Mouse Cones Adapt Fast, Rods Slowly In Vivo

Abstract

Purpose: To study rod- and cone-driven adaptation dynamics separately, we used the silent substitution technique to selectively stimulate rods or cones in the Opn1lw^LIAIS (LIAIS) mouse, in which the native M-cone pigment is replaced by a human L-cone pigment (L*).

Methods: ERG recordings were performed on anesthetized LIAIS mice. ERG stimuli were sinusoidally modulated. After 10 minutes of adaptation to 0.4 candela per square meter (cd/m²) ERGs were measured, followed by 11-minute adaptation to 8.8 cd/m² background and recordings directly after the luminance increase and every second minute. Finally, during adaptation to 0.4 cd/m² for 32 minutes, ERG responses were recorded directly after the change in background and every second minute. This protocol was repeated with rod-isolating stimuli (8 Hz; 75% rod contrast), L*-cone-isolating stimuli (12 Hz; 55% cone contrast) and white light (8 Hz and 12 Hz; 100% Michelson contrast).

Results: At 8.8 cd/m², responses directly displayed photopic response properties without further changes in either cone or white light responses. Rod-driven responses were very small. After the return to 0.4 cd/m², both rod-driven and white light responses increased over a time course of about 30 minutes. Cone-driven responses were very small. Response phases changed directly after a change in background without further alterations.

Conclusions: Rod- and cone-driven signal pathways display strongly different adaptation characteristics: adaptation of cone-driven responses to photopic conditions is very fast, whereas rod-driven responses change with a time course up to 30 minutes during scotopic conditions. Luminance responses are cone-driven at 8.8 cd/m² and rod-driven at 0.4 cd/m².

Adaptation is an important feature of the mammalian eye that enables vision under different luminance conditions ranging from very dim (10⁻⁴ candela per square meter [cd/m²] encountered at moonless nights) to very bright (more than 10⁶ cd/m² corresponding to sunny days at a white beach), thus spanning more than 10 orders of magnitude. To cover this range, adaptation processes can be found at many stages in the visual system. The pupillary reflex, for example, determines the amount of light that reaches the retina.^1,2 Rod and cone photoreceptors are active at different luminance ranges³ and divide the total luminance range into two overlapping domains (i.e., the scotopic and photopic domains; the luminance range in which both photoreceptor types are active is called mesopic). Within the rods and the cones and their postreceptoral pathways, light-dependent molecular and cellular changes (e.g., intracellular translocation of proteins, rearranging of vesicle distribution at synapses or cell coupling^4–8) are additional mechanisms that adjust their sensitivity. These mechanisms may differ in cone- and rod-driven pathways. In the mouse retina, little is known about the physiological adaptation mechanisms in the rod and cone pathways in vivo because mean luminance is generally used to separate the two, thereby precluding the investigation of its influence on adaptation mechanisms in each. The present study focuses on the dynamics of adaptation processes mediated by rods and cones by using electroretinography to analyze light-evoked potential changes in the mouse retina in vivo and by using the silent substitution stimulus technique that has been shown to reliably separate rod- and cone-driven signals independently of luminance levels in a mouse strain in which the native M-cone pigment is replaced by the human L-cone pigment. Otherwise, the strain has identical anatomical and physiological properties to those in wild-type mice.^9,10 As luminance is not the determining factor for separating rod- and cone-driven signals, it can be used as an invariant, and its influence on adaptation processes in rod- and cone-driven pathways can be studied. This is a new approach that enables studying adaption of rod- and cone-driven signals separately at high and low light levels in a mouse with normal retinal anatomy and physiology, that is, without the need for disrupting the retinal circuitry. 

Electroretinography (ERG) is a commonly used method to study retinal function in vivo by recording mass potential changes of retinal origin evoked by light stimulation. Most ERG studies are performed at a steady adaptation state.^11–20 The dynamics of adaptation in ERG responses has not been studied intensively in vivo. In most of these studies, the alterations in responses to single flashes or trains of flashes were described. In a recent study in Opn1lw^wt mice (wild-type mice on a C57Bl/6 background), we showed that ERG responses to short flashes (6.3 cd.s/m² white light) increased during light (25 or 40 cd/m² background intensity) and dark adaptation.²¹ The use of modulations around a mean luminance and chromaticity has the advantage, in comparison with flashes, that stimulus intensity (in terms of Michelson contrast) and temporal frequency can be varied independently of the state of adaptation. In our previous study,²¹ we therefore also measured the responses to sinusoidally modulated luminance stimuli. These responses increased during adaptation to low light level (1 cd/m²). A reevaluation of our previous data recorded during adaptation to a high light level (25 cd/m²) revealed that the responses to a sinusoidal modulation displayed no significant change during the observed 11 minutes. The response amplitudes decreased slightly during the first 2 minutes but returned to the initial response magnitude after 4 minutes and thereafter stayed constant during the last 6 minutes of light adaptation. In that study, we interpreted ERG responses recorded with a 25- or 40-cd/m² background to be cone driven and responses evoked with 0- or 1-cd/m² background to be rod-mediated. However, a clear separation of rod- and cone-driven responses was not possible. Furthermore, it was not possible to establish adaptation of cone-driven responses to low luminance levels or adaptation of rod-driven responses to high luminance levels. 

Detailed information about the dynamics of adaptation in the rod- and cone-driven signal pathways is necessary to understand the process of adaptation to high or low light levels. Studies that investigate the contribution of rod- or cone-mediated responses to dark and light adaptation in mice use mutant mice strains that have only one functional photoreceptor type,^22–26 adaptational methods (i.e., scotopic or photopic backgrounds),^22,27,28 or the paired-flash technique.^29,30 One method to analyze rod- or cone-based ERG responses separately is by using mutant mouse models that lack either rods or cones. The Gnat^spfl3 mouse lacks functional cones²⁵ and therefore can be used to study adaptation in rod-driven pathways. On the other hand, the Gnat1^−/− mouse has no functional rods²³ and thus can be used to study adaptation dynamics in cone pathways. However, these animals cannot be considered to be physiologically normal because pathological processes are probably involved. Even if the remaining photoreceptors and their postreceptoral pathways are intact, physiological interactions between rod- and cone-driven pathways (e.g., through mutual gap junctions or in other common retinal circuitries) are absent.^23,31,32 Therefore, ERGs recorded from these mouse strains should be interpreted with care. Another technique to isolate rod- and cone-driven responses is a temporal separation by the paired-flash technique.^3,30,33 However, this technique does not allow the investigation of the adaption dynamics of the two photoreceptor regimes in the same state of adaptation, similar to the use of scotopic or photopic backgrounds. 

