Figure 3.

Increased expression of GluN2A and GluN2C proteins in the synapses of Hipk2^−/− mouse brain. A–C, Western blots using the total cell lysates, cytosolic fraction, and the synaptosomes from 2-month-old Hipk2^+/+ and Hipk2^−/− mouse brain showed that GluN2A and GluN2C protein levels were increased in the synaptosomes of cerebral cortex in Hipk2^−/− mice, whereas GluN2B protein level was decreased, leading to a significant increase in the GluN2A/GluN2B ratio in the cerebral cortex in Hipk2^−/− mice (Figure 3-1. The relative signal intensity of GluN2A, GluN2B, and GluN2C protein bands was quantified with National Institutes of Health ImageJ software. D–I, Confocal microscopic images of the primary cortical neuron cultures showed that the number of GluN2A⁺ puncta in the dendrites of Hipk2^−/− neurons were 31.2% more, whereas the GluN2B⁺ puncta were 40% less, abundant than that in the dendrites of Hipk2^+/+ neurons. Interestingly, the percentage of GluN2A⁺;SPH⁺ synapse increased to 60.1%, whereas the percentage of GluN2B⁺;SPH⁺ synapse reduced to 10.3% on the dendrites of Hipk2^−/− neurons. The GluN2A⁺ or GluN2B⁺ puncta were quantified with National Institutes of Health ImageJ online software. J–O, Immunogold electron microscopy performed in the sensorimotor cortex of 2-month-old Hipk2^+/+ and Hipk2^−/− brains showed more GluN2A⁺ synaptic terminals, but fewer GluN2B⁺ synaptic terminals in Hipk2^−/− mutants. Overall, there was a higher percentage of GluN2A⁺ synapse, but lower percentage of GluN2B⁺ synapse in the sensorimotor cortex in Hipk2^−/− mutants. P–R, Western blots showed a significant increase in GluN2A proteins and a decrease in GluN2B proteins in the synaptosomes from the substantia nigra of 2-month-old Hipk2^−/− mouse brain. The signal intensity of GluN2A, GluN2B, and actin was quantified with National Institutes of Health ImageJ software. Data are mean ± SEM from at least three independent experiments. *p < 0.05 (ANOVA). **p < 0.01 (ANOVA). Not significant: p > 0.05.