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. Author manuscript; available in PMC: 2012 Mar 1.
Published in final edited form as: Genes Brain Behav. 2010 Nov 25;10(2):244–252. doi: 10.1111/j.1601-183X.2010.00662.x

Persistent spatial working memory deficits in rats following in utero RNAi of Dyx1c1

Caitlin E Szalkowski 1,*, James R Hinman 1, Steven W Threlkeld 2, Yu Wang 3, Ashley LePack 4, Glenn D Rosen 5, James J Chrobak 1, Joseph J LoTurco 3, R Holly Fitch 1
PMCID: PMC3041839  NIHMSID: NIHMS249783  PMID: 20977651

Abstract

Disruptions in the development of the neocortex are associated with cognitive deficits in humans and other mammals. Several genes contribute to neocortical development, and research into the behavioral phenotype associated with specific gene manipulations is advancing rapidly. Findings include evidence that variants in the human gene DYX1C1 may be associated with an increased risk of developmental dyslexia. Concurrent research has shown that the rat homolog for this gene modulates critical parameters of early cortical development, including neuronal migration. Moreover, recent studies have shown auditory processing and spatial learning deficits in rats following in utero transfection of an RNA interference vector (RNAi) of the rat homolog Dyx1c1 gene. The current study examined the effects of in utero RNAi of Dyx1c1 on working memory performance in Sprague-Dawley rats. This task was chosen based on evidence of short-term memory deficits in dyslexic populations, as well as more recent evidence of an association between memory deficits and DYX1C1 anomalies in humans. Working memory performance was assessed using a novel match-to-place radial water maze task that allows evaluation of memory for a single brief (~4–10 second) swim to a new goal location each day. A ten-minute retention interval was used, followed by a test trial. Histology revealed migrational abnormalities and laminar disruption in Dyx1c1 RNAi treated rats. Dyx1c1 RNAi treated rats exhibited a subtle, but significant and persistent impairment in working memory as compared to Shams. These results provide further support for the role of Dyx1c1 in neuronal migration and working memory.

Keywords: Brain development, Dyslexia, Neuronal migration, Working memory, RNA interference

INTRODUCTION

Neocortical migrational anomalies have been associated with learning, language, and other cognitive deficits in humans (Galaburda et al., 1994; Casanova et al., 2004; Hage et al., 2006; Boscariol et al., 2009). Studies from our lab have shown that rodents with cortical malformations exhibit deficits in rapid auditory processing abilities, which are similar to those observed in humans with language impairment (Peiffer et al., 2004a; Threlkeld et al., 2006; 2007). In fact, longitudinal assessment of infants, as well as studies of older populations, suggest that rapid auditory processing difficulties may represent one facet of the constellation of deficits associated with learning disorders such as developmental dyslexia (Tallal et al., 1993; Farmer et al., 1995; Wright et al., 1997; Tallal and Benasich, 2002; Benasich et al.., 2006; Choudhury et al., 2007).

Twin studies first suggested that developmental dyslexia was a genetic disorder, and early genetic linkage studies laid the groundwork for recent molecular genetic studies (see Fisher and DeFries, 2002 for review). Recent genetic association analyses in human populations have revealed gene variants (ROBO1, DCDC2, KIAA0319, and DYX1C1) that are associated with increased risk of developmental dyslexia (Taipale et al., 2003; Chapman et al., 2004; Francks et al., 2004; Wigg et al., 2004; Cope et al., 2005; Hannula-Jouppi et al., 2005; Meng et al., 2005; Harold et al., 2006; Paracchini et al., 2006; Shumacher et al., 2006; Brkanac et al., 2007; Marino et al., 2007; Ludwig et al., 2008; Dahdouh et al., 2009; Massinen et al., 2009; Wilcke et al., 2009). DYX1C1, which is located on chromosome 15, was the first candidate dyslexia risk gene reported, with variants linked to reading-related phenotypes in a dyslexic family in Finland. A linkage disequilibrium analysis linked the same variants of the gene to clinical disability in a larger population of Finnish dyslexics (Taipale et al., 2003).

