Data are available upon reasonable request from the lead contact, David Margolis (david.margolis@rutgers.edu) and at github.com/margolislab.

All work involving animals including housing, surgery, behavioral experimentation, and euthanasia was approved by the Rutgers Institutional Animal Care and Use Committee (protocol #: 999900197). Mice were group housed in a reverse light cycle room (lights on from 20:00 to 08:00) with food and water available ad libitum with the exception of mice undergoing water restriction during behavioral experiments. All handling and experiments were conducted within the dark phase of this light cycle. Regardless of their experimental designation, all experimental animals underwent a unilateral AAV injection or a simultaneous AAV injection and cannula implant between –5 - 65 days (average: 52.37 days, range: –7 - 61 days). Briefly, male and female double transgenic mice were used for electrophysiology experiments. To identify specific neuronal populations during electrophysiology, Ai14 mice (cre-dependent tdTomato; The Jackson Laboratory, #007914) were crossed with either (1) D1-SPN cre (B6.FVB(Cg)-Tg(DrD1cre)EY262Gsat/Mmucd; MMRRC, #030989), (2) D2-SPN cre (B6.FVB(Cg)-Tg(Adora2a-cre)KG139Gsat/Mmucd; MMRRC, #036158), or (3) PV-cre (B6.129P2-Pvalb^tm1(cre)Arbr/J; The Jackson Laboratory, #017320) mice. This permitted red fluorescence in the specific cell population for visual identification via epifluorescent illumination. Animals designated for electrophysiology were euthanized between–2 - 4.5 months. Both male and female wild-type mice (The Jackson Laboratory #000664) were used for fiber photometry and optogenetic experiments. To motivate behavioral performance, daily water intake was restricted to ∼1.5mL per mouse per day. Body weight was carefully controlled and never permitted to drop below 80% of a calculated baseline value.¹⁶⁹ The number of male and female mice were as follows, by experiment type: 6 male, 4 female (electrophysiology); 3 male, 2 female (fiber photometry); 4 male, 5 female (optogenetics). Data were not analyzed for sex differences.

Male and female double transgenic mice designated for electrophysiology experiments underwent a unilateral injection of channelrhodopsin-2 (ChR2; pAAV-hSyn-hChR2(H134R)-EYFP; Addgene #26973)¹⁶⁰ targeting the left posterior medial (POm) thalamic nucleus. Briefly, mice were anesthetized using isoflurane (4% induction, 1-2% maintenance) and moved into a stereotaxic apparatus (Stoelting/Kopf Instruments) containing a feedback-controlled heating pad on the base (maintained between 35-37°C; FHC). Ophthalmic ointment (Akorn) was applied to the eyes to prevent them from drying out. Ethiqa XR (3.25 mg/kg; Fidelis Animal Health) and Bupivacaine (0.25%, 0.1mL, Fresenius Kabi) were injected subcutaneously into the right flank and scalp, respectively. After, the scalp was sterilized by three cycles of Betadine (Purdue Products) and 70% ethanol, a midline incision was made. The exposed skull was cleared of fascia and leveled by confirming that bregma and lambda coordinates were on the same dorsoventral plane. A craniotomy was drilled (coordinates with respect to bregma: anteroposterior (AP) = -2.05 mm, mediolateral (ML) = +1.35 mm, dorsoventral (DV) = -3.25 mm) and the micropipette containing ChR2 was slowly lowered to the appropriate depth and permitted to sit for 5 minutes. Following this, ∼100 nL of ChR2 viral solution was injected over the course of ∼15 minutes via the Nanoject III system (Drummond Scientific). After an additional delay of 12 minutes, the micropipette was slowly raised, the scalp was sutured and secured with tissue glue. Immediately following surgery, mice were transferred to a clean cage on top of a heating blanket until ambulation was observed. Mice were continually monitored for 72 hours post-surgery. After this observation period, mice were permitted to recover for at least three weeks before undergoing electrophysiological experiments, permitting ChR2 expression into POm axon terminals in the striatum.

