Doc Ophthalmol (2014) 128:155–168 DOI 10.1007/s10633-014-9433-2

ORIGINAL RESEARCH ARTICLE

Contribution of retinal ganglion cells to the mouse electroretinogram Benjamin J. Smith • Xu Wang • Balwantray C. Chauhan • Patrice D. Coˆte´ Franc¸ois Tremblay



Received: 22 October 2013 / Accepted: 6 March 2014 / Published online: 23 March 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Patrice D. Coˆte´ and Franc¸ois Tremblay have contributed equally to this manuscript.

Results We confirmed in mice that CNQX intravitreal injection reduced the scotopic a-wave amplitude at high flash strength, confirming a post-receptoral contribution to the a-wave. We found that ONTx, which is more specific to RGCs, did not affect the a-wave amplitude and implicit time in either photopic or scotopic conditions while the b-wave was reduced. Both the pSTR and nSTR components were reduced in amplitude, with the balance between the two components resulting in a shortening of the nSTR peak implicit time. On the other hand, amplitude of the PhNR was increased while the OPs were minimally affected. Conclusion With an intact a-wave demonstrated following ONTx, we find that the most robust indicators of RGC function in the mouse full-field ERG were the STR components.

B. J. Smith  P. D. Coˆte´ (&) Department of Biology, Dalhousie University, PO Box 15000, Halifax, NS B3H 4R2, Canada e-mail: [email protected]

X. Wang  B. C. Chauhan  F. Tremblay Department of Physiology and Biophysics, Dalhousie University, PO Box 15000, Halifax, NS B3H 4R2, Canada

Abstract Purpose To quantify the direct contribution of retinal ganglion cells (RGCs) on individual components of the mouse electroretinogram (ERG). Methods Dark- and light-adapted ERGs from mice 8 to 12 weeks after optic nerve transection (ONTx, n = 14) were analyzed through stimulus response curves for a- and b-waves, oscillatory potentials (OPs), positive and negative scotopic threshold response (p/n STR), and the photopic negative response (PhNR) and compared with unoperated and sham-operated controls, as well as to eyes treated with 6-cyano-7nitroquinoxaline-2,3-dion (CNQX).

B. J. Smith e-mail: [email protected] X. Wang  B. C. Chauhan  F. Tremblay Retina and Optic Nerve Research Laboratory, Dalhousie University, PO Box 15000, Halifax, NS B3H 4R2, Canada e-mail: [email protected] B. C. Chauhan e-mail: [email protected] F. Tremblay e-mail: [email protected]

Present Address: X. Wang Unit on Neuron-Glia Interactions in Retinal Disease, National Eye Institute, National Institutes of Health, 6 Center Drive, 6/215, Bethesda, MD 20892, Canada B. C. Chauhan  P. D. Coˆte´  F. Tremblay Department of Ophthalmology and Visual Sciences, Dalhousie University, 1276 South Park Street, Halifax, NS B3H 2Y9, Canada

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Keywords Retinal ganglion cells  Optic nerve  Axotomy  Electroretinogram  Mouse  Scotopic threshold response  Photopic negative response

Introduction The electroretinogram (ERG) is a composite signal generated by the retina in response to light stimuli. Although originally the signal was considered to be generated almost exclusively by photoreceptor activity (a-wave) and either Mu¨ller cell K? buffering or by ON-bipolar cell activity (b-wave) (reviewed in [1]), it later became evident that other retinal cells contributed to the signal in a variety of ways. For instance, in the mid 1990s, it has been clearly demonstrated using pharmacological action of glutamate agonists and antagonists in the primate retina that post-receptoral elements contributed to the photopic a-wave [2, 3]; however, this contribution was minimal in scotopic conditions [4, 5]. In human, using double-flash technique, Friedburg [6] arrived at the same conclusion. The contribution of post-receptoral elements, and in particular of retinal ganglion cells (RGC), has been more difficult to unequivocally establish in rodents. It is known that pharmacologic block of post-receptoral contributions with cis-2,3-piperidine dicarboxylic acid (PDA) or 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) reduces the a-wave amplitude in mice [7, 8] and rats [9], but it is unclear whether the contribution blocked in these experiments originates from the RGCs or from another post-receptoral cell type. There is evidence that RGCs may contribute directly to the dark-adapted a-wave in mice. Using optic nerve transection (ONTx), Alarco´n-Martı´nez et al. [10] have recently shown in a study of albino and pigmented mice recorded under scotopic and photopic conditions that the a-wave is reduced at 2 weeks post-ONTx and that this reduction is increased at 4–12 weeks. Support for a-wave reduction after ONTx also comes from evidence in disease models and knockout mice with RGC degeneration, although there is the possibility of widespread retinal changes in these cases. In particular, an a-wave reduction has been noted in aged mouse models of glaucoma (DBA/2J) [11, 12], as well as in a mouse model of acute IOP increase with optic nerve degeneration [13], and in mice with Math5 mutation which results in a reduction in RGCs [14]. NMDA injection that blocks light responses primarily in RGCs in