Recent reports have linked variants in DYX1C1 to impairments in short term memory in dyslexic individuals (Marino et al., 2007). Verbal and non-verbal short term memory deficits — including visuospatial memory deficits — are often comorbid with the phonological impairments that characterize developmental dyslexia and specific language impairment (Archibald and Gathercole, 2006; Baddeley and Hitch, 1974; Gathercole et al., 2006; Smith-Spark and Fisk 2007).

Animal research employing embryonic (in utero) RNA interference (RNAi) has shown that knockdown of the rodent homolog for DYX1C1 (Dyx1c1) in rats results in aberrant neuronal migration, including formation of heterotopic and ectopic clusters of neurons throughout the cortex (Wang et al., 2006; Rosen et al., 2007). Behavioral assays on rats following in utero RNAi of Dyx1c1 have revealed deficits in rapid auditory processing. Spatial learning deficits were also present in a subset of animals with neuronal heterotopias in the hippocampus (Threlkeld et al., 2007).

The current study sought to explore the possibility that early cortical disruption via interference with the rodent homolog of DYX1C1 may lead to higher order memory and learning impairments paralleling those observed in developmental dyslexia. Specifically, we employed a model using E14 in utero ventricular RNAi against Dyx1c1 to characterize the working memory abilities of embryonically transfected rats.

METHODS

A total of 61 male Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA, USA) were used in these experiments (Dyx1c1 RNAi = 33; Sham = 28). All rats were pair housed in tubs with same-sex littermates, and maintained on a 12-h/12-h light-dark cycle in a temperature-controlled room. Water was available ad libitum. All procedures were performed in accordance with guidelines set forth by NIH and were approved by the University of Connecticut’s Institutional Care and Use Committee.

Transfection

Transfection of in utero RNAi of Dyx1c1 was performed by YW at the University of Connecticut. Two batches of surgeries were performed. In all Dyx1c1 treatments, plasmids encoding short hairpin (pU6DyxHPB) RNA (Dyx1c1 RNAi) were transfected into the fetal (embryonic day 14, or E14) ventricular zone (VZ) by in utero electroporation, following externalization of the uterine horn. In Batch 1, the Dyx1c1 shRNA plasmid was co-transfected with a plasmid encoding eGFP (enhanced green fluorescent protein), as well as Fast Green dye (a benign dye used to confirm injection into the lateral ventricles during the procedure). Batch 1 Shams received injections of a solution of Fast Green dye only. In Batch 2, Dyx1c1 subjects received injection of the Dyx1c1 shRNA plasmid and Fast Green dye. Sham subjects received transfection with plasmids (pCAGGS-RFP) encoding mRFP (monomeric red fluorescent protein) and Fast Green dye.

Briefly, time-mated Sprague-Dawley dams (Charles River Laboratory, Wilmington, MA, USA) were palpated to confirm gestational age as predicted by timed mating. At E14, dams were anesthetized (Ketamine/Xylazine; 100/10 mixture, 0.1 mg/g, intraperitoneally) and abdominal incisions were made through the skin and muscle. The uterine horns were exposed, and injections were made as follows: Batch 1: Dyx1c1 RNAi plasmids (1.5 μg/ μL) + eGFP plasmids (0.5 μg/ μL) + Fast Green dye, Shams received injections of Fast Green dye; Batch 2: Dyx1c1 RNAi plasmids (1.5 μg/ μL) + Fast Green dye, Shams received mRFP plasmids (0.4 μg/ μL) + Fast Green dye. All solutions were microinjected into fetal ventricles by pressure (General Valve Picospritzer, Pine Brook, NJ, USA). This was performed directly through the uterine wall. One randomly chosen lateral ventricle (left or right) of each embryo was transfected, using a pulled glass capillary (Drummond Scientific, Broomall, PA, USA). Electroporation was achieved by discharge of a 500-mV capacitor charged to 50–100 mV. A pair of copper alloy plates (1 cm × .5 cm) pinching the head of each embryo was the conduit for the voltage pulse, and voltage current was discharged through both sides of the brain, transfecting cortex bilaterally. Since embryos were unable to be sexed at this age, and only males were used for testing, equal numbers of Sham and Dyx1c1 RNAi injections were made, at roughly double the numbers needed. (Transfected females were used in other studies). Note that only males were used in this study based on previous research that has demonstrated that male rodents exhibit more robust functional deficits than females following early disruption of neuronal migration (Fitch et al., 1997; Peiffer et al., 2002b; 2004b). Additionally, there is a higher incidence of dyslexia in males than in females (Flannery et al., 2000; Katusic et al., 2002; Rutter et al., 2004).

