Detailed Visual Cortical Responses Generated by Retinal Sheet Transplants in Rats with Severe Retinal Degeneration

Transplantation procedure.

The transplantation procedure was performed according to previously described methodology (Aramant and Seiler, 2002). Line-3 rats (P24–25) were anesthetized with a mixture of ketamine (40–55 mg/kg) and xylazine (6–7.5 mg/kg; i.p.). Their pupils were dilated with 1% atropine eye drops, and local anesthesia was provided by tetracaine eye drops (0.5%). A small incision (width 1.0 mm) was made posterior to the pars plana, parallel to the limbus. The implantation instrument was inserted with extreme care to minimize disturbance of the host retinal pigment epithelium. The graft tissue (∼1 mm²) was released into the subretinal space posteriorly at the nasal quadrant near the optic disc (Fig. 1). Transplants were placed into the left eye only, leaving the right eye as a control. The incision was closed with two 10–0 sutures, and the eyes were treated with gentamycin and artificial tears ointment. For recovery, rats were given a subcutaneous injection of Ringer's saline solution, the analgesic Buprenex (0.03 mg/kg) for pain management, and placed in a Thermocare incubator. Animals with transplant misplacement and/or excessive surgical trauma were excluded from study.

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Figure 1.

Diagram of experimental setup. A retinal sheet transplant derived from fetal transgenic rats is carefully placed in the subretinal space between host-degenerated retina and retinal pigment epithelium using a custom-made surgical tool (Left). The transplant is placed close to the optic disc in the upper temporal area of the visual field (Right). The horizontal meridian (HM) bisects the upper and lower visual field representations.

Transplant verification.

Transplants were evaluated in vivo for placement, development, and integration within the host retina ∼1 month after surgery by using spectral domain optical coherence tomography. Spectral domain optical coherence tomography images of the retina were obtained using an Envisu R2200 Spectral Domain Ophthalmic Imaging System (Bioptigen). Anesthesia was induced with ketamine/xylazine as above and maintained using isoflurane (0.5%–1.5%) mixed with O₂ through a gas anesthesia mask (Stoelting). Pupils were dilated by 1% atropine sulfate ophthalmic solution. Eyes were kept moist between scans with Systane eye drops (Alcon Laboratories). Imaging was accomplished using rectangular scans of a 2.6 mm × 2.6 mm area at an imaging depth of 1.6 mm. Retina fundus scans were acquired using at least one of four parameters: 488 B scans × 488 A scans × 5 B scan averaging for fundus images; and 700 × 70 × 25, 800 × 50 × 30, and 800 × 20 × 80 for cross-sectional images (units are no. of B scans/no. of A scans/ B scan averaging value). A scans were performed along the fundus axial plane. Each A scan was then further probed by B scans to create the cross-sectional visualization of the retinal layers. B scans were then averaged together to reduce background speckle and improve resolution of low contrast and hard-to-see layers; the larger the B scan averaging value, the better the resolution. The optic disc was centered and used as a point of reference for locating the transplant and assessing surgical success.

Single-unit recordings and visual stimulation.

Rats were initially anesthetized with 2% isoflurane in a mixture of N₂O/O₂ (70%/30%) and then placed into a stereotaxic apparatus. A small, custom-made plastic chamber was glued to the exposed skull. After 1 d of recovery, reanesthetized animals were placed in a custom-made hammock, maintained under isoflurane anesthesia (1%–2% in mixture of N₂O/O₂), and a single tungsten electrode was inserted into a small craniotomy above visual cortex. Once the electrode was inserted, the chamber was filled with sterile saline. During recording sessions, animals were kept sedated under light isoflurane anesthesia (0.2%–0.4%) in a mixture of N₂O/O₂. EEG and EKG were monitored throughout the experiments, and body temperature was maintained with a heating pad (Harvard Apparatus).

