Abnormal sensory perception masks behavioral performance of Grin1 knockdown mice

Abstract

The development and function of sensory systems require intact glutamatergic neurotransmission. Changes in touch sensation and vision are common symptoms in autism spectrum disorders, where altered glutamatergic neurotransmission is strongly implicated. Further, cortical visual impairment is a frequent symptom of GRIN disorder, a rare genetic neurodevelopmental disorder caused by pathogenic variants of GRIN genes that encode NMDA receptors. We asked if Grin1 knockdown mice (Grin1KD), as a model of GRIN disorder, had visual impairments resulting from NMDA receptor deficiency. We discovered that Grin1KD mice had deficient visual depth perception in the visual cliff test. Since Grin1KD mice are known to display robust changes in measures of learning, memory, and emotionality, we asked whether deficits in these higher-level processes could be partly explained by their visual impairment. By changing the experimental conditions to improve visual signals, we observed significant improvements in the performance of Grin1KD mice in tests that measure spatial memory, executive function, and anxiety. We went further and found destabilization of the outer segment of retina together with the deficient number and size of Meissner corpuscles (mechanical sensor) in the hind paw of Grin1KD mice. Overall, our findings suggest that abnormal sensory perception can mask the expression of emotional, motivational and cognitive behavior of Grin1KD mice. This study demonstrates new methods to adapt routine behavioral paradigms to reveal the contribution of vision and other sensory modalities in cognitive performance.

Keywords: GRIN1; Morris water maze; anxiety; autism; elevated plus maze; learning & memory; mice; puzzle box; schizophrenia; tactile function; vision.

FIGURE 1

(A–D) The behavior of Grin1KD and WT mice in the “Visual Cliff” test to assess a visual depth perception. (A–B) The experimental installation, side (A) and top (B) views. (C) The percentage of time spent in the “safe” and “cliff” areas; (D) The number of entries into the “cliff” area. ***p < 0.001—in comparison with WT mice; ^### p < 0.001—in comparison with the “safe” area within genotype. N = 8–9 per genotype

FIGURE 2

(A–I) Spatial learning and memory was measured in the standard and modified Morris's water‐maze (MWM). (A–C). Schematic illustrations (images were generated via BioRender.com) of three protocols of MWM: (A) Standard protocol #1 (WT: N = 6; Grin1KD = 8), where the visual cues were distantly located on walls of the experimental room to mark south, north, east and west sides; the visual escape platform (V) was used on the 1st day of training and the hidden escape platform was used for the rest of the acquisition; (B). Modified protocol #2, where a visual platform was used during the entire acquisition session (WT; N = 9; Grin1KD = 11); (C) Modified protocol #3 was similar to the Standard protocol #1, but proximate visual cues were attached to walls of the pool (WT: N = 9; Grin1KD: N = 11). (D–F) Latency (seconds) to reach the escape platform during the acquisition session (D—standard protocol #1; E—modified protocol #2, F—modified protocol #3), where visual (V) platform was used on Day 0; ***p < 0.001—in comparison with WT mice; ^# p < 0.05; ^## p < 0.01—in comparison with protocol #1. (G–I) The percentage of time spent in the targeted quadrant of the pool or non‐targeted area (the averaged percentage of time spent in other three quadrants) during the probe day ((G)—standard protocol #1; (H)—modified protocol #2, (I)—modified protocol #3). ***p < 0.001—in comparison with WT mice; ^# p < 0.05; ^### p < 0.001—in comparison with non‐target area within each genotype

FIGURE 3

(A–E) Executive function, working memory and long‐term memory were assessed in the standard (A–B) and modified (C–D) “Puzzle Box” test. (A) Illustrations of all trials (T1–T7) used in the standard procedure; (C)—illustrations of all trials ("T1–"T9) used in the modified procedure. Latency (seconds) to enter the goal box assessed in the standard (B) and modified (D) Puzzle Box is presented. (E) The duration of digging behavior (seconds) was assessed in an independent “Digging” test. *p < 0.05; **p < 0.01; ***p < 0.001—in comparison with WT mice; ^# p < 0.05—in comparison with the standard protocol within genotype. N = 6/WT; 6/Grin1KD (standard protocol); N = 7/WT; 6/Grin1KD (modified protocol); N = 13/WT; 17/Grin1KD (“Digging” test)

FIGURE 4

(A–H) The behavior of Grin1KD and WT mice in the mouse‐sized elevated plus‐maze (mEPM) (A) and rat‐sized elevated plus‐maze (rEPM) (B). (C–D) The percentage of time spent in the open arms of the mEPM (C) and rEPM (D); (E–F) The number of defecations in mEPM (E) and rEPM (F). (G–H) The correlation analysis between behavior in the “Cliff” test and behavior in the mEPM for WT (G) and Grin1KD (H) mice. ^## p < 0.01—in comparison with mEPM within each genotype; *p < 0.05; **p < 0.01; ***p < 0.001—in comparison with WT mice; mEPM: N = 7/WT; 7/Grin1KD; rEPM: N = 6/WT; 6/Grin1KD

FIGURE 5

(A–N) The histological changes in retina' stabilization and mechanical sensors of the paw' skin (Meissner's corpuscles), in Grin1KD and WT mice. (A–B) Illustration of the central retina near the head of the optic nerve with the attached outer segment (OS) to the retina pigmented epithelium (RPE) in WT mice (A; white arrows), and less stable connections of OS to the RPE were detected in Grin1KD mice (B); magnification ×10. (C–D) Thickness of retina' layers in WT (C) and Grin1KD (D) mice. The retina of Grin1KD mice (D) is characterized by thicker outer segment (OS) layer (F) and longer non‐attached retinal pigmented epithelium (EPL) (E) than in WT mice. N = 3 mice per genotype; regions of interest (ROI): n = 9 (WT)/n = 8 (Grin1KD). (G) Schematic orientation of the hind paw for the preparation of the paraffinized samples. The analysis of Meissner corpuscles was performed on sections of the 1st digit. (H) Three ROIs are indicated by arrows on image of the hind paw (magnification ×2). Images of the glaborous footpad of WT (I) and Grin1KD (J) mice containing Meissner corpuscles. High magnification (×63) light microscopy images of Meissner corpuscles in WT (K) and Grin1KD (L) animals. N = 3 mice per genotype; 3 ROI per mouse. GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, outer segments; RPE, retinal pigmented epithelium