Visual responses in the dorsal lateral geniculate nucleus at early stages of retinal degeneration in rd¹ PDE6β mice

Abstract

Inherited retinal degenerations encompass a wide range of diseases that result in the death of rod and cone photoreceptors, eventually leading to irreversible blindness. Low vision survives at early stages of degeneration, at which point it could rely on residual populations of rod/cone photoreceptors as well as the inner retinal photoreceptor, melanopsin. To date, the impact of partial retinal degeneration on visual responses in the primary visual thalamus (dorsal lateral geniculate nucleus, dLGN) remains unknown, as does their relative reliance on surviving rod and cone photoreceptors vs. melanopsin. To answer these questions, we recorded visually evoked responses in the dLGN of anesthetized rd¹ mice using in vivo electrophysiology at an age (3–5 wk) at which cones are partially degenerate and rods are absent. We found that excitatory (ON) responses to light had lower amplitude and longer latency in rd¹ mice compared with age-matched visually intact controls; however, contrast sensitivity and spatial receptive field size were largely unaffected at this early stage of degeneration. Responses were retained when those wavelengths to which melanopsin is most sensitive were depleted, indicating that they were driven primarily by surviving cones. Inhibitory responses appeared absent in the rd¹ thalamus, as did light-evoked gamma oscillations in firing. This description of fundamental features of the dLGN visual response at this intermediate stage of retinal degeneration provides a context for emerging attempts to restore vision by introducing ectopic photoreception to the degenerate retina.

NEW & NOTEWORTHY This study provides new therapeutically relevant insights to visual responses in the dorsal lateral geniculate nucleus during progressive retinal degeneration. Using in vivo electrophysiology, we demonstrate that visual responses have lower amplitude and longer latency during degeneration, but contrast sensitivity and spatial receptive fields remain unaffected. Such visual responses are driven predominantly by surviving cones rather than melanopsin photoreceptors. The functional integrity of this visual pathway is encouraging for emerging attempts at visual restoration.

INTRODUCTION

Inherited retinal degenerations, such as retinitis pigmentosa, are the most common cause of blindness in humans, with an incidence of 1:4,000. Irrespective of etiology, most affect the outer retina and lead to progressive and irreversible death of rod and cone photoreceptors at advanced stages of the disease. In the rd¹ mouse model of retinitis pigmentosa, the retina undergoes well-defined stages of cell death and reorganization (Jones and Marc 2005; Strettoi et al. 2003). Rod photoreceptors die rapidly to be lost by postnatal day 18 (P18) (Greferath et al. 2009), and this is followed by progressive degeneration of the cone photoreceptor population (Lin et al. 2009) and remodeling of the inner retinal neurons (Marc et al. 2003; Strettoi and Pignatelli 2000; Strettoi et al. 2002). However, isolated pockets of cones can survive into the later stages of the disease (Carter-Dawson and LaVail 1979; Jiménez et al. 1996; Ogilvie et al. 1997), mirroring some human conditions.

The anatomical changes in the retina are mirrored by changes in the electrophysiological properties of residual light responses and the retinal network. During partial degeneration, residual light responses recorded from retinal ganglion cells already show a reduction in response amplitude and slower signaling kinetics (Gibson et al. 2013; Stasheff 2008; Strettoi et al. 2002). As a consequence of photoreceptor degeneration and remodeling, the remaining inner retinal neurons exhibit robust rhythmic oscillations (Choi et al. 2014; Menzler and Zeck 2011) and an increase in baseline firing at rest (Stasheff 2008). A third source of light responses (the photopigment melanopsin expressed in a specialized subset of retinal ganglion cells) is less impacted by degeneration. These cells survive retinal degeneration in adults with broadly normal retinal anatomy (Lin and Peng 2013; Vugler et al. 2008) and drive excitatory responses to light in various brain regions, including the dorsal lateral geniculate nucleus (dLGN) (Brown et al. 2010; Procyk et al. 2015).

Little is known about central responses to visual stimuli in progressive retinal degeneration. In advanced stages, responses in the visual thalamus originate solely from melanopsin and have extremely poor spatiotemporal resolution (Brown et al. 2010; Procyk et al. 2015). In this condition, spatial receptive fields for the melanopsin response of individual dLGN units can be very large, and melanopsin-driven responses to simple light steps dissipate over tens of seconds (Procyk et al. 2015). It remains unclear how disrupted visual responses are at earlier stages of degeneration, or the extent to which these responses rely on melanopsin compared with surviving cones. We address this unknown by recording visual responses in the dLGN of juvenile rd¹ mice at an age at which significant numbers of cone photoreceptors survive but rods are absent. We find a variety of light-responsive units throughout the dLGN of juvenile rd¹ mice that exhibit low amplitude and enhanced latency (as previously reported for retinal responses in such animals). Contrast sensitivity was, however, retained, and the spatial receptive fields of dLGN units in this model were at least as fine as those of wild-type mice. Application of the principles of silent substitution to bias stimuli against stimulating melanopsin indicated that dLGN light responses were driven primarily by surviving cones.

METHODS

Ethical Approval

The care and use of all mice in this study were carried out in strict accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 (revised in 2012), and approved by the local Manchester Animal Welfare and Ethical Review Board (AWERB reference 50/02506). All in vivo surgical procedures were performed under terminal urethane anesthesia, and all efforts were made to minimize suffering.

Animals

Mice were bred at the University of Manchester and housed under a 12:12-h light-dark cycle, with food and water available ad libitum. Because we aimed to use the method of receptor silent substitution to separate cone- from melanopsin-evoked responses, we undertook experiments on Opn1mw^R mice (stock no. 008619; Jackson Laboratories) in which a coding sequence for the human long-wavelength-sensitive cone opsin is knocked into the medium-wavelength-sensitive cone opsin locus. These animals have a fully functional visual system but have enhanced divergence in spectral sensitivity between cones and melanopsin, allowing for the use of carefully designed stimuli to dissect the contribution of individual photoreceptors to the light response. Our colony of Opn1mw^R mice has been backcrossed to the C57BL/6J mouse line for more than nine generations. Opn1mw^R mice homozygous for the rd¹ mutation (PDE6β^rd1/rd1) were created in house by crossing this established colony of Opn1mw^R mice with commercially available C57 rd¹ mice (stock no. 004766; Jackson Laboratories). Note that Opn1mw^R refers to the transgenic allele originally generated by (Smallwood et al. 2003) and termed simply “R” by them. For all electrophysiological experiments, Opn1mw^R and rd¹ Opn1mw^R mice were used between 3 and 5 wk of age.

