Evaluation of visual motion perception ability in mice with knockout of the dyslexia candidate susceptibility gene Dcdc2.

Developmental dyslexia is a heritable disability characterized by difficulties in learning to read and write. The neurobiological and genetic mechanisms underlying dyslexia remain poorly understood; however, several dyslexia candidate risk genes have been identified. One of these candidate risk genes-doublecortin domain containing 2 (DCDC2)-has been shown to play a role in neuronal migration and cilia function. At a behavioral level, variants of DCDC2 have been associated with impairments in phonological processing, working memory and reading speed. Additionally, a specific mutation in DCDC2 has been strongly linked to deficits in motion perception-a skill subserving reading abilities. To further explore the relationship between DCDC2 and dyslexia, a genetic knockout (KO) of the rodent homolog of DCDC2 (Dcdc2) was created. Initial studies showed that Dcdc2 KOs display deficits in auditory processing and working memory. The current study was designed to evaluate the association between DCDC2 and motion perception, as these skills have not yet been assessed in the Dcdc2 KO mouse model. We developed a novel motion perception task, utilizing touchscreen technology and operant conditioning. Dcdc2 KOs displayed deficits on the Pairwise Discrimination task specifically as motion was added to visual stimuli. Following behavioral assessment, brains were histologically prepared for neuroanatomical analysis of the lateral geniculate nucleus (LGN). The cumulative distribution showed that Dcdc2 KOs exhibited more small neurons and fewer larger neurons in the LGN. Results compliment findings that DCDC2 genetic alteration results in anomalies in visual motion pathways in a subpopulation of dyslexic patients.

Mutation of Dcdc2 in mice leads to impairments in auditory processing and memory ability.

Dyslexia is a complex neurodevelopmental disorder characterized by impaired reading ability despite normal intellect, and is associated with specific difficulties in phonological and rapid auditory processing (RAP), visual attention and working memory. Genetic variants in Doublecortin domain-containing protein 2 (DCDC2) have been associated with dyslexia, impairments in phonological processing and in short-term/working memory. The purpose of this study was to determine whether sensory and behavioral impairments can result directly from mutation of the Dcdc2 gene in mice. Several behavioral tasks, including a modified pre-pulse inhibition paradigm (to examine auditory processing), a 4/8 radial arm maze (to assess/dissociate working vs. reference memory) and rotarod (to examine sensorimotor ability and motor learning), were used to assess the effects of Dcdc2 mutation. Behavioral results revealed deficits in RAP, working memory and reference memory in Dcdc2(del2/del2) mice when compared with matched wild types. Current findings parallel clinical research linking genetic variants of DCDC2 with specific impairments of phonological processing and memory ability.

Knockdown of the dyslexia-associated gene Kiaa0319 impairs temporal responses to speech stimuli in rat primary auditory cortex.

One in 15 school age children have dyslexia, which is characterized by phoneme-processing problems and difficulty learning to read. Dyslexia is associated with mutations in the gene KIAA0319. It is not known whether reduced expression of KIAA0319 can degrade the brain's ability to process phonemes. In the current study, we used RNA interference (RNAi) to reduce expression of Kiaa0319 (the rat homolog of the human gene KIAA0319) and evaluate the effect in a rat model of phoneme discrimination. Speech discrimination thresholds in normal rats are nearly identical to human thresholds. We recorded multiunit neural responses to isolated speech sounds in primary auditory cortex (A1) of rats that received in utero RNAi of Kiaa0319. Reduced expression of Kiaa0319 increased the trial-by-trial variability of speech responses and reduced the neural discrimination ability of speech sounds. Intracellular recordings from affected neurons revealed that reduced expression of Kiaa0319 increased neural excitability and input resistance. These results provide the first evidence that decreased expression of the dyslexia-associated gene Kiaa0319 can alter cortical responses and impair phoneme processing in auditory cortex.

Mutation of the dyslexia-associated gene Dcdc2 impairs LTM and visuo-spatial performance in mice.

