Materials and Reagents

Animals

1. Double transgenic B6.Cg-Tg(APP_SWE/PSEN1_∆E9)85Dbo/Mmjax hemizygous (AD⁺) mouse strain [RRID:MMRRC_034832-JAX], initially purchased from the Mutant Mouse Resource and Research Center (MMRRC) at the Jackson Laboratory, then bred and maintained at Cedars-Sinai Medical Center.

2. Non-transgenic wild-type (WT) littermates (Jackson Laboratory, catalog number: 000664)

   The mouse colony is housed in a humidity- and temperature-controlled (21-22°C) vivarium on a 12:12-h light/dark cycle (lights on at 8:00 am; lights off at 8:00 pm) with free access to food and water.

   Three different cohorts of mice are tested, each representing a different age: 8.5-month-old WT (n=13; 9 males and 4 females) and AD⁺ (n=12; 9 males and 3 females), 13-month-old WT (n=11; 6 males and 5 females) and AD⁺ (n=11; 4 males and 7 females), and 18-month-old WT (n=19; 11 males and 8 females) and AD⁺ (n = 9; 5 males and 4 females).

   Note: The double-transgenic APP_SWE/PS1_ΔE9 is a well-established mouse strain, with early-onset pathology of AD in the hippocampus and cortex (β-amyloid plaques, severe astrogliosis, and microgliosis, as well as brain plasticity deficits and synaptic loss). We and others also reported AD manifestation in the retina of these mice (Ning et al., 2008; Koronyo et al., 2012; Mirzaei et al., 2019; Doustar et al., 2020, Shi et al., 2020a and 2020b). Importantly, cognitive deficits in these mice have been well documented and reproducible. We developed the ViS4M to further characterize visual function, especially color vision and contrast sensitivity. AD pathology in the retina of other mouse strains has been confirmed, such as the aggressive-phenotype 5xFAD or the triple-transgenic 3xTg mice (Criscuolo et al., 2018; Grimaldi et al., 2018; Habiba et al., 2020). This protocol could be implemented on different AD mouse strains, normal aging, and other neurodegenerative models to evaluate visual function.

Equipment

1. Visual-stimuli four-arm maze (ViS4M) apparatus

   Note: The visual-stimuli four-arm maze (ViS4M or Visual X-maze) that we describe below was conceived and developed in-house by our team. The prototype used to collect the present data shares the same specifications (including the color LED lights) with the now commercially available ViS4M manufactured by Maze Engineers (https://conductscience.com/maze/portfolio/visual-x-maze-vis4m/).

   1. Backbone of apparatus

      1. Custom-made x-shaped enclosure built with 15 cm-high black plexiglass walls attached to a glass base

         Each arm is perpendicular to the two adjacent arms and is 45 cm long and 10 cm wide (Figure 1A-1B).

      2. Removable transparent floor plates that can be installed at two different levels (6 cm or 11 cm above the glass base) in each arm and in the center of the ViS4M (Figure 1C)

      3. White translucent acrylic plates that can be inserted below each floor level by sliding within small track brackets made of plexiglass (Figure 1C-1D)

         Note: Behavioral testing is carried out either on the upper level (color mode) or the lower level (contrast mode) but not simultaneously on both levels.

      4. The center of the apparatus is a neutral area with no white translucent plate and no light source directly below it (Figure 1A-1C)

      5. Plastic transparent covers with perforated holes to help with breathing

         Note: Color vision and contrast sensitivity are different aspects of vision that involve different photoreceptors. While the contrast mode involves the function of rods (lower mesopic range), the color mode requires functional cones (transition between mesopic and photopic range). These two modalities of testing are complementary to appreciate the functionality of rod and cone photoreceptors.

   2. Light-emitting diode (LED) lights (for color mode)

      Each LED strip source consists of an array of surface-mounted device (SMD) 3528 LED chips evenly spaced in four rows (27 LED chips per row) and is individually inserted in each arm of the ViS4M, directly onto the glass base (Figure 1D). Spectra of the light sources were determined using an Ocean Optics USB2000 spectrometer and further validated with a Sekonic C700-U spectrometer (Figure 1E).

