Synthesis of TiO2-x NW and Au@TiO2-x NW arrays
In a typical synthesis, 0.5 mL tetrabutyl titanate (TBOT) was added into the mixture solution of 15 mL deionized water and 13 mL hydrochloric acid (HCl), and stirred to form a transparent solution. Then, the solution and fluorine-doped tin oxide (FTO) coated glass substrate with hydrophilic surface were transferred into a 50-mL Teflon-lined stainless-steel autoclave, and heated to 150℃ for 12 h. Then it was annealed at 550℃ for 3 h with a heating rate of 5℃·min-1 in air. To introduce oxygen vacancies into the TiO2 lattice, the as-synthesized TiO2 was further annealed at 350℃ in 5% H2/Ar atmosphere for 8 h, with a flowing rate of 150 ~ 200 standard cubic centimeters per minute (sccm) to obtain the TiO2-x NW arrays with oxygen vacancies. Afterwards, Au nanoparticles were coated on the TiO2 NW arrays. In brief, the FTO substrate with TiO2-x NWs was immersed into the 0.01 M HAuCl4 aqueous solution, and the pH was tuned to ~ 4.5 by adding 0.2 M NaOH aqueous solution. After incubating for 2 h, the FTO substrate was taken out, dried and annealed at 300℃ under Ar atmosphere for 2 h with a heating rate of 5 ℃·min-1.
For in vivo test, the FTO substrate was etched using a mixed solution of H2SO4/HF (40 wt%) with the volume ratio of 13:2 at 85℃ until the total thickness of the substrate was less than 100 μm.
Animals and genotyping
Mice were reared with 23 ± 2℃ room temperature, 60 ~ 65% relative humidity, and a 12 h light/12 h dark cycle. The wild-type (C57BL/6J) mice were purchased from the Slac Laboratory Animal Co. (Shanghai, China). rd1-/-/cDTA lines were maintained via crossbreeding rd1 knockout mice and cDTA transgenic mice. Cone diphtheria toxin subunit-A (cone-DTA) positive and homozygous rd1-/- mice were sorted by genotyping methods described in our previous work 25. All the mice used in this study were older than 8 weeks. All animal treatments were strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the animal facility at Fudan University.
One male rhesus monkey (Macaca mulatta) aging 12 years old and weighing 8 kg (in East China Normal University, Shanghai) was used in this study. This study was carried out under the recommendations of the Institutional Animal Care and Use Committee at East China Normal University. The protocol was approved by the Institutional Animal Care and Use Committee at East China Normal University.
Patch-clamp recording of retinal ganglion cells
Mice were anesthetized with isoflurane (1 ~ 2% at 0.5 ~ 1.0 L/min) and then treated with enucleation of the eye. The eye was placed in oxygenated Ranger’s solution (in mM, NaCl 124, KCl 2.5, CaCl2 2, MgCl2 2, NaH2PO4 1.25, NaHCO3 26, and glucose 22, pH 7.35; oxygenated with 95% O2 and 5% CO2) quickly and retina was dissected and placed in on a filter paper (Merck Millipore, Burlington, USA) in the recording chamber. Subsequently, NW arrays were attached to the inner nuclear layer of the blind retina. After a glass pipette (5 ~ 10 MΩ) was pulled by P-97 micropipette puller (Sutter Instruments, Novato, USA) filled with internal solution (in mM, potassium gluconate 105, KCl 5, CaCl2 0.5, MgCl2 2, EGTA 5, HEPES 10, Mg-ATP 4, GTP-Na 0.5, and sodium phosphocreatine 7, PH 7.4), in vitro patch clamp system was used to record action potentials of RGCs via MultiClamp 700B patch-clamp amplifier (Molecular Devices, San Jose, USA) and digitizer Digidata 1440 (Molecular Devices, San Jose, USA) under DIC microscope (Zeiss, Oberkochen, Germany) 39.
