A molecular mechanism mediating clozapine-enhanced sensorimotor gating

Abstract

The atypical antipsychotic clozapine targets multiple receptor systems beyond the dopaminergic pathway and influences prepulse inhibition (PPI), a critical translational measure of sensorimotor gating. Since PPI is modulated by atypical antipsychotics such as risperidone and clozapine, we hypothesized that p11-an adaptor protein associated with anxiety- and depressive-like behaviors and G-protein-coupled receptor function-might modulate these effects. In this study, we assessed the role of p11 in clozapine's PPI-enhancing effect by testing wild-type and global p11 knockout (KO) mice in response to haloperidol, risperidone, and clozapine. We also performed structural and functional brain imaging. Contrary to our expectation that anxiety-like p11-KO mice would exhibit an augmented startle response and heightened sensitivity to clozapine, PPI tests showed that p11-KO mice were unresponsive to the PPI-enhancing effects of risperidone and clozapine. Imaging revealed distinct regional brain volume differences and reduced hippocampal connectivity in p11-KO mice, with significantly blunted clozapine-induced connectivity changes in the CA1 region. Our findings highlight a novel role for p11 in modulating clozapine's effects on sensorimotor gating and hippocampal connectivity, offering new insight into its functional pathways.

Fig. 1. Atypical antipsychotics do not increase PPI in p11-KO mice.

A Schematic depiction of PPI experimental design. B Bar graph showing PPI after treatment with vehicle, haloperidol (0.5 mg/kg), clozapine (3 mg/kg) or risperidone (0.3 mg/kg) in WT mice (two-way rmANOVA, Prepulse: F(2,16) = 92.72, p < 0.001, Treatment: F(3,24) = 6.29, p = 0.003; Veh vs AP *p < 0.05, ***p < 0.001, Dunnet’s test). C Bar graph showing PPI after treatment with vehicle, haloperidol (0.5 mg/kg), clozapine (3 mg/kg) or risperidone (0.3 mg/kg) in p11-KO mice (two-way rmANOVA, Prepulse: F(2,10) = 8.89, p = 0.006). D Bar graph showing the mean PPI across all prepulse intensities in WT and p11-KO mice (two-way rmANOVA, Genotype × Treatment: F(3,39) = 3.82, p = 0.017; Veh vs AP **p < 0.01; WT vs p11-KO #p < 0.05, ##p < 0.01, Dunnet’s test). E Bar graph showing pulse-alone startle amplitude (two-way rmANOVA, Treatment: F(3,39) = 6.22, p = 0.002, Genotype: F(1,13) = 31.16, p < 0.001; Veh vs AP **p < 0.01, ***p < 0.001; WT vs p11-KO #p < 0.05, ##p < 0.01, ###p < 0.001, Dunnet’s test). WT n = 9 (males n = 4, females n = 5), p11-KO n = 6 (females n = 6). Data are presented as mean ± SEM. Veh vehicle, AP antipsychotic, AU arbitrary units, PPI prepulse inhibition, Hal haloperidol, Ris risperidone, Clz clozapine.

Fig. 2. Antipsychotic effects on P11-KO mice in MK-801 -induced PPI disruption paradigm.

A Schematic representation of PPI experimental design. B Bar graph showing PPI after treatment with MK-801 (0.3 mg/kg) alone or in combination with clozapine (3 mg/kg) or haloperidol (0.5 mg/kg) in WT mice (two-way rmANOVA, Prepulse: F(2,16) = 66.82, p < 0.001; Treatment: F(3,24) = 21.44, p < 0.001; post-hoc treatment comparisons **p < 0.01, ***p < 0.001, Tukey’s test). C Bar graph showing PPI after treatment with MK-801 (0.3 mg/kg) alone or in combination with clozapine (3 mg/kg) or haloperidol (0.5 mg/kg) in p11-KO mice (two-way rmANOVA, Prepulse: F(2,10) = 17.74, p < 0.001; Treatment: F(3,15) = 5.37, p = 0.01; Prepulse × Treatment: F(6,30) = 2.89, p = 0.024; post-hoc treatment comparisons *p < 0.05, **p < 0.01, ***p < 0.001; Tukey’s test). D Mean PPI across all prepulse intensities in WT and p11-KO mice (two-way rmANOVA, Treatment: F(3,39) = 19.8, p < 0.001; post-hoc treatment comparisons **p < 0.01, ***p < 0.001; Tukey’s test). E Bar graph showing pulse-alone startle amplitude (two-way rmANOVA, Treatment: F(3,39) = 3.2, p = 0.034; Genotype: F(1,13) = 6.76, p = 0.022; post hoc treatment comparisons **p < 0.01; WT vs. p11-KO #p < 0.05, ##p < 0.01; Tukey’s test). WT mice n = 9 (males n = 4, females n = 5), p11-KO n = 6 (females n = 6). Data are presented as mean ± SEM. Veh vehicle, AP antipsychotic, AU arbitrary units, PPI prepulse inhibition, MK MK-801, Hal haloperidol, Ris risperidone, Clz clozapine.

Fig. 3. Selective ablation of p11 in serotonergic neurons blunts clozapine’s effects on PPI.

