Daily visual stimulation in the critical period enhances multiple aspects of vision through BDNF-mediated pathways in the mouse retina

Abstract

Visual experience during the critical period modulates visual development such that deprivation causes visual impairments while stimulation induces enhancements. This study aimed to determine whether visual stimulation in the form of daily optomotor response (OMR) testing during the mouse critical period (1) improves aspects of visual function, (2) involves retinal mechanisms and (3) is mediated by brain derived neurotrophic factor (BDNF) and dopamine (DA) signaling pathways. We tested spatial frequency thresholds in C57BL/6J mice daily from postnatal days 16 to 23 (P16 to P23) using OMR testing. Daily OMR-treated mice were compared to littermate controls that were placed in the OMR chamber without moving gratings. Contrast sensitivity thresholds, electroretinograms (ERGs), visual evoked potentials, and pattern ERGs were acquired at P21. To determine the role of BDNF signaling, a TrkB receptor antagonist (ANA-12) was systemically injected 2 hours prior to OMR testing in another cohort of mice. BDNF immunohistochemistry was performed on retina and brain sections. Retinal DA levels were measured using high-performance liquid chromatography. Daily OMR testing enhanced spatial frequency thresholds and contrast sensitivity compared to controls. OMR-treated mice also had improved rod-driven ERG oscillatory potential response times, greater BDNF immunoreactivity in the retinal ganglion cell layer, and increased retinal DA content compared to controls. VEPs and pattern ERGs were unchanged. Systemic delivery of ANA-12 attenuated OMR-induced visual enhancements. Daily OMR testing during the critical period leads to general visual function improvements accompanied by increased DA and BDNF in the retina, with this process being requisitely mediated by TrkB activation. These results suggest that novel combination therapies involving visual stimulation and using both behavioral and molecular approaches may benefit degenerative retinal diseases or amblyopia.

Fig 1. Daily OMR testing in young mice enhances visual function.

(A) Spatial frequency threshold measurements obtained daily from P16 to P23. Thresholds continued to increase until plateauing between P19 and P23. Control mice were measured on the final day of testing (P23). By P23, daily OMR testing resulted in 1.5x greater visual acuity thresholds than controls (Student’s t-test, p<0.001). (B) On the final day of testing, contrast sensitivity was 5x increased in OMR-treated mice (Student’s t-test, p = 0.002). Data are represented as mean ± SEM. **p<0.01, ***p<0.001, a.u. = arbitrary units.

Fig 2. Electrophysiological results show selective improvement in scotopic inner retinal function.

(A) Representative dark-adapted ERG waveforms across flash stimuli illustrating that daily OMR stimulation does not improve photoreceptor and bipolar cell function—indicated by (B) dark-adapted a- and b-wave amplitudes across increasing flash stimuli, respectively. ERG measurements are indicated in the top waveform in (A): a-wave amplitude is the difference between points 1 and 2, and b-wave amplitude is the difference between points 2 and 3. (C) Representative dark-adapted ERG OP2 waveforms in response to increasing flash stimuli with an arrowhead that designates a significantly faster OP2 in OMR-treated mice in response to -2.5 log cd/sm² flash stimuli (Two-way repeated measures ANOVA F(4, 70), p = 0.004, Holm-Sidak multiple comparison at 2.5 log∙cd∙s/m², *p<0.05). This is quantified in (D), and suggests improvements in rod-driven inner retinal processing in OMR-treated mice. (E) Representative PERG waveforms in response to patterned stimuli show no significant differences between OMR-treated and control mice in either (F) amplitude or latency, suggesting no significant improvements in retinal ganglion cell function with OMR treatment. (G) Visual cortex function was also not significantly improved with OMR stimulation, as seen in representative VEP waveforms in response to 1.4 log cd s/m² stimuli. (H) VEP amplitude and implicit time were not statistically different between the control and OMR-treated mice. Data are represented as mean ± SEM.

Fig 3. Brain derived neurotrophic factor (BDNF) immunohistochemistry of retina and brain after daily OMR stimulation.

BDNF immunohistochemistry from retinas of (A, B) control and (C, D) OMR-treated mice. Scale bar represents 50 μm. (E) Comparisons of the intensity of immunofluorescence in the retinal ganglion cell layer relative to control sections showed greater BDNF labeling in OMR-treated mice (Student’s t-test, *p = 0.039). BDNF immunohistochemistry from brain sections of (F, G) control and (H, I) OMR-treated mice showed no regions of intense labeling. Scale bar represents 0.1mm. (J) Relative comparisons of image fluorescence intensity in the visual cortex indicated no statistical difference in BDNF expression. Corresponding DAPI images shown for each BDNF-labeled section. All values are represented as mean ± SEM. ONL: outer nuclear layer, INL: inner nuclear layer, IPL: inner plexiform layer, GCL: ganglion cell layer, VC: visual cortex, HC: hippocampus.

Fig 4. Improvements in visual function with daily OMR stimulation dependent on BDNF signaling.

(A) Spatial frequency thresholds measured daily from P16 to P23 were significantly reduced in mice receiving the TrkB antagonist, ANA-12, compared to vehicle-injected mice. By the second day of testing, OMR+Vehicle mice had significantly greater spatial frequency thresholds than the OMR+ANA-12 mice (Two-way repeated measures ANOVA F(7,117) = 14.95, *p<0.05 on P17) with this difference continuing until P23 (***p<0.001 on P18-P23). Control mice were measured on the final day of testing. At P23, daily OMR testing resulted in (B) 1.5x greater spatial frequency thresholds and (C) 3.9x greater contrast sensitivity in OMR+Vehicle as compared to Control+Vehicle mice (One-way ANOVA F(3,19) = 33.7, p<0.001). These enhancements were diminished in OMR+ANA-12 mice, which had only 1.2x greater spatial frequency thresholds (Kruskal-Wallis one-way ANOVA on ranks, H = 18.9, p<0.01) and contrast sensitivity indistinguishable from the Control+Vehicle mice. Data are represented as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, a.u. = arbitrary units.

Fig 5. Daily OMR is associated with increased DA and DOPAC levels.

OMR-treated mice showed increased retinal (A) DA levels compared to the control (Student’s t-test, *p = 0.035), while (B) DOPAC levels were not significantly different. Data are represented as mean ± SEM.