Sixty-one male subjects were weaned on postnatal day 21 (P21), and received right or left ear marking. Treatment could not be identified at weaning; therefore subjects were housed in same-sex littermate pairs. (Treatment was later identified post-mortem via fluorescence of GFP in Batch 1, and via fluorescence of RFP in Batch 2). Note that numbers of Sham and RNAi treated subjects was expected to be roughly equal, since equal numbers of Sham and RNAi injections were made, and only males were used. Histology later confirmed Dyx1c1 treatment (n=33) and Sham (n=28), for a total of 61.

Apparatus

The radial arm water maze was housed in a black Plexiglas pool (140 cm in diameter, 40 cm deep) filled with cool water (22 (+/− 2) °C). The maze consisted of eight removable stainless steel arms painted flat black that could be attached to a central octagonal hub (50 cm across). Each corridor was 14 cm wide and extended 36 cm to the edge of the pool (see Figure 1). A removable black plastic platform (10 cm diameter) served as the escape platform, and was submerged 4 cm beneath the surface of the water. The entire apparatus was in a large room with two empty walls, a long table, and the cage rack forming the boundaries around the maze. During testing, light was provided by a desk lamp in the northeast corner of the room, for additional spatial cues.

Figure 1.

Figure 1

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Diagram of the delayed match-to-sample radial water maze task, including sample (left) and test (right) trials. Exemplars for the first and second days of testing are given. S = start arm (location changes between sample (S1) and test trial (S2)), G = goal arm (location remains fixed). Each day of testing rats were given one forced-choice sample trial in which all arms were blocked except for the start arm and goal arm. A plastic escape platform was submerged at the end of the goal arm. During the test trial (10 minutes later), all of the arms were open and a new start arm (S2) was used, to test the animal’s memory of the spatial location of the goal arm. Each animal received one sample trial and one test trial each day of testing. The sequences of start arms and goal locations used each day varied systematically among forty-eight patterns that regulated the sequence of start and goal arms, the turn angles, and the relationship between the start and goal arms across trials.

Delayed match-to-sample testing

Beginning on P33, subjects were handled five minutes a day for the week prior to testing. On the initial testing day (P40), subjects had no prior exposure to the testing room, the water maze, or the platform. All rats were assessed on an initial acclimation trial, and were found to be capable of navigating the maze and mounting the platform. Subsequent testing of animals consisted of four daily sessions per week (four sessions per week, one session per day, one sample and one test trial per session). Each session consisted of a forced-choice sample trial and a test trial in which all arms were open (see Chrobak et al., 2008 for additional details). During the sample trial, all corridors were blocked at the intersection of the arm and central hub, except for the start and goal arms. Each rat swam out of the start arm, navigated to the only open corridor (the goal arm), and mounted the escape platform. The subject was removed immediately after mounting the platform, gently dried with a towel, and returned to the home cage. Subjects took approximately 4–20 seconds to complete the sample trial. This study employed a 10 minute delay, so the test trial was administered 10 minutes after the sample. A new start arm was used during the test trial, but the goal location remained the same. (The start arm was changed to insure navigation based on spatial memory rather than memory of turn angle). During this test trial, all maze arms were open. Subjects were tested once a day each day of a 5 day work week, over a period of 12 weeks, using a different start and goal arm each day. Sequences of start arms and goal locations were varied systematically among forty-eight patterns. This regulated the sequence of the start and goal arms, and the relationship between them, across trials. The goal location was restricted to arms 90 degrees (2 arms) or more away from the Prior (i.e. yesterday’s) goal location.

