Stereotaxic virus injections for electrophysiological experiments were performed as previously described using C57BL/6J mice between postnatal day 23–28 (Koster and Sherman, 2024). For optogenetics experiments in which cerebellar inputs to zona incerta were measured, a 250 nL (40 nL/min) infusion of AAV5-hSyn-hChR2(H134R)-EYFP (Addgene; packaged by UNC Vector Core, Chapel Hill, NC) was made into the right deep cerebellar nuclei (–1.5 ML; –5.8 AP; –3.2 DV Paxinos and Franklin, 2019) using a 1 mL Hamilton syringe (catalog: 65458–02) with a flat needle tip. Next, a small infusion (40 nL total, at 7 nL/min) of fluoro-Ruby (Thermo Fisher Scientific, Waltham, MA; catalog #: D1817) was made into left posterior medial thalamus using a 0.5 mL Hamilton syringe (Hamilton Company, Reno, NV; catalog: 65457–01). Following a two-week incubation, animals were sacrificed for recordings in acute slices (see Slice electrophysiology).

For anterograde transsynaptic labeling experiments, a dual-injection approach was utilized to specifically label the outputs of neurons in the zona incerta that receive input from the contralateral cerebellar nuclei. Flpo-recombinase (AAV1-EF1a-Flpo, item#: 55637, Addgene, Watertown, MA) was injected into left deep cerebellar nuclei as above (+1.5 ML; –5.8 AP; –3.2 DV), followed by an infusion of AAV1-Flp-on ChR2-EYFP (hSyn-Coff/Fon hChR2(H134R)-EYFP, Addgene; packaged by UNC Vector Core) into right zona incerta (–1.8 ML; –2.0 AP; –4.0 DV). Following a 5 week incubation period – the period required for anterograde transport of the AAV1 virus injected into zona incerta – animals were transcardially perfused for histology (see Tissue preparation for fluorescence microscopy and immunohistochemistry).

To visualize potential overlap between cerebellar terminals and cells in thalamic nuclei (i.e. the thalamic reticular nucleus) that project to somatosensory thalamus (ventral posteromedial nucleus and posterior medial thalamus), an orthograde virus (AAV5-hSyn-hChR2(H134R)EYFP, Addgene) was injected into the right deep cerebellar nuclei (–1.5 ML; –5.8 AP; 3.2 DV) and fluoro-Gold (2% w/v in sterile saline, Fluorochrome LLC, Denver, CO) was injected into the left somatosensory thalamus. Following a 2 week incubation, animals were transcardially perfused for histology (see Tissue preparation for fluorescence microscopy and immunohistochemistry).

Viral injections were performed to express channelrhodopsin-2 in cerebellar climbing fibers; fluorescent calcium indicators in cerebellar Purkinje cells or primary somatosensory (S1; barrel) cortex neurons; tdTomato, activating DREADDs, or inactivating DREADDs in S1 cortex; and inactivating DREADDs in interneurons in zona incerta. With respect to the inferior olive injection site (conferring viral expression in climbing fibers terminating in the contralateral cerebellar hemisphere), injections into cerebellar cortex were performed contralaterally, and injections into zona incerta and somatosensory cortex were performed ipsilaterally.

To express channelrhodopsin-2 in cerebellar climbing fibers, ChR2-EYFP (AAV9-CaMKIIahChR2(H134R)-EYFP; Addgene #26969-AAV9) or ChR2-mCherry (pAAV9-CaMKIIahChR2(H134R)-mCherry; Addgene #26975-AAV9) was injected into the left inferior olive as follows: after surgical preparation (see Two-photon calcium imaging: Cranial window surgeries), tissues attached to the occipital bone were surgically detached, followed by opening of the posterior atlanto-occipital membrane to expose the dorsal medulla. A 5 μL Hamilton Neuros syringe (catalog: 65460–03) with a 33-gauge beveled needle tip (catalog: 65461–02) was inserted into the left medulla at an angle of 58–59° to the perpendicular line (Figure 1—figure supplement 1A), 0.75 mm laterally and 1.70 mm deep from the surface of the medulla as previously described (Miyazaki and Watanabe, 2011). A total volume of 400 nL was infused at a rate of 80 nL/min, and the syringe was held in place for 5 min before removal. ChR2-EYFP or ChR2-mCherry was chosen based on the excitation and emission spectra of other fluorophores expressed in the same animal to maximize signal separation when conducting post-hoc confocal imaging.

Stereotaxic virus injections for imaging of Purkinje cell dendrites were administered as previously described using P80-120 adult C57BL/6 J mice (Lin et al., 2024). A virus mixture was prepared with 0.5% L7-Cre (pAAV-sL7-Cre-HA-WPRE, Addgene plasmid #:204488, packaged as AAV1 by the Princeton Neuroscience Institute Viral Core Facility, Princeton, NJ), and 20% Cre-dependent GCaMP6f (AAV1.CAG.Flex.GCaMP6f.WPRE.SV40; Addgene, #100835-AAV1) in saline solution. A glass pipette with a tip diameter of ~300 μm was prepared by a puller (model P-97, Sutter Instrument, Novato, CA) and used to inject a total volume of 1800 nL into two separate injection sites: 900 nL were injected into right medial Crus I (–1.8 ML; –7 AP; –1.8 DV), followed by a 900 nL injection into right medial Crus II (–2.5 ML; –7 AP; –2.2 DV). The pipette carrying the AAV was held in place for 5 min before withdrawal.