In Vivo Electrophysiology

Six juvenile C57 rd/rd Opn1mw^R mice and eight juvenile Opn1mw^R were administered with 20% urethane (1.6 mg/kg ip). Once anesthetized, mice were mounted onto a bespoke stereotaxic frame (SG-4N-S; Narishige, Japan) which was fixed onto a “lazy Susan” (RBB12A; Thorlabs, Germany). Core body temperature was maintained at 37°C with a homeothermic blanket (Harvard Apparatus, Kent, UK). An incision to expose the skull surface was made, and a small hole (~1-mm diameter) was drilled 2.2 mm posterior and 2.2 mm lateral to the bregma, targeting the dLGN. A recording probe (A4X8-5 mm-50-200-413; Neuronexus, MI) consisting of four shanks (spaced 200 μm apart), each with eight recordings sites (spaced 50 μm apart), was then positioned centrally on the exposed surface in the coronal plane and lowered to a depth of 2.5–3.3 mm to target the dLGN using a fluid-filled micromanipulator (MO-10; Narishige, Japan). Once the recording probe was in position, mice were dark adapted for 30 min to allow neuronal activity to stabilize following probe insertion. Stimuli were presented to the eye contralateral to the craniotomy, which was treated with topical atropine sulfate (1% wt/vol; Sigma-Aldrich, UK) to dilate the pupil and mineral oil to keep the cornea moist. The ipsilateral eye remained covered with blackout material throughout the entire experiment. In some experiments, following recording in one location the probe was moved 200 μm caudal and a second set of responses recorded. Neural signals were acquired using a Recorder64 system (Plexon, TX). Signals were amplified 3,000 times, high-pass filtered at 300 Hz, and digitized at 40 kHz. Multiunit activity (spikes with amplitudes >50 μV) were saved as time-stamped waveforms and analyzed offline (see Data Analysis and Statistics).

Presentation of Visual Stimuli

Light stimuli were generated in MATLAB (The MathWorks, Natick, MA) and controlled by a laptop running PsychoPy V2.6 (Peirce 2009). Light stimuli were presented via a commercially available projection system that had been modified so that each of the red, green, and blue channels was a combination of up to five independently controlled wavelengths (λ_max = 405, 455, 525, 561, or 630 nm) as previously described (Allen et al. 2017). This allowed us to present patterned stimuli that only present spatial/temporal contrast for particular photopigments in our Opn1mw^R and rd¹ Opn1mw^R mice (Fig. 1A). As such, we designed three multispectral stimuli, allowing the contribution of cone opsin and melanopsin to the rd¹ light response to be defined using receptor silent substitution (Fig. 1B). Transition from spectrum 1 to spectrum 3 was designed to provide a positive contrast for all photoreceptors in the Opn1mw^R retina (“all photoreceptor” stimulus S-cone, 51%; L-cone, 47%; rod, 34%; and melanopsin, 51%). This was matched with a “mel-less” stimulus (transition from spectrum 2 to spectrum 3) providing equivalent contrast for S-cones (50%), L-cones (49%), and rods (30%) but very low contrast for melanopsin (<5%). A full table of the effective irradiance change and Michelson contrast for each photopigment during spectral transitions is shown in Fig. 1C. All light measurements were measured using a calibrated spectroradiometer (SpectroCal; Cambridge Research Instruments, UK). Effective photon flux for each photopigment was determined using the calculated spectra and visual pigment template described by Govardovskii et al. (2000). The projector screen was positioned in the center of the visual field relative to the eye contralateral to the recording probe so that the horizontal and vertical meridians of the stimulus display subtended 72° in azimuth and 57° in elevation, respectively. To confirm these calibrated stimuli indeed had the expected photopigment selectivity, we presented 50 repeats of full-field all-photoreceptor and mel-less flashes at the beginning of each recording at a frequency of 4 Hz. As expected, visual responses to all-photoreceptor and mel-less stimuli were equivalent under these conditions in both visually intact (Fig. 1D) and degenerate mice (Fig. 1E). We characterized the responses of these units to our all-photoreceptor and mel-less conditions by quantifying the peak response amplitude (Fig. 1F) and the latency to peak response (Fig. 1G) in both Opn1mw^R and rd¹ Opn1mw^R mice. We found there to be no significant differences between the two stimulus conditions for amplitude (Opn1mw^R, P = 0.09; rd¹ Opn1mw^R, P = 0.79) or latency (Opn1mw^R, P = 0.97; rd¹ Opn1mw^R, P = 0.27).

Fig. 1.

Design and validation of silent substitution stimuli. A: the Opn1mw^R retina expresses 4 spectrally distinct opsins in the retina: S-cone opsin (maximum wavelength λ_max = 390 nm; purple), melanopsin (λ_max = 480 nm; blue), rod opsin (λ_max = 498 nm; black), and the human L-cone opsin (λ_max = 556 nm; green), which is knocked into the genome in place of the native mouse green cone opsin (λ_max = 508 nm). The rd¹ Opn1mw^R retina expresses 3 spectrally distinct and functional photoreceptors in the retina: S-cones, L-cones, and melanopsin. Rod photoreceptors are rendered functionless from birth due to the rd¹ mutation and rapidly degenerate by postnatal day 17. B: the output of 4 LEDs (peak emissions = 405, 455, 525, and 630 nm) and a laser (peak emission = 561 nm) were used to produce 3 spectra (spectrum 1, green trace; spectrum 2, pink trace; and spectrum 3, orange trace). Transition from spectrum 1 to spectrum 3 (“all-photoreceptor” stimulus) presented a positive contrast for rod opsin, cone opsins, and melanopsin. Transition from spectrum 2 to spectrum 3 (“mel-less” stimulus) provided the same contrast for rod and cone photoreceptors as the all-photoreceptor condition but had a minimal melanopsin contrast. C: effective photon flux (mean ± SE; left) and calculated Michaelson contrast (%, means ± SE; right) for each photopigment in the Opn1mw^R retina (L-cone opsin, S-cone opsin, rod opsin, and melanopsin) when presented with spectra 1, 2, and 3 for transitions in the all-photoreceptor and mel-less conditions. D and E: peristimulus time histograms (means ± SE) demonstrate the light responses of dLGN units from the Opn1mw^R and rd¹ Opn1mw^R populations, respectively, in response to 50 presentations of the all-photoreceptor (black trace) and mel-less (red trace) stimuli. Data are baseline-subtracted changes in firing rate (ΔFR; time bin = 0.01 s). F: peak response amplitude (mean ± SE) for single dorsal lateral geniculate nucleus (dLGN) units was not significantly different when the all-photoreceptor and mel-less conditions were compared for Opn1mw^R mice (26.33 ± 1.52 and 24.38 ± 1.73 spikes/s, respectively; P = 0.09) and rd¹ Opn1mw^R mice (20.25 ± 1.73 and 19.58 ± 1.84 spikes/s, respectively; P = 0.79). G: latency to peak response (mean ± SE) for single dLGN units also was not significantly different when the all-photoreceptor and mel-less conditions were compared for Opn1mw^R mice (154.26 ± 4.87 and 156.55 ± 4.68 ms, respectively; P = 0.97) and rd¹ Opn1mw^R mice (183.13 ± 5.28 and 174.93 ± 6.63 ms, respectively; P = 0.27).