Modulation around a mean luminance offers an additional advantage: using the silent substitution stimulation technique, rod- and cone-driven responses can be isolated independently of the mean luminance and chromaticity (see Kremers³⁴ and Shapiro et al.³⁵ for a description of the method). However, maximal modulation of rod and cone excitation obtained with silent substitution stimuli increases with increasing differences in absorption spectra between the two. Unfortunately, in wild-type mice rod- and M-cone-absorption spectra are quite similar (508 nm for M-cones and 498 nm for rods³⁶) so that isolated rod and cone signals with silent substitution stimuli are relatively weak (approximately 5% rod or cone contrast³⁷; rod and cone contrast is defined as the Michelson contrast of the rod or cone excitation modulation). Since the amplitude of ERG responses to flicker stimulation increases with increasing contrast, only small responses are achieved with the silent substitution stimuli in wild-type mice. We recently showed that rod- and cone-driven responses can be separated in the LIAIS mouse,³⁷ which expresses the human L-cone pigment (hereafter denoted as L*-cone pigment) instead of the murine M-cone pigment and is otherwise physiologically normal.^9,10,38 The increased difference between the maximal spectral sensitivities of the rods (498 nm) and the L*-cone pigment (561 nm) results in larger possible rod (75%) and L*-cone (55%) contrast and thus leads to larger ERG responses.³⁷ We further found that rod- and L*-cone-driven responses can be separated in the LIAIS mouse over a luminance range of three orders of magnitude. Rod-driven responses could be measured up to mean luminances of 7 cd/m² (corresponding to 7 × 10³ photoisomerizations/sec). L*-cone-driven responses were measurable at luminances of 1.4 cd/m² (10³ photoisomerizations/sec) and higher. This opens up the perspective to study the dynamics of adaptation in rod- and cone-driven ERG pathways in the LIAIS mouse using silent substitution. In the present study, we investigated the dynamics of adaptation in rod- and cone-driven responses in physiologically normal LIAIS mice using the silent substitution stimulations technique. 

Methods

Experimental Animals

ERG recordings were performed on six LIAIS mice (four hemizygous males, two homozygous females, 4 to 5 months of age; the L/M-cone pigment gene is located on the X-chromosome), two Opn1lw^wt (wild-type littermates, two males, 8 months of age), and three C57Bl/6J mice (three males, aged 4 to 5 months). The LIAIS mouse strain was originally described by Greenwald et al.¹⁰ and was sourced from Maureen and Jay Neitz from the University of Washington (Seattle, WA, USA). Hemizygous males and homozygous females of this strain express a human L-cone opsin (with the amino acids leucine, isoleucine, alanine, isoleucine, and serine on positions 153, 171, 174, 178, and 180, respectively, hence also referred to as LIAIS; murine cones expressing this gene are denoted as L*-cones) instead of the murine M-cone opsin expressed in wild-type littermates (Opn1lw^wt). Apart from this change from murine M-cone opsin to the human L*-cone opsin with no variability in absorption spectrum, LIAIS mice express the endogenous mouse S-cone opsin and rhodopsin. Different studies confirm that the substitution of the murine M-cone opsin by the L*-cone opsin results in a shift of the spectral sensitivity of the cones toward longer wavelengths with a maximum sensitivity at about 561 nm instead of 508 nm^36,39,40 without altering the cones' structure and function.^10,37 Tsai et al.³⁷ described rod- and cone-driven responses in the LIAIS mice using the silent substitution technique and showed that they can be studied with large signal-to-noise ratio (SNR). 

The animals were kept in a 12-hour dark-light cycle, and water and food were available ad libitum. All animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Society for Neuroscience. Furthermore, all methods described in this study were approved by the local animal welfare authorities (Regierungspräsidium Mittelfranken, Ansbach, Germany). 

Preparation

Prior to the ERG measurements, the animals were dark adapted overnight. Animal preparation was performed under dim red light. The animals were anesthetized by an intramuscular injection of 50:10 mg/kg ketamine/xylazine (Ketavet; Pfizer, Karlsruhe, Germany; Rompun 2%; Bayer AG, Leverkusen, Germany). A subcutaneous injection of saline (10 ml/kg, 0.9%) prevented dehydration while the animals were under anesthesia. The pupils were dilated to approximately 3-mm diameter with a drop of tropicamide (Mydriaticum Stulln, 5 mg/mL; Pharma Stulln, Stulln, Germany) and of phenylephrine-hydrochloride (Neosynephrin POS 5%; Ursapharm, Saarbrücken, Germany). Needle electrodes were placed subcutaneously at the base of the tail and on the forehead to serve as ground and reference electrodes, respectively. Active contact lens electrodes (Mayo Corporation, Inazawa, Japan) filled with ocular gel (Corneregel; Dr. Mann, Pharma, Berlin, Germany) were placed on both eyes. The anesthetized animals were placed on a heated platform to maintain body temperature during the experiment, and the platform was slid into the Ganzfeld bowl. All recordings on one animal were performed in separate sessions, with each session limited to an hour and the sessions being at least 1 week apart. After the experiments, the animals were allowed to wake up. 

Experimental Protocols

The ERG responses were evoked by sinusoidal modulation of the outputs of LED arrays in the Ganzfeld stimulator (Q450SC; Roland Consult, Brandenburg, Germany). See Figure 1 for a scheme of the experimental protocols. Three different stimulus protocols were used. Responses were recorded to 8-Hz rod-isolating stimuli in LIAIS mice (n = 6), to 12-Hz L*-cone-isolating stimuli in LIAIS mice (n = 6), and to 8- and 12-Hz luminance white light modulation in LIAIS (n = 3), Opn1lw^wt (n = 2), and C57Bl/6J (n = 3) mice. All measurements with rod- and cone-isolating stimuli in LIAIS mice were done on the same animals. The responses to luminance stimuli were measured in a subgroup of these. The measurements with the luminance stimuli were performed to compare the results in LIAIS and wild-type mice to check if the LIAIS mice were physiologically normal with respect to adaptation dynamics. Furthermore, the results of the luminance measurements in LIAIS mice were compared with the responses to rod- and L*-cone-isolating stimuli. 