For DREADD suppression of PV interneurons in zona incerta, a 1 mL Hamilton syringe was used to infuse Cre-dependent hm4D(Gi) (AAV5-hSyn-DIO-hM4D(Gi)-mCherry; Addgene #44362-AAV5) into the left zona incerta (1.8 ML; –2.0 AP; –4.0 DV) of B6 PV^cre animals.

To express GCaMP6f in primary somatosensory (barrel) cortex neurons in C57BL/6 J, Sst-IRES-Cre^tdTomato, and Vip-IRES-Cre^tdTomato animals, a 5 μL Hamilton Neuros syringe with a 33-gauge beveled needle tip was used to infuse a total volume of 800 nL of GCaMP6f (AAV1.Syn.GCaMP6f.WPRE.SV40; Addgene #100837-AAV1) at two separate injection sites: 400 nL were injected at –1.2 AP; 2.5 ML, and 400 nL at –1.5 AP; 3 ML. At each injection site, 200 nL were first injected 300 μm below the pial surface, followed by 200 nL at 150 μm below the pial surface. The injections were performed at a rate of 100 nL/min, and the syringe was held in place for 5 min before removal. To express GCaMP6f in barrel cortex neurons with co-expression of tdTomato in parvalbumin (PV)-, SST-, and VIP-expressing interneurons in B6 PV^cre, Sst-IRES-Cre, or Vip-IRES-Cre animals, respectively, GCaMP6f (Addgene #100837-AAV1) and Cre-dependent tdTomato (AAV9-FLEX-tdTomato; Addgene #28306-AAV9) were co-injected using the same technique. To optimize co-expression of the viruses, GCaMP6f with tdTomato was mixed at a 3:1 volume ratio. For DREADD activation or suppression of VIP, SST, or PV interneurons, Cre-dependent hM3D(Gq) (AAV5-hSyn-DIO-hM3D(Gq)-mCherry; Addgene #44361-AAV5) or Cre-dependent hM4D(Gi) (AAV5-hSyn-DIO-hM4D(Gi)-mCherry; Addgene #44362-AAV5) were used in place of tdTomato, respectively.

For all two-photon imaging experiments, recordings began after a 4 week incubation period after the final virus injection (see Two-photon calcium imaging: cranial window surgeries for detail).

Z-stack confocal images were taken at 5x (Olympus MPlan N 0.1NA, air; Olympus Corporation, Japan), 10x (Zeiss Achroplan 0.25NA, air; Carl Zeiss AG, Germany), 20x (Olympus UMPlanFL N 0.5NA, water), 40x (Zeiss EC Plan-Neofluar 1.3NA, oil immersion), and 63x (Zeiss W Plan-Apochromat, water) magnifications with a Zeiss LSM 900 Axio Examiner.Z1 scope. A subset of images (Figure 1E, Figure 1—figure supplement 1B) were taken with a Zeiss LSM 5 Exciter, Axioskop 2 (using the 20x Olympus UMPlanFL and 40x Zeiss EC Plan-Neofluar objectives, respectively). The images in Figure 6D (shown with channel separation in Figure 6—figure supplement 3) and Figure 6—figure supplement 2 were taken with a second Zeiss LSM 900 confocal microscope. To facilitate direct comparison, images shown in series (as in Figure 6C, Figure 6—figure supplement 2D and E) were taken with identical hardware and software configurations, available in the image metadata.

To stitch multiple images (as in Figure 1B and D, Figure 2D–F (right), Figure 4B and F, Figure 6B and C, (top), Figure 1—figure supplement 1E, Figure 6—figure supplement 1A, Figure 6—figure supplement 1B (top and bottom left), and Figure 6—figure supplement 2C–E), a series of pairwise stitches were performed using Fiji (RRID:SCR_002285). Figure 1B, Figure 1—figure supplement 1A (top) each consisted of four images (each 5x magnification, 1024×1024 pixels). Figure 1D consisted of 12 images (each 5x magnification, 512×512 pixels). Figure 2D (right) consisted of 10 images (each 10x magnification, 1024×1024 pixels). Figure 2E and F (right) each consisted of eight images (each 10x magnification, 512×512 pixels). Figure 4B and F each consisted of two images (each 20x magnification, 512×512 pixels). Figure 6B (also shown as the first image in Figure 6—figure supplement 3D), and all images in Figure 6—figure supplement 2D and E each consisted of six images (each 5x magnification, 512×512 pixels). Images in Figure 6C each consisted of nine images (each 10x magnification, 512×512 pixels). Figure 1—figure supplement 1E consisted of eight images (each 5x magnification, 1024×1024 pixels). Figure 6—figure supplement 2C consisted of 10 images (each 40x magnification, 512×512 pixels). Note that a subset of the final stitched images displayed in figures are cropped to show relevant detail.