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addition to some amacrine cells also reduces the darkadapted a-wave in mice [15, 16]. On the other hand, Germain et al. [17] showed no effect on the a-wave at the 2 week time point after optic nerve transection in albino CD1 mice. In addition, OPA1 mice, which show clear evidence of optic nerve atrophy and reduced numbers of RGCs, were documented to have a-waves comparable to their normal littermates [18, 19]. In rats, the literature is similarly inconsistent. Alarco´n-Martı´nez et al. [20] report a reduction in the a-wave in both albino and pigmented rats following ONTx. However, Bui and Fortune [21] and Mojumder et al. [22] show no changes in a-wave amplitude 4 weeks after ONTx, Gargini et al. [23], in a study of trophic factor upregulation after ONTx, documented a gradient of a-wave effects with a slight reduction in amplitude noted during the first 7 days after injury, followed by a normalization at day 21. Finally, a previous study by Bui and Fortune [21] in rat shows specifically that sham surgeries can transiently reduce the a-wave, indicating photoreceptor dysfunction following surgery without ONTx, although a specific mechanism was not explored. The effect of ONTx on photoreceptor function is important to understand since changes in photoreceptor function will alter the amplitudes of downstream responses that may be partially generated by RGC function including the positive and negative scotopic threshold response (p/n STR) [24], b-wave, photopic negative response (PhNR), and oscillatory potentials (OPs). This would cause overestimation of the RGC contribution to these ERG components. In mice, ONTx reduced both the p/n STR [10], but this was concomitant to reductions in a- and b-waves. Some recent studies in mice have suggested that the nSTR is a functional correlate of RGC loss [25, 26]; however, both these studies also show reduced a-waves. In the rat, Alarco´n-Martı´nez, et al. [20] reported a strong reduction in the p/n STR. Similarly, Mojumder et al. [22] and Bui and Fortune [21] reported a reduction in the STR in the absence of changes in a-wave. The PhNR, known to be generated by RGC activity in primates [27] and humans [28–31, reviewed in 32] has also been found to be reduced in mice and correlated with the reduction in RGC number in mouse models of dominant optic atrophy [18] and acute increase in intraocular pressure [33]. However, ablation of the RGCs with ONTx yielded more variable

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results, with reduction in PhNR reported qualitatively by Alarco´n-Martı´nez et al. [10] in mice and by Li et al. [34] in rats, while Mojumder et al. [22] could not document an effect of ONTx on the PhNR. Since the contribution of RGCs to the rodent a-wave is unclear and because photoreceptor injury during surgery would cause a reduction in all post-receptoral components of the ERG, it is necessary to determine whether the reduction in a-wave amplitude in rodents caused by ONTx represents a real RGC contribution or is a sign of surgical trauma. In the current series of experiments, using identical mouse species and the longest time points in Alarco´n-Martı´nez et al. [10], we found that ONTx in mouse does not affect a-wave amplitude. The presence of a normal a-wave in RGCdepleted retinas enables us to report on the direct contribution of RGCs to post-receptoral ERG components, including the OPs and PhNR both of which have not been previously quantified in ONTx mice. We determined that, in mice, the most reliable indicator of RGC function appears to be the amplitude of the nSTR and the reduction in IT of both pSTR and nSTR, as shown previously by Alarco´n-Martı´nez et al. [10]. We also found less consistent RGC contributions to the PhNR, OPs, and b-wave.

