No abstract

We investigated the postnatal effects of embryonic knockdown and overexpression of the candidate dyslexia gene homolog Kiaa0319. We used in utero electroporation to transfect cells in E15/16 rat neocortical ventricular zone with either 1) small hairpin RNA (shRNA) vectors targeting Kiaa0319, 2) a KIAA0319 expression construct, 3) Kiaa0319 shRNA along with KIAA0319 expression construct ("rescue"), or 4) a scrambled version of Kiaa0319 shRNA. Knockdown, but not overexpression, of Kiaa0319 resulted in periventricular heterotopias that contained large numbers of both transfected and non-transfected neurons. This suggested that Kiaa0319 shRNA disrupts neuronal migration by cell autonomous as well as non-cell autonomous mechanisms. Of the Kiaa0319 shRNA-transfected neurons that migrated into the cortical plate, most migrated to their appropriate lamina. In contrast, neurons transfected with the KIAA0319 expression vector attained laminar positions subjacent to their expected positions. Neurons transfected with Kiaa0319 shRNA exhibited apical, but not basal, dendrite hypertrophy, which was rescued by overexpression of KIAA0319. The results provide additional supportive evidence linking candidate dyslexia susceptibility genes to migrational disturbances during brain development, and extends the role of Kiaa0319 to include growth and differentiation of dendrites.

1. The afterhyperpolarization (AHP) that follows action potentials was studied in CA1 hippocampal pyramidal cells from classically conditioned and control rabbits. Measurements of the AHP were obtained with intracellular recordings from CA1 cells within hippocampal slices. 2. The AHP of rabbit CA1 pyramidal cells was found to be accompanied by a conductance increase. The AHP was reduced by bath applications of the calcium channel blockers, cadmium and cobalt, by bath application of the cholinergic agonist, carbamylcholine chloride, and intracellular injection of the calcium chelator, ethylene glycol-bis(B-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). 3. The AHP was markedly reduced in cells from rabbits that were well-trained with the nictitating membrane conditioning procedure, as compared with cells from pseudoconditioned or naive control animals. The difference in AHP amplitudes between conditioned and control groups increased as the number of spikes elicited by the stimulation pulse increased from one to four. Both the duration (measured as the time constant of AHP decay) and amplitude of the AHP were reduced in cells from conditioned animals. 4. The reduced AHP in cells from conditioned animals remained reduced in a medium that contained 0.5 microM tetrodotoxin (TTX) and 5.0 mM tetraethylammonium chloride (TEA); the AHP following calcium spikes was measured under these conditions. Since this medium eliminated synaptic transmission elicited by Schaeffer collateral stimulation, the AHP reduction in pyramidal cells from conditioned animals was not due to a modification in synaptic properties. There were no significant differences in the mean voltage thresholds, amplitudes, or durations of calcium spikes between cells from animals in the three groups. Thus the AHP reduction appears to be due to a modification of a Ca2+ -dependent K+ conductance and was not due to a secondary effect of reductions in calcium conductances underlying the spike. 5. In medium containing TTX and TEA, the amount of injected current required to elicit a calcium spike (current threshold) was significantly greater in cells from conditioned animals than in cells from control animals. This increase in current threshold persisted in 4-aminopyridine (4-AP)-containing medium and so cannot be attributed entirely to conditioning-specific increases in the A-current. 6. The conditioning-specific AHP reduction resulted in increased excitability in cells from conditioned animals versus pseudoconditioned control animals. Cells from conditioned animals fired more spikes to trains of 100-ms depolarizing current pulses than did cells from controls.

Embryonic knockdown of candidate dyslexia susceptibility gene (CDSG) homologs in cerebral cortical progenitor cells in the rat results in acute disturbances of neocortical migration. In the current report we investigated the effects of embryonic knockdown and overexpression of the homolog of DCDC2, one of the CDSGs, on the postnatal organization of the cerebral cortex. Using a within-litter design, we transfected cells in rat embryo neocortical ventricular zone around embryonic day (E) 15 with either 1) small hairpin RNA (shRNA) vectors targeting Dcdc2, 2) a DCDC2 overexpression construct, 3) Dcdc2 shRNA along with DCDC2 overexpression construct, 4) an overexpression construct composed of the C terminal domain of DCDC2, or 5) an overexpression construct composed of the DCX terminal domain of DCDC2. RNAi of Dcdc2 resulted in pockets of heterotopic neurons in the periventricular region. Approximately 25% of the transfected brains had hippocampal pyramidal cell migration anomalies. Dcdc2 shRNA-transfected neurons migrated in a bimodal pattern, with approximately 7% of the neurons migrating a short distance from the ventricular zone, and another 30% migrating past their expected lamina. Rats transfected with Dcdc2 shRNA along with the DCDC2 overexpression construct rescued the periventricular heterotopia phenotype, but did not affect the percentage of transfected neurons that migrate past their expected laminar location. There were no malformations associated with any of the overexpression constructs, nor was there a significant laminar disruption of migration. These results support the claim that knockdown of Dcdc2 expression results in neuronal migration disorders similar to those seen in the brains of dyslexics.