Visual Stimuli

Dark-adapted responses.

At the beginning of each experiment we presented 200-ms full-field flashes (irradiance = 2.50 × 10¹⁴ photons·cm⁻²·s⁻¹) from darkness with a 1-s interstimulus interval (ISI) for 50 repeats. We additionally presented 10-s light steps from darkness to the same irradiance with an ISI of 50 s over 20 repeats to identify those units which possessed a sustained component to the light response.

Contrast sensitivity.

Full-field 1-s light steps, with a 5-s ISI, were presented at eight increasing cone contrasts (1%, 2%, 5%, 16%, 20%, 30%, 40%, and 50%) from a light-adapted background (irradiance = 2.64 × 10¹⁴ photons·cm⁻²·s⁻¹). Each sequence was repeated 20 times in an interleaved manner using the all-photoreceptor stimulus settings described above.

Receptive field mapping.

Vertical bars (occupying ~13° of the visual field; irradiance = 1.04 × 10¹⁴ photons·cm⁻²·s⁻¹) from a background (irradiance = 1.55 × 10¹³ photons·cm⁻²·s⁻¹) were used to map receptive fields of dLGN neurons using the all-photoreceptor stimulus condition. Vertical bars were presented for 250 ms in a pseudorandom order in 13 (overlapping) spatial locations (4.5° separation in bar position; ISI = 1.25 s). The spectra used for these spatial stimuli did not elicit significant responses in the rd¹ population. However, because these mice do not possess functional rods, we were able to generate a new spectral transition, which allowed us to present bars with a larger calculated Michaelson contrast for both S- and L-cone opsins. Spatial receptive fields for rd¹ mice were mapped under these new settings.

Silent substitution steps.

We initially presented full-field transitions (4Hz) between our two pairs of silent substitution stimuli: all photoreceptor and mel-less. The stimulus spectra were adjusted every 50 repeats to validate the stimulus conditions. Following this, full-field 10-s light steps from a light-adapted background were presented 20 times with a 50-s ISI under the all-photoreceptor and mel-less stimulus conditions. Stimuli were presented in a pseudorandom order to determine the contribution of activating both cones and melanopsin together and cones in isolation.

Data Analysis and Statistics

Offline, neural waveforms were processed using Offline Sorter (version 2.8.8; Plexon). Cross-channel artifacts were identified and removed, and then each channel was analyzed separately. For each channel, single-unit spikes were detected and categorized on the basis of the spike waveform via a principal component analysis, whereby distinct clusters of spikes were readily identifiable and showed a clear refractory period in their interspike interval distribution (>1 ms). Single-unit data were subsequently sent to NeuroExplorer (version 4.032; Nex Technologies, MA) and MATLAB R2010a (The MathWorks) to further analyze changes in firing rate of single units in response to the different visual stimuli presented. Statistical analysis and figure generation were conducted in GraphPad Prism 7 and Corel Draw, respectively.

Identification of light responses.

In the dark-adapted state, single units were classed as light responsive if the firing rate during stimulus presentation exceeded 2 SD of the mean baseline firing rate before light exposure. Presentation of the 10-s light-step under the dark-adapted state allowed us to qualitatively categorize cells on the basis of their light-response profile. Accordingly, single units were defined as transient ON if they demonstrated a significant change in firing rate after light onset that quickly returned to baseline during the light pulse. Transient ON-OFF cells also showed an initial increase in firing rate at light onset before quickly returning to baseline, but showed a second significant increase in firing immediately after light offset. Sustained ON and sustained OFF cells were categorized if a significant increase or decrease in firing rate was maintained for more than 5 s of a 10-s light step, respectively. Under light-adapted conditions, single units were categorized on the basis of their response to the all-photoreceptor condition.

Contrast sensitivity analysis.

Single units were filtered to ensure that the firing rate at the maximum cone contrast (50%) demonstrated a significant change in firing rate that was >2 SD above the prestimulus baseline. If this criterion was met, the response of that unit at the seven lower contrasts was used for analysis regardless of whether it crossed the confidence interval. Contrast sensitivity curves were calculated by subtracting the prestimulus baseline from the average firing rate over the first 500 ms of the 1-s light step. Sensitivity curves were compared with an F test in GraphPad Prism 7 (GraphPad Software) to test whether the sensitivity of visual responses in each genotype were best fit with a single curve or two individual curves. For population data, we fitted a normalized dose-response function to individual units from Opn1mw^R and rd¹ Opn1mw^R mice and compared the cone contrast at half-maximum response (for units with R² > 0.6) using an unpaired t test.

Spatial receptive field analysis.

To qualify for inclusion in our assessment of receptive field size, single units had to show a significant change in firing rate (>2 SD above baseline) to at least one bar position over 90 repeats of the stimulus sequence. The spatial receptive field size for single units meeting this criterion was estimated by fitting a two-dimensional Gaussian fit (R² > 0.7) to the relationship between response amplitude and bar position in GraphPad Prism 7 (GraphPad Software). The receptive field size for individual cells was described as 1 SD of the best-fit Gaussian.

Silent substitution analysis.