Channel colors set during image acquisition followed conventional emission wavelength assignments (i.e. red to indicate tdTomato, green to indicate GCaMP6f, etc.). To illustrate interneuron subtypes and continuity across experiments (e.g. cyan to indicate ChR2 expression across all optogenetic stimulation experiments), channel colors were specified post-imaging using Fiji by separating channels and selecting the appropriate color when merging, ignoring source LUTs. When necessary, post-hoc brightness and contrast adjustments were applied linearly to an entire image – after stitching, if applicable – using Fiji. To facilitate direct comparison, identical linear brightness/contrast adjustments were made across all images shown in series (as in Figure 6C, Figure 6—figure supplement 2D, and Figure 6—figure supplement 2E).

To measure terminal density across the sections shown in Figure 6C, Fiji was used to rotate each image such that the axis separating ventral from dorsal zona incerta was horizontal. A 400 µm × 1 mm region of interest (ROI) was drawn around the zona incerta, and each row of pixels within this ROI was separated to facilitate calculation of the integrated density across each row of pixels. A similar method was used to quantify the terminal density of cerebellar inputs to zona incerta as in Figure 6—figure supplement 3. Briefly, images of sections matched for their rostro-caudal location were rotated as before. Then, five line-ROIs oriented vertically, each being 200 µm wide, were positioned side-by-side across the mediolateral axis of the zona incerta, and the spatial pattern of fluorescence along these lines was measured by calculating the mean gray value along their length (which was roughly 400 µm from the dorsal boundary to the ventral boundary). These were averaged for each animal used. Shading represents SEM for both measurements.

For recordings in acute slice, injected animals (see Stereotaxic injections: For electrophysiological experiments) were deeply anesthetized using the isoflurane drop method in a bell jar (with a raised platform for the animal) and immediately transcardially perfused with 8–10 mL of ice-cold oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid, which contained the following (in mM): 125 NaCl, 25 NaHCO₃, 3 KCl, 1.25 NaH₂PO₄, 1 MgCl₂, 2 CaCl₂, and 25 glucose. The brain was then extracted, glue-mounted on a vibratome (Leica Biosystems, Germany) platform and blocked for coronal slices using an agarose (5%) cube and sliced in the same solution (ice-cold). Slices were cut to 365 µm thickness. The brain slices were then transferred to 32–34°C oxygenated artificial cerebrospinal fluid that was allowed to return to room temperature over the course of 1 hr, which constituted the slice recovery period.

Slices containing the zona incerta and terminals from the cerebellar nuclei inputs were visualized using differential interference contrast with a Zeiss Axioskop 2FS microscope. Fluorescence from ChR2-EYFP expression and from retrograde fluoro-Ruby labeling was confirmed using the 5x air objective and guided recording locations in zona incerta. Recordings were made with a Multiclamp 700B amplifier and pCLAMP software (Molecular Devices, San Jose, CA). Recording glass pipettes with 7–9 MΩ resistance were filled with an internal solution as follows (in mM): 117 K-gluconate, 13 KCl, 1 MgCl₂, 0.07 CaCl₂, 10 Hepes, 0.1 EGTA, 2 Na₂-ATP, 0.4 Na-GTP, pH 7.3, 290 mOsm. Incertal cells whose morphology was clearly revealed by strong red fluorescence, indicating retrograde labeling from posterior medial thalamus, were targeted for recordings. Bath application of TTX (1 µM) was performed in a subset of recorded cells to verify the monosynaptic nature of the cerebellar input to zona incerta.

Optogenetic stimulation was delivered using a 355 nm laser (DPSS: 3505–100), controlled with galvanometer mirrors (Novanta, Bedford, MA) focused on the slice through a 5x air objective using custom software in MATLAB (The MathWorks, Inc, Natick, MA; RRID:SCR_001622). Focal photostimulation of the ChR2-expressing synaptic terminals in zona incerta was performed at 10 Hz (four pulses of 1 ms duration at 100 ms interstimulus interval).

Electrophysiological data were collected using custom MATLAB software and analyzed using Graphpad Prism (GraphPad by Dotmatics, Boston, MA; version 7.0; RRID:SCR_002798). The amplitude of excitatory responses (i.e. the first response in the stimulus train) to stimulation pulses was measured by subtracting the average value for 20 ms before the delivery of a pulse (baseline) from the maximum value of the peak in current clamp at resting membrane potential. Latency of the optogenetic response was calculated by measuring the delay in milliseconds from the onset of the first optogenetic stimulus to the moment the resulting response reached >10% of its maximum amplitude.

To minimize overall recovery burden, surgeries were performed in two stages: (1) inferior olive virus injection and headframe installation, and (2) cerebellar virus injection and cerebellar cranial window installation as previously described (Lin et al., 2024). In the first stage, mice (P60-90) were deeply anesthetized using 1–2% isoflurane and clamped by ear bars at the external acoustic foramen. The line between the clamping point and maxilla was set parallel to the horizontal plane. After trimming the fur on top of the skull with clippers, the surgical site was prepared by applying betadine and 70% ethanol three times in an alternating fashion. Meloxicam (2 mg/kg), extended-release buprenorphine (0.1 mg/kg), and 0.5 mL saline were administered subcutaneously, and depth of anesthesia was confirmed via tactile stimulation of the toe before making a 20–25 mm incision in the skin to reveal the occipital bone and attached tissues. After opening of the posterior atlantooccipital membrane and channelrhodopsin-2 virus injection into the left inferior olive (see Stereotaxic injections: For two-photon calcium imaging experiments), tissues were reattached to the occipital bone using instant adhesive, ensuring adequate space for later implantation of the cranial window over the right cerebellar cortex. A custom titanium headframe (H. E. Parmer Company, Nashville, TN) was secured with dental cement (Stoelting Co., Wood Dale, IL), completing the first stage.