Developmental reading disorder (RD) affects 5-10% of school aged children, with a heritability of approximately 60%. Genetic association studies have identified several candidate RD susceptibility genes, including DCDC2; however, a direct connection between the function of these genes and cognitive or learning impairments remains unclear. Variants in DCDC2, a member of the doublecortin family of genes, have been associated in humans with RD and ADHD and Dcdc2 may play a role in neuronal migration in rats. In this study, we examined the effect of Dcdc2 mutation on cognitive abilities in mice using a visual attention and visuo-spatial learning and memory task. We show that both heterozygous and homozygous mutations of Dcdc2 result in persistent visuo-spatial memory deficits, as well as visual discrimination and long-term memory deficits. These behavioral deficits occur in the absence of neuronal migration disruption in the mutant mice, and may be comorbid with an anxiety phenotype. These are the first results to suggest a direct relationship between induced mutation in Dcdc2 and changes in behavioral measures. Dcdc2 mutant mice should prove useful in future studies designed to further dissect the underlying neural mechanisms that are impaired following Dcdc2 mutation.

Dcdc2 knockout mice display exacerbated developmental disruptions following knockdown of doublecortin.

The dyslexia-associated gene DCDC2 is a member of the DCX family of genes known to play roles in neurogenesis, neuronal migration, and differentiation. Here we report the first phenotypic analysis of a Dcdc2 knockout mouse. Comparisons between Dcdc2 knockout mice and wild-type (wt) littermates revealed no significant differences in neuronal migration, neocortical lamination, neuronal cilliogenesis or dendritic differentiation. Considering previous studies showing genetic interactions and potential functional redundancy among members of the DCX family, we tested whether decreasing Dcx expression by RNAi would differentially impair neurodevelopment in Dcdc2 knockouts and wild-type mice. Consistent with this hypothesis, we found that deficits in neuronal migration, and dendritic growth caused by RNAi of Dcx were more severe in Dcdc2 knockouts than in wild-type mice with the same transfection. These results indicate that Dcdc2 is not required for neurogenesis, neuronal migration or differentiation in mice, but may have partial functional redundancy with Dcx.

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

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 (RNAi) vector 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 the 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 the evaluation of memory for a single brief (∼4-10 seconds) swim to a new goal location each day. A 10-min 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.

DYX1C1 functions in neuronal migration in developing neocortex.

Rodent homologues of two candidate dyslexia susceptibility genes, Kiaa0319 and Dcdc2, have been shown to play roles in neuronal migration in developing cerebral neocortex. This functional role is consistent with the hypothesis that dyslexia susceptibility is increased by interference with normal neural development. In this study we report that in utero RNA interference against the rat homolog of another candidate dyslexia susceptibility gene, DYX1C1, disrupts neuronal migration in developing neocortex. The disruption of migration can be rescued by concurrent overexpression of DYX1C1, indicating that the impairment is not due to off-target effects. Transfection of C- and N-terminal truncations of DYX1C1 shows that the C-terminal TPR domains determine DYX1C1 intracellular localization to cytoplasm and nucleus. RNAi rescue experiments using truncated versions of DYX1C1 further indicate that the C-terminus of DYX1C1 is necessary and sufficient to DYX1C1's function in migration. In conclusion, DYX1C1, similar to two other candidate dyslexia susceptibility genes, functions in neuronal migration in rat neocortex.

Impaired detection of variable duration embedded tones in ectopic NZB/BINJ mice.

Utilizing rodent models, prior research has demonstrated a significant association between focal neocortical malformations (i.e. induced microgyria, molecular layer ectopias), which are histologically similar to those observed in human dyslexic brains, and rate-specific auditory processing deficits as seen in language impaired populations. In the current study, we found that ectopic NZB/BINJ mice exhibit significant impairments in detecting a variable duration 5.6 kHz tone embedded in a 10.5 kHz continuous background, using both acoustic reflex modification and auditory event-related potentials (AERP). The current results add further support to the association between focal cortical malformations and impaired auditory processing, and the notion that these auditory effects may occur regardless of the cortical location of the anomaly.

Altered interneuron development in the cerebral cortex of the flathead mutant.