At the end of testing, all subjects were transcardially perfused for assessment of brain tissue and analysis of experimental treatment (Sham or Dyx1c1 RNAi), such that behavioral data could be analyzed as a function of Treatment. Importantly, before post mortem analyses, all behavioral assessments were performed blind to Treatment.

Control Trials

To assess the possibility that subjects might use intramaze cues (i.e., visual, olfactory, or somatosensory cues) to find the platform, we examined performance on periodic “control” trials. During these trials, no forced-choice sample trial was given, and rats had to seek and find the platform in a random new location. These trials provided a measure of “chance” performance, and consistently revealed a range of mean errors at 4.4–4.7 errors per day for both groups for all control sample trials (see Fig. 3). There were no differences between the Control Trial performance of Dyx1c1 RNAi and Sham animals.

Figure 3.

Figure 3

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Mean errors made by Sham and Dyx1c1 RNAi subjects over six 8-trial blocks (12 weeks/48 days) of testing. An overall Treatment effect on mean errors (P < .01) with no Treatment × Block interaction indicated that rats that received Dyx1c1 RNAi made significantly more errors than Sham treated rats. The shaded grey bar represents the span of average errors made by Sham and Dyx1c1 RNAi animals (4.4–4.7 errors) on the weekly Control Trials which measured chance performance on the task.

Dependent Measures

Dependent measures assessed included the number of incorrect arm entries (Errors) during the test trial, mean Latency per arm choice during the sample and test trials (total latency to reach the platform divided by the number of arms entered during the trial), and the type of the First Error made during the test trial (when an error was made). First Error types were divided into three groups: Prior Goal errors (in which the subject’s first entry was into the prior day’s goal arm); Adjacent Arm errors (in which the subject’s first entry was into one of the two arms adjacent to the goal arm); and Other errors (which describes random entry into an arm that was not the Prior Goal nor an adjacent arm).

Histological Analysis

Upon the completion of testing, subjects were weighed, deeply anesthetized with ketamine/xylazine (100 mg/kg/15 mg/kg), and transcardially perfused with phosphate buffered saline followed by chilled 4% paraformaldehyde. Heads were removed and brains were extracted, bottled in paraformaldehyde, and shipped to GDR at Beth Israel Deaconess Medical Center for histological preparation. Brains were placed into 30% sucrose buffer prior to being cut in the coronal plane at 40um section thickness. A 1-in-10 series of sections were mounted and stained with Thionin for Nissl substance, while an adjacent series of free-floating sections were mounted and screened using fluorescence microscopy for the presence of GFP or RFP. Another series of sections was immunohistochemically processed for visualization of RFP or GFP (Chemicon, 1:200) using ABC protocols. Light microscopic analysis was used to visualize the disposition of transfected cells, and to identify dysplasia in RNAi treated and Sham subjects.

Data Analyses

Multivariate analyses of variance (ANOVA) were used to analyze both error and latency data for order and trend. The pattern of error types was analyzed using a Chi-square analysis on the frequency distribution of Prior, Adjacent, and Other errors. All reported p-values are two-tailed. All statistical analyses were conducted using SPSS or Microsoft Excel.

RESULTS

Histology

Fluorescence microscopy and immunohistochemical staining were used to confirm the presence or absence of GFP and/or RFP. Analysis revealed 33 experimental (RFP-negative and/or GFP-positive) and 28 control (RFP-positive and/or GFP-negative) subjects. Further histological examination revealed five categories of cortical characterization: (1) no visible malformations (RNAi treated n=18, Sham n = 23), which describes any subject whose brain tissue was free of the gross malformations defined in the other categories; (2) injection site ectopia (RNAi treated n = 10, Sham n = 5), resulting from the injection puncture wound, forming an ectopic collection of cells in Layer 1; (3) non-injection site ectopia (RNAi treated n = 2, Sham n = 0; an ectopic collection of neurons in areas of Layer 1 that were distal to the injection site); (4) unmigrated neurons (RNAi treated n = 12, Sham n = 0), in which collections of neurons failed to migrate to their target layers and instead formed heterotopic pockets in the white matter near the border of the ventricular zone; and (5) hippocampal dysplasia (RNAi treated n = 5, Sham n = 0), or unmigrated neocortical neurons that primarily disrupted the dentate gyrus (see Figure 2). The various malformations varied in size and number, and some subjects had multiple types of disruption. It is also worth noting that, other than focal injection site ectopia (n=5), Sham animals did not display any cortical disruption. Note that in addition to being analyzed as a function of Treatment (Dyx1c1 RNAi vs. Sham); all behavioral results were analyzed as a function of these histological subgroups.