Data were acquired using a 32-channel Scout recording system. The spike signal was bandpass filtered from 500 Hz to 7 kHz and stored in a computer hard drive at 30 kHz sampling frequency. Spikes were sorted online in Trellis while performing visual stimulation. Visual stimuli were generated in MATLAB (The MathWorks) using Psychophysics Toolbox (Brainard, 1997; Pelli, 1997; Kleiner et al., 2007) and displayed on a gamma-corrected LCD monitor (55 inches, 60 Hz) at resolution 1920 × 1080 pixels and 52 cd/m² mean luminance. Stimulus onset times were corrected for LCD monitor delay using a photoresistor and microcontroller (in-house design).

For recordings of visually evoked responses, cells were first tested for visual responsiveness with 100 repetitions of a 500 ms bright flash stimulus (105 cd/m²). Receptive fields for visually responsive cells were located using square-wave drifting gratings, after which optimal orientation/direction and spatial and temporal frequencies were determined using sine wave gratings. Spatial frequencies tested were from 0.001 to 0.5 cycles/°. Temporal frequencies tested were 0.1–10 cycles/s. With these optimal parameters, size tuning was assessed using sizes of 1°-110°, and 100% contrast. With the optimal size, temporal and spatial frequencies, and at high contrast, the orientation selectivity of the cell was tested again using 16 directions stepped by 22.5° increments. This was followed by testing of contrast.

Virus injections.

Upon completion of V1 recordings, a subset of rats were given injections of mCherry and/or GFP-expressing versions of a glycoprotein-deleted rabies virus (Wickersham et al., 2007a,b). Viruses were grown in the laboratory from existing stock and concentrated to a titer range of ∼5 × 10⁹ infectious units/ml following published protocols (Wickersham et al., 2010; Osakada et al., 2011). For injections, animals were anesthetized with isoflurane and placed in a stereotaxic head-holder. Under sterile conditions, the existing craniotomy from the recording procedure was accessed. Glass pipettes with tips broken to ∼20 μm were filled with virus and inserted using a computer-controlled micro-positioner attached to a stereotaxic arm (Kopf). Pressure injections of ∼0.5 μl were made at depths of ∼500 and 1200 μm using a pico-pump. Following injections, artificial dura (Tecoflex, Microspec) was placed over the craniotomy, the skull sealed with dental acrylic, the animals revived and given a 7–10 d survival before perfusion and histology.

Histology.

Upon completion of recordings and/or following virus injection survival time, animals were deeply anesthetized with Euthasol (390 mg/ml of sodium pentobarbital and 50 mg/ml of sodium phenytoin; i.p.; Vedco) and perfused transcardially, first with saline and then with 4% PFA in phosphate buffer (PB), pH 7.4. Brains were removed and cryoprotected in 30% sucrose for ∼48 h before sectioning. Eyes were also removed and postfixed in 4% PFA overnight.

Whole brains were cut coronally into 40 μm sections and then mounted and coverslipped using PVA-DABCO to preserve rabies virus fluorescence. Brain sections were examined under fluorescent microscopy (Carl Zeiss Axioplan) with 10× (0.45 NA) objectives where rabies-infected cell positions were reconstructed using Neurolucida software (MicroBrightField). To limit bleaching of fluorescence, analysis was done offline. Images of whole sections were captured with a high-power black and white digital camera (SensiCam QE, Cooke) and stitched together through the Virtual Slide module. For each animal, one of every four sections was used to identify the number of infected neurons in four visual structures: the LGN, lateral posterior nucleus (LP), V1, and higher visual cortex, confirmed through the atlas by Paxinos and Watson (2001). Based on these counts, the percentage of labeled cells found in each area was calculated for each case (see Results). In animals where both the GFP and mCherry-expressing rabies viruses were injected, infected cells were counted separately.