Mice were briefly induced (via 3% isoflurane), deeply anesthetized with an intraperitoneal injection of ketamine-xylazine (300/30 mg/kg, respectively), and transcardially perfused with recovery artificial cerebrospinal fluid (ACSF), which contains the following (in mM): NMDG 103, KCl 2.5, NaH₂PO₄ 1.2, NaHCO₃ 30, HEPES 20, Glucose 25, HCl (1N) 101, MgSO₄ 10, Thiourea 2, Sodium Pyruvate 3, N-Acetyl-L-Cysteine 12, and CaCl₂ 0.5 (saturated with 95% O₂ and 5% CO₂).^89,109 Following decapitation, the brain was rapidly extracted and submerged in recovery ACSF before being mounted onto a VT-1200S vibratome (Leica). The vibratome chamber was filled with oxygenated recovery ACSF, and 300µm slices were cut. Slices were immediately transferred to recovery ACSF that was heated to 35°C for ∼5 minutes. After, slices were transferred to RT external ACSF which contained (in mM): NaCl 124, KCl 2.5, NaHCO₃ 26, NaH₂PO₄ 1.2, Glucose 10, Sodium Pyruvate 3, MgCl₂ 1, and CaCl₂ 2 (saturated with 95% O₂ and 5% CO₂), and slices were allowed to recover for at least ∼45 minutes before recording. Once the hippocampus began to appear, vibratome sectioning was terminated, and the posterior tissue block containing the injection site was transferred into 10% neutral-buffered formalin for post-hoc confirmation.

Whole-cell patch clamp recordings were acquired from slices that were constantly superfused (2-4mL/min) with oxygenated external ACSF at ∼34°C. Slices and cells were visualized by infrared differential interference contrast (IR-DIC) microscopy using a CCD camera (Hamamatsu) mounted onto a BX-51WI upright microscope (Olympus) fitted with a swinging objective holder containing two switchable lenses: a 4X air lens and a 40X water-immersion lens. Patch pipettes (3-5 MΩ) were pulled from borosilicate glass micropipettes (2mm O.D., Warner Instruments) using a P-1000 horizontal puller (Sutter Instruments).

Current-clamp recordings were obtained from unidentified and identified striatal neurons in mice expressing tdTomato in either D1-SPNs, D2-SPNs, or PV cells within pDLS (-0.34 to - 1.22mm relative to bregma), which is known to receive POm projections.^55,56,105 The internal pipette solution for current-clamp experiments contained (in mM): K Methanesulfonate 130, KCl 10, HEPES 10, MgCl₂ 2, Na₂ATP 4, Na₂GTP 0.4, at pH 7.25 and 285-290 mOsm/L. Further, 2% biocytin was freshly dissolved in the internal solution on the recording day. ChR2 in the POm axon terminals was stimulated via illumination with a 2.5 ms, 470 nm LED light pulse (∼0.6 mW measured after the objective; Thorlabs) delivered through the 40X objective lens. The illumination spot size had a diameter of ∼550 µm. After patching, the internal solution was permitted to dialyze for ∼5 minutes. At this point, the resting membrane potential was recorded. All cells were held at -80 ± 2mV to ensure equal driving forces when studying synaptic strength and short-term synaptic plasticity. Baseline voltages that drifted outside of this range were excluded from analysis. Patched cells were held for ∼25-35 minutes while being run through a standardized set of protocols: (1) hyperpolarizing/depolarizing current steps, (2) single pulse (SP), (3) paired pulse ratio (PPR), and (4) train stimulation. Occasionally, unidentified cells within the same FOV were sequentially patched following the initial identified cell patch to control for injection site volume and location. After breaking in, the cell was allowed to recover for ∼5 minutes before being subjected to hyperpolarizing and depolarizing current steps (-300 pA to +400 pA, 50 pA steps, 500 ms, 15 sweeps) for cell health and intrinsic parameter confirmation. For the SP protocol, a single 2.5 ms blue light pulse was presented once every 15s for 20 sweeps. For the PPR protocol, five 2.5 ms pulses, separated by 50 ms inter-pulse intervals (IPI), were presented once every 15s for 20 sweeps. For the train protocol, thirty 2.5 ms pulses, separated by 64.2 ms IPI (15 Hz), were presented once every 30s for 5 sweeps. While these protocols were being run, biocytin within the internal solution diffused into the cell for subsequent 3D morphological reconstructions. Data were acquired via a EPC10USB amplifier and digitized at 20 kHz in Patchmaster Next (HEKA). Liquid junction potential was not corrected in these traces.