      1. Red monochromatic light: wavelength (λ) = 628 nm, full width at half maximum (FWHM) = 17 nm

      2. Green monochromatic light: λ = 517 nm, FWHM = 31 nm

      3. Blue monochromatic light: λ = 452 nm, FWHM = 22 nm

      4. White light: λ1 = 441 nm, FWHM = 19 nm and λ2 = 533 nm, FWHM = 104 nm

         The white LED is made of a blue-emitting diode that also excites a yellow-emitting phosphor [cerium doped yttrium aluminum garnet (Ce:YAG-Y3Al5O12) crystals] embedded in the epoxy dome.

         Note: The criterion for choosing the LED colors is as follows: the red light as a dark-space control arm with low- to no-color stimulus; the green light to stimulate the mouse retinal M-opsin in M-cones, without stimulating S-opsin; and the blue light to stimulate the mouse retinal S-opsin in S- and M-cones, in addition to the M-opsin.

         The following components control the brightness of the light stimuli.

      5. LED single color dimmers using pulse width modulation (PWM) technology

      6. Individual remote devices to control the dimmers

         
         

      
      Figure 1. Characteristics of the ViS4M in color mode. (A) Photograph and (B) illustration of the x-shaped ViS4M in color mode with apparatus measurements. (C) Photograph of the ViS4M in color mode, E condition, showing the illuminated arms. (D) Photograph of an arm with the opening for the red lights and with the white translucent plate inserted at the upper level. (E) Illustration of the positioning of the clear floor and white translucent diffuser plates within the arms of the maze, paired with a photograph of the outside front-view of an arm. (F) Spectral distribution of the LED sources according to mouse M- and S-opsins sensitivity with table showing the characteristics of the light stimuli (wavelength, FWHM).

      
      

   3. Grayscale objects (for contrast mode)

      Four objects have the following characteristics (Figure 2A).

      1. Identical shape of a right-angled triangular prism

      2. Dimensions: base (adjacent side) = 5 cm; height (opposite side) = 1 cm

      3. Different shades (black, gray, white, and clear) that create different contrasts with the black walls and white floors of the maze (Figure 2B)

         The luminance ratios of the objects against the black walls and/or white floors as measured with a light meter are indicated below.

      4. Black object against the black walls = 1.06 (minimal to no contrast)

      5. Gray object against the black walls = 6.00

      6. White object against the black walls = 9.69 (high contrast against the black walls but minimal contrast with the white floor)

      7. Clear object against the black walls = 6.56

      8. Flexible positioning and location for the placement of the grayscale objects to accommodate investigator’s goals

      
      

      
      Figure 2. Characteristics of the ViS4M in contrast mode. (A) Photograph of the ViS4M in contrast mode with measurements for positioning of the grayscale objects. (B) Photographs of the objects within each arm as seen from the center of the ViS4M. Objects, black walls, and white floors are delineated to better assess contrasts. (A-B) B, Black; C, Clear; G, Gray; W, White.

      
      

2. Other accessories

   1. Sekonic Flashmate L-308S light meter (Sekonic, catalog number: 401-309)

   2. Timer

   3. Digital USB camera

      Note: A color camera is preferable to identify the colored arms. However, a black and white camera can be used, provided the position of each arm on the screen is recorded. A short clip of the apparatus positioning can be made prior to each testing session with the lights of the room turned on and the arms identified by name.

   4. USB camera varifocal lens with 2.8-12 mm focal length

Software

1. Rodent Toolbox V1 (freely available at https://www.ndcn.ox.ac.uk/team/stuart-peirson)

2. ANY-maze behavior tracking software 6.3 (www.stoeltingco.com/anymaze/video-tracking/software.html) or other video tracking software, such as EthoVision XT (www.noldus.com/ethovision-xt

3. ViS4M Toolbox V1 (Supplementary file: ViS4M Toolbox V1)

4. GraphPad Prism 9.0

5. ParallelSets V2 (freely available at Parallel Sets (eagereyes.org))

6. Circos online (freely available at mkweb.bcgsc.ca/tableviewer/)

Procedure