Behavioral Assessment of Visual Function via Optomotor Response and Cognitive Function via Y-Maze in Diabetic Rats

Summary

Neural degeneration in both eyes and brain as a result of diabetes can be observed through behavioral tests carried out on rodents. The Y-maze, a measure of spatial cognition, and the optomotor response, a measure of visual function, both provide insight into potential diagnoses and treatments.

Abstract

The optomotor response and the Y-maze are behavioral tests useful for assessing visual and cognitive function, respectively. The optomotor response is a valuable tool to track changes in spatial frequency (SF) and contrast sensitivity (CS) thresholds over time in a number of retinal disease models, including diabetic retinopathy. Similarly, the Y-maze can be used to monitor spatial cognition (as measured by spontaneous alternation) and exploratory behavior (as measured by a number of entries) in a number of disease models that affect the central nervous system. Advantages of the optomotor response and the Y-maze include sensitivity, speed of testing, the use of innate responses (training is not needed), and the ability to be performed on awake (non-anesthetized) animals. Here, protocols are described for both the optomotor response and the Y-maze and examples of their use shown in models of Type I and Type II diabetes. Methods include preparation of rodents and equipment, performance of the optomotor response and the Y-maze, and post-test data analysis.

Introduction

Over 463 million people live with diabetes, making it one of the largest global disease epidemics¹. One of the serious complications that arises from diabetes is diabetic retinopathy (DR), a leading cause of blindness for working-age American adults². In the next 30 years, the percentage of the population at risk for DR is projected to double, so it is crucial to find new ways of diagnosing DR in its earlier stages to prevent and mitigate DR development³. DR has conventionally been thought to be a vascular disease^4,5,6. However, now with evidence of neuronal dysfunction and apoptosis in the retina that precedes vascular pathology, DR is defined to have neuronal and vascular components^4,5,6,7,8,9. One way to diagnose DR would be to examine neural abnormalities in the retina, a tissue that may be more vulnerable to oxidative stress and metabolic strain from diabetes than other neural tissue¹⁰.

Declines in cognitive and motor function also occur with diabetes and are often correlated with retinal changes. Older individuals with Type II diabetes portray worse baseline cognitive performance and show more exacerbated cognitive decline than control participants¹¹. Additionally, the retina has been established as an extension of the central nervous system and pathologies can manifest in the retina¹². Clinically, the relationship between retina and brain has been studied in the context of Alzheimer’s and other diseases but is not commonly explored with diabetes^{12,13,14,15,16}. Changes in the brain and retina during the progression of diabetes can be explored using animal models, including the STZ rat (a model of Type I diabetes in which the toxin, streptozotocin or STZ, is used to damage pancreatic beta cells) and the Goto-Kakizaki rat (a polygenic model of Type II diabetes in which animals develop hyperglycemia spontaneously at around 3 weeks of age). In this protocol, a description for the Y-maze and the optomotor response to assess cognitive and visual changes in diabetic rodents, respectively, is provided. The optomotor response (OMR) assesses spatial frequency (similar to visual acuity) and contrast sensitivity by monitoring characteristic reflexive head tracking movements to gauge visual thresholds for each eye¹⁷. Spatial frequency refers to the thickness or fineness of the bars, and contrast sensitivity refers to how much contrast there is between the bars and the background (Figure 1E). Meanwhile, the Y-maze tests short-term spatial memory and exploratory function, observed through spontaneous alternations and entries through the arms of the maze.

Both tests can be performed in awake, non-anesthetized animals and have the advantage of capitalizing on innate responses of the animals, meaning that they do not require training. Both are relatively sensitive, in that they can be used to detect deficits early in the progression of diabetes in rodents, and reliable, in that they produce results that correlate with other visual, retinal, or behavioral tests. Additionally, using the OMR and the Y-maze in conjunction with tests such as electroretinogram and optical coherence tomography scans can provide information on when retinal, structural, and cognitive changes develop relative to each other in disease models. These investigations could be useful in identifying neural degenerations that occur due to diabetes. Ultimately, this could lead to new diagnostic methods that effectively identify DR in early stages of progression.

The OMR and the Y-maze systems used to develop this protocol are described in the Table of Materials. Previous research on the OMR, by Prusky et al.¹⁸, and the Y-maze, by Maurice et al.¹⁹, was used as the starting point to develop this protocol.