Materials and methods Animals and ethics statement All procedures complied with the standards of the Canadian Council of Animal Care and ethics approval was obtained from the Dalhousie University Committee on Laboratory Animals. Adult C57Bl/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Mice were housed and bred in a 12-h light–dark cycle environment with luminance inside the room at 180 lux and at 13 lux inside the cages (determined from the average of 5 measurements near the cage rack or from inside five different cages, respectively). Food and water were available ad libitum. Optic nerve transection Optic nerve transection surgeries (n = 14) were performed by X.W. who also performed the surgeries in a similar study by Chauhan et al. [35]. This technique yielded a 92 ± 4 % reduction in RGCs in

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Adult Thy1-CFP transgenic mice (Strain: B6.CgTg(Thy1-CFP)23Jrs/J) twenty days after ONTx. Internal controls with Brn-3a immunostaining (Fig. 2) confirmed the severe reduction in RGCs 8 weeks after ONTx. Briefly, under anesthesia using a cocktail of ketamine (90 mg kg-1) and xylazine (10 mg kg-1), the globe was rotated downward with a 9–0 conjunctival suture. An incision was made in the skin near the supraorbital ridge after which the intraorbital subcutaneous tissues were dissected to expose the optic nerve. The optic nerve dura was cut longitudinally along the superior aspect, and the optic nerve was completely transected approximately 0.5 mm from the globe. Care was taken to ensure there was no damage to the ophthalmic artery located underneath the transected nerve. After the incision was closed, the fundus was examined with an operating microscope to confirm the absence of ischemic damage. Sham surgeries (n = 5) were performed exactly as described above except that the optic nerve was left intact following the dissection of the dura. Intravitreal injection Following application of a topical anesthetic to the cornea of anesthetized mice 0.5 ll of 200 lM CNQX disodium salt (TOCRIS Bioscience, Bristol, UK) dissolved in phosphate-buffered saline pH 7.4 (n = 5), or PBS alone (sham, n = 6), was injected into the vitreous via a 30-gauge needle mounted on a Hamilton syringe under dim red light. The injection site was located approximately 0.5 mm behind the ora serrata. Following injection mice were further darkadapted for approximately 30 min to allow for diffusion of the drug before recording. Drug concentration at the retina was calculated using an estimated vitreal volume of 5 ll in mouse [36]. Immunohistochemistry Animals at 8 weeks post-surgery were perfused with 0.1 M phosphate buffer saline (PBS, pH 7.4) followed by 4 % paraformaldehyde in 0.1 M PBS. Operated (ONTx) and unoperated fellow (control) eyes were post-fixed with 4 % paraformaldehyde for 3 h. The retinas were dissected, flattened with four radial cuts, collected in 24-well plate and permeabilized with 0.5 % Triton-9100 in PBS (PBS-TX) by freezing for

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15 min at -70 °C. Free-floating retinas were washed for 10 min in PBS-TX, then incubated overnight at 4 °C with Brn3a (dilution 1:100, Santa Cruz Biotechnology, Dallas, TX) in blocking buffer (2 % BSA and 2 % Triton-9100 in PBS). After incubation, the retinas were washed with PBS for 1 h and incubated with Alexa Fluor 546 Donkey anti-goat IgG (dilution 1:200, Molecular Probes, Eugene, OR) at room temperature for 2 h. Retinas were then washed with PBS, placed on a glass slide, coverslipped with antifade medium (Citifluor; Marivac, Halifax, NS), and imaged with confocal microscopy (Nikon C1, Nikon Canada Inc., Mississauga, ON). Electroretinography C57Bl/6 mice between 8 and 16 weeks of age were dark-adapted for at least 8 h before being anesthetized under dim red light by intraperitoneal injection of ketamine–xylazine. The pupils were dilated with cyclopentolate HCl 0.5 % (Alcon, Fort-Worth, TX) and 0.5 % proparacaine hydrocloride (Alcon) was applied as a topical anesthetic. Body temperature was maintained at 37 °C with a heated pad and monitored rectally. Mice were killed by anesthetic overdose followed by cervical dislocation. The active electrode was a Dawson-Trick-Litzkow-plus microconductive fiber (Diagnosys, Littleton, MA) placed on the corneal surface and hydrated with 2.5 % hydroxypropyl methylcellulose solution to maintain conductivity. Platinum subdermal electrodes (Grass Instruments, Quincy, MA) were placed in the base of the nose (reference) and in the tail (ground). The signal from the corneal electrodes was amplified 10,000-fold using a differential amplifier with a bandwidth of 0.3–300 Hz (P511, Grass instruments) and acquired at 1 kHz by an A/D board (PCI 6281, National Instruments, Austin, TX, USA) and displayed and stored for processing using LabVIEW 9.0 dedicated software (National Instruments, Austin, TX). Flash stimuli were generated by a Ganzfeld stimulator (LKC Technologies, Gaithersburg, MD) that produced a maximum illumination of 10.1 log cd s m-2 at the corneal surface. Flash strengths were attenuated by up to 7.0 log units by manual interposition of neutral density filters (Kodak Wratten, Rochester, NY). The stimulus interval between flashes varied from 5 s at the lowest stimulus strengths to 30 s at the highest ones. Repeated flashes did not attenuate the ERG even at high