Single units were first classified as sustained or transient on the basis of their response to a 10-s light-step under the all-photoreceptor condition. Single units were classified as sustained if they maintained their change in firing rate >2 SD above baseline for >5 s over the course of the 10-s light step. Comparisons between the total number of spikes (calculated by integrating under the peristimulus time histogram (PSTH) from 2 to 10 s during light exposure) in the all-photoreceptor and mel-less conditions in both genotypes was used to determine the contribution of melanopsin signaling to the dLGN and were analyzed using two-way ANOVA (with post hoc Bonferroni correction) in GraphPad Prism 7 (GraphPad Software).

Tissue Preparation

Following electrophysiological recordings, mice were transcardially perfused with 0.9% saline followed by cold 4% methanol-free paraformaldehyde (Sigma-Aldrich, UK). The brain was removed and postfixed overnight in 4% paraformaldehyde before cryoprotection for 24 h in 30% sucrose in 0.1 M PBS. Coronal sections (100 μm) were cut using a sledge microtome and mounted onto glass slides, and coverslips were applied using Vectashield (Vector Laboratories). Electrode placement in the dLGN was confirmed by visualization of a fluorescence dye (Cell Tracker CM-DiI; Invitrogen, Paisley, UK) applied to the probe before recording and compared with the stereotaxic mouse atlas. Images were collected on an Olympus BX51 upright microscope using a ×4/0.30 Plan Fln objective and captured using a CoolSNAP ES camera (Photometrics) through MetaVue Software (Molecular Devices). Specific bandpass filters set for DAPI, FITC, and Texas red prevented bleed-through of channels.

RESULTS

We set out to describe the impact of partial retinal degeneration on dLGN visual responses using young rd¹ mice in which loss of cones is incomplete. To facilitate attempts to determine whether surviving responses originated with cones or the inner retinal photoreceptor, melanopsin, we used animals further manipulated to shift the spectral sensitivity of cones expressing medium-wavelength-sensitive opsin to longer wavelengths far from those favored by melanopsin (Opn1mw^R; Smallwood et al. 2003). We first presented full-field 200-ms flashes (2.50 × 10¹⁴ photons·cm⁻²·s⁻¹) from darkness to eight Opn1mw^R and six rd¹ Opn1mw^R mice (3–5 wk of age) and recorded responses in the dLGN using extracellular multichannel recording electrodes. Light-evoked changes in activity were recorded across the anatomical extent of the dLGN in rd¹ Opn1mw^R mice (shown for a representative individual in Figs. 2, A and B). Although we could detect visual responses in all animals, we found the number of light-responsive units per electrode placement to be negatively correlated with age in the rd¹ Opn1mw^R (linear regression slope = −1.35; P = 0.003) but not visually intact animals (linear regression slope = 0.67; P > 0.05; Fig. 2C).

Fig. 2.

Dark-adapted light-responses in the rd¹ Opn1mw^R dorsal lateral geniculate nucleus (dLGN). A: representative image of DiI-labeled electrode tract (blue) superimposed with channels of the A4X8-5 mm-50-200-413 recording electrode (gray circles) in an rd¹ Opn1mw^R mouse, confirming placement of recording electrode (Paxinos and Watson mouse atlas used to confirm placement of the recording electrode in the dLGN, outlined by black dotted line). B: representative reconstruction of light-responsive channels found in the rd¹ Opn1mw^R dLGN recording from A in response to full-field 200-ms flashes (2.50 × 10¹⁴ photons·cm⁻²·s⁻¹) from darkness. C: plotting the number of light-responsive units per electrode placement as a function of age demonstrated a significant decrease in light-responsive units in the rd¹ Opn1mw^R population (green crosses; slope = −1.35; P = 0.003) compared with Opn1mw^R mice (black crosses; slope = 0.67; P = 0.06). D: single-unit light responses could be categorized as transient or sustained in response to a 10-s light step (irradiance = 2.50 × 10¹⁴ photons·cm⁻²·s⁻¹) from darkness (n = 135 light-responsive units from 8 Opn1mw^R mice and 90 light-responsive units from 6 rd¹ Opn1mw^R mice). Transient cells could be further subdivided into transient ON and transient ON-OFF responses to light, whereas sustained cells demonstrated a sustained ON or sustained OFF response to light (percentage of cells in each genotype with response type shown at top right). E: peak ON response amplitude was calculated for transient (transient ON, transient ON-OFF; circles) and sustained (sustained ON; triangles) units for both Opn1mw^R (green symbols) and rd¹ Opn1mw^R mice (black symbols). There was no significant difference between transient and sustained populations for Opn1mw^R (P = 0.83) and rd¹ Opn1mw^R units (P = 0.15), but there was a significant difference between transient units in Opn1mw^R and rd¹ Opn1mw^R mice (*P = 0.026) and between sustained units in the Opn1mw^R dLGN and transient units in the rd¹ Opn1mw^R dLGN (***P = 0.0002; 2-way ANOVA with post hoc Bonferroni correction). F: time to peak response was significantly faster for Opn1mw^R dLGN units. Transient units in the Opn1mw^R dLGN were significantly faster than transient (****P < 0.0001) and sustained units (**P = 0.0085) in the rd¹ Opn1mw^R units. Sustained units in the Opn1mw^R dLGN were also significantly faster than transient units in the rd¹ Opn1mw^R dLGN (***P = 0.0009), but were not significantly faster than sustained units (P = 0.20; 2-way ANOVA with post hoc Bonferroni correction). G: the integrated peristimulus time histogram of the sustained component of the light response (total spikes, means ± SE) was significantly larger in Opn1mw^R compared with rd¹ Opn1mw^R units (112.1 ± 14.52 and 20.36 ± 3.28 spikes, respectively; **P = 0.0028; unpaired t test). H: normalized power spectrum density (PSD) of light-responsive units during a 10-s light pulse demonstrates that a robust peak can be identified in the Opn1mw^R population (green trace; 31.3 ± 0.39 Hz, mean ± SE) but that there is no discernible peak in the in the rd¹ Opn1mw^R population (black trace).