Visual stimuli for patch clamp recording of RGCs were provided by mercury lamp (X-Cite 120PC Q, LUMEN DYNAMICS, Ontario, Canada) and Digital Micromirror Device (DMD) (wavelength: 400 nm). To investigate the response of RGCs to light with different wavelengths, mercury lamp generates UV, blue and green light, and provides the photo-stimulation via a × 40 water-immersive objective. DMD was used to present moving, static and flash visual stimuli. The moving light bar appears 10 times in 30 sec, while static light bar remains bright for 30 sec. The velocity of the moving light bar used is 6.85 degrees per sec. Different widths of the moving light bar (0.64, 1.28, 1.92, 2.56, 3.20 and 3.84 degrees) were used to study the spatial resolution capacity of mice’s RGCs. Different temporal frequencies (5.00, 2.5, 1.57, 1.25, 1.00, 0.5 Hz) of a flash stimulus (180 × 180 μm2) were used to study the temporal resolution capacity of mice’s RGCs.
Clampfit (Axon, Scottsdale, USA) was used to analyze the action potentials of RGCs and Prism 6 (Graph pad, San Diego, USA) was used to analyze firing rates and the fraction of responses. Firing rate is the number of action potentials, which occurred during visual stimulation, over the stimulation time (1.4 sec). Fraction of responses is calculated as the rate of the action potentials in ten times of visual stimulation.
Implant surgery in mice
The rd1-/-/cDTA mice (blind mice) aged 8 to 10 weeks were anesthetized with isoflurane (1 ~ 2% at 0.5 ~ 1.0 L/min). The eye-ball was fixed in a suitable position for implantation by suturing the upper and lower eyelids. A small incision was made on the dorsal sclera, and 1 ~ 2 μL of 0.9% sodium chloride solution was injected rapidly and carefully with nano project to create the space for NW arrays implantation. This step was prone to bleeding and required prompt compressions and other effective methods for hemostasis. Expanding the incision at an angle of 45 ~ 60 degrees to the nasal-temporal axis to leave enough space for NW arrays implant, which was depended on the size of the substrate size of NW arrays. The typical size of implant NW arrays was 0.5 ~ 1 mm2, which were sterilized by steam under high pressure. NW arrays were clamped by tweezers and inserted carefully into the cut along the direction of the incision under the microscope. After the implant, washed the eye-ball with 0.9% sodium chloride solution and removed the suture, applied erythromycin eye ointment finally.
Choice-box-based Behavioral Test
The choice-box (240 mm × 240 mm × 180 mm) is composed of acrylic sheets, a water reward system (Kamoer, Shanghai, China), which is composed of water pipe, water port, and water pump, and an audio signal system, including a buzzer. Visual stimuli appear at two positions in the visual stimulation panel (one side of the box opposite to the water port). LED visual stimulation, water reward system and the audio signal system are all controlled by an Arduino board.
Choice-box-based behavior test is divided into adaptation phase, training phase and testing phase 40. During the adaption phase, mice undergo water restriction 3 days before the training phase. During the training phase, mice can first freely explore and acclimatize themselves to the environment of the choice box for 5 ~ 10 min before the test starts. Subsequently, an audio signal is sent to indicate the mice of the upcoming visual stimulus, which then appear at two random positions in the visual stimulation panel. The stimulation duration and reaction time are both 20 sec. Within the stimulation duration, the mice need to touch the corresponding position of the target visual stimulus either by their paws or noses. Afterward, another audio signal will be given and the mice can obtain a water reward at the water port. If the mice touch the visual stimulation panel before the stimulation appears, the next trial will pause until the mice leave the panel. If the mice do not touch the visual stimulation panel within the stimulation duration, another visual stimulation trial will proceed. The interval of each trial is at least 10 sec, and 40 trials are carried out daily. The testing phase is similar to the training phase, except that the stimulation duration and the reaction time are reduced to 10 sec, and the interval between each trial is at least 5 sec.