Figure 1

 

Stimulation protocols and recording procedure.

Stimuli and adaptation conditions were generated with the Ganzfeld bowl containing four identical LED arrays, each containing six different LED types. In order to achieve the required mean luminances, two of these LED arrays were blocked and the remaining two LED arrays were covered by gelatin filters (ND 1; Göttinger Farbfilter GmbH, Bovenden-Lenglern, Germany). The mean luminance and chromaticity of the adaptation lights were the same as those of the stimuli (luminance stimuli: white LEDs; cone- and rod-isolating stimuli: ratio of the output of the red, green, and blue LED arrays, 6:6:1; see Table 1 and below for more details), ensuring that the adaptation process was not interrupted during the measurements (assuming that the 8- and 12-Hz modulations do not induce additional adaptation). 

Table 1

 

Stimulus Settings for the Isolation of L*-Cone-Driven, Rod-Driven, and Luminance Responses

 

After preparation, the animals were adapted to a 0.4 cd/m² mean luminance (low light level) for 10 minutes. A baseline recording was then performed. We previously described that this adaptation time is sufficient to reach a steady state.²¹ After the baseline measurement, the mean luminance was increased to 8.8 cd/m² mean luminance for 11 minutes (high light level), and the responses were recorded every 2 minutes, with the first recording starting directly after the mean luminance was increased. In total, six recordings were performed. Thereafter, the mean luminance was reduced to 0.4 cd/m², and again we recorded periodically every 2 minutes up to 32 minutes, the first recording starting directly after the mean luminance was reduced. In total, 17 measurements were performed during the adaptation to the low light level. A recording session generally lasted about 54 minutes, which was feasible without additional injections of anesthesia. After the recording session, the animals were allowed to wake up. The responses to the different stimuli (see below) were recorded in separate sessions. 

Stimuli

As mentioned above, we used four different stimulus conditions to measure rod-driven (8 Hz), L*-cone-driven (12 Hz), and luminance (8 and 12 Hz) responses. Each recording averaged the responses to 33 stimulus cycles of 1 second each. To avoid onset artifacts, the responses to the first three cycles were discarded. 

Luminance (100% Michelson contrast; 8 or 12 Hz) stimuli were created by sinusoidal modulation of the output of white LEDs. As mentioned above, adaptation was provided by the same white LEDs. 

To isolate rod- or cone-driven responses we used the silent substitution method that is explained in detail in the review by Kremers³⁴ and for the LIAIS mouse by Tsai et al.³⁷ Briefly, this method enables recording ERG responses that are driven by a single photoreceptor type by silencing all other photoreceptor types. The stimuli therefore were generated by red, green, and blue LED arrays with peak wavelengths of 625, 525, and 470 nm, respectively. By modulating the three different LED types with preset contrasts and phases, it is possible to modulate the excitation of only one photoreceptor type while the excitation of the other photoreceptor types is not modulated. Table 1 summarizes the different stimulus conditions. 

As a control experiment, we measured the responses to a steady background where we repeated the adaptation protocol in the absence of LED modulation. Hence, the spectral composition of the stimulus was the same as for the rod- and L*-cone-isolating stimuli, but neither rod nor L*-cone excitations were modulated. This allowed an independent estimate for noise (see below). 

Signal Acquisition and Analysis

Visual stimulation and signal acquisition were controlled using a system for measuring bioelectrical signals (RetiPort; Roland Consult). ERG signals were amplified (100,000 times), band-pass filtered (1 to 300 Hz), and digitized with a sampling rate of 1024 Hz. The averaged ERG responses of each animal were Fourier transformed to analyze amplitude and phase of the first harmonic component (also referred to as fundamental, i.e., at the stimulus frequency). Noise was defined as the mean amplitude of the Fourier transformed at ±1 Hz of the stimulus frequency. If the signal amplitude was smaller than two times the noise (i.e., SNR < 2), then the amplitude was set to 0 μV and the corresponding response phase was discarded from further analysis. If the signal amplitude was larger than two times the signal noise, then the signal amplitudes were corrected by subtracting the noise. 

To define a baseline noise, we Fourier transformed the signals of the recordings in the absence of a stimulus (0% LED contrast) and isolated the amplitude at 8 and 12 Hz (amplitudes measured in two animals were averaged). These measurements were done to compare the resulting noise estimate with the noise obtained as described above (mean amplitude at stimulus frequency ±1 Hz). We found that both noise definitions resulted in similar values. Furthermore, the noise level was stable throughout the whole experiment, and neither a change in light level nor the duration of adaptation had any effect on the noise level. 

The response amplitudes and phases in the 32 minutes after a change from 8.8 to 0.4 cd/m² conditions were plotted as a function of adaptation time. If the data showed a trend to increase or decrease during adaptation, then these data were fitted with a function describing an inverted exponential rise to maximum or a linear function in the case of the rod phase and the response amplitudes to 12-Hz luminance stimulation in LIAIS and C57Bl/6J mice (see below):  

