup was controlled by Bpod (Sanworks) through scripts in Python .Habituation to head-fixation and water restrictionMice (P40-P50) were handled using carton tubes and the cupping technique until they were comfortable in the experimenter’s hands, attested by eating while handled. Mice were gradually habituated to the experimental setup and head fixation for 5 days. The third day of habituation, a water-restriction protocol was implemented, where mice had access to liquid water in the setup and to a solid water supplement (Hydrogel, BioServices) in their home cage. In total, the animals received 1.5-2 ml of water per day, which corresponds to 50-65% of their ad libitum consumption, while ensuring that they did not lose more than 10% of their weight. Each mouse received 6-8 g of Hydrogel (ad libitum) during the weekend. This water restriction protocol was maintained throughout behavioral training and until the end of behavioral testing.Go/No-Go vibrotactile task training and testingHabituated mice (8 weeks old) were trained to associate vibrotactile stimulus delivery (pure sinusoid, 500 ms duration, 15µm amplitude, 40 Hz frequency) with a water reward (8 µl). Go trials consisted of stimulus delivery followed by a 2 s response window during which the mice could lick to receive the reward. No-Go (catch) trials included no stimulation and licking during these trials resulted in a 5 s timeout before the beginning of the following trial. Inter-trial intervals were variable (5-10 s). Training was subdivided in 3 phases: (a) automatic water delivery at the beginning of the response window (b) Pre-training: lick-triggered water delivery during Go trials and timeout delivery during No-Go trials (c) Training: lick-triggered water delivery during Go trials when the mouse did not lick during the inter-trial interval (3-8 s) before the stimulation. No stimulus delivery and timeout (5 s) if the animal licked during the inter-trial interval (3-8 s) before the stimulus and timeout delivery (5 s) during No-Go trials. All sessions consisted of 300 trials with 70-80% Go and 20-30% No-Go proportion. All trials were presented in a pseudorandom sequence. Pilot experiments with an extra sensor to monitor forepaw placement confirmed that the mice did not remove their forepaws from the bar before stimulus delivery. To complete Pre-training, mice needed to complete 80% of successful Go trials and reduce their spontaneous licking during No-Go trials below 40%. To complete Training, mice needed to reach the criterion of more than 80% successful Go-trials and less than 30% unsuccessful No-Go trials as an average for 3 consecutive days. All mice that fulfilled this criterion were tested for the detection of vibrotactile stimuli of differing amplitudes that they haven’t experienced before. During testing a 90% Go:10% No-Go ratio was delivered. Go trials consisted of 6 vibrotactile stimuli (pure sinusoid, 500 ms duration, 10 Hz frequency, 2-4-6-8-10-12 µm amplitude), delivered in a pseudorandom manner.Go/No-Go vibrotactile task analysisAll analysis was performed with custom-made Python scripts. Behavior was quantified based on the lick events and four main outcomes were measured: Hit and Miss rate for successful and unsuccessful Go trials respectively (number of Hit or Miss divided by the total number of Go trials where a stimulus was delivered), Correct Rejection and False Alarm rate for successful and unsuccessful No-Go trials respectively (number of Correct Rejections or False alarms divided by the total number of No-Go trials). Prestimulus spontaneous licking was calculated as Timeout events during Go trials divided by the total number of Go trials. Training duration was calculated based on the total number of days each animal passed in pre-training and training. For testing, only sessions with less than 40% unsuccessful No-Go trials were analyzed. Psychometric curves were fitted on the Hit rate for each stimulus amplitude using a general linear model. An average of 140 repetitions for each amplitude was used to calculate Hit rates. Detection thresholds were calculated based on the psychometric curves, as the stimulus that the mice detect 50% of the time.Adhesive tape removal testMice (11-13 weeks old) were handled for one day and underwent three days of gradual habituation to restriction and to a round Plexiglas arena (14 cm diameter) Two patches of adhesive tape were attached to their forepaws as described before (Bouet et al., 2009). The time before the first contact of each tape and the total time necessary for their removal was calculated manually using a Python script.<br>Two-photon calcium imagingSetupMeasurements were performed with a Femtonics FEMTOSmart microscope coupled with a widely tunable, femtosecond Ti:Sapphire Laser (Chameleon Ultra, Coherent) pumped by a solid-state laser (Verdi 18W, Coherent). Excitation at 920 nm was used for GCaMP8m and at 1000 nm for mRuby3and two photomultiplier tubes were used for the collection of green and red fluorescence. Laser power was modulated with pockels cells. A 20x/1 NA objective (Zeiss) was used to obtain512 × 512 pixel images covering 480 × 480 μm of cortex were acquired at 30.96 Hz using the resonant scanner system (Femtonics). Measurements were written as series of 32 bit images and saved as ome files. Analog and digital inputs sent by Bpod and the piezoelectric sensor controller were collected and saved as timestamps by the microscope and were used to synchronize the behavioral events with the calcium traces from imaging.