After monitoring recovery for a period of 4 days, mice underwent the second surgery. A circular craniotomy over right cerebellar cortex with a diameter of 4 mm, centered at –2.7 ML; –6.9 AP, was performed using a dental drill. The dura was carefully removed to expose lobules Crus I and anterior Crus II before administering GCaMP6f injections (see Stereotaxic injections: For two-photon calcium imaging experiments). Following injection, a two-layer glass window was installed using C&B Metabond dental cement (Patterson Dental Company, Saint Paul, MN), completing the second stage. The glass windows consisted of a 4 mm glass window (Tower Optical Corp, Boynton Beach, FL, # 4540–0495) adhered to a 5 mm glass window (Warner Instruments, Holliston, MA # CS-5R) using ultraviolet light-activated glue (Norland Optical Adhesive 71; Norland Products Inc, Jamesburg, NJ). Experiments were conducted 4.5 weeks after inferior olive injection and 4 weeks after GCaMP6f injection.

Surgeries were again separated into two stages: (1) inferior olive virus injection, headframe installation, and cerebellar cranial window installation; and (2) S1 cranial window installation and virus injection. First, channelrhodopsin-2 was injected into the left inferior olive as above (see Cranial window surgeries for imaging of Purkinje cell dendrites and Stereotaxic injections: For two-photon calcium imaging). After reattaching tissues to the occipital bone using instant adhesive, a dental drill was used to perform a circular craniotomy over right cerebellar cortex (lobules Crus I and II), followed by removal of the dura and installation of the glass window as above. Finally, the custom titanium headframe was secured using dental cement, completing the first stage.

After monitoring recovery for a period of 4 days, mice underwent the second surgery. After surgically detaching a small portion of the left temporalis muscle attached to the parietal bone, a second circular craniotomy over left primary somatosensory cortex with a diameter of 4 mm (centered at –1.5 AP; 3 ML) was performed. The dura was removed to expose the neocortical surface before administering the appropriate viral injections (see Stereotaxic injections: For two-photon calcium imaging experiments). Following injection, a glass window was installed over the craniotomy using dental cement. To ensure optical isolation during recordings, a custom sheath was used to couple an optical patch cable (used for LED stimulation of cerebellar climbing fibers; see Optogenetic and tactile stimulation) to the cerebellar window. This sheath was adhered over the cerebellar window using Metabond dental cement, completing the second stage. For experiments involving DREADD manipulation of neurons in zona incerta, viral injections into left zona incerta were administered three days prior to the first surgery. Thus, experiments were conducted approximately 5 weeks after zona incerta injection, 4.5 weeks after inferior olive injection, and 4 weeks after primary somatosensory cortex injections.

After completion of surgeries, postoperative care was provided for 5–7 days. Once animals exhibited exploratory behavior without signs of pain or distress, habituation began with handling of mice for 15 mins and transportation from the animal facility to the laboratory. After two days, anxiety during handling typically diminished, and the handling period increased to 30 min in the vicinity of the recording area. Following two days of handling, the mice were introduced to a free-running treadmill and head restraint via clamping of the head frame on left and right sides. The duration of time spent on the treadmill increased from 10 min to 2 hr per day depending on the mouse’s comfort. The relative time spent running on the treadmill typically decreased after two days. To habituate mice to air puff stimulation during this time, receptive field mapping of the field of view was conducted by delivering 8 psi air puffs through a capillary tube while observing epifluorescence signals and calcium responses in real time. Air puffs were delivered at irregular frequencies, with at least 30 s between stimuli, to avoid influencing subsequent plasticity experiments. In total, mice were habituated for a period of 1–2 weeks until they showed no aversive responses to air puff stimulation, spent the majority of time in the recording environment at rest, and reduced movement initiation (whisking/running) with air puff stimulation. Animals used for DREADD manipulation experiments were familiarized with the DCZ injection procedure by receiving DMSO/saline injections during habituation for two days prior to recording.