Protocol

All procedures were approved by the Atlanta Veterans Affairs Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications, 8^th edition, updated 2011).

1. The optomotor response (OMR)

1. Set up the OMR apparatus (details on apparatus and software in Table of Materials)
   1. Choose the appropriate-sized platform for the rodent: mouse, rat, or large/impaired rat (Figure 1A).
   2. Open the OMR software, which should open to a window with several tabs of options and a live video feed of the inside of the OMR/virtual drum (Figure 1B). Zoom in or out with the video camera as needed so that the platform and its surroundings are visible.
   3. Note the icons along the left-hand side of the live image (Figure 1C). Click on the asterisk icon and the rotating stripes icon so that both the green asterisk and green rotating stripes disappear from the live feed.
   4. Click on the compass icon so that a green circle and two perpendicular lines appear. Stretch the green circle so that it aligns perfectly with the black circle on the platform, which will ensure that the OMR is perfectly aligned.
   5. Click on the compass icon because it is not necessary to see the circle during testing. Click on the green asterisk icon and the green rotating stripes icon to make these reappear. Note that the green stripes rotate in the same direction as the stripes in the drum, allowing the researcher to know the direction of the stripes.
   6. Click on the Testing tab. Under Testing, click on the Psychophysics tab. Under Threshold, select Frequency to measure spatial frequency.
      NOTE: The OMR software uses a staircase paradigm to automatically calculate spatial frequency (SF). Contrast will be maintained at 100%.
   7. Under Testing, click on the Presets tab. Select the default settings for Mouse¹⁸ or Rat²⁰.
   8. Under Testing, click on the Blanking tab. Check the Blank on Tracking box, which will pause the stripes/blank out the computer screens in the drum whenever the mouse is right-clicked.
   9. Click on the Results tab, which is where the results of the test will be displayed.
2. Evaluate spatial frequency
   1. Place the rodent on the circular platform in the center of the virtual reality chamber comprising four computer monitors showing vertical sine wave gratings circling the chamber at a velocity of 12˚/s (Figure 1D).
   2. Note that the video camera positioned at the top of the chamber is projecting the rodent’s behavior live onto the computer monitor.
   3. Look for the presence or absence of reflexive actions by the rodent’s head as the gratings move in a clockwise or counterclockwise direction. Make sure illustrated bars are visible in the program—these will show the direction of the grating movement.
      1. Watch for the rodent’s head to move in the same direction as the gratings. Wait until there is a smooth pursuit, not erratic bursts of head motion, to count it as tracking.
      2. Click on Yes or No as appropriate. Note that SF will start with 0.042 cyc/deg and adjust with each yes and no to become easier or more difficult (Figure 1E). Click on Reset if the test needs to be reset due to accidental or incorrect clicking of yes and no.
   4. As the rodent is tested, make sure to keep the asterisk positioned over the rodent’s head.
      NOTE: This has two effects: 1) It maintains the correct spatial frequency. If the asterisk is positioned between the shoulders, for example, the spatial frequency will be lower and the bars will be easier to see, resulting in a falsely high score. 2) For rodents with slight head movements, the asterisk makes it easier to gauge whether the head is actually moving.
   5. Watch for the system to say “Done” when the rodent’s spatial frequency is reached. Note that the Yes and No buttons will no longer be clickable.
   6. Click on the Results tab, which will display the spatial frequency for the left eye, right eye, and combined eyes.
      NOTE: Sometimes the software is set such that the results are flipped, i.e., the right eye is reported as the left eye and the left eye is reported as the right eye. This was discovered when assessing rodents that had only one eye lesioned in a glaucoma model.
3. Evaluate contrast sensitivity
   NOTE: Contrast Sensitivity testing can be performed immediately after the spatial frequency measurement step or on its own on the same day or a different day if the rodent appears fatigued after spatial frequency testing (follow steps 1–2.2 if only testing contrast sensitivity).
   1. Click on the Testing tab and then on the Psychophysics tab. Under Threshold, select Contrast (single) to measure contrast sensitivity.
   2. Also using a staircase paradigm, start gratings with SF constant at the peak of the Contrast Sensitivity (CS) curve. To do this, click on the Stimulus tab and then on the Gratings tab. In the Spatial Frequency box, type 0.064 for rats and 0.103 for mice.
   3. Begin the contrast at 100% and look for the same reflexive head movements as seen during spatial frequency testing. Note that the contrast will decrease as the testing progresses until the rodent no longer has reflexive head movements in response to the stimulus (Figure 1E).
   4. Watch for the system to say “Done” and the Yes and No buttons to no longer be clickable once the rodent no longer responds to the visual stimulus and the contrast sensitivity threshold has been reached. Click on the Results tab, where the contrast sensitivity for the left eye, right eye, and combined eyes will be listed.
4. Perform post-testing analysis
   1. For diabetic retinopathy studies, where both eyes are expected to have similar deficits, use the combined score (average of right and left eyes) for analysis. For models that cause differential damage to eyes (i.e., blast injury or glaucoma), keep the left and right eye data separate.
   2. For Spatial Frequency, use raw scores (the data from the Results tab) for analysis and average these scores together by group (i.e., diabetic, control, etc.).
   3. For Contrast Sensitivity, use the raw value to calculate the reported contrast sensitivity by the Michelson contrast from a previous measurement of the screen’s luminance.