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flash strengths indicating that inter stimulus light adaptation did not occur. Two to ten responses were averaged depending on flash strength for flash stimuli between 1.0 and -4.4 log cd s m-2. Lower flash strengths usually used between 10 and 40 responses, with the stimulus repeated until the average waveform no longer showed any changes with successive stimuli. Analysis ERG waveforms were analyzed using a custom ERG analysis toolbox in Matlab (Mathworks, Natick, MA). Prior to analysis of the a- and b-waves, the OPs were eliminated with a low-pass eighth-order Butterworth filter at 50 Hz for both photopic and scotopic responses. The a-wave amplitude was measured from the baseline to the maximum negative trough, and the b-wave was measured from the a-wave trough to the maximum positive peak. The PhNR and nSTR were taken to be the local minimum following the b-wave (and i-wave when present) in photopic and scotopic conditions, respectively. We considered the pSTR and nSTR to be generated by flash strengths below -3.6 log cd s m-2, similar to Alarco´n-Martı´nez et al. [10], and the b-wave to be generated by higher flash strengths. However, Saszik et al. [24] proposed a positive scotopic response (pSR) in the mouse to better explain the proximal contribution to the b-wave at these low flash strength; we did not specifically isolate the two responses in this study. The OPs were isolated by high-pass filtering of the ERG using an eighthorder Butterworth filter at 50 Hz [37]. Data for the control and treated eyes were collected simultaneously and results compiled with Microsoft Excel for Mac (version 14.2; Microsoft Corporation, Redmond, WA) and analyzed with SPSS software (version 20 for Mac; International Business Machines, Armonk, NY). Stimulus series for individual ERG components were submitted to repeated measures ANOVA (rmANOVA) analysis with within-subject factor including flash strength matched for treatment effect (Control vs ONTx). When the model was found significant at the 0.05 level, results were contrasted using post hoc analysis with Sidak adjustment for multiple comparisons. Because of the limited sample size, normality was difficult to assess so we confirmed the significance level of the rmANOVA using the nonparametric Wilcoxon signed-ranked test. In all cases, the nonparametric approach supported the rmANOVA

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Table 1 Summary of the statistically significant effects of ONTx on various ERG components

Implicit time

Amplitude

a-wave b-wave p100 pSTR/pSR n233 nSTR PhNR OP 1 OP 2 OP 3 OP 4 OP 5 a-wave b-wave pSTR nSTR PhNR

1.0

0.6

0.2

-0.2

# *

1.0

# *

0.6

-1.2

# *

0.0

-1.8

# *

-0.6

-3.0

# *

Photopic

-2.4

-4.2

-3.6

-4.6

-5.0

-5.4

-5.8

-6.2

-6.6

Scotopic (log cd•m -2)

*

# #

# # # #

# # #

# # #

# # # #

# #

# # # #

*

* *

* *

*

*

*

* * *

* # #

# #

# #

# #

# #

# #

# # *

Asterisks indicate statistical differences between ONTx and control (Student’s t test; p \ 0.05) * Indicates that rmANOVA rejected the hypothesis of significance within-subject effect, while pair-wise comparisons identified a few significant differences # Indicates a significant rmANOVA within-subject effect, supported by pair-wise contrasts

findings that are reported in Table 1. Significance level for the rmANOVA was set at 0.05. A summary of the post hoc analysis of the rmANOVA results is presented in Table 1. Results Glutamatergic blockade to post-receptoral cells reduces the a-wave Blockade of post-receptoral contributions with CNQX results in a reduced a-wave in rats [9] and mice [7]. Intravitreal injection of 200 lM CNQX was performed (n = 8) and compared with uninjected and vehicleinjected controls and ONTx eyes (Fig. 1A, B). The a-wave amplitude at flash strengths above 0.0 log cd s m-2 was found to be significantly (p \ 0.05) reduced to 89.2 ± 0.07 % of uninjected control. At flash strengths above 0.0 log cd s m-2, the a-wave from the CNQX-injected eyes was also found to be significantly different from the ONTx eyes (p \ 0.05). Contribution of RGCs to the Scotopic ERG The efficacy of the ONTx procedure was documented previously [35] and in the present study by the loss of