We next presented 10-s full-field pulses from darkness (irradiance = 2.50 × 10¹⁴ photons·cm⁻²·s⁻¹) and could categorize light-responsive units into four qualitatively distinct groups: transient ON, transient ON-OFF, sustained ON, and sustained OFF. Transient ON units showed an initial increase in firing rate at light onset but quickly returned to baseline (Fig. 2D, first row). Transient ON-OFF units showed a transient increase in firing at both light onset and offset (Fig. 2D, second row). Sustained ON units demonstrate an initial increase in firing rate at light onset, and firing remained elevated above baseline throughout the duration of the light stimulus (Fig. 2D, third row). Conversely, sustained OFF units show a reduction in firing rate maintained over the duration of the light stimulus (Fig. 2D, fourth row). In visually intact Opn1mw^R mice of equivalent age, 24% of light-responsive units were transient ON, 23% transient ON-OFF, 45% sustained ON, and 8% sustained OFF. Light-responses in rd¹ Opn1mw^R mice were more transient in nature, with 65% of light-responsive units having a transient ON, 15% a transient ON-OFF, and only 20% a sustained ON response. We did not find a single example of a sustained OFF responses in the rd¹ Opn1mw^R population.

We then set out to characterize the transient ON component of these visual responses. We found that there was a significant difference in the peak response amplitude of light responses when comparing transient units in Opn1mw^R mice and rd¹ Opn1mw^R (9.07 ± 0.44 and 6.21 ± 0.53 spikes/s, respectively, means ± SE; P = 0.026, 2-way ANOVA with post hoc Bonferroni correction; Fig. 2E). Sustained units in Opn1mw^R mice also exhibited larger amplitude responses compared with transient units in rd¹ Opn1mw^R mice (10.45 ± 0.9 and 6.21 ± 0.53 spikes/s, respectively; P = 0.0002, 2-way ANOVA with post hoc Bonferroni correction). Sustained units in the rd¹ Opn1mw^R mice (9.70 ± 1.56 spikes/s) were not significantly different from transient units (P > 0.99) or sustained units (P > 0.99) in Opn1mw^R mice. Response latency was also significantly increased for units in rd¹ Opn1mw^R mice compared with Opn1mw^R mice (P < 0.0001; Fig. 2F). Latency was calculated for transient and sustained populations separately and showed that the time to peak response was significantly increased for transient units in rd¹ Opn1mw^R mice compared with Opn1mw^R mice (208.6 ± 5.25 and 153.6 ± 6.27 ms, respectively; P <0.0001, 2-way ANOVA with post hoc Bonferroni correction), but not for sustained units (168.2 ± 10.50 and 202.4 ± 9.21 ms, respectively; P = 0.20, 2-way ANOVA with post hoc Bonferroni correction). Sustained units in Opn1mw^R mice also showed significantly faster responses (168.2 ± 10.50 ms) compared with transient units in rd¹ Opn1mw^R mice (208.6 ± 5.25 ms; P = 0.0009, 2-way ANOVA with post hoc Bonferroni correction). Turning our attention to the sustained component of visual responses, we calculated the strength of the sustained response in both Opn1mw^R and rd¹ Opn1mw^R mice by integrating under the PSTH from the end of the transient increase in firing to the end of the light pulse (0.25–10 s). We found that the total number of spikes was significantly greater for sustained units in the Opn1mw^R dLGN (112.1 ± 14.52 spikes) compared with the rd¹ Opn1mw^R dLGN (20.36 ± 3.28 spikes; P = 0.0028, unpaired t test; Fig. 2G). Irradiance steps induce narrowband oscillations in the mouse dLGN (Saleem et al. 2017; Storchi et al. 2017). Power spectrum density analysis of firing rates on presentation of these 10-s light steps revealed such behavior (a robust oscillation at 31.3 ± 0.39 Hz; Fig. 2H) in Opn1mw^R mice, but not in rd¹ Opn1mw^R mice.

We next sought to characterize the sensory capabilities of juvenile rd¹ Opn1mw^R mice in more detail. For this purpose, we used the approach of receptor silent substitution to separately interrogate responses driven by cones vs. melanopsin under light-adapted conditions (see Fig. 1 in methods for stimuli descriptions). Concentrating first on cone-driven responses, we presented 1-s light steps from a light-adapted background (irradiance = 2.64 × 10¹⁴ photons·cm⁻²·s⁻¹) with cone contrasts ranging from 1 to 50%, but with minimal predicted melanopsin contrast. We identified 61 units that showed a significant change in firing rate following light onset at the highest contrast in the Opn1mw^R population and 54 units in the rd¹ Opn1mw^R population. The PSTHs (means ± SE) at each contrast for the Opn1mw^R and rd¹ Opn1mw^R are shown in Fig. 3A. Plotting the average change in firing rate over the first 500 ms after light onset at each cone contrast demonstrated that the rd¹ Opn1mw^R mice had a significantly reduced change in firing rate across this contrast range compared with Opn1mw^R mice and is best fit by two individual curves (Fig. 3B; P < 0.0001; F = 44.41). Normalizing these changes in firing rate to the maximum response amplitude in each genotype demonstrated that these cells retain contrast sensitivity similar to that of visually intact controls, given that both populations are best fit by the same dose-response curve (Fig. 3C; R² = 0.94; P = 0.34, F test). We confirmed this by plotting the normalized dose-response function for individual units in visually intact and degenerate mice, which showed there to be no significant difference in the cone contrast at half-maximum response between Opn1mw^R mice (15.39 ± 1.21%) and rd¹ Opn1mw^R mice (15.41 ± 0.97%; P = 0.988, unpaired t test; Fig. 3D).

Fig. 3.

Contrast sensitivity in the rd¹ Opn1mw^R dorsal lateral geniculate nucleus (dLGN). A: peristimulus time histograms (means ± SE) of light-responsive units in the dLGN of Opn1mw^R (green traces; n = 61 units) and rd¹ Opn1mw^R (black traces; n = 54 units) in response to 20 repeats of a 1-s light step at 8 increasing cone contrasts (1, 2, 5, 16, 20, 30, 40, and 50%) presented against a background of irradiance = 2.64 × 10¹⁴ photons·cm⁻²·s⁻¹ (time bin = 0.01 s; interstimulus interval = 5 s). B: change (Δ) in firing rate (mean ± SE) over the first 500 ms of the light step plotted as a function of cone contrast (mean of contrast for L-cone and S-cone). Opn1mw^R mice (green trace) showed significantly larger amplitude response than rd¹ Opn1mw^R mice (black trace) since both populations are best fit by 2 separate dose-response curves (P < 0.001, F = 44.1; Opn1mw^R, R² = 0.95; rd¹ Opn1mw^R, R² = 0.97). C: normalizing peak response amplitude of the data in B to maximum response for that genotype allowed the data for Opn1mw^R (green trace) and rd¹ Opn1mw^R mice (black trace) to be best fit by a single curve (F = 0.798; R² = 0.94). D: normalizing peak response amplitude of the data and fitting a dose-response curve for individual units (R² > 0.6) showed there was no significant difference in the cone contrast at half-maximum response (mean ± SE) between Opn1mw^R mice (15.39 ± 1.21%; green trace) and rd¹ Opn1mw^R mice (15.41 ± 0.97%; black trace; P = 0.988, unpaired t test).