Figure 2.

Figure 2

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Histology of Dyx1c1 RNAi subjects. Histology revealed four categories of gross cortical disruption: (A) Injection site ectopia (RNAi treated n = 10, Sham n = 5), resulting from the injection puncture wound, forming an ectopic collection of cells in Layer 1 (black arrowheads) and a characteristic cell-free streak in the subjacent layers (black arrow). Scale bar = 150 μm. (B) Non-injection site ectopia (RNAi treated n = 2, Sham n = 0), characterized by an ectopic collection of neurons in areas of Layer 1 that were distal to the injection site (black arrowheads). (Note the absence of the cell-free streak associated with the injection site). Scale bar = 150 μm. (C) Unmigrated neurons (RNAi treated n = 12, Sham n = 0), which describes collections of neurons that failed to migrate to their target layers and instead formed heterotopic pockets in the white matter, near the border of the ventricular zone (black arrowheads). Scale bar = 500 μm. (D) Hippocampal dysplasia (RNAi treated n = 5, Sham n = 0), which primarily affects the dentate gyrus (black arrowheads). Scale bar = 500 μm.

Errors to find the platform (Test Trials)

Analyses of overall errors revealed an overall significant effect of Treatment, with Dyx1c1 RNAi treated subjects (n=33) showing impaired acquisition and performance of the delayed match-to-sample radial water maze task as compared to Shams [F(1,59) = 7.826, P = .007] (See Figure 3). We also found a significant effect of Two Week Block (referring to two week blocks of testing) [F(1,59) > 20, P < .001], with improved performance (fewer errors) over testing. There was no Two Week Block × Treatment interaction [F(1,59) < 1, NS], indicating learning for both groups. Yet these data show that rats that received RNAi targeted against Dyx1c1 exhibited significant and sustained impairments across all twelve weeks of testing as compared to Shams.

Distribution of First Error Types

We divided the type of first errors made into three categories (for each test trial in which an error was made). Interestingly, in spite of the fact that the Dyx1c1 RNAi treated subjects made more errors than Shams throughout testing, the distribution of first error types for this group did not differ from Shams (see Figure 4). That is, the distribution of the types of first error during the last four week block of testing indicated no Treatment difference (χ2 = .93, df(2), NS). Recall that a Prior Goal error indicates proactive interference from the previous testing day, while an Adjacent Arm error indicates that there is a weakened representation of the current goal location. In fact, both Shams and Dyx1c1 RNAi treated rats showed greater than 50% of errors in the last block of testing being made to the prior goal or to an adjacent arm. This finding indicates that errors in all subjects, regardless of Treatment, were mainly due to proactive interference or weakened goal representation (rather than no memory of the goal at all, which was measured by “Other” errors). Thus, while the spatial working memory system was apparently impaired or weakened in the Dyx1c1 RNAi treated rats, they nonetheless performed the task using a similar strategy to Shams.

Figure 4.