All data were analyzed offline using custom-written MATLAB and Python scripts. DAT files from Patchmaster Next (HEKA) were imported, organized, and saved as a mat variable. The mat variable data was imported into Python for post-processing using the electrophysiology feature extraction librarIFEL) created by the Blue Brain Project.¹⁶¹ All pertinent intrinsic value parameters were calculated at the +350pA current step. Key values pertaining to every action potential in a sweep were calculatIia eFEL including the action potential threshold value and index, the peak value and index, and the corresponding minimum afterhyperpolarization (AHP) value and index. Each action potential threshold value and index were captured using a derivative threshold method (dV/dt ≥ 10mV/s). Action potential properties were assessed for all spikes in a sweep and averaged together. The action potential peak was defined as the difference between the action potential threshold and its maximum positive peak. Half-height width (HHW) and rise time were both calculated via interpolation. HHW was measured at 50% of the action potential peak, while rise time was measured from 10-90% of the action potential peak. AHP amplitude was calculated as the difference between the action potential threshold value and the minimum AHP value. Maximum frequency

For all optogenetically-evoked postsynaptic potentials (PSPs), a baseline measure was calculated by averaging the first 10,000 sampling points for each individual sweep. This measured value was subtracted from every value in each individual sweep, setting the baseline equal to zero. The maximum PSP amplitude, relative to the zero baseline value, was calculated from the average of 20 sweeps during SP and PPR protocols, and 5 sweeps during Train protocols. The latency to maximum PSP amplitude was measured as the difference between photostimulation onset and the maximum PSP index. For PPR and Train protocols, an exponential function was fitted to the decay component of each PSP, and their amplitudes were extracted after subtracting the decay of preceding PSPs.

For male and female wild-type in the designated fiber photometry cohort, a 400µm core optical cannula (ferrule OD = 2.5mm, length = 2mm, nA = 0.50; Thorlabs #CFM15L02) was implanted directly above the left pDLS along with a ∼100nL unilateral injection of axon-jGCaMP8s (pAAV-hSynapsin-axon-jGCaMP8s-P2A-mRuby3; Addgene #172921)¹⁶² into the ipsilateral POm. Male and female wild-type mice designated for optogenetic manipulation were split into two cohorts: (1) the No Stim or (2) the JAWS cohort. Both optogenetic cohorts were implanted with a 200µm core optical cannula (ferrule OD = 1.25mm, length = 2mm, nA = 0.50, CFMLC52L02, Thorlabs) above the left pDLS along with a ∼100nL AAV injection into the ipsilateral POm. However, the No Stim cohort was injected with the excitatory optogenetic actuator, ChR2 (pAAV-hSyn-hChR2(H134R)-EYFP, Addgene #26973),¹⁶⁰ while the JAWS cohort was injected with the inhibitory optogenetic actuator, JAWS (pAAV-hSyn-JAWS-KGC-GFP-ER2, Addgene #65014).^{123,124,125,163} Further, an HHMI head plate¹⁶⁴ was fitted to each mouse using methods previously described.^{89,117,165,166,167} Briefly, mice were anesthetized with isoflurane (4% induction, 0.8 – 1.5% maintenance) and mounted within a stereotaxic frame (Stoelting/Kopf Instruments) with a feedback-controlled heating pad (FHC) maintaining the body temperature between 35 – 37°C. Following administration of an analgesic (Ethiqa XR, 3.25mg/kg; Fidelis Animal Health) to the left flank and a local anesthetic (0.25% Bupivacaine; Fresenius Kabi) under the scalp, the scalp was sterilized with a triple cycle of Betadine (Purdue Products) followed by 70% ethanol (Fisher). A midline incision was made, and a circular piece of scalp was removed to expose the skull. Both lateral muscles and the nuchal muscle were separated from the skull. The skull was cleaned by gently scraping away the periosteum. A dental etch bonding agent (iBond; Heraeus Kulzer) was applied to the clean skull and cured with blue light for 20s twice. A ring of dental composite (Charisma; Heraeus Kulzer) was applied to the outer edge of the skull and cured with blue light. After ensuring that bregma and lambda coordinates were equal in the dorsoventral plane, two craniotomies were made: one above the left posterior striatum (AP = -0.80mm, ML = +2.8mm, DV = 1.8mm) for cannula implantation and the other above left POm (AP = -2.05mm, ML = +1.35mm, DV = -3.25mm) for the corresponding AAV injection. The AAV injection was always performed first. The micropipette containing axon-jGCaMP8s (fiber photometry cohort), ChR2 (No Stim cohort), or JAWS (JAWS cohort) was slowly lowered to the appropriate depth and permitted to sit for 5 minutes. ∼100nL of AAV solution was injected over the course of ∼15 minutes via the Nanoject III system (Drummond Scientific). After an additional delay of 12 minutes, the micropipette was slowly raised. Once the optic cannula was secured in the stereotaxic frame and lowered to the appropriate DV coordinate, it was secured with dental composite (Tetric Evoflow; Heraeus Kulzer) that was cured with blue light. The HHMI headpost was delicately secured on the posterior area of the charisma ring with cyanoacrylate and dental composite. Finally, a single layer of dental composite was used to build and secure the rest of the headcap before being cured four times with blue light for 20s each. The scalp was closed around the headcap by using cyanoacrylate.