To optogenetically stimulate cerebellar climbing fibers, the tip of a Ø200 µm core, 0.39 NA rotary joint patch cable (Thorlabs catalog: RJPSL2; Thorlabs, Inc, Newton, NJ) was inserted into the sheath attached to the cranial window over the right cerebellar cortex (see Two-photon calcium imaging: Cranial window surgeries). For experiments in which Purkinje cell responses to optogenetic climbing fiber stimulation were also measured, the patch cable was inserted into a custom light shield that fit over both the objective and the cable. The tip of the patch cable was centered over Crus II in all optogenetic stimulation experiments (–2.7 ML; –6.9 AP). Blue light pulses were delivered with a 470 nm Fiber-Coupled LED (Thorlabs catalog: M470F3). The absolute power output was adjusted to 2.5 mW. Given the tip of the LED was positioned 1 mm over the surface of the brain (0.7 mm over the surface of the optical window), the estimated spot size was 0.6 mm and the estimated power density was 8 mW/mm² at the pial surface. To deliver whisker stimuli, a 0.86 mm diameter glass capillary tube was positioned over the right C-row whiskers 5 mm from the right whisker pad at an angle of 45° in the X and Y planes (adjusted slightly during receptive field mapping for each animal). Air puffs were delivered at 8 psi (Picospritzer III, Parker Hannifin, Cleveland, OH). For all imaging sessions, calcium activity was measured in trials lasting 20 s, with a stimulus delivered once per trial. Trials began at random intervals, with an interval of at least 5 s between trials. When testing Purkinje cell responses to optogenetic climbing fiber stimulation, 470 nm pulses were delivered with the following durations: 1×20 ms, 1×50 ms, 3×15 ms at 8 Hz, and (3×15 ms at 8 Hz)*2 with a 250 ms interval between epochs. The 1×50 ms light pulse was selected for all subsequent experiments given this stimulation duration evoked responses resembling spontaneous events in the same Purkinje cells (Figure 1—figure supplement 1). To test the basic transmission of the climbing fiber signal to S1 (i.e. the immediate influence of optogenetic stimulation of climbing fibers on acute responses to whisker stimulation shown in Figure 2), 100 ms air puff test pulses were delivered with or without optogenetic co-stimulation of climbing fibers. For paired test pulse stimuli, the 50 ms blue light stimulus was delivered with a delay of 45 ms with respect to the onset of the whisker stimulus. This delay was chosen to mimic the natural latency of climbing fiber responses to whisker stimulation, which peaks approximately 50 ms after stimulus onset (Bosman et al., 2010). For plasticity experiments, whisker test pulses were delivered during a baseline period of at least 40 min, followed by a 5 min period of rhythmic whisker stimulation (RWS) at 8 Hz – the speed at which mice naturally sample objects (Diamond et al., 2008). For plasticity experiments with optogenetic co-activation of climbing fibers (RWS+CF), the same protocol was followed with the addition of 50 ms blue light pulses at 1 Hz during plasticity induction. The 8 Hz whisker stimulation and 1 Hz CF stimulation occurred at a consistent time relative to one another, with a blue light pulse always occurring at a 45 ms delay relative to the onset of a whisker stimulus (the same delay used for the paired test pulses). After RWS or RWS+CF, whisker test pulses were delivered for a period of at least 60 min. To coordinate precise stimulus delivery, a Cygnus Digital Stimulator (catalog: PG4000A, Cygnus Technology, Inc, Delaware Water Gap, PA) was interfaced with an Arduino Uno (Qualcomm, San Diego, CA) to trigger both the LED driver and picospritzer.

Calcium imaging of the genetically encoded indicator GCaMP6f was conducted in Crus I/II of the right hemisphere of awake, head-fixed mice using a laser scanning two-photon microscope (Neurolabware, Los Angeles, CA, USA) and Scanbox software (Scanbox, Los Angeles, CA). Calcium images were obtained at a frame rate of 30.98 fps with a pixel dimension of 512 × 796, using an 8 KHz resonant scanning mirror with bidirectional scanning. A Mai Tai DeepSee (Spectra-Physics, Milpitas, CA) laser source was used to excite GCaMP6f at 920 nm. The fluorescence emission was collected through a 16x water immersion objective (Nikon LWD 0.8NA, 3 mm WD) using a GaAsP PMT (Hamamatsu Photonics, Shizuoka, Japan). A 2.0 or 2.8x digital zoom was applied during imaging, generating a field of view of 760 × 613 or 551 × 444 μm, respectively. To minimize background noise originating from ambient light, a custom light shield was fitted around the brain window and objective. This shield had a small opening such that the Thorlabs patch cable used for LED stimulation of climbing fibers could be positioned over the cerebellar window. The laser power was set to 1% with a PMT gain between 0.79 and 0.81, allowing for prolonged recording while minimizing phototoxicity.

Calcium imaging of GCaMP6f was conducted in barrel cortex of the left hemisphere using the same hardware as above. Images were obtained at a frame rate of 30.98 Hz with a pixel dimension of 512 × 796. A 2.4x digital zoom was applied during imaging, generating a field of view of 638 × 515 μm. Two of 11 RWS+CF experiments and one of 10 RWS experiments were conducted using a 2.0x digital zoom (generating a field of view of 760 × 613 μm) and were added to the experimental cohorts, as results were not significantly different across any reported measures. A custom light shield was fitted around the brain window and objective to reduce background noise originating from ambient light. Note further isolation of the LED light used for optogenetic stimulation of climbing fibers in these recordings was provided by the sheath attached to the cerebellar window (see Cranial window surgeries for imaging of barrel cortex neurons). While observing the epifluorescence signal, small vessels on the putative surface of the brain were put into focus in the z plane and matched to a field of view selected during receptive field mapping (see Habituation). After adjusting the focus 250 μm below the putative pial surface, small adjustments were made in the x-y planes while observing calcium signal in real time to maximize responsivity to whisker stimulation and overlap between GCaMP6f and tdTomato or mCherry in the field of view. GCaMP6f was excited at 920 nm and tdTomato or mCherry (for DREADD experiments) at 1040 nm, with emitted fluorescence collected using two GaAsP PMTs. The laser power was set to 2%, and adjustments to the PMT gain were made due to slight variance in the level of GCaMP6f and tdTomato/mCherry signal across animals. PMT gain in the green channel was 0.73 (SD: 0.07), while PMT gain in the red channel was 0.83 (SD: 0.14), which were not significantly different across experimental groups. Responsivity to whisker stimulation was determined by intermittently delivering 8 psi air puffs at random intervals, with a minimum of 30 s between stimuli, to avoid influencing plasticity experiments. If more than 10 min were spent determining an optimal field of view on the day of recording, the experiment was aborted and instead conducted the following day. Mice were occasionally used for multiple recording configurations but were sacrificed immediately after performing plasticity experiments, and selected animals were used for histological characterization (see Tissue preparation for fluorescence microscopy and immunohistochemistry). Note DREADD expression was histologically verified in all animals to ensure expression was restricted to neocortex.

