Overexpression of calcium-activated potassium channels underlies cortical dysfunction in a model of PTEN-associated autism - PubMed (original) (raw)

Overexpression of calcium-activated potassium channels underlies cortical dysfunction in a model of PTEN-associated autism

Pablo Garcia-Junco-Clemente et al. Proc Natl Acad Sci U S A. 2013.

Abstract

De novo phosphatase and tensin homolog on chromosome ten (PTEN) mutations are a cause of sporadic autism. How single-copy loss of PTEN alters neural function is not understood. Here we report that Pten haploinsufficiency increases the expression of small-conductance calcium-activated potassium channels. The resultant augmentation of this conductance increases the amplitude of the afterspike hyperpolarization, causing a decrease in intrinsic excitability. In vivo, this change in intrinsic excitability reduces evoked firing rates of cortical pyramidal neurons but does not alter receptive field tuning. The decreased in vivo firing rate is not associated with deficits in the dendritic integration of synaptic input or with changes in dendritic complexity. These findings identify calcium-activated potassium channelopathy as a cause of cortical dysfunction in the PTEN model of autism and provide potential molecular therapeutic targets.

Keywords: SK; gain; mTOR; sensory processing; visual cortex.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Intrinsic excitability is altered by Pten mutation. (A) Example voltage traces elicited by current injection in a L2/3 pyramidal neuron from a control and Pten heterozygote (HET) mouse in acute slice preparations. (B) Intensity–Frequency (I-F) plot in response to somatic current injection (10 steps of 50 pA, 500 ms duration) in control and Pten+/− (HET) neurons. Note the decreased firing rate in HET neurons. Two-way ANOVA test, Bonferroni post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Input resistance measured in pyramidal neurons of cortical slices taken from control (white bar) and HET (gray bar) mice. Note the decreased input resistance in HET neurons (Student t test, P = 0.0053). (D) Action potential after-hyperpolarization (AHP) measurements in control (white bar) and HET (gray bar) neurons. Note the increased AHP in HET neurons measured 25 ms after action potential onset (Student t test, P = 0.0045). (E) (Left) Action potential average (30 ms) from control (black trace) and HET (gray trace) neurons. (Right) Average of AHP traces from both genotypes. Note the increased AHP in the HET trace (gray) compared with control trace (black). In all traces, error bars indicate SEM.

Fig. 2.

Fig. 2.

Calcium-mediated conductances are responsible for the decrease in intrinsic excitability. (A) I-F plots in response to somatic current injection in control and Pten+/− (HET) neurons in presence of BAPTA (20 mM). Note there are no significant differences in firing rate in control–BAPTA (blue trace) versus Pten HET–BAPTA (red trace) neurons. Dotted lines represent control situation. (B) Input resistance recovery after BAPTA application. Note how the differences in IR are corrected with BAPTA in HET neurons (gray trace) without affecting control neurons (black trace). ANOVA test, Bonferroni post hoc test (*P < 0.05). (C) I-F plot in response to somatic current injection in control and HET neurons in presence of apamin (200 nM). Note there are no significant differences in firing rate between control–apamin (blue trace) and Pten HET–apamin (red trace) neurons after bath application of apamin. Dotted lines represent control situation. (D) Input resistance recovery after apamin bath application. Note how the differences in IR are corrected with apamin in HET neurons (gray trace) without affecting control neurons (black trace). ANOVA test, Bonferroni post hoc test (*P < 0.05). (E) No differences in AHP after SKCa channel blockade. Apamin decrease AHP in control (black trace) and HET (gray trace) neurons, suppressing previous differences in control situation. ANOVA test, Bonferroni post hoc test (**P < 0.01). (F, Left) Action potential average (30 ms) from control–apamin (blue trace) and Pten HET–apamin (red trace) neurons. (F, Right) Resized plot of boxed region showing average AHP traces from both genotypes. Note the same decreased AHP in control vs. HET. Dotted lines represent control situation. (G) Representative Western blot of cortical protein extracts made from control (WT) and Pten HET mice. Immunoblot was probed with PTEN and SK2 antibodies, as well as β-actin as a loading control. (H) Quantification of PTEN and SK2 proteins. Densitometry values indicate relative expression of PTEN and SK2 proteins, normalized to β-actin. (Pten HET: n = four mice; control; n = three mice; P < 0.05 Mann Whitney). In all traces, error bars indicate SEM.

Fig. 3.

Fig. 3.

Single- and double-copy loss of Pten decrease evoked firing rates in vivo without altering tuning. (A) Example traces of visually evoked responses of a neuron in control, Pten+/−, and _Pten_−/− mice. Recordings were made in loose cell-attached mode. Drifting gratings were presented in 12 directions, shown at the bottom of each panel. Each gray bar represents 3 s of visual stimulation. White bars are responses to a gray screen. (B) Spontaneous (white bar) and peak evoked (black bar) firing rates in control and mutant mice. (C) Distribution of orientation tuning preferences in control (black bars) and Pten mutant (gray bars) mice (homozygous and heterozygous mutants are pooled for this panel and for D and E). Tuning is measured as 1 minus circular variance. (D) Distribution of direction selectivity indices. (E) Distribution of bandwidth measurements in control (black circles) and mutant (gray circles) mice. Mean and SEM are shown in red. (F) Average background subtracted spike rate plotted for all orientations and directions for control (black), Pten+/− (blue), and _Pten_−/− (red) mice. Tuning for all cells has been circularly rotated so that all cells have best tuning at 90°(vertical bars drifting to the right). Note the preservation of direction and orientation selectivity despite the decreased firing rate in the mutant neurons.

Fig. 4.

Fig. 4.

Tuning of evoked subthreshold membrane depolarization is unaffected by Pten mutation. (A and D) Maximum-intensity projection of a L2/3 pyramidal neuron from a control mouse (A) and Pten HET mouse (D) filled with Alexa 594 in whole-cell patch configuration and imaged with two-photon laser-scanning microscopy. The patch pipette is seen entering the image from the Left. (Scale bar, 100 μm.) (B and E) Single-trial membrane potential (MP) responses elicited by 3 s of visual stimulation with sine wave-drifting gratings (red line represents visual stimulus with the grating) at the preferred, opposite, and orthogonal direction from a whole-cell recording from a control (B) and Pten HET (E) L2/3 neuron. Spikes have been truncated for display purposes. (C and F) Average DC (F0) (Upper) and AC (F1) (Lower) modulation of the membrane potential for the control neuron (C) and the Pten HET neuron (F). (G) DC (F0) (Upper) and AC (F1) (Lower) modulation of subthreshold responses as a function of direction for control (black) and mutant (orange) neurons. Plots are mean and SE. Peak orientation is normalized to 90° for all cells. (H) Distribution of orientation selectivity indices for the DC (F0) (Upper) and AC (F1) (Lower) modulations for control and Pten mutant neurons. The bar shows the median OSI, and error bars represent the range.

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