We continued to ask whether the spatial resolution of cone-driven dLGN responses was impacted by retinal degeneration by mapping receptive fields with the use of a vertical bar minimally visible to melanopsin but with ~50% cone contrast for Opn1mw^R mice (Fig. 4A) and ~70% cone contrast for rd¹ Opn1mw^R mice (Fig. 4B). We identified 38 single units from Opn1mw^R mice and 48 single units from rd¹ Opn1mw^R mice responsive to this stimulus. The response of two representative units is shown in Fig. 4C. For all single units we defined receptive field by a best-fit Gaussian to the relationship between bar position and response amplitude (Fig. 4D; R² > 0.7; mean = 0.87 for both rd¹ Opn1mw^R and Opn1mw^R mice). Receptive field diameter was significantly smaller in rd¹ Opn1mw^R mice than in Opn1mw^R mice (Fig. 4E; 9.96° ± 0.3 and 12.17° ± 0.5, respectively; P = 0.0005, unpaired t test). Similarly to the dark-adapted condition, we found the amplitude of these responses to be significantly reduced in rd¹ Opn1mw^R mice (7.02 ± 0.8 spikes/s) compared with Opn1mw^R mice (10.1 ± 1.2 spikes/s; P = 0.03, unpaired t test), even when using stimuli with a higher effective cone contrast in the degenerate mice (Fig. 4F). rd¹ Opn1mw^R mice also demonstrated a significantly slower time to peak response (177.9 ± 5.4 ms) compared with Opn1mw^R mice (112.3 ± 4.46 ms; P < 0.0001, unpaired t test) for cells in which we could record a spatial receptive field (Fig. 4G).

Fig. 4.

Spatial receptive fields in the rd¹ Opn1mw^R dorsal lateral geniculate nucleus (dLGN) A and B: effective photon flux (mean ± SE) of the background and bar stimuli used for receptive field mapping in the Opn1mw^R and rd¹ Opn1mw^R mice, respectively, with calculated Michaelson contrast (%, mean ± SE) for each photopigment. Note that rod contrast is not relevant for rd¹ mice because these animals lack rods at the age of recording. C: heat map for representative single units from the dLGN of an Opn1mw^R (top) and rd¹ Opn1mw^R (bottom) mouse showing change in firing rate (spikes/s; scale at right) in response to the appearance of vertical bars (250 ms starting at time 0; 13° width at 4.5° resolution) as a function of location on azimuth of bar center. D: peak response amplitude (change in firing rate, mean ± SE) as a function of bar position for the 2 units in C, fit with a Gaussian function. E: box-and-whisker plot showing that receptive field diameter (mean ± SE) for all light-responsive units was significantly larger in Opn1mw^R (12.17 ± 0.5°; n = 38 units; green bar) than in rd¹ Opn1mw^R mice (9.96 ± 0.3°; n = 48 units; black bar; ***P = 0.0005, unpaired t test). Box shows interquartile range; line in box is the median; cross is the mean; and whiskers indicate minimum to maximum range. F: peak response amplitude (change in firing rate, mean ± SE) was significantly larger in Opn1mw^R (10.1 ± 1.2 spikes/s) than in rd¹ Opn1mw^R mice (7.02 ± 0.8 spikes/s; *P = 0.03, unpaired t test). G: response latency (mean ± SE) was significantly increased in rd¹ Opn1mw^R (177.9 ± 5.4 ms) compared with Opn1mw^R mice (112.3 ± 4.46 ms; ****P < 0.0001, unpaired t test).

We finally turned our attention to whether light responses were driven disproportionately by melanopsin at this stage of retinal degeneration. We used the silent substitution approach to present longer (10-s full field) steps using our all-photoreceptor stimulus (which is known to activate melanopsin in the adult wild-type retina; Allen et al. 2017) compared with our mel-less stimulus, which has an equivalent contrast for cones but minimal contrast for melanopsin. We found 11 units with a sustained OFF phenotype in the Opn1mw^R population but none in the rd¹ Opn1mw^R population, and as such, these units were excluded from any further analysis. Of units excited by the stimuli, 16/76 in Opn1mw^R and 14/68 in rd¹ Opn1mw^R populations showed a “sustained” response with maintained firing throughout the light step (Fig. 5, A and B), and the remaining “transient” units excited only at the start and/or end of the light step (Fig. 5, D and E). Overall, the response profiles of each population to all-photoreceptor and mel-less stimuli were comparable (Fig. 5, A and B, compared with Fig. 5, D and E). On the basis of previously published work (Allen et al. 2017; Brown et al. 2012), we expected any melanopsin contribution to be apparent in the maintained response of sustained units. Although we found there to be a trend for the magnitude of the all-photoreceptor sustained response to be larger than the mel-less response in both genotypes, we found no significant difference in the total number of spikes throughout the sustained component of the light step (2–10 s) of the sustained population in the all-photoreceptor and mel-less conditions in either the Opn1mw^R mice (18.30 ± 3.0 and 13.67 ± 3.10 total spikes, respectively; P = 0.50, 2-way ANOVA with post hoc Bonferroni correction; Fig. 5C, left) or rd¹ Opn1mw^R mice (17.58 ± 3.25 and 10.05 ± 2.08 total spikes respectively; P = 0.17, 2-way ANOVA with post hoc Bonferroni correction; Fig. 5C, right). As expected, the transient population showed no significant difference in their response between the all-photoreceptor and mel-less conditions over the same duration in Opn1mw^R mice (1.19 ± 0.59 and 0.47 ± 0.59 total spikes, respectively; P = 0.84, 2-way ANOVA with post hoc Bonferroni correction; Fig. 5F, left) or rd¹ Opn1mw^R mice (3.89 ± 0.78 and 3.28 ± 0.71 total spikes, respectively; P > 0.99, 2-way ANOVA with post hoc Bonferroni correction; Fig. 5F, right). The lack of a detectable melanopsin contribution to the sustained population in either genotype was surprising in view of previous description of melanopsin signals in the adult wild-type dLGN (Allen et al. 2017; Brown et al. 2012; Davis et al. 2015) and could be an effect of this particular developmental stage or simply a limitation in the statistical power of these experiments. In either event, these findings confirm that melanopsin does not make a disproportionate contribution to dLGN light responses at this stage of degeneration in rd¹ mice.

