Neuroscience

The compound eyes of Drosophila are widely used to gain valuable insights into genetics, developmental biology, cell biology, disease biology, and gene regulation. Various parameters, such as eye size, pigmentation loss, formation of necrotic patches, and disorientation, fusion, or disruption of ommatidial arrays, are commonly assessed to evaluate eye development and degeneration. We developed an improved imaging method named low-angle ring illumination stereomicroscopy (LARIS) to capture high-contrast images of the Drosophila compound eye. Different optical alignments were tested to capture the fly compound eye image under the stereomicroscope; the highest contrast with minimal reflection was achieved through the LARIS method. The images captured using LARIS clearly showed ommatidial fusion, disorientation, and pigmentation loss, which were hardly visible with a conventional imaging method in the degenerating compound eyes of Drosophila. In addition to its research applications, this protocol is cost-effective due to the low expenses associated with supplies and equipment. We anticipate that LARIS will facilitate high-contrast imaging of the compound eyes in Drosophila and other insects.

Vestibulo-ocular reflexes (VORs) are compensatory ocular reflexes that maintain stable vision during head movements. In research, VORs encompass angular VOR (aVOR) and off-vertical axis rotation (OVAR) tests, which various groups have employed to assess vestibular function in mice. This protocol outlines the process for measuring VORs in mice, including eye rotation calibration, immobilizing the mouse with a noninvasive setup, configuring the aVOR and OVAR stimulus modes, and interpreting the obtained waveforms to derive VOR values. As technology advances, VORs are expected to yield more qualitative and quantitative insights into the function of the horizontal semicircular canal cristae (HSCC) and the otolith organs. This methodology can serve as a standard for evaluating common vestibular deficits in mice.

Visual impairments, notably loss of contrast sensitivity and color vision, were documented in Alzheimer’s disease (AD) patients yet are critically understudied. This protocol describes a novel visual-stimuli four-arm maze (ViS4M; also called visual x-maze), which is a versatile x-shaped maze equipped with spectrum- and intensity-controlled light-emitting diode (LED) sources and dynamic grayscale objects. The ViS4M is designed to allow the assessment of color and contrast vision along with locomotor and cognitive functions in mice. In the color testing mode, the spectral distributions of the LED lights create four homogenous spaces that differ in chromaticity and luminance, corresponding to the mouse visual system. In the contrast sensitivity test, the four grayscale objects are placed in the middle of each arm, contrasting against the black walls and the white floors of the maze. Upon entering the maze, healthy wild-type (WT) mice tend to spontaneously alternate between arms, even under equiluminant conditions of illumination, suggesting that cognitively and visually intact mice use both color and brightness as cues to navigate the maze. Evaluation of the double-transgenic APP_SWE/PS1_ΔE9 mouse model of AD (AD⁺ mice) reveals substantial deficits to alternate in both color and contrast modes at an early age, when hippocampal-based memory and learning is still intact. Profiling of timespan, entries, and transition patterns between the different arms uncovers variable aging and AD-associated impairments in color discrimination and contrast sensitivity. The analysis of arm sequences of alternation reveals different pathways of exploration in young WT, old WT, and AD⁺ mice, which can be used as color and contrast imprints of functionally intact versus impaired mice. Overall, we describe the utility of a novel visual x-maze test to identify behavioral changes in mice related to cognition, as well as color and contrast vision, with high precision and reproducibility.

Graphic abstract:

Exploratory behavior of AD⁺ mice versus age- and sex-matched WT mice is tracked (top left: trajectory from a 5-min video file) in a novel visual-stimuli four-arm maze (ViS4M; also named visual x-maze) equipped with spectrum- and intensity-controlled LED sources or grayscale objects. Consecutive arm entries reveal that APP_SWE/PS1_ΔE9 (AD⁺) mice alternate less between arms, as opposed to WT mice. Sequence analysis, according to the three alternation pathways (depicted by white, yellow, and brown arrows) under different conditions of illumination, uncovers specific deficits linked to color vision in AD⁺ mice, evidenced by a color imprint chart.

Magnetic resonance spectroscopy (MRS) can be used to measure in vivo concentrations of neurometabolites. This information can be used to identify neurotransmitter involvement in healthy (e.g., perceptual and cognitive processes) and unhealthy brain function (e.g., neurological and psychiatric illnesses). The standard approach for analyzing MRS data is to combine spectral transients acquired over a ~10 min scan to yield a single estimate that reflects the average metabolite concentration during that period. The temporal resolution of metabolite measurements is sacrificed in this manner to achieve a sufficient signal-to-noise ratio to produce a reliable estimate. Here we introduce two analyses that can be used to increase the temporal resolution of neurometabolite estimates produced from MRS measurements. The first analysis uses a sliding window approach to create a smoothed trace of neurometabolite concentration for each MRS scan. The second analysis combines transients across participants, rather than time, producing a single “group trace” with the highest possible temporal resolution achievable with the data. These analyses advance MRS beyond the current “static” application by allowing researchers to measure dynamic changes in neurometabolite concentration and expanding the types of questions that the technique can be used to address.