2. The Y-maze

1. Prepare rodents for testing
   1. Adapt rodents to the room for 30 min prior to testing.
      NOTE: The researcher can remain in the room with the lights on but should remain silent during this time.
   2. Clean the Y-maze with sanitizing solution safe for animals and wipe away all sanitizing solution with paper towels. Ensure that the maze is dry.
2. Conduct the Y-maze
   1. Label the initial arm of the Y-maze as B and the other 2 arms as A and C (Figure 2A). Place one rodent in the arm closest to the researcher (arm B) near the center of the Y-maze. Once the rodent has been placed, start the timer (details on maze and timer in Table of Materials).
      1. Allow each rodent to explore the Y-maze for 8 min. Take recordings during this time and note any observations. Sit several feet away from the maze while keeping it in sight and avoid making any noise.
      2. Record the starting location as A, and each time the rodent makes an entry into a new arm, record the new location of the rodent (Figure 2B). Define an entry as all four limbs of the rodent being in one of the arms.
      3. Watch for rodents to hide and remain stationary in one arm of the maze. If the rodent remains in the same spot for more than 60 s and does not appear to show exploratory behavior, move the rodent toward the center of the Y-maze, and continue the trial.
   2. After each rodent, remove any feces and clean the maze with sanitizing solution.
      1. Ensure that all sanitizing solution is wiped away with paper towels and the maze is completely dry before placing the next rodent in the maze.
3. Calculate spontaneous alternation and exploratory behavior
   1. Calculate exploratory behavior as the total number of entries made during 8 min.
   2. Calculate spatial cognition as measured by spontaneous alternation:
      the number of successful alternations/(the total number of entries - 2)
      1. Define a successful alternation as the rodent moves into three different locations sequentially (Example: ABC, CAB, BCA, etc.). Note each successful alternation (Figure 2B).
      2. If the movements were recorded as ACABCABABCABC, disregard the two initial starting locations when calculating spontaneous alternation (such that there are 11 movements in the denominator). Count the number of accurate movements (accurate movements = 8). Calculate the percent accuracy as: 8/(13 - 2) = 72.7%.

Results

The OMR is considered successful if spatial frequency and contrast sensitivity thresholds can be obtained from a rodent. Here, the use of the OMR to assess spatial frequency is illustrated in naïve control Brown-Norway and Long-Evans rats, both young (3–6 months) and aged (9–12 months). Brown-Norway rats typically show a higher baseline spatial frequency than Long-Evans rats. Additionally, an aging effect on spatial frequency was observed in the Long-Evans rats (Figure 3A). Data were an...

Discussion

The OMR and the Y-maze allow for the non-invasive assessment of visual function and cognitive function deficits in rodents over time. In this protocol, the OMR and the Y-maze were demonstrated to track visual and cognitive deficits in rodent models of diabetes.

Critical steps in the protocol

The OMR

Some important points to consider when performing the OMR to assess visual function are the testing parameters used, experim...

Materials

Name	Company	Catalog Number	Comments
OptoMotry HD	CerebralMechanics Inc.		OMR apparatus & software
Timer	Thomas Scientific	810029AR
Y-Maze apparatus	San Diego Instruments	7001-043	Available specifically for rats