One approach to defining mechanisms essential to neocortical development is to analyze the phenotype of novel spontaneous mutations that dramatically affect the generation and differentiation of different neocortical neurons. Previously we have shown that there is a large decrease in the total number of cortical neurons in the flathead mutant rat, and in this paper we show that the flathead (fh/fh) mutation causes an even larger decrease in the number of interneurons. The decrease in relative interneuron number is different in different cortical lamina and for different interneuron subtypes. Specifically, the percentage of GABA and calretinin- positive cells in upper layers of somatosensory cortex is not appreciably decreased in homozygous mutants, while other interneuron subtypes in somatosensory cortex and all GABA-positive interneuron types in entorhinal cortex are decreased. In addition, the soma and dendritic arbors of interneurons in flathead are greatly hypertrophied, while those of pyramidal neurons are not. Furthermore, we found that at embryonic day 14, flathead mutants display high levels of cell death throughout both the neocortical and ganglionic eminence (GE) proliferative zones with a larger increase in cell death in the GE than in the neocortical VZ. In addition, we provide evidence that there is widespread cytokinesis failure resulting in binucleate pyramidal cells and interneurons, and the number of binucleate interneurons is greater than the number of binucleate pyramidal neurons. Together, these results demonstrate that the fh mutation causes dramatic alterations in interneuron development, and suggest that the flathead mutation causes differential cytokinesis failure and cell death in different types of neocortical progenitors.

Electrophysiological and morphological characterization of neurons within neocortical ectopias.

Focal developmental abnormalities in neocortex, including ectopic collections of neurons in layer I (ectopias), have been associated with behavioral and neurological deficits. In this study, we used infrared differential interference contrast microscopy and whole cell patch-clamp to complete the first characterization of neurons within and surrounding neocortical ectopias. Current-clamp recordings revealed that neurons within ectopias display multiple types of action potential firing patterns, and biocytin labeling indicated that approximately 20% of the cells in neocortical ectopias can be classified as nonpyramidal cells and the rest as atypically oriented pyramidal cells. All cells had spontaneous excitatory (glutamatergic) and inhibitory (GABAergic) postsynaptic currents. Exhibitory postsynaptic currents consisted of both N-methyl-D-aspartate (NMDA) receptor-mediated and AMPA/kainate (A/K) receptor-mediated currents. The NMDA receptor-mediated component had decay time constants of 15.35 +/- 2.2 (SE) ms, while the A/K component had faster decay kinetics of 7.6 +/- 1.7 ms at -20 mV. GABA(A) receptor-mediated synaptic currents in ectopic cells reversed at potentials near the Cl- equilibrium potential and had decay kinetics of 16.65 +/- 1.3 ms at 0 mV. Furthermore we show that cells within ectopias receive direct excitatory and inhibitory input from adjacent normatopic cortex and can display a form of epileptiform activity.

In vitro biocytin injection into perinatal mouse brain: a method for tract tracing in developing tissue.

Injection of biocytin provides an effective method for labeling axonal projections. Several difficulties arise when this technique is employed in fetal or early postnatal animals in vivo, including limited access to injection sites and extended post-injection survival periods. To circumvent these problems, we adapted the technique of extracellular biocytin injection for use in explanted brain hemispheres of developing mice. Briefly, entire brain hemispheres from perinatal mice (E16-P9) were removed and placed in oxygenated aCSF in a brain slice recording chamber. Following visually guided injection of biocytin (2%) into the prelimbic cortex, the brains were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) for varying periods of time and then immersion-fixed in 4% paraformaldehyde and 0.5% glutaraldehyde. The next day, the brains were sectioned and processed for biocytin histochemistry using the avidin-biotin-complex method. We examined the method of injection, electrode type, time of injection, and post-injection incubation period. We found that in E16-P9 animals iontophoresis of biocytin using 8- to 12-megaohm patch clamp electrodes for a duration of 10 min provides optimal axonal labeling. Post-injection incubation times of four or more hours are sufficient for labeling fine caliber collaterals as well as axon bundles that reach distances over 3 mm. In vitro injection of biocytin into explanted brain hemispheres provides a quick and easy method for tract tracing in developing brains.

The flathead mutation causes CNS-specific developmental abnormalities and apoptosis.