\(\def\upalpha{\unicode[Times]{x3B1}}\)\(\def\upbeta{\unicode[Times]{x3B2}}\)\(\def\upgamma{\unicode[Times]{x3B3}}\)\(\def\updelta{\unicode[Times]{x3B4}}\)\(\def\upvarepsilon{\unicode[Times]{x3B5}}\)\(\def\upzeta{\unicode[Times]{x3B6}}\)\(\def\upeta{\unicode[Times]{x3B7}}\)\(\def\uptheta{\unicode[Times]{x3B8}}\)\(\def\upiota{\unicode[Times]{x3B9}}\)\(\def\upkappa{\unicode[Times]{x3BA}}\)\(\def\uplambda{\unicode[Times]{x3BB}}\)\(\def\upmu{\unicode[Times]{x3BC}}\)\(\def\upnu{\unicode[Times]{x3BD}}\)\(\def\upxi{\unicode[Times]{x3BE}}\)\(\def\upomicron{\unicode[Times]{x3BF}}\)\(\def\uppi{\unicode[Times]{x3C0}}\)\(\def\uprho{\unicode[Times]{x3C1}}\)\(\def\upsigma{\unicode[Times]{x3C3}}\)\(\def\uptau{\unicode[Times]{x3C4}}\)\(\def\upupsilon{\unicode[Times]{x3C5}}\)\(\def\upphi{\unicode[Times]{x3C6}}\)\(\def\upchi{\unicode[Times]{x3C7}}\)\(\def\uppsy{\unicode[Times]{x3C8}}\)\(\def\upomega{\unicode[Times]{x3C9}}\)\(\def\bialpha{\boldsymbol{\alpha}}\)\(\def\bibeta{\boldsymbol{\beta}}\)\(\def\bigamma{\boldsymbol{\gamma}}\)\(\def\bidelta{\boldsymbol{\delta}}\)\(\def\bivarepsilon{\boldsymbol{\varepsilon}}\)\(\def\bizeta{\boldsymbol{\zeta}}\)\(\def\bieta{\boldsymbol{\eta}}\)\(\def\bitheta{\boldsymbol{\theta}}\)\(\def\biiota{\boldsymbol{\iota}}\)\(\def\bikappa{\boldsymbol{\kappa}}\)\(\def\bilambda{\boldsymbol{\lambda}}\)\(\def\bimu{\boldsymbol{\mu}}\)\(\def\binu{\boldsymbol{\nu}}\)\(\def\bixi{\boldsymbol{\xi}}\)\(\def\biomicron{\boldsymbol{\micron}}\)\(\def\bipi{\boldsymbol{\pi}}\)\(\def\birho{\boldsymbol{\rho}}\)\(\def\bisigma{\boldsymbol{\sigma}}\)\(\def\bitau{\boldsymbol{\tau}}\)\(\def\biupsilon{\boldsymbol{\upsilon}}\)\(\def\biphi{\boldsymbol{\phi}}\)\(\def\bichi{\boldsymbol{\chi}}\)\(\def\bipsy{\boldsymbol{\psy}}\)\(\def\biomega{\boldsymbol{\omega}}\)\(\def\bupalpha{\unicode[Times]{x1D6C2}}\)\(\def\bupbeta{\unicode[Times]{x1D6C3}}\)\(\def\bupgamma{\unicode[Times]{x1D6C4}}\)\(\def\bupdelta{\unicode[Times]{x1D6C5}}\)\(\def\bupepsilon{\unicode[Times]{x1D6C6}}\)\(\def\bupvarepsilon{\unicode[Times]{x1D6DC}}\)\(\def\bupzeta{\unicode[Times]{x1D6C7}}\)\(\def\bupeta{\unicode[Times]{x1D6C8}}\)\(\def\buptheta{\unicode[Times]{x1D6C9}}\)\(\def\bupiota{\unicode[Times]{x1D6CA}}\)\(\def\bupkappa{\unicode[Times]{x1D6CB}}\)\(\def\buplambda{\unicode[Times]{x1D6CC}}\)\(\def\bupmu{\unicode[Times]{x1D6CD}}\)\(\def\bupnu{\unicode[Times]{x1D6CE}}\)\(\def\bupxi{\unicode[Times]{x1D6CF}}\)\(\def\bupomicron{\unicode[Times]{x1D6D0}}\)\(\def\buppi{\unicode[Times]{x1D6D1}}\)\(\def\buprho{\unicode[Times]{x1D6D2}}\)\(\def\bupsigma{\unicode[Times]{x1D6D4}}\)\(\def\buptau{\unicode[Times]{x1D6D5}}\)\(\def\bupupsilon{\unicode[Times]{x1D6D6}}\)\(\def\bupphi{\unicode[Times]{x1D6D7}}\)\(\def\bupchi{\unicode[Times]{x1D6D8}}\)\(\def\buppsy{\unicode[Times]{x1D6D9}}\)\(\def\bupomega{\unicode[Times]{x1D6DA}}\)\(\def\bupvartheta{\unicode[Times]{x1D6DD}}\)\(\def\bGamma{\bf{\Gamma}}\)\(\def\bDelta{\bf{\Delta}}\)\(\def\bTheta{\bf{\Theta}}\)\(\def\bLambda{\bf{\Lambda}}\)\(\def\bXi{\bf{\Xi}}\)\(\def\bPi{\bf{\Pi}}\)\(\def\bSigma{\bf{\Sigma}}\)\(\def\bUpsilon{\bf{\Upsilon}}\)\(\def\bPhi{\bf{\Phi}}\)\(\def\bPsi{\bf{\Psi}}\)\(\def\bOmega{\bf{\Omega}}\)\(\def\iGamma{\unicode[Times]{x1D6E4}}\)\(\def\iDelta{\unicode[Times]{x1D6E5}}\)\(\def\iTheta{\unicode[Times]{x1D6E9}}\)\(\def\iLambda{\unicode[Times]{x1D6EC}}\)\(\def\iXi{\unicode[Times]{x1D6EF}}\)\(\def\iPi{\unicode[Times]{x1D6F1}}\)\(\def\iSigma{\unicode[Times]{x1D6F4}}\)\(\def\iUpsilon{\unicode[Times]{x1D6F6}}\)\(\def\iPhi{\unicode[Times]{x1D6F7}}\)\(\def\iPsi{\unicode[Times]{x1D6F9}}\)\(\def\iOmega{\unicode[Times]{x1D6FA}}\)\(\def\biGamma{\unicode[Times]{x1D71E}}\)\(\def\biDelta{\unicode[Times]{x1D71F}}\)\(\def\biTheta{\unicode[Times]{x1D723}}\)\(\def\biLambda{\unicode[Times]{x1D726}}\)\(\def\biXi{\unicode[Times]{x1D729}}\)\(\def\biPi{\unicode[Times]{x1D72B}}\)\(\def\biSigma{\unicode[Times]{x1D72E}}\)\(\def\biUpsilon{\unicode[Times]{x1D730}}\)\(\def\biPhi{\unicode[Times]{x1D731}}\)\(\def\biPsi{\unicode[Times]{x1D733}}\)\(\def\biOmega{\unicode[Times]{x1D734}}\)\begin{equation}\tag{1}y\left( t \right) = {y_0} + {\rm{\alpha }}\left( {1 - {e^{ - {\rm{\beta }}t}}} \right)\qquad inverse{\rm{\ }}exponential{\rm{\ }}change{\rm{\ }}to{\rm{\ }}reach{\rm{\ }}a{\rm{\ }}plateau\end{equation}

