Measurement of Electroretinograms and Visually Evoked Potentials in Awake Moving Mice

<div><p>The development of new treatments for intractable retinal diseases requires reliable functional assessment tools for animal models. <i>In vivo</i> measurements of neural activity within visual pathways, including electroretinogram (ERG) and visually evoked potential (VEP) recordings, are commonly used for such purposes. In mice, the ERG and VEPs are usually recorded under general anesthesia, a state that may alter sensory transduction and neurotransmission, but seldom in awake freely moving mice. Therefore, it remains unknown whether the electrophysiological assessment of anesthetized mice accurately reflects the physiological function of the visual pathway. Herein, we describe a novel method to record the ERG and VEPs simultaneously in freely moving mice by immobilizing the head using a custom-built restraining device and placing a rotatable cylinder underneath to allow free running or walking during recording. Injection of the commonly used anesthetic mixture xylazine plus ketamine increased and delayed ERG oscillatory potentials by an average of 67.5% and 36.3%, respectively, compared to unanesthetized mice, while having minimal effects on the a-wave and b-wave. Similarly, components of the VEP were enhanced and delayed by up to 300.2% and 39.3%, respectively, in anesthetized mice. Our method for electrophysiological recording in conscious mice is a sensitive and robust means to assess visual function. It uses a conventional electrophysiological recording system and a simple platform that can be built in any laboratory at low cost. Measurements using this method provide objective indices of mouse visual function with high precision and stability, unaffected by anesthetics.</p></div

Comparison of VEPs recorded under awake and anesthetized conditions in the same mice.

<p>Representative traces of scotopic (top) and photopic (bottom) VEPs from the same mouse recorded in the awake (left) and anesthetized (right) condition. Amplitudes of scotopic (top) and photopic (bottom) VEPs from the same mice recorded in the awake (black) and anesthetized (red) condition. Implicit times of scotopic (top) and photopic (bottom) ERGs from the same mice recorded in the awake (black) and anesthetized (red) condition. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p

Stability of the ERG and VEP recording.

<p>A, B, Amplitude time courses of ERG and VEP waveforms recorded in anesthetized mice (<i>N</i> = 4 for each time point). The flashes (0 log cd/m²) were given in the scotopic condition. The bars indicate mean ± S.E.M.</p

Comparison of OPs extracted from ERGs acquired under awake and anesthetized conditions.

<p>Representative ERG traces before and after the extraction of OPs from a scotopic ERG (0 log cd s/m²). Schematic illustration showing the amplitude (a_n) and implicit time (t_n) of each OP wavelet (OP_n). C, D. Amplitudes (C) and implicit times (D) of individual OP wavelets (OP₁–OP₄) recorded in the awake (black) and anesthetized (red) condition from the same mice. E, F. Summed amplitudes (C) and implicit times (D) of all OP wavelets (OP₁–OP₄) from ERGs recorded under awake (black) and anesthetized (red) conditions from the same mice. * <i>P</i> < 0.05. Data from seven mice. The bars indicate mean ± S.E.M. anes: anesthetized.</p

Analysis of Macular Drusen and Blood Test Results in 945 <i>Macaca fascicularis</i>

<div><p>Age-dependent formation of macular drusen caused by the focal accumulation of extracellular deposits beneath the retinal pigment epithelium precede the development of age-related macular degeneration (AMD), one of the leading causes of blindness worldwide. It is established that inflammation contributes to the pathogenesis of drusen and AMD. However, development of a preemptive therapeutic strategy targeting macular drusen and AMD has been impeded by the lack of relevant animal models because most laboratory animals lack macula, an anatomic feature present only in humans and a subset of monkeys. Reportedly, macular drusen and macular degeneration develop in monkeys in an age-dependent manner. In this study, we analyzed blood test results from 945 <i>Macaca fascicularis</i>, 317 with and 628 without drusen. First, a trend test for drusen frequency (the Cochran–Armitage test) was applied to the quartile data for each parameter. We selected variables with an increasing or decreasing trend with higher quartiles at P < 0.05, to which multivariate logistic regression analysis was applied. This revealed a positive association of age (odds ratio [OR]: 1.10 per year, 95% confidence interval [CI]: 1.07–1.12) and white blood cell count (OR: 1.01 per 1 × 10³/μl, 95% CI: 1.00–1.01) with drusen. When the monkeys were divided by age, the association between drusen and white blood cell count was only evident in younger monkeys (OR: 1.01 per 1 × 10³/μl, 95% CI: 1.00–1.02). In conclusion, age and white blood cell count may be associated with drusen development in <i>M</i>. <i>fascicularis</i>. Systemic inflammation may contribute to drusen formation in monkeys.</p></div

Multiple logistic regression analysis and associated factors for retinal drusen.

<p>Multiple logistic regression analysis and associated factors for retinal drusen.</p

Fundus of a monkey eye with drusen.

<p>Fundus photograph of a 21-year-old male <i>Macaca fascicularis</i> with drusen (open triangle; upper panel). Note retinal hemorrhage is seen inferiorly (filled triangle). Histologic section of an eye with drusen from another 34-year-old monkey. Note an accumulation of extracellular material underneath the retinal pigment epithelium consistent with drusen formation (*).</p

Basic biologic and hematologic data for monkeys with and without drusen.

<p>Basic biologic and hematologic data for monkeys with and without drusen.</p

Multiple logistic regression analysis and associated factors for retinal drusen in 232 male and 713 female monkeys.

<p>Multiple logistic regression analysis and associated factors for retinal drusen in 232 male and 713 female monkeys.</p

Quartile analyses and trend tests in 945 monkeys.

<p>Quartile analyses and trend tests in 945 monkeys.</p