Immediately after the optical implant and viral injection surgery, mice were placed in a sterile, clean cage that was half-on/half-off a heating pad until ambulation was observed. Mice were diligently monitored for 72 hours post-surgery. After this monitoring period, mice were transferred to a clean cage on a ventilated rack for at least three weeks prior to handling and water restriction.

After a three-week waiting period, mice were placed under citric acid water restriction¹⁶⁸ during which mice were handled twice daily for one week. The bitterness of citric acid naturally causes mice to reduce their water consumption and, consequently, their weight while still having access to water. Initially, mice were acclimated to handling as researchers placed their hands into the cage for increasing amounts of time (e.g., 5 minutes to 10 minutes to 15 minutes). After mice became comfortable, they were held for increasing amounts of time (e.g., 2 minutes to 5 minutes to 10 minutes). Additionally, mice were acclimated to head fixation by holding their HHMI headposts (e.g., 30 seconds to 1 minute to 2 minutes), and they were allowed to freely explore the behavioral tube for 5 minutes per handling session. Finally, mice were headfixed for increasing amounts of time in the behavioral setup (e.g., 5 minutes to 10 minutes to 15 minutes). Water was provided via transfer pipette to comfortably acclimate mice to head fixation. The head-fix apparatus contained a tube (length = 15cm; inner diameter = 4cm) that was affixed to a custom metal platform (length = 17cm; width = 12cm). The platform also contained HHMI mounting arms and holders for head fixation.¹⁶⁴ During the final day of handling, mice were transitioned to full water restriction as this permits greater control over motivation level. During behavioral testing, daily water intake was limited to ∼1.5mL per day to motivate performance on the Go/NoGo paradigm described below. The baseline body weight was measured daily during water restriction, and overall body weight was not permitted to drop below 80% of the baseline weight, consistent with levels of restriction used to motivate performance.¹⁶⁹

Fiber photometry data were collected using a RZ10x lock-in amplifier within the Synapse suite (Tucker-Davis Technologies). This amplifier and accompanying Synapse software was used to control a custom-built optical benchtop through drivers (LEDD1B, Thorlabs) to modulate LED signals. Briefly, this optical benchtop consisted of a self-contained system of four 30mm cage cubes with integrated filter mounts (CM1-DCH/M, Thorlabs). A 405nm LED (M405L4, Thorlabs) and a 470nm LED (M470L3, Thorlabs) were mounted onto the first 30mm cage cube. The 405nm LED was used to extract the calcium-independent isosbestic signal, and the 470nm LED was used to acquire calcium-dependent axonal GCaMP signals during the Go/NoGo paradigm. The 405nm isosbestic signal was modulated at 210Hz, and the 470nm GCaMP signal was modulated at 330Hz. A 425nm dichroic longpass filter (DMLP425R, Thorlabs) in the first cage cube reflected the 405nm excitation light and permitted the 470nm light to pass through. As the light entered the second cage cube, both excitation lights were reflected by a 495nm dichroic longpass filter (495DCLP, 67-079, Edmund Optics) into the third cage cube. A 460/545nm bandpass filter (69013xv2, Chroma) reflected both excitation wavelengths down to the subject via a low autofluorescence patch cable (MAF3L1, core = 400µm, nA = 0.50, length = 1m, Thorlabs). This cable was attached onto the implanted optical cannula (see above) by a ceramic mating sleeve (Thorlabs). Isosbestic and axon-jGCaMP8s emissions were collected via the optic cannula and passed through the 460/545nm bandpass filter (69013xv2, Chroma)into the fourth. Finally, the emission fluorescence passed through the detection pathway to reach the RZ10x photosensors for online observation.