To activate the hM4D(Gi) and hM3D(Gq) receptors, we used deschloroclozapine dihydrochloride (DCZ, MedChemExpress, Monmouth Junction, NJ), which was chosen over clozapine N-oxide (CNO) as an agonist due to its heightened selectivity for hM4D(Gi) and hM3D(Gq) receptors over endogenous receptors, a more rapid onset than CNO, and significantly increased potency (Nagai et al., 2020). DCZ was dissolved in DMSO at a 0.02 mg/mL concentration and stored at −80°C. On the day of recording, DCZ aliquots were thawed to room temperature and diluted to 0.01 mg/mL with DMSO/saline. Two-photon calcium imaging experiments began with recordings of baseline responses to whisker stimulation for at least 40 min (with and without optogenetic stimulation of climbing fibers in a subset of experiments), followed by an injection of DCZ (0.1 mg/kg) intraperitoneally. For DREADD inhibition of PV neurons, a dose of 0.02 mg/kg was used instead, as the 0.1 mg/kg dose caused significant increases in neocortical excitability (note PV animals that had received a dose of 0.1 mg/kg were not used for any subsequent experiments or in any of the datasets shown here). Injections were carefully administered to ensure the field of view would not be disturbed; experiments were aborted if this occurred, as responses in each period could no longer be directly compared. Activation of receptors was allowed to take effect for 15 min prior to resumption of recording, after which a second period of baseline activity was recorded. This period lasted between 15 and 30 min, such that a period of fifteen minutes of stable activity was recorded prior to plasticity induction. Plasticity induction protocols were followed as above (see Optogenetic and tactile stimulation), and whisker test pulses were delivered for at least 40 min after plasticity induction. To ensure observed plasticity phenomena were not the result of prolonged changes in activity related to hM4D(Gi) or hM4D(Gq) activation, control experiments lasting the same duration as the plasticity experiments were conducted similarly, except without induction of plasticity (see Figure 4—figure supplement 1). To ensure DREADD expression was localized to the neocortex, viral spread was histologically characterized in all animals (see Tissue preparation for fluorescence microscopy and immunohistochemistry).

Calcium images taken in the green (PMT0) and red (PMT1) channels were first merged, trial-averaged, and concatenated using Fiji. The average image was then calculated, followed by identification of all cells expressing tdTomato or mCherry (applicable to all recordings except the zona incerta manipulation experiments). ROIs were manually drawn around each cell co-expressing GCaMP6f and tdTomato or mCherry. All images across a session were then concatenated and trial-averaged before calculating the maximum image. ROIs were manually drawn around all putative pyramidal neurons, which were identified with the following criteria: a triangular or pyramidal soma, the presence of a single apical dendrite extending in a similar orientation to other pyramidal cells in the same imaging session, and the presence of basal dendrites smaller in diameter than the apical dendrite. In a subset of experiments in which apical dendrites could not be clearly distinguished in the imaging plane, z-stack images were taken at the end of the imaging session, averaged, and referenced to visualize apical dendrites. After the identification of fluorescently tagged interneurons and pyramidal neurons, relevant putative interneurons were morphologically identified and compiled (e.g. putative VIP and SST neurons in PV-tagged mice). Putative interneurons were identified by the presence of an elongated soma and bipolar dendritic arbors (likely VIP), round somas smaller in diameter than pyramidal cells, and neurons with multiple perisomatic dendrites (usually highly branched; likely PV). The morphology of untagged interneurons was visually compared to the morphology of tagged interneurons in relevant mice (e.g. morphologically identified interneurons in B6 PV^cre mice were compared to fluorescently tagged interneurons in Sst-IRES-Cre and Vip-IRES-Cre mice). The average (11.07 µm, SD 1.64 µm) and maximum (18.14 µm, SD 2.74 µm) soma diameter for morphologically identified pyramidal neurons (n=3358) were significantly higher than in morphologically identified interneurons in the same mice (n=3522; average: 9.90 µm, SD 1.96; maximum: 16.37 µm, SD: 3.39 µm; p<0.001 for both values, unpaired t-test) and tagged VIP, SST, and PV neurons (n=543; average: 8.96 µm, SD 1.87 µm; maximum: 14.98 µm, SD 3.51 µm; p<0.001 for both values, unpaired t-test).