 

\begin{equation}\tag{2}y\left( t \right) = {y_0} + {\rm{\delta }}t\qquad linear{\rm{\ }}change\end{equation}

 

Statistics

Statistical analysis was done using software (SPSS 21; IBM, Armonk, NY, USA). A Shapiro-Wilk test confirmed that the data were normally distributed. To test if the data showed a significant trend during adaptation to the high or low light level, a Friedman test was performed. To test if two groups (e.g., two different stimulus settings or different genotypes) were significantly different regarding amplitude and phase, an unpaired t-test was performed at four distinct time points. We chose to compare means of the first and the last recording at each light level (e.g., for high light level, at 11 and 21 minutes after the start of the experiment; for low light level, at 22 and 54 minutes after start of the experiment). An α value of 0.05 was adopted for significance. The α value of the t-test was corrected after Bonferroni for multiple testing. 

Results

Rod- and Cone-Driven Responses During Adaptation to High and Low Light Levels

The responses of LIAIS mice to rod- and L*-cone-isolating stimuli at chosen time points during the adaptation to 8.8 and 0.4 cd/m² mean luminance are displayed in Figure 2. L*-cone-driven responses, shown in Figure 2A, were substantially larger at 8.8 cd/m² than at 0.4 cd/m² mean luminance. An increase in mean luminance led to a rapid increase in response amplitude with no further alteration of the response during light adaptation. L*-cone-driven responses strongly decreased directly after the mean luminance was decreased to 0.4 cd/m², and the response amplitude did not change further during the adaptation to the low light level. Rod-driven responses (see Fig. 2B) showed a rapid decrease in amplitude and a phase shift after the mean intensity was increased to 8.8 cd/m². During the adaptation to the 8.8-cd/m² background, the responses were small and showed no change in amplitude or phase. The subsequent decrease of the mean luminance to 0.4 cd/m² rapidly led to an increased and phase-shifted response. The responses further increased during the following 32 minutes of adaptation to the lower light level. Interestingly, the response recorded after 32 minutes of adaptation to 0.4 cd/m² was larger than the response to the 0.4 cd/m² reference measurement at the beginning of the session. 

Figure 2

 

ERG responses evoked by L*-cone-isolating (left), rod-isolating (middle), and luminance-modulating (right) stimuli. (A) Averaged L*-cone-driven responses of LIAIS mice (n = 6; 12 Hz) at different time points (given on the left together with mean luminance and the time after change in mean luminance). (B) Averaged rod-driven responses of LIAIS mice (n = 6; 8 Hz). (C) Averaged luminance responses of LIAIS mice evoked by 8-Hz (recordings at 0.4 cd/m² mean luminance; n = 3) or 12-Hz (recordings at 8.8 cd/m² mean luminance; n = 3) stimuli.

The amplitudes and phases of the first harmonic response components were further analyzed to obtain a quantitative description of the adaptation dynamics of rod- and L*-cone-driven responses. The first harmonic amplitude and phase as a function of the adaptation time during the whole experiment are displayed in Figure 3. The plots in Figure 3A show the time course of the first harmonic amplitudes (upper plot) and phases (lower plot) of L*-cone-driven responses. The amplitudes were small (<1 μV), with a 0.4-cd/m² mean luminance, but they directly increased by a factor of three after the light level was increased. During the following 11 minutes of light adaptation, the amplitude of the L*-cone-driven response did not change (mean change ± SD: −0.03 ± 0.53 μV; P = 0.794; Friedman test, see Table 2). The subsequent luminance reduction led to a rapid decrease in amplitude, and no further change was observed afterward (mean ± SD: +0.08 μV ± 0.32, Friedman test not possible). The first harmonic phase of L*-cone-driven responses shifted by 120° directly after the luminance was increased and showed no further change during the following light adaptation (mean ± SD: −4.04° ± 11.49; P = 0.549; Friedman test). The subsequent luminance decrease had only a minor effect on the phase, and during the adaptation to 0.4 cd/m², the phase of the L*-cone-driven responses did not change significantly (mean ± SD: −26.43° ± 21.56, Friedman test not possible). In pilot experiments, we did not find any indication for further amplitude and phase changes. The phase data at low luminance conditions were not reliable because the SNR was smaller than two for many responses (thus a statistical analysis was not possible), and the remaining phases were variable. 

Figure 3

 

Amplitudes and phases of the first harmonic for rod- and L*-cone-driven responses. (A) First harmonic amplitudes and phases of L*-cone-driven responses plotted against adaptation time (n = 6). (B) Amplitudes and phases of the first harmonic of rod-driven responses as a function of time (n = 6). Luminance levels are indicated in the bottom plots. Friedman tests were performed on the data to determine significant changes during the adaptation to high and low light level. Results are included in the graphs (n.s.; *** P < 0.001; t.n.p). First harmonic amplitudes and phases of rod-driven responses during the adaptation to the low light level were fitted with the equations indicated in the plots. ML, mean luminance; n.s., not significant; t.n.p, test not possible.

Table 2

 

Statistical Analysis of Response Changes During Adaptation With the Friedman Test

 

Figure 3B displays the first harmonic amplitudes and phases of rod-driven responses. The amplitude of the reference measurement was about 10 μV. The luminance increase led to a rapid and strong decrease in amplitude to about 0.6 μV. During the adaptation to the 8.8-cd/m² light level, the amplitudes did not change (mean ± SD: −0.26 μV ± 0.57; P = 0.969; Friedman test). After returning to the 0.4-cd/m² luminance level, the rod-driven responses directly increased in amplitude by a factor of six (to about 4 μV). This was followed by a gradual and significant increase during the subsequent 32 minutes of adaptation (mean ± SD: +13.97 μV ± 2.89, P < 0.0001, Friedman test). The amplitude data during adaptation to the low light level showed a hint of a discontinuity between 34 and 44 minutes, which was neither present in phase data nor in other responses at low light levels (see below). Therefore, the data were fitted with an inverse exponential function (see Table 3). However, after 32 minutes of adaptation, a plateau was not yet reached, indicating that rod-driven adaptation is a slow process. Furthermore, the responses after 32 minutes of adaptation were larger than those in the reference measurements. One explanation may be that the animals were not completely dark adapted during the reference measurement. Indeed, the presence of the L*-cone pigment of the LIAIS mice probably results in an increased sensitivity to the red light used during animal preparation. However, we found the same effect, that responses after 32 adaptations to scotopic conditions exceeded those in the reference measurements, also in Opn1lw^wt and C57Bl/6J animals (see below), which are insensitive to red light. Possibly, light regimes, preceding the adaptation period, influence the response amplitudes. 

