Syngap1 haploinsufficiency damages a postnatal critical period of pyramidal cell structural maturation linked to cortical circuit assembly – biological psychiatry brain anoxia

Figure 1

Pathogenic syngap1 mutations accelerate the rate of normal neocortical pyramidal cell growth and maturation. (A) three-dimensional reconstructed confocal imaging sections of somatosensory cortex obtained in postfixed brain slices in syngap1+/− and wild-type ( wt) (postnatal day [PND] 21 and PND 60). (B) representative example of three-dimensional reconstruction layer V pyramidal neurons (PND21 and PND60), scale bar = 5 μm. (C–E) graphs depicting the total length, intersections, and volume of traced neurons, * p .05 within genotype group comparison, # p .05 within age group comparison. (F) scatter plot graphs showing the correlation index between cell body volume and the total length of the neuronal dendrites measured by sholl analysis quantification. (G) immunostaining for phosphorylated S6 (p-S6) (red) and neun (green) in L5 neurons in prefrontal cortex of PND4 syngap1+/− ( n = 5 animals, 150 cells) mice and wt ( n = 3 animals, 90 cells) littermates. * p .05, student t test.Brain anoxia


values represent means; error bars indicate sems. Complete statistical reporting can be found in table S1 in supplement 1 .

Figure 4

Premature cellular maturation causes altered spine dynamics in syngap1 heterozygous knockouts. (A) schematic demonstrating the sequential time-lapse recording experiment. (B) representative dendritic segments. In vivo time-lapse imaging of the same dendritic segments over 2 hours and 4 hours in the primary somatosensory cortex (scale bar = 5 μm). (C–E) graphs depicting filopodia density (C), proportion of stable spines over 2 hours and 4 hours recording (D), or proportion of filopodia that converted into spines in syngap1+/− ( n = 5) and wild-type ( wt) ( n = 5); # p .05. (F–I) diagram showing time-lapse recording experimental design at postnatal day (PND)21 to PND23 and PND30 to PND32. (G–L) 2-photon laser scanning microscope imaging session of the same dendritic segments over 2 days (scale bar = 5 μm). (H, I) PND21 to PND23 dynamic properties of dendritic spines.Brain anoxia graphs showing the proportion of new formed spines and spine eliminated detected over 2 days each time point (H) and turnover index (I), student t test ( t 5 = 3.223, p = .023) syngap1+/− ( n = 4) and wt ( n = 3) animals. (J–L) spine formation and elimination from PND30 to PND32. Graphs showing the dynamic plasticity of spine imaged over 2 days (K) and turnover index (L), student t test ( t 6 = 2.46, p = .0492) syngap1+/− ( n = 4) and wt ( n = 3) animals. Arrowheads: green = new formed; yellow = lost; red = stable spines; white = filopodia; purple = filopodia converted to spines. Values represent mean, error bars indicate SEM. Least significant difference post hoc test was used for multiple comparisons.Brain anoxia complete statistical reporting can be found in table S4 in supplement 1 . * p .05; # p .05; A.U., arbitrary unit; YFP, yellow fluorescent protein.

Figure 5

Widespread spine enlargement in syngap1 mutants is caused by a disrupted developmental critical period. (A) representative dendrites of postnatal day (PND)14 layer 2 neurons. Cumulative frequency curves depict spine head diameter in layer 2 and layer 5 apical branches (located in layer 1) in PND14 syngap1+/− and wild-type ( wt). (B) representative electron microscopy images of presynaptic (yellow) and postsynaptic (green) area at PND14 in which postsynaptic density (PSD) area is outlined. Cumulative frequency curves depict PSD area observed in layer 1. (C) cumulative frequency curves depicting spine head diameter observed in layer 5 at PND60. (D) cumulative frequency curves showing spine head diameter of newborn spines detected during a second in vivo transcranial imaging (i.E., spines present at PND23 but not at PND21). (E) representative dendrites of PND 90 layer 2 neurons in syngap1+/− and syngap1+/floxed adult-induced haploinsufficiency model.Brain anoxia cumulative frequency curves show that wider spine heads persist throughout adulthood in conventional (conv.) syngap1 heterozygous knockout ( het.) mutants, while this effect does not occur in animals with syngap1 haploinsufficiency induced in adulthood. Complete statistical reporting can be found in table S5 in supplement 1 . N.S., not significant.

Figure 6

Gross anatomical long-distance synaptic connectivity appears normal in syngap1 mutants. (A) experimental design. (B) representative coronal sections in the anterior to posterior axis of long-range connectivity to the medial prefrontal cortex (mpfc). Scale bar, 1 mm. (C) quantitative characterization of the injection sites and total number of direct-labeled inputs to the mpfc. (D) relative hemispheric representations of the contributions of inputs form ipsilateral (ipsi) and contralateral (contra) sides. (E) relative representation of inputs clustered by cortical (cortex), subcortical (subcx.), and limbic brain areas. (F) relative representation of primary (1ry) and secondary (2ry) cortical (cx) inputs including areas from ipsilateral motor, auditory, visual and somatosensory cortex, and contralateral motor and somatosensory cortices.Brain anoxia values represent means, error bars indicate sems. P values indicate two-tailed unpaired t test comparisons of wild-type ( wt) versus syngap1+/− mice. Data are presented as a proportion of the total number of direct inputs to the mpfc between wt and syngap1+/− mice. A.U., arbitrary unit; cg1, cingulate cortex, area 1; IL, infralimbic cortex; M2, secondary motor cortex; prl, prelimbic cortex; RV-GFP, rabies virus-green fluorescent protein.

