Protein Phosphatase 2a and glycogen synthase kinase 3 signaling modulate prepulse inhibition of the acoustic startle response by altering cortical M-Type potassium channel activity

Abstract

There is considerable interest in the regulation of sensorimotor gating, since deficits in this process could play a critical role in the symptoms of schizophrenia and other psychiatric disorders. Sensorimotor gating is often studied in humans and rodents using the prepulse inhibition of the acoustic startle response (PPI) model, in which an acoustic prepulse suppresses behavioral output to a startle-inducing stimulus. However, the molecular and neural mechanisms underlying PPI are poorly understood. Here, we show that a regulatory pathway involving protein phosphatase 2A (PP2A), glycogen synthase kinase 3 beta (GSK3beta), and their downstream target, the M-type potassium channel, regulates PPI. Mice (Mus musculus) carrying a hypomorphic allele of Ppp2r5delta, encoding a regulatory subunit of PP2A, show attenuated PPI. This PPP2R5delta reduction increases the phosphorylation of GSK3beta at serine 9, which inactivates GSK3beta, indicating that PPP2R5delta positively regulates GSK3beta activity in the brain. Consistently, genetic and pharmacological manipulations that reduce GSK3beta function attenuate PPI. The M-type potassium channel subunit, KCNQ2, is a putative GSK3beta substrate. Genetic reduction of Kcnq2 also reduces PPI, as does systemic inhibition of M-channels with linopirdine. Importantly, both the GSK3 inhibitor 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)1H-pyrrole-2,5-dione (SB216763) and linopirdine reduce PPI when directly infused into the medial prefrontal cortex (mPFC). Whole-cell electrophysiological recordings of mPFC neurons show that SB216763 and linopirdine have similar effects on firing, and GSK3 inhibition occludes the effects of M-channel inhibition. These data support a previously uncharacterized mechanism by which PP2A/GSK3beta signaling regulates M-type potassium channel activity in the mPFC to modulate sensorimotor gating.

Figure 1.

Characterization of Ppp2r5δGt gene trap mice. A, Schematic of the Ppp2r5δ locus disrupted by gene trap insertion between exons 2 and 3 of the gene. Exon number and size of locus in kilobases are indicated. βgeo, β-Galactosidase/neomycin resistance markers within the gene trap insertion. B, C, Western blot analysis of brain lysates from Ppp2r5δGt/+ and wild-type control mice quantifying protein levels. “+” denotes wild-type mouse, and “−” denotes Ppp2r5δGt/+ mouse. PPPP2R5δ protein is reduced by ∼50% in Ppp2r5δGt/+ mice, whereas pGSK3β (Ser-9) levels are increased by ∼30%. Ppp2r5δ disruption does not appear to affect pGSK3α (Ser-21) phosphorylation or total amounts of GSK3α or GSK3β. D, E, Ppp2r5δGt/+ and Gsk3β(tm1JW)/+ mice show reduced PPI relative to wild-type controls. F, GSK3 inhibitor, SB216763 (10 mg/kg), attenuates PPI in wild-type mice when administered as a single, intraperitoneal injection. Drug was administered 15 min before behavioral testing. Error bars represent SEM. *p < 0.05.

Figure 2.

M-type potassium channels regulate PPI. A, Schematic of the Kcnq2(tm1Dgen) locus. The gene trap insertion disrupts the Kcnq2 locus between exons 1 and 2. Exon number and size of locus in kilobases are indicated. βgeo, β-Galactosidase/neomycin resistance markers within the gene trap insertion. B, Kcnq2 mRNA is reduced in total brain tissue from Kcnq2(tm1Dgen)/+ mice by ∼50%. C, Kcnq2(tm1Dgen)/+ mice show reduced PPI relative to wild-type controls. D, Acute systemic administration of the M-channel blocker, linopirdine (5 mg/kg), attenuates PPI in wild-type mice when administered as a single, intraperitoneal injection. Drug was administered 15 min before behavioral testing. Coadministration of SB216763 (10 mg/kg) and linopirdine (5 mg/kg) conferred a similar effect on PPI as either drug alone. Error bars represent SEM. *p < 0.05.

Figure 3.

GSK3β and KCNQ2 proteins colocalize to mPFC. A, Triple-channel confocal images of mPFC. Individual apical dendrites of pyramidal cells were identified with the anti-MAP2 antibody (blue). Some dendrites were positive for both GSK3β (red) and KCNQ2 (green) indicating coexpression of both proteins in mPFC neurons (arrowheads point to same areas on all panels). Merge (1) panel shows overlay of anti-GSK3β, anti-KCNQ2, and anti-MAP2 staining in low-magnification panels. The inset shows the area of mPFC from which images were taken. B, Higher magnification images of areas shown in A, indicated in boxed region, illustrate colocalization of all three markers; the dashed lines outline the same dendrite fragment on all panels. Merge (2) panel shows overlay of anti-GSK3β and anti-KCNQ2 staining in high magnification panels; merge (3) panel shows overlay of anti-GSK3β, anti-KCNQ2, and anti-MAP2 staining in high-magnification panels. Scale bars: A, 20 μm; B, 5 μm.

Figure 4.

GSK3β and M-type potassium channels function in the mPFC to regulate PPI. A, PPI is reduced in wild-type mice administered SB216763 (5 pm) bilaterally in mPFC compared with vehicle-infused controls. B, PPI is reduced in wild-type mice administered linopirdine (5 pm) bilaterally in mPFC compared with vehicle-infused controls. C, Schematic depicting placement of cannulae targeting mPFC (between bregma +1.8 and bregma +2.8) in subjects from drug infusion experiments. Subjects were tested for PPI immediately after drug infusion. Error bars represent SEM. *p < 0.05.

Figure 5.

GSK3 and M-type potassium channel inhibition have similar effects on AP firing and accommodation in layer II/III prelimbic neurons in vitro. A, Example traces showing that linopirdine and SB216763 exposure reduce accommodation, measured at the current step at which eight APs were generated, and that linopirdine has no effect in SB216763-pretreated neurons. The inset for the control/linopirdine trace shows the increase in firing at the same current step shown for the control/baseline trace. Number of APs per current step was determined for each neuron in relation to rheobase, the minimum current required to evoke an AP in that neuron. B, Linopirdine and SB216763 enhance AP firing, and linopirdine has no effect on firing in SB216763-pretreated neurons. C, Linopirdine and SB216763 reduce accommodation, and linopirdine has no effect on accommodation in SB216763-pretreated neurons. Accommodation was assayed at the current step with eight APs and was determined by converting ISI values for each AP pair in an AP train to IFF, and then normalizing IFF values for each AP pair in the AP train to the IFF value for the second AP pair in the AP train. D, E, Bar graphs showing statistical differences between the APs generated at the 100 pA step above rheobase (D), and the IFF values for the seventh AP pair in the AP train (E). ctl, Control; freq., frequency; instant., instantaneous; lin, linopirdine; n.s., not significant, p > 0.05; SB, SB216763. *p < 0.05 or **p < 0.001.

Figure 6.

Proposed model of how M-channel inhibition in mPFC attenuates PPI. Under normal conditions, mPFC neuron in the PPI circuitry fires in response to the prepulse stimulus. M-channel activation renders the neuron less responsive to the subsequent startle stimulus and startle reactivity is gated. mPFC neurons in which M-channels have been inhibited, either through reduced PP2A/GSK3 signaling or direct inhibition of the M-channel, are hypothesized to fire normally (or with increased probability) in response to a prepulse stimulus but remain responsive to the startle stimulus. “B56δ” denotes the PP2A regulatory subunit, PPP2R5δ.