Table 3

 

Summary of the Fitting Parameter for First Harmonic Components After the Change to 0.4 cd/m²

 

The first harmonic phase of the rod-driven responses decreased by about 110° directly after the luminance increase and did not change significantly during the 8.8 cd/m² illumination (mean ± SD: +24.97° ± 24.82, Friedman test not possible). After the luminance decrease to 0.4 cd/m², the rod-driven phase directly increased about 110° and thereafter showed a slow and small, but significant, increase by +18.47° (±4.80; P < 0.0001; Friedman test) during the next 32 minutes of adaptation. The phase change was approximately linear, indicating that the phase changed by 0.6° per minute (see Table 3). 

Comparison Between Single Photoreceptor Type–Driven and Luminance Responses

Figure 4 displays the ERG response amplitudes (A) and phases (B) measured in LIAIS mice to luminance stimuli. The responses to 8- and 12-Hz luminance modulation were recorded during adaptation to 0.4- and 8.8-cd/m² mean luminance. To compare the luminance data with rod- and L*-cone-driven responses, the 8-Hz luminance responses and rod-driven responses (also measured at 8 Hz) are shown for the lower luminance level, and the 12-Hz luminance responses are shown for the high mean luminance levels, together with the responses to 12-Hz L*-cone-isolating stimuli. At 8.8 cd/m², the luminance and L*-cone-driven responses resembled each other, whereas the luminance and rod-driven ERG responses were similar at 0.4-cd/m² mean luminance. One exception is that the rod-driven and luminance responses had phase differences of about 180°. This difference, however, can be fully explained by the fact that the rod-isolating stimuli were 180° phase shifted relative to the internal trigger of recording equipment (see Table 1, Methods section). These data indicate that responses to luminance modulation are nearly completely rod-driven at low luminances and L*-cone-driven at high luminances without substantial rod intrusion. Statistical analysis confirmed that L*-cone-driven and 12-Hz luminance responses were not significantly different either directly after the mean luminance was increased to 8.8 cd/m² (P = 0.515, unpaired t-test; see Table 4) or after 10 minutes of adaptation to 8.8 cd/m² (P = 0.694, unpaired t-test). 

Figure 4

 

Comparison of first harmonics of responses to luminance modulation with rod- and L*-cone-driven responses (see Fig. 3). (A) First harmonic amplitudes versus adaptation time. Amplitudes at low light level are fitted with equation (1), that is, an exponential rise to maximum. To compare the single photoreceptor type–driven data to luminance responses directly after luminance increase, after 10 minutes of light adaptation, directly after luminance decrease, and after 32 minutes of adaptation to the low light level, t-tests were performed. Results of these tests are indicated in the graph (n.s.). (B) First harmonic phases versus adaptation time. Values for reference measurements are shown for all four stimuli: (i) 12-Hz cone-isolation (red upward triangles, n = 6, mean ± SD), (ii) 8-Hz rod isolation (blue downward triangles, n = 6, mean ± SD), (iii) 8-Hz luminance modulation (open squares, n = 3, mean ± SD), and (iv) 12-Hz luminance modulation (open circles, n = 3, mean ± SD). Cones and 12-Hz luminance data are shown at high light levels; data of rods and 8-Hz luminance modulation are shown for low light levels. Light levels are indicated in the bottom graphs.

Table 4

 

Summary of the Statistical Comparison of Response Amplitudes Between Different Groups and Stimulation Protocols at Four Distinct Time Points Throughout the Experiment

 

Effect of Stimulus Frequency on Response Amplitudes

In this study, two different luminance frequencies were used (8 and 12 Hz). Figure 5A shows original ERG responses to 8- and 12-Hz luminance modulation after 10 minutes of adaptation to 8.8 cd/m² and after 32 minutes of adaptation to 0.4 cd/m². In both conditions, the 8-Hz responses were larger than the 12-Hz measurement. Figure 5B shows the first harmonic amplitudes of the 8- and 12-Hz responses plotted versus recording time, showing that generally the responses to 8-Hz stimuli were larger than those to 12-Hz stimuli over the whole recording period (directly after luminance increase to 8.8 cd/m²: P = 0.034, unpaired t-test; after 10 minutes of 8.8 cd/m²: P = 0.030, unpaired t-test; directly after luminance decrease to 0.4 cd/m²: P = 0.344, unpaired t-test; after 32 minutes of 0.4 cd/m²: P = 0.040, unpaired t-test; see Table 4). This is agreement with previous data,¹¹ where it was found that rod- and cone-driven responses have low pass characteristics (more pronounced in rod-driven than in cone-driven responses). 

Figure 5

 

Effect of stimulus frequency on luminance response amplitude. (A) Averaged ERG responses (n = 3) to 8- (thin lines) and 12-Hz (thick lines) luminance modulation recorded 10 minutes after adaptation to 8.8 cd/m² (upper plots) and 32 minutes after adaptation to 0.4 cd/m² (lower plots). (B) First harmonic amplitudes for 8- and 12-Hz luminance modulation (n = 3, mean ± SD; squares: 8 Hz; circles: 12 Hz). Amplitudes measured at low light levels were fitted with equation (1) for 8-Hz stimulation and with equation (2) for 12-Hz stimulation. To compare the 8- and 12-Hz data directly after luminance increase, after 10 minutes of light adaptation, directly after luminance decrease, and after 32 minutes of adaptation to the low light level, t-tests were performed. Results of these tests are indicated in the graph.