POm activity has been well correlated with whisking and pupil activity.^18,26 To analyze these dynamics, an LED driver controlled an IR spotlight that illuminated the contralateral eye and mystacial pad, and facial recordings were captured through an autofocusing USB webcam (NexiGo N660P) at ∼20fps within the Synapse software. To limit light pollution from outside sources (e.g., LED illumination within the brain/eye), an IR filter was placed in front of the webcam. Also, a shortpass emission filter at 750 nm (Chroma #ET750sp-2p8) was placed between the third and fourth cage cubes to prevenI recorded IR light from overloading the photosensors.

The Synapse software suite (Tucker-Davis Technologies) permitted the synchronous recording of both emitted (isosbestic and calcium) signals and video frames. Furthermore, the LabVIEW system-controlled paradigm-related components (e.g., texture movement, lick thresholds, trial type, trial outcome, etc.) and recorded the resulting behavioral parameters (e.g., licking activity, trial type, and trial outcome). To synchronize these two data streams for post-hoc analysis, TTL pulses relating to the texture arrival at its endpoint (which indicates the start of the presentation time window) from the LabVIEW system were captured within the Synapse software.

For the JAWS cohort, a high-powered 617nm LED (M617F2, Thorlabs) and current driver (LEDD1B, Thorlabs) were used for photoinactivation of POm axons in the striatum.^{123,124,125,163} LED stimulation was provided at a probability of 0.50 for every session from the start of the Learning phase until mice reached the Expert phase.²⁶ The light intensity was measured at ∼7mW at the tip of the fiber. Stimulation intensity was kept consistent between mice and days by measuring the intensity with an optical power meter (PMD-100D, Thorlabs) prior to the first session every day. Light was delivered to the thalamic afferents in pDLS through an optical fiber patchcord (M95L01, fiber core diameter: 200µm, length: 1m, nA: 0.50) connected to a 200µm core optical cannula (described above) via a mating sleeve (Thorlabs). A small piece of black heat shrink tubing (Qualtek) was placed over the cannula during LED testing to prevent stray light from illuminating the presented texture during the task. The LabVIEW system controlled a Pulse Pal¹⁷⁰ that activated the LED current driver to provide constant illumination for two seconds, evenly split 1s before and 1s after texture presentation, corresponding to the increased calcium activity observed during fiber photometry recordings.

For the No Stim cohort, a high-powered 470nm LED (Prizmatix) and current driver (Prizmatix) were used for optogenetic activation. Note that no LED stimulation was provided during the Learning or Expert phase. Testing occurred during the first 4 sessions after the 5 initial Shaping sessions and the last 4 sessions after mice reached Expert status. Sessions consisted of 50 baseline trials followed by 10 alternating blocks of 10 OFF and 10 ON trials. The light intensity was measured at ∼5mW at the tip of the fiber and was kept consistent between mice and days by measuring the intensity with an optical power meter (PMD-100D, Thorlabs) prior to the first session on testing days. Light was delivered to the thalamic afferents in pDLS through an optical fiber patchcord (M73L01, fiber core diameter: 200µm, length: 1m, nA: 0.50, Thorlabs) connected to a 200µm optical cannula (described above) via a mating sleeve (Thorlabs). A small piece of black heat shrink tubing (Qualtek) was placed over the cannula during LED testing to prevent stray light from illuminating the presented texture during the task. The LabVIEW system controlled a Pulse Pal¹⁷⁰ that activated the LED current driver to provide pulsed illumination for 2s at 15Hz (matching the electrophysiology train photostimulation paradigm), evenly split 1s before and 1s after texture presentation.