Figure 4

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Distribution of error types for the first error made in the first and last 8-trial blocks of testing. We divided the first errors into three categories for each test trial in which an error was made (Prior, Adjacent, Other). A Chi-square on the distribution of the type of first error between the first two weeks and last two weeks of testing showed no Treatment effect. For each group, greater than 50% of the errors being made at the end of testing were either to the Prior goal or to an Adjacent arm, indicating that errors were mainly due to proactive interference or weakened goal representation, rather than no memory of the goal at all (which was measured by the percent of Other errors). Thus, while the spatial working memory system was apparently impaired in the Dyx1c1 RNAi treated rats, they performed the task in a similar manner (i.e., using a similar strategy), to Shams

Latencies to mount the platform

An ANOVA was used to analyze the average latency per arm choice during the sample and test trials (see Table 1). Results showed a significant Two Week Block effect [F(1,59) > 10, P = .001] indicating that rats swam and entered arms more rapidly over the 12 weeks of testing. There was not a significant Treatment effect [F(1,59) = 1.845, NS], nor Block × Treatment interaction [Fs (1,59) < 1, NS]. This finding shows that the Dyx1c1 RNAi treated rats performed the motor component of the task (i.e., swimming/entering arms) comparably to Shams.

Table 1.

Average latency per arm choice (in seconds) during the test trial

8 Trial Blocks (1 trial/day over 8 days)
8 16 24 32 40 48
Dyx1c1 RNAi (n=33) 7.4 (1.9) 6.2 (1.4) 6.5 (2.3) 6.4 (2.4) 5.8 (1.8) 5.6 (1.9)
Sham (n=28) 6.6 (1.9) 6.3 (1 .9) 6.0 (1.8) 5.9 (2.2) 5.2 (1.1) 4.8 (1.0)
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Means +/− SEM. All data are reported in seconds. Mean latencies as a function of the number of arms entered during the test trial. No significant differences.

Anatomical Subgroups

The subjects were divided into three groups to determine the effect of the anatomical phenotype on performance. An ANOVA was used to compare the average errors made by Shams (n=28), Dyx1c1 RNAi animals with No Visible Malformations (n=18), and Dyx1c1 RNAi animals with Gross Malformations (i.e., ectopias or heterotopias; all animals with gross malformations were pooled into one group because of the small number of animals within each individual anatomy subgroup) (n=15). A significant overall difference in the number of errors made among the three groups was detected [F(1,58) = 4.742, P =.012] (see Figure 5). There was a significant Block effect [F(1,58) = 52.903, P < .001] indicating significant improvement among all three groups over the 12 weeks of testing. There was not a significant Block × Group interaction [F(2,58) < 1, NS]. Interestingly, when compared separately, the Dyx1c1 RNAi animals with Gross Malformations did not differ significantly from Shams in the average number of errors made across 12 weeks of testing [F(1,44) = 2.476, P =.123]. However, the Dyx1c1 RNAi animals with No Visible Malformations did differ significantly from Shams, consistently making more errors across 12 weeks of testing [F(1,44) = 8.536, P =.005]. The Dyx1c1 animals with No Visible Malformations did not differ from Shams on any of the other behavioral measures.

Figure 5.

Figure 5

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Mean errors per day as a function of anatomical phenotype. A repeated measures ANOVA reveals a significant Group effect on mean errors (P = .012) among Shams (n=28), Dyx1c1 RNAi animals with No Visible Malformations (n=18), and Dyx1c1 RNAi animals with Gross Malformations (n=15). When compared separately, a repeated measures ANOVA revealed a significant difference between the Dyx1c1 RNAi animals with No Visible Malformations and Shams (P = .005), with the Dyx1c1 RNAi animals with No Visible Malformations consistently making more errors than Shams. There was not a significant difference in performance between the Dyx1c1 RNAi animals with Gross Malformations and Shams (P = .123). This result suggests that knockdown of Dyx1c1 may lead to formation of subtle malformations that consequently result in memory deficits.

DISCUSSION

Here we report that in utero disruption of Dyx1c1 is associated with impaired learning and memory on a delayed match-to-sample radial water maze task. Moreover, impairments were present even after twelve weeks of testing, indicating that the disruptions to higher order working memory induced by RNAi of Dyx1c1 were persistent.