We describe a new mutation, flathead (fh), that arose spontaneously in an inbred colony of Wistar rats. The mutation is autosomal recessive, and the behavioral phenotype of fh/fh rats includes spontaneous seizures, tremor, impaired coordination, and premature death. A striking feature of the fh mutation is a dramatic reduction in brain size (40% of normal at birth). In contrast, no abnormalities are evident in the peripheral nervous system or in other tissues outside of the CNS. Although bromodeoxyuridine incorporation assays indicate that the rate of cell proliferation in the fh/fh cortex is similar to that of unaffected animals, in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling assays reveal a dramatic increase in apoptotic cell death beginning after embryonic day 16 (E16). At E18 there is a 20-fold increase in cell death in the ventricular zone of fh/fh neocortex, and at postnatal day 1 (P1), the number of apoptotic cells is still two times that of normal. However, by P8 the extent of cell death in fh/fh is comparable to that of unaffected littermates, indicating that the reduction in brain growth is caused by abnormally high apoptosis during a discrete developmental period. Late-developing structures such as the cerebellum, neocortex, hippocampus, and retina are most severely affected by the fh mutation. Within these structures, later-generated neuronal populations are selectively depleted. Together, these results suggest that the flathead gene is essential for a developmental event required for the generation and maturation of late-born cell populations in the brain.

Neocortical ectopias are associated with attenuated neurophysiological responses to rapidly changing auditory stimuli.

Developmental dyslexia has been separately associated with the presence of ectopic collections of neurons in layer I of neocortex (ectopias) and with alterations in processing rapidly changing stimuli. We have used BXSB/MpJ-Yaa mice, some of which have neocortical ectopias, to directly test the hypothesis that ectopias may alter auditory processing. Auditory event related potentials (AERPs) were elicited by pairs of 10.5 kHz tones separated by silence, 0.99 kHz, or 5.6 kHz tones of variable duration. Half of the mice tested had 1-3 ectopias in frontal or parietal cortex, and half had no ectopias. Mice with ectopias showed a reduced response to the second 10.5 kHz stimuli only when it was preceded by short duration 5.6 kHz tones. These results indicate that BXSB mice are an excellent model for determining how focal neocortical anomalies alter sensory processing.

Noggin is a negative regulator of neuronal differentiation in developing neocortex.

Bone morphogenetic proteins (BMPs) trigger neuronal differentiation of neocortical precursors within the ventricular zone (VZ) [Li et al. (1998): J Neurosci 18:8853-8862]. BMP-2/4 protein is concentrated at the VZ surface and BMPs rapidly promote the differentiation of neocortical precursors in both dissociated cell and explant cultures. Noggin binds to BMP-2/4 with high affinity, and prevents binding to cell surface receptors. In the present study, we used human recombinant noggin protein to determine whether endogenous BMP-2/4 triggers neuronal differentiation in dissociated cell culture. We find that noggin inhibits the differentiation of neocortical neurons: noggin decreases the number of MAP-2- and TUJ1-positive cells after 24 h of treatment, yet has no effect on either proliferation or cell survival. Noggin also significantly decreases neurite growth of MAP-2-positive cells. In addition, using Western blot analysis we show that noggin protein is present in developing cortex at E15. These results are consistent with previous results showing that endogenous BMPs trigger neuronal differentiation in the neocortical VZ and also indicate that a balance of noggin and BMP may regulate the differentiation of neocortical neurons in vivo.

Characterization of seizures in the flathead rat: a new genetic model of epilepsy in early postnatal development.

PURPOSE: Disorders in normal central nervous system (CNS) development are often associated with epilepsy. This report characterizes seizures in a novel genetic model of developmental epilepsy, the Flathead (FH) rat. METHODS: Animals (n = 76) ages P0-22 were monitored for clinical and electrographic seizure activity. The effects of various AEDs on seizure frequency and duration also were assessed: phenobarbital (PB; 40 mg/kg), valproate (VPA; 400 mg/kg), or ethosuximide (ESM; 600 mg/kg). RESULTS: FHs display episodes of behavior characterized by whole-body tremor, strub tail, alternating forelimb clonus, and complete tonus. EEG recordings from neocortex reveal that FH seizures are bilateral and begin around P7. Seizures occur at a frequency of approximately six per hour from P7 to P18 and the average duration of seizures increases through development. PB, VPA, and ESM failed to prevent seizures; however, PB significantly increased the interval of seizures but had no effects on the duration of seizures, whereas VPA decreased the duration of seizures and not the interval. CONCLUSIONS: Seizures in FH rats occur at a constant and high frequency through a defined period in early postnatal development, and these seizures are not completely blocked by high doses of PB, VPA, or ESM. Because FH is a single-locus mutant displaying a highly regular pattern of seizure activity, it is an ideal model for examining the process of epileptogenesis in the developing brain, evaluating new AED therapies, and determining the identity of a gene essential to the normal development of cortical excitability.