Different visual stimulations were used in our study in different experiments. LED light with different wavelengths and light intensities was used. LED visual stimuli appear at one of two positions randomly in the visual stimulation panel. Mice need to touch within 6 cm in diameter centered at the on-LED in the visual stimulation panel as the center to get water reward. In the testing phase, UV LED light (375/15 nm, 6.24 μW·mm-2), blue LED light (465/25 nm, light intensity: 6.75, 3.31, 2.93, 1.53 and 0.64 μW·mm-2), and green LED light (535/28 nm, light intensities: 7.83, 4.20, 2.04, and 1.15 μW·mm-2) were used.
Moreover, both moving and static light bar were used, presented by a projector (CB-S41, EPSON, Suwa Japan). The moving light bar stimulus appears at one of the two positions randomly in the visual stimulation panel, and a static light bar stimulus appears at the other position simultaneously. Mice can get a water reward if they touch within the moving range of the moving light bar. In the testing phase, static or moving light bar stimulus with different widths and velocities (width of light bar: 2.63, 5.25, 7.57, 7.88, and 3.94 degrees; velocity of moving light bar: 5.23, 6.54, 7.85, 9.18, and 10.47 degrees/sec) were used.
Finally, a flash stimulus and constantly bright stimulus were used, presented by a projector (CB-S41, EPSON, Suwa, Japan). A flash stimulus (6 cm × 6 cm square) appears at one of the two positions randomly in the visual stimulation panel, and a constantly bright stimulus appears at the other position simultaneously. Mice need to touch the position of flash stimulus to get a water reward. In the testing phase, temporal frequencies of 1, 1.25, 2, 3.33 and 5 Hz were used for the flash stimulus.
If mice touch the corresponding area of the visual stimulation panel within the required time, it is a correct trial. If mice do not touch the visual stimulation panel, it is considered as a miss trial. The wrong trial refers to the case when mice touch the non-corresponding position of the visual stimulation panel. When the correct rate of rd1-/-/cDTA mice, which have been implanted with Au@TiO2-x NW arrays, reaches 70% and maintains stability for 3 days, the testing phase starts.
Visual acuity and contrast sensitivity measurement
To measure the visual acuity and contrast sensitivity, we developed a system with 4 LCD screens arranged in a quadrangle, a cylindrical mice platform (height: 12 cm, radius: 4 cm) and a camera on the top of the system to record the experiment. Visual stimulus was generated by Python, using the Psychopy toolbox 41. There are two alternating moving directions (0 degrees, 180 degrees) of the stimulus, lasting 10 sec for 3 times.
Mice were placed one at a time on the platform and the animals were allowed to move freely. When a grating perceptible to the mouse was showed on the screen, the mouse normally stopped moving its body and would begin to track the grating with reflexive head movements accompanying the rotation. Before the test started, all animals were habituated by handling and placing them on the platform for several minutes at a time. If the animal slipped or jumped off the platform during the course of testing, it was simply returned to the platform and testing was resumed.
Immunohistochemistry
Mice were deeply anesthetized using overdose isoflurane for euthanasia and perfused transcardially with physiological saline, followed by 4% paraformaldehyde (PFA) using a perfusion pump. Brains were kept in 4% PFA for fixation at 4℃ overnight and then dehydrated in 30% sucrose until the sample sunk to the bottom. After embedded and frozen, brains’ tissue was sectioned into 30-μm coronal slices in a cryostat (Leica CM 1950, Leica, Wetzlar, Germany). The slices were washed with Tris-buffered saline (TBS) 5 times (5 min each), covered with coverslips and mounted to photograph by a fluorescence microscope (A1R, Nikon, Tokyo, Japan).