Brn3a-positive RGCs at 8 weeks (Fig. 2). The effects RGC loss on the ERG was examined 8–12 weeks postONTx (n = 14). ONTx did not significantly change the amplitude (p = 0.71) or implicit time (p = 0.79) of the dark-adapted a-wave at any stimulus strength (Fig. 3A–D; Table 1 for rmANOVA with matched data, post hoc contrasts with p \ 0.05). The b-wave (Fig. 3E, F, I, J) was defined as the positive potential generated by flash strengths above -3.6 log cd s m-2, while the pSTR (Fig. 3G–J) was discriminated as the positive potential occurring below -3.6 log cd s m-2, which likely includes as well the positive scotopic response (pSR) referred to in Saszik et al. [24]. The amplitude of the b-wave measured at the peak of the response was significantly reduced (p = 0.001). Post hoc analysis (Table 1) revealed that this reduction was significant only at flash strengths below -0.6 log cd s m-2, where the b-wave is predominately generated by the rod pathway [38, 39], with the proportion of the b-wave generated by RGCs shrinking as stimulus strength increased (Fig. 3I, average 19.2 ± 2.5 % reduction between -3.6 and -0.6 log cd s m-2). At higher stimulus strengths, where cone-dominated pathways begin to contribute, the b-wave was not significantly reduced in ONTx eyes. The implicit time of the b-wave was not

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Fig. 1 Blocking ionotropic glutamate receptors suppresses the amplitude of the dark-adapted mouse a-wave. A Example waveforms of ERG recordings from CNQX (gray traces) and uninjected fellow eye (black traces). Flash strength values are indicated in log cd s m-2. B Intensity response curves from CNQX (gray, n = 5), ONTx (dashed, n = 14), sham-injected eye (dotted, n = 6) and control uninjected fellow eyes of the CNQX group (black). Asterisks indicate significance of CNQX versus uninjected control (p \ 0.05). No significant difference was found between sham-injected and uninjected fellow eyes

significantly changed after ONTx (p = 0.38). After ONTx, the pSTR amplitude was significantly reduced at most flash strengths where the component is present

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(p = 0.006; Fig. 3G, H); this reduction was maximal at -5.0 log cd s m-2, where it reached 54.7 ± 20.1 % of controls. The percentage of the b-wave/ pSTR generated by the RGCs (Fig. 3I) was found to be relatively stable between -5.4 and -3.6 log cd s m-2. Between these stimulus strengths, ONTx ˆ ± 3.9 % of control. At reduced the pSTR to 56.4 A higher flash strengths, corresponding to the b-wave, the percent of the positive component generated by ˆ ± 5.6 (-3.6 RGCs decreased rapidly from 47.4 A -2 ˆ ± 3.9 % (-2.4 log cd s log cd s m ) to 9.0 A m-2). The pSTR IT was faster on average by ˆ ± 2.7 ms, which was highly significant (p \ 7.2 A 0.001). For comparison to previous work investigating inner retinal contributions to the b-wave, pSR, and pSTR in mice [24] where changes to the pSTR were measured at fixed times, we measured amplitude changes at the averaged implicit time for stimulus strengths above -2.4 log cd s m-2 where the IT of the positive component is relatively steady (99.6 ± 0.7 ms, rounded to the nearest ms). Similar to measurements at the peak of the b-wave, significant reductions were found in b-wave amplitude for stimulus strengths below 0.0 log cd s m-2 (average 18.4 ± 2.5 % reduction). In order to show the size of the contribution made by RGCs to the positive component of the ERG as a function of flash strength, the amplitude of positive component of the ERG measured at 100 ms (Fig. 4A, B) in the transected eye from control eyes was subtracted (Fig. 4D). The RGC component has a relatively smooth increase in amplitude reaching a

Fig. 2 Loss of Brn3a-positive RGCs 8 weeks post-ONTx. Micrographs of flat-mounted retinas dissected from axotomized (ONTx) or unoperated fellow (control) eyes stained with anti-Brn3a antisera were imaged with a confocal microscope. Bar is 200 lm

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Fig. 3 Effects of ONTx on the dark-adapted ERG at component peak. A, B Example waveforms of ERG recordings obtained from ONTx (gray) and control fellow (black) eyes over a range of high (A) and low (B) stimulus strengths. C–F Dark-adapted intensity response curves for a-wave amplitudes (C) and implicit times (D) and b-wave amplitudes (E) and implicit times (F).