Mice underwent the same surgical procedures described in Two-Photon calcium imaging: Cranial window surgeries for imaging of barrel cortex neurons. To facilitate manipulation of single whiskers, mice were lightly anesthetized with 10 mg/kg ketamine and placed on a stereotaxic frame. Using a custom GUI written in MATLAB, small vessels were visualized under a green LED (525 nm wavelength) with a high-speed camera (Genie Nano GigE, Teledyne DALSA, Waterloo, Ontario, Canada). Focus in the z-plane was adjusted approximately 250 μm below the putative pial surface before taking a reference image of the vasculature. For measurement of intrinsic signals, the field of view was illuminated with red light (625 nm). An Arduino Uno and MATLAB were used to trigger trial onset, the device used to mechanically deflect whiskers (a glass capillary tube attached to a rotary motor), a blue (470 nm) LED to stimulate climbing fibers, and recording of intrinsic signals at 30 Hz. Trials consisted of a 1 s baseline period, followed by a 1 s test pulse in which whiskers contralateral to the optical window were mechanically deflected at 8 Hz (RWS). This was repeated with an inter-trial interval of 18 s for 30 min prior to plasticity induction, after which whiskers were stimulated at 8 Hz for 5 min. Test pulses were delivered for 40 min after plasticity induction. For RWS+CF and control experiments, 50 ms blue light pulses were delivered at 1 Hz during plasticity induction with or without 8 Hz co-stimulation of whiskers, respectively.

Evoked signals were calculated similarly to Vasquez et al., 2023. Briefly, images were smoothed with a Gaussian filter and spatially downsampled by a factor of four. For each trial, an average baseline reflectance image (R0) was generated by calculating the average across the 1 s (30 frame) period before stimulus onset. Post-stimulus reflectance images were temporally averaged across 200 ms bins for 600 ms total, starting 400 ms after stimulus onset. The change in reflectance, ∆R/R, was calculated by subtracting R0 from each post-stimulus image and dividing the result by R0. Each of the three bins (400–600 ms, 600–800 ms, and 800 ms-1 s post stimulus onset) were averaged across a minimum of 30 trials and finally summed to yield a single total stimulus-evoked ∆R/R image. The reference image taken with green light illumination was used to draw a region of interest around the cranial window. Pixels outside this ROI mask were set to zero. Binary images were generated by calculating z-scores of the ∆R/R image and thresholding values below a z-score of –1.5 (note negative changes in reflectance indicate an increase in the presence of deoxyhemoglobin caused by increased oxygen consumption by active neurons). Binarized images were overlaid onto reference images. To calculate the areal extent of single whisker-evoked responses, clusters of active pixels were identified by applying a median filter with a 5×5-pixel neighborhood size, and the number of pixels above threshold was calculated. To reduce the impact of residual noise (i.e. detected pixels outside the area reliably activated by a single whisker), values were normalized by the area of the cranial window.

Processing of Purkinje cell calcium traces was conducted as previously described (Busch and Hansel, 2023; Lin et al., 2024). Individual trials from each session were concatenated along the z-axis, representing a time series across the entire recording session, to ensure consistency among trials after motion correction. The concatenated file underwent motion correction using a custom MATLAB script (MATLAB R2017b) based on whole frame cross-correlation (provided by the lab of Mark Sheffield, University of Chicago). Cellular regions of interest were manually selected in Fiji based on the average field of view across trials. Trials were then separated for further processing. In a subset of trials, optogenetic stimulus artifacts were visible in a portion of the field of view in 1–3 nonconsecutive frames, depending on the stimulus duration. These frames were manually identified and excluded using Fiji before further processing.

Using custom MATLAB script (version R2022a), ΔF/F values were calculated from the raw trace using the following equation: (Ft − F0)/F0, where Ft represented the raw calcium intensity of the time series, and the baseline fluorescence (F0) was set as the 20^th percentile of the fluorescence trace from each trial. ΔF/F values were then subjected to low-pass filtering using a five-frame moving window smoothing function. Finally, traces were normalized to a pre-stimulus baseline period by subtracting the average ΔF/F value from the 5-frame period prior to stimulus onset from the entire trace. To visualize population average responses to optogenetic stimulation of climbing fibers, calcium signals from each ROI were first trial-averaged, followed by concatenation and averaging of the signal across cells and mice. To calculate maximum amplitude of responses in the evoked period, the maximum in a window 0–700 ms after stimulus onset was calculated for each ROI’s trial-averaged trace, then concatenated across cells and mice. Area under the curve (AUC) was calculated similarly by integrating the ΔF/F values over the same window. To identify spontaneous events, frame indices in which the first and second derivatives of the ΔF/F signal reached 0.04 were identified (F^idx). Peaks were detected using a peak prominence threshold of 0.1 ΔF/F, with spontaneous events defined as peaks occurring within 200 ms of F^idx. The onset of each spontaneous event was determined as the first frame in which the first derivative of the ΔF/F dropped below 0.05 (working backward from frame index of the detected peak). A 1 s window following event onset was extracted. Finally, after averaging all detected spontaneous events within ROIs, spontaneous events were compiled across neurons to generate the population average trace.

