Fig 2: Quantitative analyses for PSD-95 in Slitrk2 KO brains(A) Representative images for PSD-95 (green) and VGlut1 (red) immunostaining in the hippocampus CA1 stratum radiatum (CA1), CA3 stratum lucidum (CA3), dentate gyrus (DG), granule cell layer of Xth lobule (CBL), pontine gray matter (PG), and tegmental reticular nucleus (TRN) from the para sagittal section (lateral to 0.60–0.72 mm). Scale bars, 5 μm.(B) Quantitative analysis of PSD-95 and VGlut1 punctate signal sizes in WT and Slitrk2 KO mice. 9–13 M-old mice were used for the analysis. Data are presented as means ± SD. ∗, p < 0.05. n = 6 or 7 mice (CA1, CA3, DG) or n = 7 mice (CBL, PG, TRN) per genotype. Independent 8 images from 4 sections were averaged for a mouse. ∗, p < 0.05. n = 6 or 7 mice (CA1, CA3, DG) or n = 7 mice (CBL, PG, TRN) per genotype. 8 images from 4 sections were averaged for a mouse. †, p < 0.05; †††, p < 0.001 in t-test where values for each image were used for the statistical tests (n = 48 or 56 images, Table S1). See also Table S1 and Figure S5.

Fig 3: Electrophysiological analysis of hippocampal granule cells in Slitrk2 KO mice(A) Schematic diagram of hippocampal excitatory neuronal circuits.(B–D) Analyses of the medial perforant path–dentate gyrus granule cell synapse. (B) Input–output relationship. n = 10 slices from 6 mice per genotype. (C) Paired–pulse ratio. n = 10 or 9 slices from 7 mice per genotype. (D) LTP induced by tetanic stimulation delivered at 0 min. Sample recordings show averaged consecutive EPSPs at baseline and 30–40 min after the tetanic stimulation. Scale bar: 10 ms, 0.5 mV n = 10 or 12 slices from 7 or eight mice per genotype.(E–J) Analyses of mossy fiber–CA3 synapses. (E) EPSP to presynaptic fiber volley ratio. n = 21 or 22 slices from 9 or 8 mice per genotype. (F) Paired pulse facilitation. n = 7 slices from 5 mice per genotype. (G) Time course of frequency facilitation induced by 1 Hz stimulation. (H) Summary of the magnitude of frequency facilitation induced at 0.2, 1, and 2 Hz ∗, p = 0.035. 0.2 Hz, n = 7 or 6 slices from 6 or 5 mice per genotype; 1 Hz, n = 8 or 9 slices from 5 or 4 mice per genotype; 2 Hz, n = 7 slices from 4 mice per genotype. (I) Modulation of mossy fiber–CA3 synaptic transmission by serotonin (5-HT) applied at 1 μM (left) or 5 μM (right) from 0 to 5 min. Sample recordings show averaged consecutive EPSPs at baseline and the peak of potentiation. Scale bar: 10 ms, 0.2 mV. (J) Summary of the magnitude of serotonin-induced synaptic potentiation. ∗, p = 0.0413, ∗∗, p = 0.0095. 1 μM, n = 8 or 9 slices from 4 or 5 mice per genotype; 5 μM, n = 8 or 10 slices from 8 or 9 mice per genotype. Data are presented as means ± SEM, except E, H, and J where error bars represent SD.

Fig 4: Distribution of Slitrk2 protein(A) Immunoblot analysis was performed using antibodies against Slitrk2, actin, and PSD-95. In each lane, 5 μg of protein was electrophoresed. (left top) Distribution of Slitrk2 protein in the adult CNS. (left bottom) Distribution of actin as a reference. (right top) Subcellular distribution of Slitrk2 protein. (right bottom) Subcellular distribution of PSD-95 protein as a reference. Mouse brain lysate was biochemically fractionated. P1, cell nuclei and debris; P2, crude synaptosome; P3HDM, fraction enriched with Golgi apparatus; P3LDM, microsome fraction enriched with ER and transport vesicles; LP1, synaptosomal membrane; LS1, synaptic vesicle and synaptosomal cytosol; LP2, synaptic vesicle; LS2, soluble synaptosomal fraction.(B) Immunostaining of Slitrk2. Arrows indicate nonspecific signals in the cerebral cortex that remained in the Slitrk2 KO brain (Figure S2).(C) Coronal sections through 9th (IX, left) and 8th (VIII, right) lobules of the cerebellar vermis from WT and Slitrk2 KO immunoreacted with anti Slitrk2.(D) Co-immunostaining of Slitrk2 and PSD-95. Sagittal sections (hp) or coronal sections (TRN) from WT and Slitrk2 KO mice immunoreacted with anti-Slitrk2 (green) and anti-PSD-95 (red) antibodies. DG, hippocampus dentate gyrus; hp, hippocampus; LRN, lateral reticular nucleus; NPP, nucleus prepositus; PG, pontine gray; TRN, tegmental pontine reticular nucleus; VIII, IX, X, cerebellar lobules. Thin scale bars, 200 μm; thick scale bars, 1 mm. See also Figures S1, S2, and S10.

Fig 5: Serotonergic dysregulation and location of serotonergic cells(A) Monoamine contents in Slitrk2 KO brain regions. (top) Each brain region was punched out from frozen brain sections. AMY, amygdala; HPC, hippocampus; NAC, nucleus accumbens; PFC, medial prefrontal cortex; STR, striatum. Scale bar, 1 mm. (bottom) Serotonin (5-HT) concentrations, serotonin metabolite (5-HIAA) concentrations, serotonin turnover (5-HIAA/5-HT), dopamine (DA) and its metabolites (DOPAC and HVA) concentrations; n = 6 mice per genotype.(B) Morphometric analysis of serotonergic neurons. (top) Representative SERT or Tph2 immunostaining images (bregma −4.5 mm) for dorsal raphe (DR) and median raphe nuclei (MnR) from WT and Slitrk2 KO mice. The boxed regions in low magnification views are shown below. Thick scale bar, 1 mm; thin scale bar, 100 μm. (bottom) Estimated total particle count per mouse (left), mean particle size (middle), and mean signal intensity of particles (right) were quantified. 8–10 M-old mice were used for the analysis. n = 7 mice per genotype. Values from 5 to 13 images (DR) or 3–8 images (MnR) were summed or averaged per mouse.(C) SERT-positive fiber-like signals were quantified in hippocampal CA1 stratum lacunosum moleculare, CA3 stratum radiatum; and dentate gyrus (DG) stratum moleculare (dorsal lip). n = 48–51 images from 8 mice per genotype (4–8 images per mouse). Data are presented as means ± SD. ∗, p < 0.05; ∗∗, p < 0.01 in t-test. See also Figure S7.