Headfixed, water restricted mice were trained to perform a whisker-based discrimination paradigm as two textures were presented unilaterally to the right whiskers in a randomized order based on a custom-written LabVIEW code (National instruments). This code used transistor-transistor logic (TTL) pulses to control all aspects of the paradigm including a water delivery spout connected to a piezo film sensor (MSP1006-ND; Measurement Specialties), and a motorized linear stage (T-LSM100A; Zaber) with a stepper motor (T-NM17A04; Zaber) containing two windmill arms holding two different sandpaper textures (Go texture = 100 grit sandpaper, P100; NoGo texture = 1200 grit sandpaper, P1200; 3M) as previously described.^{89,117,165,166,167} Mice were trained to discriminate between these two textures by licking the piezo film sensor spout. Mechanical spout displacement resulted in transient voltage changes, and a lick was defined as voltage changes crossing either an upper or lower threshold once. After a lick was detected within the appropriate response window, the LabVIEW software immediately delivered the appropriate output: for Go trials, mice received a small water reward via a solenoid valve (0127; Buerkert) through the piezo spout, and, for NoGo trials, mice received a timeout period with co-occurring white noise. This paradigm occurred within a darkened room to minimize non-tactile related cues. Both textures and the headfix apparatus were cleaned with 70% ethanol between each mouse. If mice were not performing the task, sessions could be ended early. Water could be automatically delivered (AutoReward or AR) by the experimenter following 20 consecutive trials without a response. Finally, a session could be terminated if a mouse did not lick when water was present on the end of the spout after three ARs.

Trials began with a 1000ms pre-task interval followed by a brief cue tone (100ms, 2039Hz) that accompanied windmill texture movement towards the mice. Once the windmill texture had reached a predetermined distance within the whisking field, mice had to respond within a 2000ms presentation time (PT) window. For the first 500ms of the PT window, a grace period was present where responses did not trigger appropriate outcomes to reduce impulsivity. If mice licked in response to the Go texture, the trial was considered a Hit and resulted in a water reward accompanied by a correct tone (2793Hz). If mice licked in response to the NoGo texture, the trial was considered a False Alarm (FA) and resulted in punishment parameters including a time-out period (12000ms) and an accompanying white noise during the time-out period. Air puffs were eschewed as a punishment parameter to permit continuous pupil dynamic recording. If mice did not lick to the Go or the NoGo texture, nothing occurred, and the trial was considered a Miss or a Correct Rejection (CR), respectively. Immediately after a lick was recorded or the 2000ms PT window had elapsed, the windmill texture retreated to its original position where the current texture either remained or the other texture was rotated into position. Finally, trials were separated by a 2000ms intertrial interval. Behavioral performance was tracked across texture discrimination sessions by computing multiple behaviorally-related parameters including Hit Rate, FA Rate, Sensitivity (d’) and Bias.¹⁷¹ For the fiber photometry cohort, the 405nm and 470nm LEDs provided constant illumination throughout all trials. For the JAWS cohort, LED photoinactivation occurred at a trial probability of 0.50 for every session from the start of the Learning until the end of the Expert phase.²⁶ For the No Stim cohort, LED stimulation did not occur during training (i.e., during either the Learning or Expert phases). It only occurred for 4 sessions after the initial 5 shaping sessions, and 4 sessions after mice reached expert status.

This whisker-based discrimination paradigm lasted up to 3 weeks. The FP cohort were only tested once per day to limit photobleaching. The JAWS and No Stim cohorts were tested twice daily. Each session consisted of 150 trials. All cohorts progressed through three behavioral phases (Shaping, Learning, and Expert) that were segmented into five analytical time points (Shaping, Early Learning, Late Learning, and Expert). The Shaping time point was the same for all mice. For the first three sessions, mice were acclimated to reliably trigger water delivery by licking the water spout. During these sessions, neither texture was presented to the whiskers. After, mice proceeded to texture discrimination training still under the Shaping phase. For the last two sessions, both textures were presented simultaneously with a Go texture probability of 0.90 and 0.75, respectively. Go and NoGo trials were interleaved in a pseudorandom order determined by the LabVIEW software. After the 0.75 probability session, mice progressed into the Learning phase. For all following sessions, the Go texture probability was set to 0.50 with a maximum of three consecutive presentations of the same texture. Discrete behavioral time points were determined as follows. The Early Learning time point was considered the first two sessions of the Learning phase. The Late Learning time point was considered the last two sessions of the Learning phase before achieving expert status. This occurred when mice had a Hit Rate ≥ 0.80 and a FA Rate ≤ 0.30 for two consecutive sessions. A strict sensitivity threshold was not used due to artificially increased sensitivity (discrimination) values as Hit Rate and/or FA Rate approach their extremes (e.g., see FPOm-18 sessions 8 to 15 in Figure 6). After discrimination training (i.e., achieving expert status), mice in the fiber photometry cohort were subjected to a single Reward session to assess calcium, licking, and pupil activity in the absence of texture input and licking-related outcomes. During this session, the Go trial probability remained at 0.50, and the Zaber motor moved the windmill texture holder towards the whisker field, but the textures were rotated out of whisker range. Further, the upper and lower thresholds were set so that licking could not trigger outcomes before the end of the PT window. A water reward was automatically delivered at the end of the PT window during Go trials only. A whisker trim control session was performed in a subset of mice to confirm that mice used their whiskers to discriminate as previously observed.⁸⁹