Figure 7

Evidence of macroscale disruptions in long-distance intracortical synaptic connectivity in syngap1 mutants. (A–D) comparison of the relative strength of monosynaptic inputs to the medial prefrontal cortex in the ipsilateral cortical (A), contralateral cortical (B), subcortical (C), and limbic (D) areas.Brain anoxia values represent means, error bars indicate sems. P values indicate two-tailed unpaired t test comparisons between wild-type ( wt) ( n = 9) and syngap1+/− ( n = 9) animals. (E) monosynaptic inputs to the medial prefrontal cortex (cx.) labeled with RV-GFP in wt and syngap1+/− for brain areas located in cortical (layer 5 pyramidal neurons in the somatosensory cortex), subcortical (thalamus), and limbic (amygdala) regions. Pia is indicated with a dotted line. Scale bar shown in all figures, 200 μm. (F) cumulative frequency curves of numbers of primary somatosensory neurons in both groups; p .0001; two-sample kolmogorov-smirnov test. (G) relative strength in connectivity represented as a measure of the location of specific brain regions.Brain anoxia location of the brain area biased the strength of connectivity of cortical inputs to the prefrontal cortex ( n = 19 brain areas, p = .006 for linear regression of connectivity ratio and anatomical location). The connectivity ratio was established as the differential inputs between wt and syngap1+/− relative to the number of inputs in wt. Gray indicates 95% confidence interval for the regression line. * p .05; ** p .01. AMY, amygdala; A/P, antero/posterior; au1, primary auditory cortex; au2, secondary auditory cortex; cg1/2, cingulate cortex, area 1/2; ect, ectorhinal cortex; ent, entorhinal cortex; fra, frontal association; HPC, hippocampus; hyp, hypothalamus; IC, insular cortex; IL, infralimbic cortex; M1, primary motor cortex; M2, secondary motor cortex; MPA, medial preoptic area; OFC, orbitofrontal cortex; pir, piriform cortex; prh, perirhinal cortex; prl, prelimbic cortex; RS, retrosplenial cortex; RV-GFP, rabies virus green fluorescent protein; S1, primary somatosensory cortex; S1BF, primary somatosensory cortex, barrel field; str, striatum; tea, temporal association cortex; thal, thalamus; V1, primary visual cortex; V2, secondary visual cortex.Brain anoxia

Figure 8

Early, but not late, postnatal repair of a pathogenic syngap1 mutation prevents cognitive and behavioral abnormalities. Four groups of mice (syngap1+/+; CAG-creer−/−, syngap1+/lox-stop; CAG-creer−/−, syngap1+/+; CAG-creer+/−, syngap1+/lox-stop; CAG-creer+/−) were administered either a single injection (SQ) of tamoxifen (TMX) (20 mg/kg) on postnatal day (PND)1 or five daily injections (intraperitoneal [IP]) of TMX starting on PND21. Behavioral batteries consisting of elevated plus maze (EPM), open field (OF), and remote (30d) contextual fear conditioning (FC) measured by activity suppression ratio tests (first 2 min of the test)/[(first 2 min of the test) + (first 2 min of the training)], respectively, were conducted starting at 12 weeks.Brain anoxia hippocampi were dissected and processed for immunoblot of syngap protein levels in the four genotype groups after conclusion of the behavioral batteries. (A) genotypic reversal of syngap1 is induced in syngap1+/lox-stop; CAG-creer+/− mice administered TMX at PND1. Densitometric values were normalized to β-tubulin (TUBB) levels and subsequently transformed. (B–D) performances of syngap1+/lox-stop; CAG-creer+/− mice are rescued relative to that of syngap1+/+; CAG-creer+/− mice in EPM, OF, and FC tests. (E) genotypic reversal of syngap1 is induced in syngap1+/lox-stop; CAG-creer+/− mice administered TMX at PND21. Densitometric values were normalized to β-tubulin levels and subsequently transformed. (F–H) performances of syngap1+/lox-stop; CAG-creer+/− mice are not rescued relative to that of syngap1+/+; CAG-creer+/− mice in EPM, OF, and FC tests.Brain anoxia multiple comparisons within each independent factor were calculated. * p .05, ** p .01, *** p .001, difference within cre; # p .05, ## p .01, ### p .001, difference within genotype. Numbers of animals are indicated within bar graphs. Complete statistical reporting can be found in table S6 in supplement 1 . N.S., not significant.