A luminance increase led to an amplitude reduction at both frequencies. During the following adaptation to the high light level, the amplitudes of both recordings were constant, compatible with the above-mentioned conclusion that luminance responses at 8.8 cd/m² are cone driven. After the subsequent luminance decrease to 0.4 cd/m², there was no sudden change in amplitude, but during the following 32 minutes of adaptation to the low light level, the amplitudes increased strongly, again in agreement with the conclusion that the responses were rod-driven. This increase was stronger for the 8-Hz stimulation (mean ± SD: +11.11 μV ± 1.66, P = 0.022, Friedman test; see Table 2) than for the 12-Hz stimulation (mean ± SD: +7.52 μV ± 1.09, P = 0.010, Friedman test). The increase was described by an inverse exponential rise for 8-Hz stimulation and by a linear fit for 12-Hz stimulation (see Table 3 for fitting parameters). 

Comparison Between LIAIS and Wild-Type Mice

Figure 6 shows the ERG responses to 12-Hz luminance modulation measured in LIAIS mice and two wild-type strains (i.e., Opn1lw^wt and C57Bl/6J) using the same adaptation procedure as described above. The responses of all three strains showed the same response characteristics, including an rapid decrease in response amplitude after the increase of mean luminance, the absence of any changes during the subsequent 10 minutes of adaptation, no immediate amplitude changes after the decrease in mean luminance, and a slow response increase in the following 32-minute adaptation period. From this we conclude that the adaptation processes, described above for the LIAIS mouse, are a general feature also present in wild-type mice. 

Figure 6

 

Comparison between responses measured in LIAIS and wild-type mice. (A) Averaged ERG responses to 12-Hz luminance modulation in LIAIS (thick line, n = 3), Opn1lw^wt (dashed line, n = 2), and C57Bl/6J (thin line, n = 3) mice shown for different adaptation time points (indicated on the left). (B) First harmonic amplitudes and phases of 12-Hz luminance responses in LIAIS (open circles, n = 3, mean ± SD), Opn1lw^wt (black squares, n = 2), and C57Bl/6J (gray triangles, n = 3) mice. The amplitudes of LIAIS and Opn1lw^wt mice during low light level were fitted with an equation for exponential rise to maximum, whereas the amplitudes of the C57Bl/6J mice were fitted with a linear regression. To compare the data of the three strains directly after luminance increase, after 10 minutes of light adaptation, directly after luminance decrease, and after 32 minutes of adaptation to the low light level, t-tests were performed. Results of these tests are indicated in the graph.

The LIAIS mice have seemingly smaller amplitudes compared to the wild-types (see Fig. 6B). However the LIAIS and the Opn1lw^wt mice did not differ significantly at any of the four tested time points of the recording (i.e., directly after the mean luminance was increased to 8.8 cd/m², 10 minutes after adaptation to 8.8 cd/m², directly after the mean luminance was decreased to 0.4 cd/m², and 32 minutes after adaptation to 0.4 cd/m²; see Table 4). The difference between LIAIS and C57Bl/6J mice after 32 minutes of adaptation to 0.4 cd/m² was also not significant after Bonferroni correction (P = 0.046; unpaired t-test; Table 4). In general, differences between genotypes were small. 

Also, the phases changed in a similar manner in the three strains, strengthening the above notion that similar adaptation processes are involved. The first harmonic phase of the response showed an immediate increase after a luminance increase and a phase decrease after a luminance decrease without strong subsequent changes. However, a Friedman test revealed that the response phase in the Opn1lw^wt mice increased significantly (mean ± SD:+13.83° ± 4.19; P = 0.020; see Table 2) during the 32 minutes of adaptation to 0.4 cd/m². 

In conclusion, the comparison of the responses obtained from the three mouse strains confirms previous conclusions^9,10 that the LIAIS mouse is physiologically normal. 

Discussion

The purpose of the present study was to describe adaptation dynamics of rod- and L*-cone-driven responses in vivo in physiologically normal mice. To separate the signals of the two photoreceptor types we used the silent substitution technique in which the number of isomerizations in a single photoreceptor type is modulated. Thus, the technique does not require an adaptation-dependent desensitization of photoreceptor types. Theoretically, the silent substitution technique can achieve perfect isolation, whereas adaptation techniques can only achieve biases toward certain photoreceptor systems. In practice variations in preretinal absorption and in pigment properties may result in small deviations in the silent substitution conditions. Furthermore, as adaptation is not required to isolate the responses of one photoreceptor type, its influence on these responses can be studied. As the response strength in the isolated photoreceptor types increases with decreasing overlap of the involved pigment absorption spectra, we used mice that express a human L-cone opsin variant instead of the murine M-cone opsin (LIAIS mice). It was therefore possible to study the kinetics of both rod- and L*-cone-driven responses during adaptation to high and low light levels. 

We define adaptation as a change in response to equal stimuli owing to a change in mean luminance or chromaticity. Implicit in this definition is the quantification of equal stimuli. We express stimulus strength in terms of Michelson contrast, that is, the modulation in luminance or photoreceptor excitation normalized to their mean. Because the stimuli had equal Michelson contrasts at 0.4 and 8.8 cd/m², an equal response at the two luminances indicates the absence of adaptation. However, particularly the strength of flashed stimuli is often defined by the absolute modulation or the Weber fraction.⁴¹ In our experiments, the absolute modulation of photoreceptor excitation was proportional to the mean luminance. If the responses were proportional to the absolute excitation modulation, the absence of adaptation would lead to a response change that is proportional to the mean luminance change (i.e., by a factor of about 22). Rod-driven responses increase strongly after a decrease in mean luminance. L*-cone-driven responses increase by a factor of about three when luminance is increased from 0.4 to 8.8 cd/m². Thus, even with absolute modulation depth as quantification of stimulus strength, the phenomena described in the present study would be indicative for the presence of adaptation effects. In addition, changes in response phase are generally a result of adaptation processes. 

The results might be influenced by contrast adaptation. In this study, the sinusoidal stimulus was presented for 30 seconds every 2 minutes, resulting in a 1.5-minute time period without stimulation. We therefore assume that any effects of contrast adaptation would disappear during the 1.5 minutes between stimuli. Contrast adaptation during the 30-second stimulation interval was not observed. 