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G, H pSTR amplitudes (G) and implicit times (H). I, J Amplitude ratios of the b-wave (I) and amplitudes of the RGC pSTR and b-wave component (J, control amplitude minus ONTx amplitude). K, L nSTR amplitudes (K) and implicit times (L). Bracket depicts the stimulus intensity range corresponding to the nSTR

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Fig. 4 Effects of ONTx on the dark-adapted ERG at 100 ms and 233 ms post-stimulus. Amplitude of ONTx (gray ) or control (black) at 100 ms following A weak (-6.6 to -4.2 log cd s m-2) or B strong (-3.6 to 1.0 log cd s m-2) stimuli. C Ratio of ONTx amplitude vs control amplitude at

100 ms. D Amplitude of the RGC component at 100 ms. E Amplitude of control or ONTx at 233 ms. F Ratio of ONTx amplitude versus control amplitude at 233 ms. G RGC component at 233 ms

maximum of 19.1 ± 6.7 lV at -1.8 log cd s m-2 and saturating. The percentage of the contribution generated at 100 ms by the RGCs (Fig 4C) was found to be stable between -4.6 and -3.0 log cd s m-2. Between these stimulus strengths, ONTx reduced the contribution to ˆ ± 3.5 % of control. 67.6 A We then examined the nSTR and found it to be reduced by ONTx (p = 0.034). The average amplitude reduction at the peak (Fig. 3K) was, however, only 14.2 ± 2.4 % of ˆ ± 10.0 % control on average, with a maximum of 17.8 A at flash strength of -5.0 log cd s m-2. The IT of the nSTR (Fig. 3I) on the other hand was also significantly reduced for all values (p [ 0.001) with an average reduction in 36.55 ± 8.2 ms. Like the pSTR, we measured the nSTR at a fixed time (233 ms), which revealed a more substantial and consistent reduction compared with measuring at the peak, with significant reductions in amplitude for stimulus strengths below -4.6 log cd s m-2 (Fig. 4E, average 54.2 ± 8.7 % reduction). The individual scotopic OPs (Fig. 5A, B) were also measured and found not to be significantly affected (OP1: p = 0.15; OP2: p = 0.21; OP3: p = 0.12; OP4: p = 0.25; OP5: p = 0.36). However, examining contrasts revealed that OP1 was affected most (significantly different at the three strongest stimuli), while OP3, OP4, and OP5 were affected only with one bright stimulus. When the OPs were summed (not shown), there was no significant overall change in OP amplitude.

Contribution of RGCs to the photopic ERG

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ONTx had no effect on the amplitude (Fig. 6B, p = 0.27) or IT (Fig. 6C, p = 0.91) of the photopic a-wave. The amplitude of the photopic b-wave was not significantly affected (Fig. 6D, p = 0.49) by ONTx nor was the IT (Fig. 6E, p = 0.61). The amplitude (Fig. 6F) and IT (Fig. 6G) of the PhNR were measured at the maximum depression following the photopic b-wave. Overall, ONTx had a significant effect on the amplitude (p = 0.05) and a noticeable change in IT (p = 0.06) was observed for the PhNR when all stimuli were considered. At the brightest stimulus strength used, no significant change in the amplitude of the PhNR was observed but surprisingly, as the stimulus strength decreased the loss of RGCs caused the amplitude of the PhNR ˆ to increase significantly relative to controls (159.1 A ± 7.5 %) at flash strength dimmer than 0.6 log cd s m-2. The IT changed as well, becoming significantly reduced by 40 ± 14 ms but only at the highest stimulus strength. The amplitude and IT of the five OPs identified in photopic conditions were quantified (Fig. 5C, D); no significant change in the amplitude (OP1: p = 0.49; OP2: p = 0.58; OP3: p = 0.37; OP4: p = 0.61; OP5: p = 0.62) and ITs (not shown for brevity) of any of the photopic OPs following ONTx.