These findings are complementary to a recent study by Marino et al. (2007) that revealed a link between memory performance and DYX1C1. Marino’s group studied associations between DYX1C1 and several dyslexic phenotypes in a large, family-based study of Italian dyslexics. Results of the study revealed a significant association between specific variants (single nucleotide polymorphisms, or SNPs) within the DYX1C1 gene, and performance on the Single Letter Backward Task (a measure of auditory short term memory). Dyslexic individuals who had a copy of the DYX1C1 gene that contained the variants of interest made significantly more errors on the task than those without the variants.

A recent study by Dahdouh et al. (2009) found an association between DYX1C1 variants and short term memory in female dyslexics. This finding is interesting in light of a recent report that the DYX1C1 protein interacts with alpha and beta estrogen receptors, which are present in the brain during development (Massinen et al., 2009). In the current study, only males were used for testing based on the higher incidence rates of dyslexia in males, as well as previous research which indicates that the functional deficits in male rodents are more robust than those in females following early disruption of neuronal migration (Fitch et al., 1997; Flannery et al., 2000; Katusic et al., 2002; Peiffer et al., 2002b; 2004b; Rutter et al., 2004). Given the emerging evidence of sex-specific effects of DYX1C1, future studies will assess the effects of early interference with candidate dyslexia susceptibility genes in female rodents in addition to males.

The current results are interesting in view of the fact that phonological deficits associated with developmental dyslexia consistently occur in parallel with verbal and non-verbal short term memory deficits (Smith-Spark and Fisk, 2007). For example, when tested on tasks such as word-list recall (which is thought to tap into phonological short term memory), dyslexic individuals show significant deficits as compared to age-matched controls (Baddeley and Hitch, 1974). Language-impaired individuals also show deficits in higher order processing of new and stored phonological information, which is required for sentence processing. These deficits are typically characterized as “verbal working memory deficits” (Gathercole et al., 2006, Archibald and Gathercole, 2006, Smith-Spark and Fisk, 2007).

Additionally, deficits in visuospatial short term memory have occasionally been reported in dyslexics. For example, dyslexic individuals show impairment on tasks such as the Corsi block span task, and the Visual Patterns Test - both of which test pattern sequence recall abilities (Gathercole et al., 2006; Smith-Spark and Fisk, 2007). Thus, a potential core deficit in central executive working memory systems in developmental dyslexia has recently been characterized (Smith-Spark and Fisk, 2007). Such a cross-modal processing deficit would account for the wide range of working memory impairments observed in developmental dyslexia.

Concomitant evidence from animal models suggests that rapid auditory processing deficits (as seen in cortically disrupted animals) are aggravated when difficulty and complexity of the task are increased (Peiffer et al., 2004a; Fitch et al., 2008). Data from these studies suggest evidence of higher order learning and memory deficits in cortically disrupted animals, prompting questions about a possible relationship between cortical dysgenesis and other cognitive deficits associated with developmental dyslexia (i.e. working memory; Smith-Spark and Fisk, 2007). In fact, deficits in working memory have been reliably replicated in the same animal models that showed difficulties in rapid auditory processing (Boehm et al., 1996, Waters et al., 1997, Hoplight et al., 2001, Threlkeld et al., 2007; Fitch et al., 2008). The fact that deficits in both rapid auditory processing (RAP), and also learning and memory, can be reliably elicited from rodent models employing different forms of cortical disruption (e.g., microgyria, ectopia, hypoxic-ischemic injury) suggests that cortical disruption during vulnerable periods early in development appears to cause robust long-term impairments across an array of processing modalities (Threlkeld et al., 2007; Fitch et al., 2008).

Higher order working memory disorders have previously been demonstrated in other models of early cortical disruption. For example, BXSB/MpJ mice — a strain in which 40–60% of mice exhibit spontaneously occurring molecular layer ectopias — have shown deficits in performance on a delayed match-to-sample Morris water maze, inverted Lashley III maze, and two versions of the Hebb-Williams maze (all of which emphasize working memory demands; Boehm et al., 1996; Waters et al., 1997; Hoplight et al., 2001). Our present findings are consistent with these reports, all of which suggest that early disruption of cortical development may lead to robust memory deficits.