A Illustration (upper) showing the presynaptic and postsynaptic action of atypical antipsychotics on the 5-HT system, along with RNAscope images (lower) depicting the overlap of p11 transcripts with Tph2+ cells in the DR (scale bar: 100 μm). B Illustration (left) of the brain areas dissected from p11-flx and Sert-cp11KO mice for HPLC analysis, and a bar graph (right) showing the 5-HT concentration in the PFC, CP, and HIP of p11-flx and Sert-cp11KO mice (*p < 0.05, unpaired t test). C Schematic representation of PPI experimental design using Sert-cp11KO mice. D Bar graph showing PPI after treatment with vehicle, clozapine (3 mg/kg) or risperidone (0.3 mg/kg) in p11-flx control mice (two-way rmANOVA, Prepulse: F(2,28) = 46.67, p < 0.001; Treatment: F(2,28) = 6.61, p = 0.005; Veh vs. AP *p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s post-hoc test). E Bar graph showing PPI after treatment with vehicle, clozapine (3 mg/kg) or risperidone (0.3 mg/kg) in Sert-cp11KO mice (two-way rmANOVA, Prepulse: F(2,32) = 25.5, p < 0.001; Treatment: F(2,32) = 4.41, p = 0.02; Veh vs. AP *p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s post hoc test). F Bar graph showing the mean PPI across all prepulse intensities (two-way rmANOVA, Treatment: F(2,60) = 7.59, p = 0.001; Veh vs. AP *p < 0.05, **p < 0.01, Dunnett’s post hoc test). G Bar graph showing the pulse-alone startle amplitude (two-way rmANOVA, Treatment: F(2,60) = 23.24, p < 0.001; Genotype: F(1,30) = 4.26, p = 0.048; Veh vs. AP *p < 0.05, **p < 0.01, ***p < 0.001; p11-flx control vs. Sert-cp11KO #p < 0.05, Dunnet’s test). Sample size: HPLC: p11-flx n = 6 (females n = 6), Sert-cp11KO n = 7 (females n = 7). PPI: p11-flx n = 15 (males n = 8, females n = 7), Sert-cp11KO n = 17 (males n = 7, females n = 10). Data are presented as mean ± SEM. 5-HT serotonin, DR dorsal raphe, Tph2 tryptophan hydroxylase 2, Sert serotonin transporter, HPLC high pressure liquid chromatography, Veh vehicle, AP antipsychotic, AU arbitrary unit, PPI prepulse inhibition, Ris risperidone, Clz clozapine.

Fig. 4. P11-KO mice display increased DG but reduced BLA volumes.

A Schematic depiction of hypothesis and experimental design. B Heatmap showing the expression of p11 throughout the brain. C Whole brain MR template showing the regions with volume changes in color (red-increases, blue-decreases) in p11-KO compared to WT mice. Voxelwise differences data are displayed on the study-specific template image using the dual coding approach: differences are mapped to color hue, and associated p-values are mapped to color transparency. Family-wise error rate controlled p > 0.5 in areas with completely transparent colour overlay. Contours delineate statistically (adjusted p < 0.05) significant difference. Sample sizes: WT mice: n = 10, males: n = 5, females: n = 5; p11-KO mice: n = 10, males: n = 5, females: n = 5. MRI magnetic resonance imaging, DMV/XII dorsal motor nucleus of vagus/hypoglossal nerve nucleus, IO inferior olive, VII facial motor nucleus, DR dorsal raphe, CA1 cornu ammonis 1, L5a cortical layer 5a, BLA basolateral amygdala, DG dentate gyrus.

Fig. 5. Clozapine induced decrease in CA1 functional connectivity is blunted in p11-KO brains.

A Schematic depiction of the experimental design and selected brain regions analyzed in the fUS experiments. B Left: Heatmap showing the standardized responses of the selected brain areas in WT mice after clozapine administration (4 mg/kg). Right: Dot plot displaying the p-values of these responses on a log scale. C Correlation matrix illustrating functional connectivity differences between drug treatment and baseline in the selected brain regions of WT mice (*FDR < 0.05). D Left: Heatmap showing the standardized responses of the selected brain areas in p11-KO mice after clozapine administration (4 mg/kg). Right: Dot plot displaying the p-values of these responses on a log scale. E Correlation matrix illustrating functional connectivity differences between drug treatment and baseline in the selected brain regions of p11-KO mice (*FDR < 0.05). Left: Line graphs showing functional connectivity changes between CTX (F) or CA1 (G) and the rest of the selected brain regions over time. Right: Bar graphs showing mean functional connectivity between CTX (F) or CA1 (G) and the rest of the selected brain regions at baseline and during the last 15 min of clozapine treatment. Statistical analysis: two-way repeated-measures ANOVA, CTX (Treatment: F(1,20) = 23.49, p < 0.0001; Genotype: F(1,20) = 8.586, p = 0.0083), CA1 (Treatment: F(1,20) = 13.64, p = 0.0014; Genotype: F(1,20) = 13.69, p = 0.0014); post-hoc comparisons (Veh vs. Clz: *p < 0.05, ***p < 0.001; WT vs. p11-KO: ##p < 0.01, ###p < 0.001; Sidak’s post hoc test). H Left: Line graph showing functional connectivity changes between CA1 and CTX over time. Right: Bar graphs showing mean functional connectivity between CA1 and CTX at baseline and during the last 15 min of clozapine treatment. Statistical analysis: two-way repeated-measures ANOVA (Genotype × Treatment: F(1,20) = 4.882, p = 0.039); post hoc comparisons (Veh vs. Clz: ***p < 0.001; WT vs. p11-KO: ###p < 0.001; Sidak’s post hoc test). Sample sizes: WT mice: n = 11 (males: n = 7, females: n = 4); p11-KO mice: n = 11 (males: n = 6, females: n = 5). Data are presented as mean ± SEM. Veh vehicle, HIP hippocampal region, CA1 cornu ammonis 1, CTX cortex, fUS functional ultrasound, pfUS pharmacological functional ultrasound, fc functional connectivity, Clz clozapine, Dex dexmedetomine.