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Fig. 5 Effects of ONTx on oscillatory potentials. A Example waveforms of dark-adapted ERGs digitally high-pass filtered at 50 Hz from transected (gray) and control fellow (black) eyes. B Dark-adapted intensity response curves of individual OPs. C Example waveforms of light-adapted ERGs digitally high-pass filtered at 50 Hz. D Lightadapted intensity response curves of individual OPs

Discussion RGCs do not contribute directly to the mouse scotopic a-wave Historically, the a-wave was believed to be solely a reflection of photoreceptor function [1]. However, recent reports examining functional correlates of RGC loss in mice and rats suggested the possibility that

lesion of RGCs may cause a reduction in the a-wave of the ERG [10, but see 17, 20, 22, 40, 41]. This was supported by the observation that the a-wave can be reduced by blocking glutamatergic neurotransmission to post-receptoral cells in rats [9], a phenomenon which we also observed in mice [7]. Reports indicating that ONTx could have long-range effects on the function of horizontal [42, 43] and amacrine cells [44, 45] also corroborated this possibility. In addition,

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Fig. 6 Effects of ONTx on light-adapted ERG. A Example waveforms of ERG recordings from transected (gray) and control fellow (black) eyes to increasing flash intensities in light-adapted conditions. B–G Graphs of ERG recordings from transected (gray) and control fellow (black) showing a-wave

amplitudes (B) and implicit times (C), as well as b-wave amplitudes (D) and implicit times (E), and also PhNR amplitudes (F) and implicit times (G) in light-adapted conditions

ONTx has been shown to transiently down-regulate phototransduction-related genes in rats [46]. Other research, however, suggests that a-wave amplitude is largely independent of RGC activity. Bui and Fortune [21] found that while the a-wave was reduced in ONTx eyes, it was also transiently reduced in sham-operated eyes, thus showing that surgery but not ONTx alone could cause a reduction in the scotopic a-wave. In mice, Germain et al. [17] saw no change in the a-wave amplitude of CD1 mice 2 weeks after ONTx and while an a-wave reduction was initially observed by Gargini et al. [23] in rats shortly after ONTx, the amplitude returned to normal levels 3 weeks post-transection.

Our findings highlight the difficulty in performing ONTx to avoid long-term non-specific damage in addition to RGC loss. In the present study, the amplitude and IT of the a-wave were unchanged when recorded at 8–12 weeks post-ONTx, when the large majority of RGCs have died [35], which suggests, as in Bui and Fortune [21], that surgical complications, e.g., ischemia, might be a significant contributor in the previous observation of a-wave reduction post-ONTx. In the absence of a confounding a-wave reduction, the variations observed in the other ERG components could now be interpreted as a direct consequence of RGC death, and therefore the contribution of RGCs to the ERG can now be more clearly interpreted.

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Contribution of RGCs to the scotopic b-wave and STR The positive components of the ERG (b-wave and/or pSTR) were significantly reduced at flash strengths below -1.2 log cd s m-2, implying that RGCs contribute to the b-wave in addition to the pSTR, although the contribution was limited to flash stimuli too weak to stimulate cones [38, 39]. This confirms the presence of an RGC component in the positive responses obtained in scotopic conditions. The pSTR was reduced by *45 %, which agrees with previous results in mice [10, 14, 17], suggesting the pSTR in mice is not entirely generated by the RGCs. In comparison, the rat pSTR is eliminated completely by ONTx below -5.12 log cd s m-2 [21, 22]. Germain et al. [17] reported that ONTx caused significant differences only in pSTR, leaving the b-wave intact, while Alarco´n-Martı´nez et al. [10] reported an overall reduction in the b-wave of about 20 % at all stimulus strengths tested. Our results confirmed the RGC contribution to the pSTR and to the b-wave at lower stimulus strengths where cones do not contribute to the b-wave. The reduction in b-wave amplitude at stimulus strengths below 0.0 log cd s m-2 may be due to a reduction in the pSR reported by Saszik et al. [24]; however, the pSR is confined to relatively dim stimulus strengths. These differences may be explained by the relatively short time period (2 weeks) in the Germain et al. [17] study, and the reduction in photoreceptor function in the Alarco´n-Martı´nez et al. [10] study indicated by the reduction in the a-wave. Consistent with Alarco´n-Martı´nez et al. [10] and Holcombe et al. [26], we found that the IT of the pSTR was reduced after ONTx. In addition to the pSTR, we also analyzed the nSTR, which is reduced post-ONTx in rats [20–22] and mice [10] and is reduced in a mouse models of glaucoma with chronic high intraocular pressure [26]. In the present study, the nSTR amplitude measured at peak showed an overall significant reduction in amplitude, but analysis of contrast revealed that this reduction was not expressed consistently at all stimulus strengths; in addition, the reduction was small (maximum \20 % reduction or effect size lower than 0.30), making the peak nSTR amplitude an unreliable marker of RGC death. On the other hand, the IT was consistently shorter in ONTx eyes, probably due to the weaker pSTR unmasking the non-RGC component of