Motion correction and ROI curation were performed as above (see Processing of cerebellar data and Two-photon calcium imaging: Characterization of neurons). Residual optogenetic stimulus artifacts were observed in a single frame in a subset of trials, typically at or immediately after stimulus offset. These artifacts were detected using the findpeaks function in MATLAB and corrected by replacing the artifact frame’s value with a linear interpolation of the two adjacent frames before further processing. Background subtraction was performed by calculating the bottom 1st percentile of the raw fluorescence trace collected from an ROI that did not contain cell bodies or neuropil, then subtracting this value from all trials. Smoothing, expression of raw signals as ΔF/F, calculation of baseline noise (σ), and calculation of the signal-to-noise ratio (SNR) were performed as in Ayaz et al., 2019. Smoothing was performed with a 51-point 1st-order Savitsky-Golay filter, followed by calculation of the relative percent change of fluorescence ((Ft − F0)/F0) using the 1st percentile of the smoothed trace as F0. Given background subtraction prior to calculation of F0 may generate negative F0 values and subsequent signal inversion, ROIs with F0<0 were rejected from further analyses (44/7423 cells). To calculate σ for each neuron, a 5 s (155 frame) sliding window was first used to calculate the standard deviation of the fluorescence change during each 5 s period within a session. After compiling all values, the 1st percentile value was taken as σ. For each neuron, the SNR was defined as the 95^th percentile of ΔF/F signals recorded across all trials divided by the baseline noise.

To detect whisker–evoked events, we first applied OASIS deconvolution to the ΔF/F traces using an event detection threshold of 3σ to generate a binary trace containing event times. An autoregressive model with order p=1 was used to ensure the number of events (‘spikes’) for each calcium transient varied with the amplitude of the calcium transient as previously observed (Theis et al., 2016). Next, for each frame, the sum of all events was calculated and divided by the number of trials to determine the proportion of trials with an event in each frame (P^event). P^event was averaged across frames, and consecutive frames with P^event three standard deviations above the mean were selected. This generated an evoked event window of 387 ms (Figure 1—figure supplement 2B). Finally, for each neuron, the calcium traces for all trials containing an event within the evoked event window were compiled for further analysis. To visualize population average traces for all experiments, evoked calcium signals for each neuron were first averaged across trials, followed by averaging across neurons. For visualizations between the same neural populations in different conditions, only those cells that responded to both conditions, i.e., having an evoked event in each condition, were included (following the definition of ‘Persistent cells’ used in Williams et al., 2025). Shaded regions in population average traces represent SEM.

For all calculations of AUC in S1 recordings, the ∆F/F signal was integrated over a 700 ms window after stimulus onset unless otherwise specified (details below). Note that AUC is equal to the average response in the 700 ms window, multiplied by the 700 ms window duration (thus, AUC is directly proportional to the mean). We choose to report AUC, a descriptive statistic, rather than the mean within this 700 ms window.

To quantify plasticity phenomena caused by RWS or RWS+CF, all evoked events measured within 50 min prior to plasticity induction (‘Pre’) or 60 min after plasticity induction (‘Post’”) were extracted for each cell and trial-averaged before calculating AUC. Only the neurons with evoked events both pre- and post-plasticity induction were included in subsequent analyses. AUC were averaged both across all neurons or within animals to perform statistics across neurons or mice (see Statistics and quantifications). To visualize the duration of plasticity effects, evoked events were binned into 10 min epochs and trial-averaged within cells, followed by calculation of the AUC and averaging across neurons. To verify the robustness of plasticity effects, AUC and time course measurements were also analyzed across all cells and trials (i.e. those that did not have stimulus-evoked events; Figure 1—figure supplement 2) and in the absence of animal movement (Figure 1—figure supplement 3; see Determination of active and rest trials). To quantify plasticity phenomena in DREADD manipulation experiments, response amplitude was calculated in addition to AUC before and after plasticity induction. Response amplitude was taken as the maximum ∆F/F value in the same 700 ms window used for AUC calculations.

To quantify the immediate effect of climbing fiber activation on basic whisker responses in S1 cortex, AUC was calculated similarly for whisker stimulation (W) or whisker and CF coactivation (W+CF) trials. An additional measurement was taken 650–850 ms after stimulus onset for pyramidal neurons and VIP interneurons, as the effect of CF modulation was present in the later phase of the calcium response. Note that while W and W+CF test pulses were delivered prior to DCZ administration in DREADD experiments to verify functional opsin expression, these mice were excluded from this analysis due to an insufficient number of trials in each animal.

To verify that hM4D(Gi) and hM3D(Gq) expression in SST neurons indeed suppressed or increased their activity, respectively, AUC was calculated before and after DCZ administration in a window 1750 ms after stimulus onset. This larger window was used to capture the significantly increased duration of whisker-evoked calcium responses in SST neurons expressing hM3D(Gq). To verify that hM4D(Gi) and hM3D(Gq) expression in VIP neurons indeed suppressed or increased their activity, respectively, AUC was calculated before and after DCZ administration in a window 650–850 ms after stimulus onset. Given VIP neurons are modulated by climbing fiber stimulation in this phase of the calcium response (Figure 2G), this later window was chosen to ensure DREADD manipulation of VIP neurons suppressed or enhanced VIP responses in the physiologically relevant response period.