Knockdown of the candidate dyslexia susceptibility gene homolog dyx1c1 in rodents: effects on auditory processing, visual attention, and cortical and thalamic anatomy

Abstract

The current study investigated the behavioral and neuroanatomical effects of embryonic knockdown of the candidate dyslexia susceptibility gene (CDSG) homolog Dyx1c1 through RNA interference (RNAi) in rats. Specifically, we examined long-term effects on visual attention abilities in male rats, in addition to assessing rapid and complex auditory processing abilities in male and, for the first time, female rats. Our results replicated prior evidence of complex acoustic processing deficits in Dyx1c1 male rats and revealed new evidence of comparable deficits in Dyx1c1 female rats. Moreover, we found new evidence that knocking down Dyx1c1 produced orthogonal impairments in visual attention in the male subgroup. Stereological analyses of male brains from prior RNAi studies revealed that, despite consistent visible evidence of disruptions of neuronal migration (i.e., heterotopia), knockdown of Dyx1c1 did not significantly alter the cortical volume, hippocampal volume, or midsagittal area of the corpus callosum (measured in a separate cohort of like-treated Dyx1c1 male rats). Dyx1c1 transfection did, however, lead to significant changes in medial geniculate nucleus (MGN) anatomy, with a significant shift to smaller MGN neurons in Dyx1c1-transfected animals. Combined results provide important information about the impact of Dyx1c1 on behavioral functions that parallel domains known to be affected in language-impaired populations as well as information about widespread changes to the brain following early disruption of this CDSG.

Fig. 1

Timeline of the behavioral testing of the male and female Wistar rats (a) and schematics of auditory processing tasks (b–d). b A schematic of the silent gap detection task. Silent gaps were of variable durations between 2 and 100 ms. c A schematic of uncued and cued trials in the FM sweep task. In the cued trial, the cue is a reversal of the background frequency sweep from high-to-low to low-to-high, as depicted. The high frequency is 2,300 Hz and the low frequency is 1,100 Hz. The intrastimulus duration progressed from long to short across days of testing, making the frequency sweep (and thus the cue) more difficult to discriminate. The between-sequence ISI was always 200 ms greater than the intrastimulus duration. d A schematic of the 3-tone oddball task. The intra-stimulus duration and between-sequence ISI varied across weeks of testing, progressing from long to short to increase the temporal demands of the task.

Fig. 2

Coronal Nissl-stained brain sections from male (a, b) and female (c–e) Dyx1c1 shRNA animals. Histology revealed 3 categories of gross cortical malformation. a, c Injection site ectopia (female Dyx1c1 shRNA, n = 3, male Dyx1c1 shRNA, n = 4). These anomalies resulted from the injection puncture wound and were observed as collections of ectopic cells in layer I of the neocortex (arrows). b, d Unmigrated neurons in the white matter (female Dyx1c1 shRNA, n = 5; male Dyx1c1 shRNA, n = 2). These anomalies were observed as small and large collections of unmigrated cells forming heterotopic pockets in the white matter subjacent to the cortex (arrows). b, e Hippocampal dysplasia (female Dyx1c1 shRNA, n = 1; male Dyx1c1 shRNA, n = 2). These anomalies were observed as pockets of unmigrated cells that disrupted the structure of the hippocampal formation (most often the dentate gyrus) (arrowheads). Scale bars = 500 μm.

Fig. 3

Auditory testing performance by male and female Dyx1c1 shRNA and sham rats. a, b Juvenile silent gap attenuation scores. These graphs illustrate similar performance between males (a) and females (b) and between sham and Dyx1c1 shRNA animals. Paired-samples t tests indicated that animals could successfully detect gaps down to 5 ms in length. c, d FM sweep attenuation scores. These graphs illustrate FM sweep performance by males (c) and females (d) in the juvenile period and adulthood. A significant age × sweep duration × treatment interaction indicates that at the short sweep duration, sham animals consistently perform better than Dyx1c1 animals (as indicated by their lower attenuation scores), across both age and sex (* p < 0.05). (e, f 3-Tone oddball attenuation scores. These graphs illustrate the 3-tone oddball performance by males (e) and females (f) in the adult period. Attenuation scores were averaged across 5 days for each of the 3 versions of this task. There was a marginal effect of treatment across the 3 tasks suggesting worse overall performance by Dyx1c1 shRNA animals as compared to shams (across sex) (^# p = 0.09). Separate analyses revealed significant treatment effects on the 60/260 and 10/210 versions of the task (* p < 0.05), with shams outperforming Dyx1c1 shRNA animals across sexes. Values represent means ± SEM.

Fig. 4

MWM data. a The average latency to mount a visible platform during the water escape task, given the day before the start of MWM testing. There were no main effects of sex or treatment, indicating that male and female Dyx1c1 shRNA and sham animals were all able to see, swim, and mount the platform. b The average time spent in each of the 4 maze quadrants during the probe trial. All animals spent significantly more time in the target quadrant during the probe trial (indicated by ** p < 0.01), which indicates that all animals successfully learned the location of the goal platform across the 4 prior days of testing. There were no main effects of sex or treatment on the probe trial data. c MWM average path length to the platform. A significant effect of sex (* p < 0.05) indicates that females swam longer paths than males before reaching the platform across 4 days of testing. No main effect of treatment was found, indicating that sham and Dyx1c1 shRNA animals swam similar distances before finding the goal platform. Values represent means ± SEM.

Fig. 5

Visual attention testing performance by male Dyx1c1 shRNA and sham rats. a Overall percent correct responses as a function of visual attention task. Note that Dyx1c1 shRNA animals had consistently lower percent correct responses than sham animals, although this difference did not reach statistical significance. VITI = Variable intertrial interval. b Average percent correct responses across 3 days of testing on the 60, 60, 5 version of the 5CSRT task (60-second stimulus duration, 60-second response window, 5-second ITI). A repeated measures ANOVA revealed a significant effect of treatment, with Dyx1c1 shRNA animals performing significantly worse than shams (* p = 0.05, one-tailed). c Bivariate correlation between visual attention testing performance and rapid auditory testing scores. Shams demonstrated a significant correlation between average percent correct during visual attention testing (averaged across all tasks) and average ATT scores on adult short FM sweep testing (p < 0.05). Dyx1c1 shRNA animals’ performances on the 2 tasks were not significantly correlated. Values represent means ± SEM.

Fig. 6

Cell size distribution in the MGN of the thalamus. This frequency histogram represents the distribution of cell size in the MGN of Dyx1c1 shRNA (black bars) and sham (white bars) animals. Note that only male brains were analyzed. The average percent of cells in each size bin for each treatment group was calculated for 14 consecutive bins based on the cell area. A significant bin × treatment interaction (p = 0.05) indicates that Dyx1c1 shRNA animals have more small MGN neurons than their sham counterparts. Post hoc testing revealed that the biggest contributor to this interaction was a significant difference at the fourth size bin, where the Dyx1c1 shRNA animals had significantly more cells than shams (* p < 0.05).