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the nSTR. If we measured the nSTR at a fixed time point (233 ms), a more robust reduction in the nSTR was uncovered (*50 % reduction). In previous studies where consistent nSTR reductions were observed, it is not possible to totally eliminate widespread damage that would have affected the retina upstream of the RGCs, whether the model is using ONTx or IOP increase [see 40, 47]. It is possible that the significantly reduced nSTR (*50 % reduction measured at peak at 12 weeks) in ONTx mice found in Alarco´nMartı´nez et al. [10] was partly driven by the reduction in the photoreceptor function (represented by reductions in a-wave amplitude) or non-specific cell death. This is supported by the significant reductions in both a-wave and nSTR seen in Holcombe et al. [26]. These results show that there is a RGC contribution to the nSTR, but it is necessary to measure at a fixed time to observe it reliably. RGCs contribute little to the dark-adapted OPs Experimental models that result in RGC loss [10–14, 17, 48] suggest that RGCs contribute to the generation of OPs in mice. In addition, there have been suggestions that the OPs may be generated by feedback from amacrine cells coupled to RGCs [49, 50]. We did not find a significant contribution of RGCs to the amplitude of the OPs when they are summed or when they are considered individually. When the OPs are considered separately, OP1 was reduced at higher stimulus strengths while OPs three and four were slightly increased in the absence of RGCs. The increase in OPs three and four was surprising but not without precedent as Bui and Fortune [21] also found that in response to weak stimuli (-2.3 to -1.2 log cd s m-2) ONTx increased the summed amplitude of the OPs. Albeit surprising, this result could be explained by considering that at least a subset of the RGCs generate rhythmic responses to light stimuli [49, 51] that could be out of phase relative to oscillations generated in the inner retina. The removal of this destructive interference would potentially occur following ONTx. In any case, it appears that the OPs are not a strong indicator of RGC function in mice. Contributions of RGCs to the photopic ERG Previous results have indicated the possibility of RGCgenerated components in the rodent photopic ERG

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[10, 21]. Similar to the qualitative data reported by Miura et al. [52] after optic nerve crush in mice, we found no significant RGC contribution to the photopic a- and b-wave. Furthermore, we documented that the photopic OPs were unaltered by the ONTx. A reduction in PhNR following RGC lesion was qualitatively documented in mice [10, 52] and quantitatively in rats [22, 34]. In addition, a mouse model of dominant optic atrophy (OPA1) shows a specific loss of PhNR [53], and a recent study [33] shows that the PhNR is reduced in a model of acute increase in IOP. We found that ONTx had a weakly significant contribution to the PhNR, but surprisingly this difference was an increase in amplitude rather than the expected decrease. A close inspection of the example waveforms reveals a faster decay of the b-wave after ONTx (Fig. 6A), which may explain the observed increase in amplitude of the PhNR. Furthermore, it is possible that the PhNR is more influenced in rodents by the activity of inner retinal cells other than the RGCs as proposed previously [22, 52, 54]. This explanation agrees with the results of Chrysostomou and Crowston [33] since the same model causes amacrine cell degeneration [55].

Conclusions In conclusion, we assessed the contribution of RGCs to a number of ERG parameters associated with inner and outer retinal function. We show that RGCs do not contribute substantially to the photopic and scotopic a-waves, as well as to the photopic b-wave. Mild, inconsistent effects were observed with the PhNR and nSTR. There are small contributions of RGCs to the scotopic b-wave at low flash strengths, but the most reliable measures of RGC function in mice appear to be the amplitude of the pSTR as well as the reshaping of the late negative potentials of the ERGs (nSTR and PhNR). The expression of the RGC signal in the murine ERG is different than in primate, and this should be taken into account whenever results from mouse studies are used as a representation of human retinal function in normal and diseased states. Acknowledgments Funding for this work was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) to PDC and FT and the Canadian Institutes of Health Research (CIHR) to BCC (Grant No. MOP-89808).

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Contribution of retinal ganglion cells to the mouse electroretinogram.

To quantify the direct contribution of retinal ganglion cells (RGCs) on individual components of the mouse electroretinogram (ERG)...
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