Fig. 5.

Melanopsin signals are absent from the juvenile mouse dorsal lateral geniculate nucleus (dLGN). A and B: peristimulus time histograms (PSTH) for change (Δ) in firing rate (means ± SE) of single units with a sustained response phenotype from the dLGN of Opn1mw^R (n = 16 single units from 8 mice) and rd¹ Opn1mw^R (n = 14 single units from 6 mice) mice, respectively, associated with all-photoreceptor and mel-less conditions (black and red traces, respectively; stimulus onset at time 0; duration 10 s). C: total number of spikes (integrated sum of spikes between 2 and 10 s during the light pulse; means ± SE) for the sustained population of cells showed no significant difference between all-photoreceptor and mel-less conditions for Opn1mw^R mice (18.3 ± 3.0 and 13.67 ± 3.1 spikes, respectively; P = 0.5, 2-way ANOVA with post hoc Bonferroni correction) or rd¹ Opn1mw^R mice (17.58 ± 3.25 and 10.05 ± 2.08 spikes, respectively; P = 0.17, 2-way ANOVA with post hoc Bonferroni correction). D and E: PSTH for change in firing rate (means ± SE) of transient units in Opn1mw^R mice (n = 60 units) and rd¹ Opn1mw^R mice (n = 50 units), respectively, associated with all-photoreceptor and mel-less conditions (black and red traces, respectively; stimulus onset at time 0; duration 10 s). F: total number of spikes (integrated sum of spikes between 2 and 10 s during the light pulse, means ± SE) for the transient population of cells showed no significant difference between all-photoreceptor and mel-less conditions for Opn1mw^R mice (1.19 ± 0.59 and 0.47 ± 0.59 spikes, respectively; P = 0.84, 2-way ANOVA with post hoc Bonferroni correction) or rd¹ Opn1mw^R mice (3.89 ± 0.78 and 3.28 ± 0.71 spikes, respectively; P > 0.99, 2-way ANOVA with post hoc Bonferroni correction). Graphs A, B, D, and E show baseline-subtracted firing rate (in spikes/s, means ± SE) in 0.25-s time bins.

DISCUSSION

To date, much of our understanding of the progress of retinal degeneration has come from anatomical studies (Carter-Dawson et al. 1978; Jones and Marc 2005; Strettoi et al. 2002) and more recent electrophysiological recordings (Gibson et al. 2013; Stasheff 2008; Stasheff et al. 2011) from the degenerate retina. Few studies have investigated what quality of information these residual light responses support in downstream visual centers in the brain (Chen et al. 2016; Dräger and Hubel 1978), and none have recorded from the dLGN, the major retinorecipient of visual information in mammals (Grubb and Thompson 2003; Huberman and Niell 2011). Addressing this deficit is important to understand disease progression and how central vision changes as a function of retinal degeneration. Characterizing the residual light responses in this nucleus also provides a context for attempts to restore vision by re-photosensitizing the retina (Bi et al. 2006; Cehajic-Kapetanovic et al. 2015; De Silva et al. 2017; Lagali et al. 2008; Mandai et al. 2017; McLelland et al. 2018; Tochitsky et al. 2018). If central remodeling processes substantially degrade the visual response in the dLGN, this might provide an additional barrier to success in these approaches. Alternatively, if response properties are largely intact, this would suggest that the early visual system, at least up until the level of the dLGN, remains capable of taking advantage of such interventions to restore not only sensitivity to light but also the ability to resolve spatial patterns at realistic levels of contrast. The retention of spatial receptive fields in the rd¹ retina in this study is especially encouraging, because it indicates that remodeling has not fundamentally degraded the early visual system’s potential for spatial acuity. An important question for future work will be whether receptive fields are similarly retained at later stages in degeneration at which there has been more scope for remodeling. That would inform whether therapeutic interventions should be applied early in degeneration in the hope that they can co-opt and maintain functional circuits or can still be applied in late degeneration.

In many aspects we found our electrophysiological recordings in the dLGN were consistent with previous reports of visual responses in the degenerate retina. Light responses could be readily elicited up to ~4 wk of age in the rd¹ dLGN; however, there was a rapid decline in the frequency of encountering light-responsive cells between P18 and P33, consistent with previous anatomical (Carter-Dawson et al. 1978; Jiménez et al. 1996; LaVail et al. 1997; Lin et al. 2009) and electrophysiological (Dräger and Hubel 1978; Gibson et al. 2013; Stasheff 2008) descriptions of the progression of cone photoreceptor death in this animal. The variety of the identified light responses in the rd¹ dLGN (transient ON, transient ON-OFF, sustained ON) were also qualitatively similar to those previously described in the juvenile degenerate retina (Stasheff 2008), although we did find a proportional shift toward responses being more transient in the rd¹ dLGN. This indicates that visual information can cross the retinogeniculate synapse at these early stages of degeneration. To interrogate this circuitry in more detail, we recorded spatial receptive fields from dLGN neurons and found these to have a mean diameter of 9.96° ± 0.3°, which is at least as small as in our parallel recordings from age-matched visually intact mice and in agreement with previous recordings from the tectum of young rd¹ mice (11.5°; Dräger and Hubel 1978). It is also within the range previously reported in the dLGN of visually intact adult mice (2–10°; Grubb and Thompson 2003). One caveat to the interpretation of these data is that we only used vertical bars to map spatial receptive fields in the dLGN. Given that some dLGN neurons in the mouse exhibit orientation selectivity (Piscopo et al. 2013; Scholl et al. 2013; Zhao et al. 2013), our recordings may in fact underestimate the total number of units for which we could record a spatial receptive field. Nonetheless, our ability to record significant responses to complex spatial stimuli under light-adapted conditions in the rd¹ dLGN indicates that not only is the retinal circuitry linking remaining cones, horizontal cells, and bipolar cells, at least superficially intact for those dLGN neurons for which we could record spatial receptive fields, but also that there is no detectable gross change in the number of retinal ganglion cells converging to an individual dLGN neuron at these early stages of degeneration.