With regard to immunohistochemistry of mice’s retina, the retina was fixated in 4% PFA for 4 ~ 7 h at 4℃, after enucleation of eye and dissection of the retina. After fixation, the retina was dehydrated using 10%, 20% and 30% sucrose solution respectively. After dehydration, the retina was embedded in OCT compound at -80℃ for more than 2 h, and subsequently sectioned into 14 mm slices in a cryostat (Leica CM 1950, Leica, Wetzlar, Germany). After rinsed by TBS and immersed in 0.5% Triton-X-100 for 20 min, slices were incubated in a blocking solution consisting of DST for 20 h. Afterward, slices were incubated by primary antibody (anti-Choline Acetyltransferase and anti-Brn3a, Millipore, Massachusetts, USA, 1:200) diluted in blocking solution overnight. The next day, slices were washed 3 times (5 min each), and secondary antibody (Donkey anti-Goat conjugated to Alexa Flour 594, Jackson ImmunoResearch, West Grove, USA, 1:300) diluted in blocking solution was used to cover the slices for 2 h in darkness. Slices were then stained with 1:3000 DAPI after rinsing, and finally rinsed, air-dried, and coverslipped. A fluorescence imaging microscope (A1R, Nikon, Tokyo, Japan) was used to obtain fluorescence images of the slices. The fluorescence image was then processed using Adobe Photoshop CC 2018.
Intravitreal injection
The mice were anesthetized with isoflurane (1 ~ 2% at 0.5 ~ 1.0 L/min). Subsequently, 69 nL Dil Stain Perchlorate (1.5mg/mL, D282, Invitrogen, USA) was injected into the nasal dorsal retina of rd1-/-/cDTA mice, in which the NW arrays were typically implanted, using a Nanoject II (Drummond scientific company, Broomall, USA). Following surgery, mice were placed on a heating pad to recover and monitored for postoperative health.
Surgery for CTB-488 and GCaMP6s virus injections in primary visual cortex
CTB-488 (2 mg/µL, C22841, Life Technology, USA) was injected into the medial V1 in rd1-/-/cDTA mice which had been treated with intravitreal injection. Four coordinates used for targeting V1 were as follows: 3.2 mm posterior and 2.0 mm lateral, 3.6 mm posterior and 2.0 mm lateral, 3.2 mm posterior and 2.5 mm lateral, 3.6 mm posterior and 2.5 mm lateral of bregma, at depths of 0.45, 0.33 and 0.20 mm depth (60 nL in each deep) using a Nanoject II (Drummond scientific company, Broomall, USA). Following surgery, mice were placed on a heating pad to recover and monitored for postoperative health.
To prepare animals for recording, we injected an adeno associated virus (AAV) that expresses GCaMP into the visual cortex 42,43 in rd1-/-/cDTA mice. At the time of surgery, all mice were 2 months old and anesthetized with isoflurane (1 ~ 2% at 0.5 ~ 1.0 L/min). We removed the skin above the surface of the brain and dried the skull using air. To express GCaMP specifically in visual cortex neurons, we unilaterally injected AAV2/8-hSyn-GCaMP6s (Taitool Bioscience Co., LTD, Shanghai, China) into the right V1. To target the V1 layer 2/3, we made a small craniotomy and injected virus in three sites (2.1 mm lateral and 3.2 mm posterior, 2.5 mm lateral and 3.2 mm posterior, 2.3 mm lateral and 3.6 mm posterior of bregma) at depths of 0.40 and 0.25 mm using a Nanoject II (Drummond scientific company, Broomall, USA). At each depth, 100 nL was delivered manually in 25 × 4 nL steps, with approximately 20 sec pauses between steps. Virus injection lasted several minutes, and then the injection pipette was slowly removed over the course of several minutes.