For electrophysiology experiments, the tissue block containing the injection site was stored overnight in 10% neutral-buffered formalin at 4°C. After, it was transferred into 0.2% PBS Azide at 4°C until sectioning. The tissue block was mounted onto a stage and sectioned in 0.1M PBS at a thickness of 100µm using a VT-1000 vibratome (Leica). Slices were mounted onto microscope slides using DAPI Fluoromount-G (Southern Biotech #0100-20) and coverslipped before confocal imaging.

Following all behavioral experiments, mice were anesthetized with an intraperitoneal injection of Ketamine-Xylazine (120mg/kg Ketamine, 24 mg/kg Xylazine) and transcardially perfused with PBS followed by 10% neutral-buffered formalin. The brain was delicately extricated and stored in 10% neutral-buffered formalin overnight at 4°C. Tissue was mounted onto a stage and sectioned at 100µm using a VT-1000 vibratome (Leica). Sections were mounted and coverslipped using DAPI Fluoromount-G (Southern Biotech #0100-20). Confocal images were acquired using a LSM800 confocal laser scanning microscope (Zeiss) for injection site location verification, cannula placement, and viral expression in POm axons within pDLS and stereotypical POm-cortical projections in S1 L1 and L5a of all experimental mice.^{4,25,26,47,49} All data were acquired using the Zen software suite (Zeiss).

Behavioral performance was tracked across texture discrimination sessions by computing multiple behaviorally-related parameters including Hit Rate, FA Rate, Sensitivity (d’) and Bias.¹⁷¹ Hit Rate was calculated as follows: [Hit / (Hit + Miss)], where hit is the number of correct Go trials and Miss is the number of incorrect Go trials. The FA Rate formula is similar to the Hit Rate formula except it replaces Hit with FA and Miss with CR: [FA / (FA + CR)]. Sensitivity illustrates the ability to discriminate between the signal (Go texture) and the noise (NoGo texture), and it is derived from signal detection theory.¹⁷¹ It is calculated as follows: [normalized inverse–(Hit Rate) - normalized inverse (FA Rate)]. Finally, Bias illustrates the overall responding bias, independent of trial type. It is calculated as follows: [0.5 * (normalized inverse (Hit Rate) + normalized inverse (FA Rate)].

After all data were captured, the recorded orofacial videos were analyzed using DeepLabCut, a deep learning model for pose estimation, to estimate pupil dynamics as mice learned to discriminate between the two textures.^118,119 Briefly, a model¹²⁰ was created with eight markers circumscribing the pupil, permitting the estimation of the approximate pupil area for each frame. All videos were cropped to a smaller dimension (230 x 275 pixels) that focused on each mouse’s face. For each mouse, one video was selected at varying behavioral time points, and 30 frames were extracted and manually labeled with the eight markers. Each marker corresponded to a pupil location: top, top right, right, bottom right, bottom, bottom left, left, and top left. Additionally, another marker was placed on a static location (e.g., the water spout) to label frames when blinking occurred. Overall, the initial training dataset contained 150 labeled frames, and the model was trained on this dataset with a ResNet50-based neural network for 250,000 iterations. After, the initial training videos were analyzed to assess the performance of the model from its marker estimations. 25 outlier frames from the initial training videos were extracted, manually corrected, and merged with the initial dataset. The model was trained again on this 275 labeled frame dataset with a ResNet50-based neural network for 250,000 iterations. After evaluating the network, the train error was calculated at 1.24 pixels, and the test error was measured at 1.25 pixels. Once the model was successfully trained, all behavioral videos of the five mice were analyzed. To estimate pupil area, a python library (scikit-image)¹⁷² was used to fit an ellipse on the estimations of the eight markers generated by DeepLabCut. This ellipse model was used to predict the values of the vertices and co-vertices on the ellipse, permitting the calculation of the major and minor axes. Thus, these measurements were used to approximate the area of the pupil for each frame. To detect blinking behavior, the maximum pupil size in non-blinking conditions was calculated and used as a threshold to identify abnormally high pupil predictions. As such, pupil areas greater than 450 pixels were removed via interpolation. The pupil area data were saved as a csv file for importing into MATLAB.