Differential regulation of connexin 26 and 43 in murine neocortical precursors.

Proliferating cells of the developing murine neocortex couple together into clusters during neurogenesis. Previously, we have shown that these clusters contain neural precursors in all phases of the cell cycle except M phase, and that they extend a nestin-expressing process from the cluster to the pial surface. In addition, coupling within neocortical cell clusters is a dynamic process related to the cell cycle, with maximal coupling in S/G2 phase, uncoupling in M phase and then recoupling during G1 and S phases of the cell cycle. In the present study, we use immunohistochemistry to demonstrate that cycling neocortical cells as well as radial glial cells express the gap junction proteins connexin 26 and connexin 43. Furthermore, we demonstrate that biocytin labeled clusters extend processes to the pial surface that express the glial cell antigen RC2. Lastly, by combining bromodeoxyuridine and connexin immunohistochemistry on acutely dissociated neocortical cells, we show that the percentage of cycling cells immunoreactive to connexin 26 and connexin 43 changes through the cell cycle. These results indicate that radial glial cells as well as neural precursors couple into clusters, and suggest that through differential regulation of connexins, neocortical precursors may compartmentalize as they progress through the cell cycle.

Neuronal differentiation of precursors in the neocortical ventricular zone is triggered by BMP.

Neocortical neurons begin to differentiate soon after they are generated by mitoses at the surface of the ventricular zone (VZ). We provide evidence here that bone morphogenetic protein (BMP) triggers neuronal differentiation of neocortical precursors within the VZ. In cultures of dissociated neocortical neuroepithelial cells, BMPs increase the number of MAP-2- and TUJ1-positive cells within 24 hr of treatment. In explant cultures, BMP-4 treatment leads to an increase in the number of TUJ1-positive cells within the ventricular zone. Furthermore, truncated, dominant-negative, BMP type I receptor, introduced into neocortical precursors by retrovirus-mediated gene transfer, blocks neurite elaboration and migration out of the VZ. Finally, immunocytochemistry indicates that BMP protein is present at the VZ surface. Together, these results indicate that BMP protein is present within the VZ, that BMP is capable of promoting neuronal differentiation, and that signaling through BMP receptors triggers neuronal precursors to differentiate and migrate out of the VZ.

Early patterning of prelimbic cortical axons to the striatal patch compartment in the neonatal mouse.

The striatum receives excitatory input from virtually the entire cerebral cortex. In the adult, this input is segregated into two functionally distinct compartments of the striatum, the patch (striosome) and matrix regions. This study determined whether the patterning of corticostriatal afferents from the prelimbic cortex to the striatal patch compartment develops during the early period of collateral formation or instead at the time of peak synaptogenesis. Initial formation of corticostriatal axon collaterals was observed by embryonic day (E) 19. Quantification of corticostriatal collaterals revealed a significant increase in the number and complexity of collateral branches at postnatal day 6 as compared to E19. Concomitant with the increase in collateral branching, a heterogeneous pattern of collateralization consisting of parallel rows of corticostriatal collaterals was observed in the medial striatum. In addition to the rows, clusters of corticostriatal axons occurred more laterally. These clusters colocalized with patches of dense tyrosine hydroxylase-positive fibers, a marker for the striatal patch compartment in the neonatal mouse. Together, these data indicate that corticostriatal patterning occurs during the period of early axon collateralization resulting in a segregation of corticostriatal axon collaterals from the prelimbic cortex to the striatal patch compartment.

A gene essential to brain growth and development maps to the distal arm of rat chromosome 12.

A recently discovered, spontaneous, autosomal recessive mutation in rats, flathead (fh), results in greatly reduced brain growth beginning in late fetal development. In this study we have mapped the fh mutation by determining the pattern of segregation of polymorphic microsatellite markers with respect to fh in 51 affected F2 offspring from a single interstrain intercross. Two markers on chromosome 12, D12Rat80 and D12Mgh6, cosegregated with the fh mutation in all 51 affected animals. The distribution of six additional markers in 40 informative meioses further localizes fh approximately 2 cM teleomeric to nos1. There are no known mutations in homologous regions of either mouse or human genomes that result in deficits in late neurodevelopment similar to that observed in fh/fh animals. The unique phenotype of fh/fh animals and the location of fh suggests the presence of a novel gene essential to normal brain development on the distal end of rat chromosome 12.