Cranial window implantation
Surgery for cranial window implantation was performed 6 ~ 8 weeks after virus injections when most neurons exhibited cytosolic-only GCaMP6 expression 44,45. Mice were anesthetized with isoflurane (1 ~ 1.5% at 1 ~ 1.5 L/min). The depth of anesthesia was monitored by the pinch withdrawal reflex throughout the surgery. Core body temperature maintained at 37 ~ 38℃ using a heating pad. Eyes were protected from dehydration during the surgery with eye ointment. The scalp overlaying the visual cortex was removed, the center of the glass coverslip was positioned above right V1, 2.3 mm lateral and 1.3 mm anterior of lambda. To gain optical access to the cortex, a 2.5 mm diameter craniotomy was performed. After removing the skull flap, the cortical surface was kept moist with physiological saline (0.9% NaCl). The dura was left intact and any occasional bleedings were immediately stopped with Gelfoam (Fu Kang Sen Medical Equipment Co., LTD, Guilin, China). A 3 mm diameter glass coverslip sterilized in ethanol (0.1 mm thickness) was fixed on the brain using cyanoacrylate-based glue (3M Animal Care Products, St. Paul, USA) to gently compress the underlying cortex. After waiting for fifteen minutes for this glue to dry, we applied dental cement (Super Bond C&B, Japan) to attach a fixed bar allowing for subsequent head fixation during subsequent imaging, and glass coverslip to the cranium. After waiting for twenty minutes for the dental cement to cure, the surgery was completed. Mice were recovering on a heating pad for one hour and returned to their home cage after being injected with ceftiofur sodium by intraperitoneal (5 mg/kg body weight, Quan Yu Biotechnology Animal Pharmaceutical Co., LTD, Shanghai, China) and dexamethasone sodium phosphate by intramuscular (0.1 mg/kg body weight, Quanyu Biotechnology Animal Pharmaceutical Co., LTD, Shanghai, China) mixture dissolved in saline.
Two-photon microscope setup and neuronal imaging
Recordings of neuronal activity were performed with Olympus FluoView FVMPE-RS upright two-photon laser-scanning system with an Olympus XL Plan N25 × /1.05 WMP ∞/0-0.23/FN/18 dipping objective (Olympus, Tokyo, Japan). Two-photon excitation was performed using 920 nm MAITAI eHPDS-OL laser (Mai Tai, Spectra-Physics, Santa Clara, USA), and emitted fluorescence was detected through a 495 ~ 540 nm bandpass filter. For imaging of GCaMP6s expression, fields of view were imaged with a resolution of 512 × 512 pixels at 30 Hz. Imaging sessions lasted 2 ~ 3 h and included 1 ~ 2 h of effective imaging time.
On the day of blue LED stimulus recording mice were placed in a stereotaxic apparatus, in which the animal’s head was rigidly held in a specially designed holder in the awake state, during the imaging sessions. Anesthesia was maintained throughout receptive field stimuli recording with isoflurane (0.5 ~ 1.0% at 1 ~ 1.5 L/min), and body temperature was monitored continuously to judge the animal’s health and maintain proper anesthesia. We administered topical 3% sodium hyaluronate eye drops to prevent drying eyes during the recording.
Visual stimulus for two-photon Calcium imaging.
Blue LED was used as light sources for the light-responsive experiment. LED was located 6 cm away from the mouse eye. Each experiment trial started with a blank period for 20 ~ 25 sec randomly, and a LED stimulation was subsequently turned on for 1 sec, repeated 6 times.
Stimuli used in receptive field mapping were generated by Python, using the Psychopy toolbox 41. The screen was shown adjusted to be 45 degrees from the anteroposterior axis, 12 cm from the mouse’s eye, subtending 96 and 80 degrees of the horizontal and vertical visual field, respectively. We used a sparse noise stimulus, consisting of blue squares presented on a black background along a grid of 6×5 squares to map RFs, each square is ~ 16×16 degrees. The squares were presented for either 1 sec with a 10-sec interval for the anesthetized mice with 8 repetitions.