It is worth noting, however, that when using the delayed match-to-sample Morris water maze, the inverted Lashley III maze, and the Hebb-Williams maze, performance of ectopic and non-ectopic BXSB/MpJ mice was found to converge rather quickly. That is, although the ectopic mice initially performed worse than their non-ectopic littermates, their performances converged within one to two weeks of testing (Boehm et al., 1996; Hoplight et al., 2001). This is in contrast to the current results, which demonstrate persistent working memory deficits even after 12 weeks of testing. Thus, the current results provide support for the use of the delayed match-to-sample radial arm water maze as a more demanding working memory task capable of eliciting sustained working memory deficits.

The neurophysiological underpinnings of the behavioral deficits induced by knockdown of Dyx1c1 remain unknown. The observation that roughly half of the Dyx1c1 RNAi transfected animals exhibit a lack of gross, visible malformations while the others exhibit large disruptions that can be observed with the naked eye is a paradox. Although there are not visible malformations in the brains of some of the Dyx1c1 treated animals, this does not imply normal cortex. The behavioral data suggest that there is some disruption of functional connectivity in the brains of the RNAi transfected animals lacking gross malformations (see Figure 5). This suggestion parallels overwhelming evidence of functional activation differences between dyslexic and typical samples during language processing tasks, even though anatomic anomalies are not always observed in these same dyslexic samples (Maisog et al., 2008; Beneventi et al., 2010; Wolf et al., 2010; also see Webster et al., 2008). Differential patterns of activation under fMRI indicate that subtle differences in circuitry or regional specialization are present, even though current methods of in vivo anatomic quantification cannot detect them. Previous studies have demonstrated that, in addition to creating gross malformations such as ectopias and heterotopias, knockdown of Dyx1c1 can lead to subtle disruption of neuronal migration, causing individual neurons to migrate to Layer 2 instead of reaching their appropriate target destination in Layer 3 (Wang et al., 2006; Rosen et al., 2007). Thus, it is possible that such subtle laminar disruption exists in the Dyx1c1 RNAi animals with no visible malformations, which could account for the significant impairment in this specific subgroup of animals. Future studies will further characterize the subtle disruption that exists in the Dyx1c1 RNAi brains at the microscopic and electrophysiological level.

Finally, hippocampal malformations were observed in five Dyx1c1 RNAi animals. In a previous study assessing the behavioral impacts of Dyx1c1 RNAi, hippocampal malformations were specifically linked to a robust spatial learning impairment (Threlkeld et al., 2007). In the current study effects seen for this sub-group were no more significant than those for the other Dyx1c1 RNAi animals without hippocampal malformations. This suggests that the radial arm maze taps into a circuit that is different than hippocampal-dependent spatial navigation abilities. However, the relative contribution of hippocampal anomalies to these behavioral effects cannot be discounted.

The results of the current study have significant implications for our understanding of the role of the DYX1C1 gene in the development of dyslexia. As previously mentioned, working memory deficits (both verbal and non-verbal) have been associated with dyslexia (Jeffries and Everatt, 2004; Smith-Spark and Fisk, 2007). Working memory is implicated in the storage of relevant representations that allow grapheme-to-phoneme conversion and phoneme assembly, both of which are necessary for reading. In addition to the previously mentioned study by Marino et al.(2007), studies by Wigg et al. (2004) and Smith et al. (2005) showed similar associations between working memory impairment and variants within the coding region of the DYX1C1 gene in dyslexic and language-impaired populations, using the Nonword Repetition task which measures phonological abilities. Thus, DYX1C1 variants are associated with both phonological and working memory deficits in dyslexic populations. Animal studies have provided a parallel to clinical work, and have similarly shown that Dyx1c1 knockdown is associated with both rapid auditory processing and working memory deficits (Threlkeld et al., 2007). The convergent data on neuronal migration abnormalities in dyslexics, the discovered role of Dyx1c1 in neuronal migration, and the apparent links between DYX1C1/Dyx1c1 and a range of dyslexic phenotypes in both human and animal studies (respectively), all provide support for a potential role of DYX1C1 in the etiology of dyslexia.

Acknowledgements

This work was supported by NIH Grant HD20806.

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