Although many fundamental aspects of thalamic vision were thus substantially intact at early stages of degeneration, there was, of course, a marked effect of retinal degeneration. The most notable impact was on response amplitude and latency. We found that the magnitude (change in firing) of responses to simple light pulses from darkness and contrast steps were significantly reduced in rd¹ mice, whereas latency was significantly increased. These observations are in agreement with previous electroretinogram (ERG) recordings demonstrating that both a-waves and b-waves in rd¹ mice are significantly reduced and delayed as early as at P14 (Gibson et al. 2013; Strettoi et al. 2002) and with multielectrode array recordings from P15 rd¹ retinas (Stasheff 2008). They likely reflect not only the loss of the rod population but also the poor state of surviving cones, which progressively lose their outer segments (Jones et al. 2003; LaVail et al. 1997; Lin et al. 2009) and have the opsin protein redistributed to be expressed in the plasma membrane of the inner segment (Nir et al. 1989), indicating a loss of efficient phototransduction. Importantly, the changes in response amplitude we observed under light-adapted conditions did not significantly alter contrast sensitivity (which was similar in the intact and rd¹ dLGN), indicating that it need not have a simple consequence for vision under natural light-adapted conditions.

A second substantial abnormality of the dLGN light response in rd¹ mice was that we failed to identify a single sustained OFF response. The origin of this deficit is unclear. Whereas anatomical remodeling occurs much later in disease progression (Marc et al. 2003), neurochemical remodeling, most notably of glutamatergic receptors, has been reported in a number of degenerate strains during the early stages of retinal degeneration (Chua et al. 2009; Puthussery et al. 2009). These include the downregulation of both metabotropic and ionotropic glutamate receptors (Marc et al. 2007; Strettoi et al. 2002) and the aberrant expression of ionotropic glutamate receptors on cone bipolar cells (Chua et al. 2009). The cone OFF pathway employs ionotropic glutamate receptors on the dendrites of OFF cone bipolar cells (Thoreson and Witkovsky 1999). However, the sustained component of the OFF responses derives from crossover inhibition with ON cone bipolar cells via GABAergic amacrine cells (Rosa et al. 2016). These GABAergic amacrine cells also exhibit abnormal receptor expression at early stages of degeneration (Chua et al. 2009; Srivastava et al. 2015) and as such could result in the creation of corrupted circuitry that fails to faithfully transmit this visual response. Furthermore, it is possible that the segregation of ON and OFF retinogeniculate synapses never fully matures in rd¹ mice. In visually intact animals, the correlated spike timing of pre- and postsynaptic neurons is crucial to this segregation and happens within a narrow time window during development (Lee et al. 2002; Myhr et al. 2001; Wong and Oakley 1996). However, in rd¹ mice, retinal waves show significant abnormalities in their mean firing rate and interburst interval before photoreceptor death, in addition to exhibiting sustained hyperactivity and rhythmic oscillations in their firing rate (Stasheff 2008), which could affect the normal refinement of ON-OFF segregation in the dLGN.

Although spatial receptive fields were substantially intact in the rd¹ dLGN, our side-by-side comparison with age-matched visually intact Opn1mw^R mice reveals them to be significantly reduced in diameter (by ~3°). One simple potential origin for this effect is the reduced response amplitude, which would make it harder to detect relatively small responses to stimuli located on receptive field margins. This may explain our findings, but we found no correlation between response amplitude and receptive field diameter between degenerate or visually intact mice (data not shown). A second possibility is that although the retinal mosaic of horizontal cells develops normally in degenerate mice, their synaptic connections with photoreceptors never completely mature (Rossi et al. 2003) and therefore modestly alter the spatial receptive field structure of individual retinal ganglion cells.

The final impact of degeneration on dLGN responses that we observed was in the temporal distribution of spike firing. Irradiance steps induce narrowband oscillations in the dLGN of visually intact mice (Storchi et al. 2017). We found similar light-induced narrowband oscillations at a frequency of ~30 Hz in visually intact juvenile mice, but no discernible peaks in the power spectrum across a wide range of frequencies (0–50 Hz) in the degenerate dLGN. Oscillations in the dLGN, and those recorded from the visual cortex in visually intact mice (Saleem et al. 2017), are believed to be at least in part inherited from network interactions in the retina (Storchi et al. 2017) and to play a role in improving the signal-to-noise ratio of neighboring neurons in the dLGN network. Thus the lack of any narrowband oscillations in the degenerate dLGN suggests the impairment of some retinal networks at these early stages, which may have significant implications for visual processing (Koepsell et al. 2009), and is supported by the loss of ERG signals by P14 in rd¹ mice (Strettoi et al. 2002) and the appearance of correlated firing and spontaneous hyperactivity recorded in retinal ganglion cells in these mice (Goo et al. 2016; Menzler and Zeck 2011; Stasheff et al. 2011).

As the prospect of restoring photosensitivity to the degenerate retina increasingly becomes a reality, it is important to turn attention to the central response to these new signals because abnormalities in the functioning of downstream visual circuits may impose a significant constraint on the quality of restored vision. Our data overall support an optimistic view of this problem for potential therapies. Thus, although aspects of the dLGN light response are certainly abnormal in the juvenile rd¹ mouse, they are not obviously more disrupted than has been reported in the retina, and key features, especially contrast sensitivity and receptive field size, are retained. This implies that, at least at the level of the dLGN, central reorganization or secondary degeneration need not pose a barrier to the efficacy of restored photoreception. An important caveat to this conclusion, however, is that the rd¹ mouse has very rapid retinal degeneration that begins during visual system development. It therefore may not be the most suitable model to study more gradual changes in circuitry that could occur in humans, who would typically experience progressive degeneration over many years.

GRANTS

This research was supported by European Research Council Grant 268970 (to R. J. Lucas).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

C.A.P., A.E.A., F.P.M., and R.J.L. conceived and designed research; C.A.P. performed experiments; C.A.P. and A.E.A. analyzed data; C.A.P., A.E.A., and R.J.L. interpreted results of experiments; C.A.P. prepared figures; C.A.P. drafted manuscript; C.A.P., A.E.A., and R.J.L. edited and revised manuscript; C.A.P., A.E.A., F.P.M., and R.J.L. approved final version of manuscript.