Analysis of in vivo calcium imaging data
In vivo calcium movies were pre-processed in python using a custom-built pipeline based on CaImAn package 46 for large-scale calcium imaging data analysis. Movies were motion-corrected using a rigid registration method to remove motion artifacts 47. Calcium activity traces of single neuron were extracted from the registered movie using a constrained non-negative matrix factorization (CNMF) framework 48. Spatial correlation thresholds for ROI detection were set to 0.85, and the signal to noise ratio for accepting a component was set to 2.50. The automatic detection was manually screened to ensure correct segmentation of somatic calcium activity. This pipeline generated a set of spatial footprints and temporal traces for each animal on each day of recording. The relative percentage changes in fluorescence (ΔF/F) trace were used in further analyses.
Light responsive neurons identification
To identify light-responsive neurons, we defined a pre-stimulus period as the 2 sec window (60 frames) preceding the stimulus onset, and the baseline of the trial is the mean ΔF/F during the pre-stimulus period. We also defined a post-stimulus period as the 5 sec window (150 frames) following the stimulus onset. A neuron was considered responsive to the stimulus if the maximum ΔF/F during the post-stimulus period was more than five times SD above the baseline and the time to decay half-peak must over 10 frames in more than 50% of the trials. Mean ΔF/F amplitude, latency. To estimate an ROI’s ΔF/F mean amplitude during its activation, we isolated the ΔF/F traces during each light-evoked spiking segment and used the maximum ΔF/F value at each segment as a measurement of the ΔF/F amplitude during that segment. The time point after the stimulus and firstly exceeded the threshold was used as the latency. The average of these maximal values and time points for light-responsive neurons were computed as the mean ΔF/F and latency. To compare the changes of latency and mean ΔF/F amplitude, we took the data recorded before implantation and the 3rd day after implantation as the standard of latency and mean ΔF/F amplitude, respectively. Fitting spatial RFs. The retinotopic organization of single neuron was assessed by measuring the average ΔF/F response to each of the 30 stimulus positions (6 × 5 grid) with 8 repetitions. First, these data were interpolated by a 2D bilinear interpolation. Afterward, and fit by least-squares regression with a two-dimensional Gaussian Model. Neurons whose receptive field center within 5 degrees from the edge of the screen were selected for further analysis.
Surgical procedures for NW arrays implantation in monkey
Monkey (Macaca mulatta) was anesthetized with intramuscular tiletamine hydrochloride and zolazepam hydrochloride (Zoletil 50, 0.1 mL/kg body weight, Virbac S.A., France) after intramuscular atropine (0.5 mg/mL, 0.1 mL/kg body weight, Healton Animal Pharmaceutical Co., LTD, Sichuan, China) 20 min later and isoflurane inhalation maintained general anesthesia during retinal implant surgery. Mydriasis in the right eye was induced by tropicamide (5 mg/mL, Santen Pharmaceutical Co., LTD, Osaka, Japan). Lateral canthotomy was performed to allow 23-gauge trocars (Carl Zeiss Meditec AG, Jena, Germany) for vitrectomy and a retinal bleb was created by subretinal injection of balanced salt solution 16. NW implants (5 pieces of implantation in all, 0.4 mm width and 1.8 mm length for each piece) were inserted subretinally through 3 mm-wide sclerotomies (Constellation vision system, Fort Worth, USA). The detached retina was reattached with injection perfluorocarbon liquid (Bausch & Lomb, Rochester, USA) and silicone oil (Arciolane3000, Arcadophta SARL, France). Retinal laser photocoagulation was operated at the border of the retinotomy 49,50.
Eyes Examinations for Monkey
One to two drops of Compound Tropicamide Eye Drops (Santen Pharmaceutical Co., LTD, Shiga Plant, Japan) were instilled into monkeys’ eyes to dilute its pupil 3 times and 1 h before the examination began. Monkey was injected with atropine (0.5 mg/mL, 0.1 mL/kg body weight, Healton Animal Pharmaceutical Co., LTD, Sichuan, China) and then anesthetized with intramuscular injection of tiletamine hydrochloride and zolazepam hydrochloride (Zoletil 50, 0.1 mL/kg body weight, Virbac S.A., France). After anesthetization, Monkey was then placed prone in a supporting apparatus to receive eye examinations.