After washing with PB, eye cups were dissected along the dorsoventral axis, embedded in Tissue-Tek optimum cutting temperature compound (Sakura Finetek), and frozen in isopentane on dry ice. The 10 μm sections were cut on a cryostat and stored at −20°C. Every fifth slide was analyzed for the presence of the transplant by staining with BCIP/NBT substrate (B1911; Sigma-Aldrich). BCIP/NBT-stained slides were imaged on an Olympus BXH10 (Olympus Scientific Solutions) using an Infinity 3–1U camera (Lumenera). For immunofluorescence, cryostat sections underwent antigen retrieval at 70°C with Histo-VT One (Nacalai) and blocked for at least 30 min in 10% donkey serum. Primaries were left on sections overnight at 4°C. Antibody vendor and concentration are as follows: rabbit α-hPAP (1:200; Epitomics, Abcam), mouse α-hPAP (1:25, clone A89; Thermo Fisher Scientific), mouse α-PKCα (1:500; Stressgen Biotechnologies), rabbit α-rhodopsin (1:100; kind gift of Dr. Robert Molday, University of British Columbia), and rabbit α-red/green opsin (1:100; Millipore Bioscience Research Reagents, Fisher Scientific). After several PBS washes, slides were incubated for at least 30 min at room temperature in fluorescent secondary antibodies: AlexaFluor-488 donkey anti-rabbit IgG (H+L), rhodamine × donkey anti-rabbit IgG (H+L), AlexaFluor-488 donkey anti-mouse IgG (H+L), or rhodamine × donkey anti-mouse IgG (H+L) (1:400; Jackson ImmunoResearch Laboratories). Fluorescent sections were coverslipped using Vectashield mounting media (Vector Labs) with 5 μg/ml DAPI (Thermo Fisher Scientific). Fluorescence was imaged using an LSM700 confocal microscope (Carl Zeiss) taking tiled stacks of 5–8 μm thickness at 40× (selected images). Zen 2012 software (Carl Zeiss) was used to extract confocal images. 3D images were extracted separately for each channel and combined in Photoshop CS6 software (Adobe).

Data analysis.

Tuning curves were calculated based on average spike rate. Optimal visual parameters were chosen as the maximum response value. Orientation selectivity index (OSI) was calculated as follows (Cavanaugh et al., 2002; Hashemi-Nezhad and Lyon, 2012):

where θ_n is the nth orientation of the stimulus and R_n is the corresponding response.

For tuning width, the orientation responses were fitted to Gaussian distributions (Carandini and Ferster, 2000; Alitto and Usrey, 2004; Y. J. Liu et al., 2015, 2017) using the following:

where O_s is the stimulus orientation, R_Os is the response to different orientations, O_p is the preferred orientation, R_p and R_n are the responses at the preferred and nonpreferred direction, σ is the tuning width, and baseline is the offset of the Gaussian distribution. Gaussian fits were estimated without subtracting spontaneous activity, similar to the procedures of Alitto and Usrey (2004). The orientation tuning bandwidth of each tuning curve was measured in degrees as the half-width at half-height (HWHH), which equals 1.18 × σ based on the equation above.

Size tuning curves were fitted by a difference of Gaussian function (Y. J. Liu et al., 2011a) as follows:

in which R_s is the response evoked by different aperture sizes. The free parameters, K_e and r_e, describe the strength and the size of the excitatory space, respectively; K_i and R_i represent the strength and the size of the inhibitory space, respectively; and R₀ is the spontaneous activity of the cell.

The optimal spatial and temporal frequencies were extracted from the data fitted to Gaussian distributions using the following equation (DeAngelis et al., 1993; Van den Bergh et al., 2010):

Where R_SF/TF is the estimated response, R_pref indicates response at preferred spatial or temporal frequency, SF/TF indicates spatial or temporal frequency, σ is the SD of the Gaussian, and baseline is Gaussian offset.

The contrast tuning was fitted by using the Naka-Rushton equation (Naka and Rushton, 1966; Albrecht and Hamilton, 1982; Van den Bergh et al., 2010; Przybyszewski et al., 2014):

where g is the gain (response), C₅₀ is a contrast at mid response, and n is the exponent. For each fit, the background spontaneous activity was subtracted from the response curve and values below background SD were changed to 0 (Van den Bergh et al., 2010). The contrast threshold was defined as the contrast value exceeding 10% of maximum response.

The cumulative distribution function (CDF) was used to show and compare data distributions between animal groups. The CDF indicates the probability that the sample value will be less than or equal to the given parameter.