L*-Cone-Driven Responses Adapt Rapidly

L*-cone-driven responses increased, and the phase shifted rapidly after the mean luminance was increased from 0.4 to 8.8 cd/m², with no further changes in either amplitude or phase during the following 10 minutes of adaptation. The reversed change in mean luminance resulted in an equivalent decrease in response amplitude and a phase shift (although the responses were small and thus the phases not very reliable at 0.4 cd/m²). From this we conclude that adaptation in L*-cone-driven responses probably is completed during the first measurement after the change in mean luminance and, therefore, probably within seconds. A higher temporal resolution might give more information about the time course of L*-cone-driven adaptation to high luminance levels. Yeh et al.⁴² also found that the responses of primate ganglion cells in the cone pathway adapt rapidly (i.e., within seconds). Responses to luminance stimuli were equally fast when adapting to the 8.8 cd/m², indicating that these responses were mainly L*-cone driven and that the responses to luminance stimuli rapidly switch from rod- to L*-cone driven. The similar adaptation dynamics recorded with L*-cone-isolating (55% contrast) and luminance (100% contrast) stimuli at 8.8 cd/m² suggest that the speed of adaptation does not depend on the stimulus contrast. As cone-driven responses reflect the activity of both ON and OFF bipolar cells, adaptation effects of both cell types may interfere. This complicates the interpretation of the ERG data. Our data reveal that the responses to L*-cone and luminance stimuli change very fast after an increase in luminance without any further changes in amplitude or phase, indicating that cones fully adapt within the first recording (30 seconds) within the used range of luminance levels. In a recent study, we showed that the responses to sine-wave luminance stimuli (100% contrast) hardly changed after a change from 1 to 25 cd/m² white background (after previous adaptation to 1 cd/m²) in LIAIS mice.²¹ Adaptation of photopic (i.e., mainly cone driven) flash ERGs to 25 and 40 cd/m² (i.e., 1.4 and 1.6 log cd/m²) backgrounds (after complete dark adaptation) took several minutes. Possibly, the different light levels and different stimulus types employed in the previous study caused the differences in results. Cameron and Lucas²³ showed in Gnat1^−/− mice, lacking functional rods, that flash ERG responses increased during a 20-minute period of light adaptation to a 5.7 log cd/m² background after a period in the dark. This indicates that different adaptation processes may be involved in ERG responses to flashes upon a background and to temporal modulations around a mean luminance and chromaticity. 

Rod-Driven Responses Adapt Over Several Minutes to Low Light Levels

Rod-driven responses increased in amplitude and phase during the adaptation from 8.8 to 0.4 cd/m² mean luminance. Neither amplitude nor phase reached a plateau during the observed 32 minutes of adaptation to the low luminance level. Additionally, the fits of the first harmonic amplitudes and phases indicated that a plateau was not yet reached. In a previous study, we showed that it takes at least 30 minutes for the parameters of flash- and flicker-ERG to reach a steady state to low luminances.²¹ The present study suggests an even longer time period for rods to fully adapt. DeMarco et al.²² found the retinal sensitivity in wild-type mice to increase over a time course of about 40 minutes during dark adaptation after previous light adaptation to 2.4 log cd/m² (≈250 cd/m²) for 10 minutes and was even much more delayed in Gnat2^cplf3 mice lacking functional cones. Also, in normal human observers and in rod monochromats, dark adaptation of rod-mediated perception is a slow process.⁴³ In conclusion, these data are in agreement with ours showing that adaptation in rod-driven pathways is slow. Our data also confirm that the sensitivity increase of intact rod circuitries during adaptation to low light levels is comparable with the sensitivity dynamics in isolated rods as investigated by Baylor et al.⁴⁴ 

L*-Cone and Rod Dependency of Luminance Responses Is Modulated by Luminance Level

Our study reveals that luminance responses are driven exclusively by rods at 0.4 cd/m² and by cones at 8.8 cd/m². However, the luminance levels were deliberately chosen so the responses driven by cones (at 0.4 cd/m²) and rods (at 8.8 cd/m²) were negligible at steady state.¹¹ Therefore, with these luminances, interactions between rod- and cone-driven signals are not expected. At intermediate luminances, combined rod- and cone-driven responses can be expected. 

When rods and cones are both active, their signals may interact. Cameron and Lucas²³ found that rods have an effect on cone-mediated light adaptation measured with flash ERGs. They found that Gnat1^−/− mice, which have no functional rods, have larger b-wave amplitudes than wild-type animals during light adaptation and suggested that rods suppress cone-driven responses. DeMarco et al.²² found that dark adaptation was delayed in Gnat2^cplf3 mice that lack functional cones compared to wild-type mice, indicating that cones may also influence rod-based dark adaptation. The rod-cone interactions as described by DeMarco et al.²² and by Cameron and Lucas²³ in wild-type animals are possibly also present in LIAIS mice that are physiologically normal (see below). The silent substitution technique not only offers the possibility to stimulate the rods and cones differently, but they can also be adapted differently.⁴⁵ Thus, the suppressive rod-cone interactions may also be studied in the LIAIS mouse. 

LIAIS Mice Are Physiologically Normal

Although the LIAIS mice showed slightly (but nonsignificantly) smaller responses than Opn1lw^wt and C57Bl/6J mice, the adaptation dynamics in the responses to luminance stimuli were similar in the three strains. These results are in agreement with the conclusions from previous studies showing that the retina of the LIAIS mice is structurally and physiologically normal.^9,10 The shift in the absorption spectra of the M/L*-cones leads to a sensitivity to long wavelengths that is not present in wild-type mice. The advantage compared to previous studies on mutant mice with only one functioning photoreceptor type^22–26 is that all retinal networks are intact, with the result that all interactions can be studied. 

Conclusions

In the present study, for the first time adaption dynamics of rod- and L*-cone-driven signals were measured in vivo in mice that are physiologically normal. The LIAIS mouse model that expresses a long-wavelength-shifted L*-cone pigment instead of the endogenous M*-cone pigment enabled the use of the silent substitution stimulation technique. This technique provided the great advantage of isolating L*-cone and rod-driven signals without using luminance condition or temporal frequency to separate the signal pathways of L*-cones and rods. We therefore were able to investigate rod- as well as L*-cone-driven ERG responses during adaptation to low and high luminance levels in the intact retina in vivo. Rod-driven ERGs adapt much slower than L*-cone-driven ERGs. The adaptation to luminance increases and to luminance decreases are not symmetric. 

Acknowledgments

Disclosure: A. Joachimsthaler, None; J. Kremers, None