Custom-written MATLAB scripts were used for post processing of the fluorescent signals. Photobleaching was corrected in both the isosbestic and GCaMP signals using detrended lines of best fit and subtracting the line from all values. After, the isosbestic and GCaMP median absolute deviation of z-score (ZMAD) signals were calculated before subtracting the GCaMP signal from the isosbestic to remove calcium-independent artifacts.

A custom-written MATLAB script imported the lick-related and overall responding data, the processed ZMAD GCaMP signal, and the processed pupil csv for alignment. The overall responding data (containing trial elements such as time of PT window start which is the TTL flag within Synapse) was converted from UNIX timecode into seconds to match the Synapse (containing the pupil video and processed GCaMP signal) time. Pupil data was resampled to align with GCaMP signal using rational fraction approximation. Furthermore, the length of each trial, as determined by the overall responding data, was recorded and used to capture the GCaMP and pupil data within each trial window. Licks were identified by setting upper and lower thresholds, and detecting when either was crossed. At this point, all parameters (e.g., GCaMP, pupil, and licking activity) were captured and aligned within the time window of each trial. These parameters could now be segmented by trial type (e.g., Go vs. NoGo texture) and by trial outcome (e.g., Hit, Miss, FA, CR) for more advanced analysis.

auROC is an analysis commonly applied to calcium imaging data to characterize the stereotypy of neuronal responses.^121,122 Briefly, a baseline window is set within a non-task-related component of the overall calcium signal and compared to the rest of the signal via a sliding window. The maximal value of each signal is captured at each behavioral time point and averaged across the cohort. auROC values equal to 0.50 indicate no differences between the baseline signal and the task-related signal.

To assess longitudinal changes in calcium activity, two windows were established: a Control Window located from trial start to the sound cue indicating trial start, and a Target Window located 2s before and after PT start (overall = 4s). For each trial, all calcium peaks were measured, and onl^y peaks ≥ 90th percentile with a minimum peak prominence of 2 were selected. Finally, the maximum values within the Control and Target Windows (if present) were captured and averaged for every session.¹⁷³

All data are reported as mean ± SEM unless otherwise noted. Statistical analyses were performed in GraphPad Prism (USA). All data were tested using the Shapiro-Wilk normality test. The means of different data distributions were analyzed and compared using two-tailed Student’s t-test (Figures S1O, S1R, 2E FA Rate, 2E Bias, 3F, 4E Hit Rate, 4E Sensitivity, 4E Bias, 4G FA Rate, 4G Bias, 4K Learning/Expert Hit Rate, 4K Learning/Expert FA Rate, 4K Learning/Expert Sensitivity, 4K Learning/Expert Bias, 4L, S5C Hit Rate, S5C FA Rate, S5C Sensitivity, S5C Bias), Wilcoxon signed rank test (Figures S1N, S1Q, 2E Hit Rate, 2E Sensitivity, 4E FA Rate, 4G Hit Rate, 4G Sensitivity), ordinary one-way ANOVA with Tukey’s multiple comparison correction (Figures S1G, S1L), Kruksal-Wallis with Dunn’s multiple comparison correction (Figures 1E, 1F, 1I, 1L, S1H, S1I, S1J, S1K, 4I), linear regression (Figure S1E), Repeated measures one-way ANOVA with Tukey’s multiple comparison correction (Figures 3B, 3C), Repeated measures mixed-effects analysis with Tukey’s multiple comparison correction (Figures S4D Top, S4D Bottom), Repeated measures two-way ANOVA with Sidak’s multiple comparison correction (Figures 3K, 3L), Repeated measures mixed-effects analysis with Sidak’s multiple comparison correction (Figure 3Q). For all statistical tests, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.