Color, Red-free, and autofluorescence fundus photography (TRC-50DX, TOPCON, Tokyo, Japan) of monkey’s right eye were taken before photocoagulation, 11 months after photocoagulation, 4 days, 3, 4, 6 and 8 weeks after implantation with Au@TiO2-x NW arrays. Optic Coherence Tomography (OCT) (Cirrus HD-OCT 4000, Zeiss, Oberkochen Germany) was also used to scan the retinal structure and the corresponding places of Au@TiO2-x NW arrays in the retina of the right eye of the monkey. OCT images were taken 4 days, 3, 4, 6 and 8 weeks after implantation with Au@TiO2-x NW arrays. Silt lamp (Oculus Optikgerate GmbH, Wetzlar, Germany) was used to examine the monkey’s lenses, cornea and anterior chamber by providing direct visualization.
Recording of eye position and visually-guided saccade task
The monkey was seated in a chair which placed on a platform in front of the screen (the distance between the monkey’s eyes and the screen is 30 cm) and looked flat at the central fixation point. A lightweight acrylic cap was implanted for head stabilization chronically. Monkey’s head was fixed by a custom-made head holder to keep the head stable during experiment. Eye position was monitored and digitalized by an infrared eye system, including an eye-tracking camera (above the center of the screen), infrared illuminator and Wise Center software for real-time recording of traces of pupil’s position. Visual stimulations were presented by red 642/18 nm), blue (465/25 nm) and UV (360/15 nm), the stimuli in this task were generated by TEMPO experiment control system (Reflective computing, Olympia, WA, USA), Neuron task software and Arduino. A variable DC power supply was used to control the light intensity of the LED light.
During visual-guided saccade task trials, the monkey maintained fixation for 1000 msec after fixation onset. After the fixation point (0.5º ´ 0.5º) disappeared, a red, blue, or UV target point (0.5 degrees), with a centrifugal rate from the fixation point of 17 to 21 degrees (interval 2 deg) and a pole angle of 0 to 350 degrees (interval 10 degrees) appeared. The animal needed to make a saccade to the target point within 1 sec of the onset of the target point for a fluid reward.
To calculated the saccadic endpoint, we calculated the velocity changes of eye movement trajectories using self-written python code and determined the mean eye position in a time window when the eye position was stationary (50 ~ 100 msec after the peak velocity) as the previous study 20. The experiment animal was considered to have completed one correct saccade if the angle between the endpoint of the saccade and the target point was less than 40 degrees and the pitch was within 15 degrees. The animal was rewarded immediately when it made a correct saccadic eye movement. With regards to normalized saccadic endpoints location indicated by θ and ρ in the polar coordinate, θ is the angular difference between the saccadic endpoints and target points, and ρ is the normalized distance between the saccadic endpoints and target points
Statistical analysis
The differences were tested by Prism (Graphpad, San Diego, CA). Comparisons between two groups were made by unpaired and paired two-tailed student’s t-test, and comparisons between three or more groups were made by one-way ANOVA. Results of the choice-box-based behavioral test were analyzed by two-way repeated-measures (RM) ANOVA followed by the Tukey post-hoc test. Results of patch-clamp recording and visually-guided saccade test were analyzed by one-way repeated-measures ANOVA followed by the Dunnett post-hoc test. With regards to the fitting model, we used log-linear regression to fit the data. P < 0.05 was considered statistically significant. Data are presented as mean ± SEM.
Data availability
Data used from this study are available from the corresponding author upon request.
Code availability
Custom-written codes used to analyze data from this study are available from the corresponding author upon request.