Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders

Abstract

Both genetic and environmental factors are thought to contribute to neurodevelopmental and neuropsychiatric disorders with maternal immune activation (MIA) being a risk factor for both autism spectrum disorders and schizophrenia. Although MIA mouse offspring exhibit behavioral impairments, the synaptic alterations in vivo that mediate these behaviors are not known. Here we employed in vivo multiphoton imaging to determine that in the cortex of young MIA offspring there is a reduction in number and turnover rates of dendritic spines, sites of majority of excitatory synaptic inputs. Significantly, spine impairments persisted into adulthood and correlated with increased repetitive behavior, an ASD relevant behavioral phenotype. Structural analysis of synaptic inputs revealed a reorganization of presynaptic inputs with a larger proportion of spines being contacted by both excitatory and inhibitory presynaptic terminals. These structural impairments were accompanied by altered excitatory and inhibitory synaptic transmission. Finally, we report that a postnatal treatment of MIA offspring with the anti-inflammatory drug ibudilast, prevented both synaptic and behavioral impairments. Our results suggest that a possible altered inflammatory state associated with maternal immune activation results in impaired synaptic development that persists into adulthood but which can be prevented with early anti-inflammatory treatment.

Keywords: Anti-inflammatory; Autism; Dendritic spines; Excitation; Inflammation; Inhibition.

Fig. 1

Reduced cortical dendritic spine density in young MIA offspring. Confocal images of layer 5 pyramidal neuron apical tuft dendrites from P17 offspring of control (a) and MIA (b) YFP-H mice. (c) MIA results in a reduction in total dendritic spine density (control: 1.014 ± 0.07 spines/micron, n = 4; MIA: 0.78 ± 0.04 spines/micron, n = 6, P = 0.018, unpaired t-test). (d) MIA results in a general reduction in density of all dendritic spine categories although these differences did not reach significance following multiple comparison corrections (mushroom P = 0.27, stubby P = 0.06, thin P = 0.185 and filopodia P = 0.11, multiple t-test with Sidak–Bonferroni correction. Data are represented as mean ± SEM.

Fig. 2

Reduced density and dynamics of dendritic spines in MIA offspring. Multiphoton imaging of cortical neurons from P17 YFP-H mice through a thinned skull window in control (a) and MIA (b) offspring. Images were collected every 12 min for a period of 1.5 h. Note the reduced density of dendritic protrusions in the MIA offspring. Dendritic spine formation (red arrows) and elimination (yellow arrows) is prevalent in control but not MIA offspring. Spines that appear but then disappear are marked with an orange arrow. (c–f) Quantification of spine parameters imaged in vivo in 5 mice for each condition (multiple t-tests with Bonferroni corrections). Spine density is reduced in MIA offspring (control: 0.92 ± 0.02 spines/micron; MIA: 0.72 ± 0.02 spines/micron, P = 0.0001). Turnover rate (TOR) is reduced in MIA offspring (control: 0.091 ± 0.01; MIA 0.057 ± 0.004, P = 0.013) due to reduction in both fraction of spines gained (control: 0.1 ± 0.003; MIA: 0.064 ± 0.004, P < 0.0001) and fraction of spines lost (control: 0.08 ± 0.01; MIA: 0.05 ± 0.005, P = 0.028). Data are represented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Altered presynaptic input in the cortex of MIA offspring. (a and b) Cortical P17 sections from YFP-H mice immunostained with VGluT1 (blue) and GAD-65 (red) in control (a) and MIA offspring (b). (c and d) Quantification of VGluT1, VGluT2 and GAD-65 puncta do not show a reduction in number of puncta in MIA offspring. VGluT1, control (black): 307 ± 29.57, n = 8; MIA (red): 285.3 ± 28.15, n = 8, P = 0.60; VGluT2, control: 162.5 ± 32.54, n = 8; MIA: 138.3 ± 18.56, n = 6, P = 0.57; GAD-65, control: 174.5 ± 12.7, n = 8; MIA: 154.2 ± 20.56, n = 8, P = 0.41). No significant difference in the volume of the puncta was observed. VGluT1, control: 0.305 ± 0.04, n = 6; MIA: 0.264 ± 0.035, n = 8, P = 0.46; VGluT2, control: 0.258 ± 0.048, n = 8; MIA: 0.239 ± 0.012, n = 6, P = 0.75; GAD-65, control: 0.448 ± 0.073, n = 6; MIA: 0.336 ± 0.027, n = 8, P = 0.13 unpaired t-test). (e and f) Colocalization analysis of VGluT1 and GAD-65 with YFP labeled apical tuft dendrites and spines shown in (a) and (b). Arrowheads and arrows denote colocalization of a punctum with a dendritic shaft or spine respectively. Red and blue arrows mark colocalization with GAD-65 and VGluT1 puncta respectively. A yellow arrow marks a dendritic spine in a putative contact with both GAD-65 and VGluT1 puncta. (g) High magnification images of colocalization of presynaptic puncta on dendritic spines showing examples of spines with VGluT1, GAD-65 and VGluT1 + GAD-65 puncta. (h) Quantification of percent spines contacted by puncta. A non-significant increase in spines contacted by VGluT1, VGluT2 or GAD-65 in the MIA offspring (VGluT1, control: 30.24 ± 2.1%, n = 9; MIA: 39 ± 3.5%, n = 9, P = 0.048; VGluT2, control: 25.46 ± 2.84%, n = 6; MIA: 23.92 ± 4.66%, n = 6, P = 0.78 and GAD-65, control: 13.02 ± 1.47%, n = 11, MIA: 20.4 ± 2.56%, n = 12, P = 0.024). Quantification of percent of dendritic spines in putative contact with both VGluT1 and a GAD-65 puncta demonstrates an increase in MIA offspring (control: 7.15 ± 1.1%, n = 9; MIA: 14.53 ± 1.78%, n = 9, P = 0.003). Multiple t-tests with Sidak–Bonferroni corrections were used and only the VGluT1 + GAD category survived the correction. Data are represented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

Altered cortical excitatory and inhibitory synaptic transmission in the MIA offspring. (a) mEPSCs recorded from layer 2 pyramidal neurons in somatosensory cortex from control and MIA offspring. (b) Reduced mEPSCs frequency (control (black): 1.22 ± 0.05 Hz, n = 9 mice, 28 cells; MIA (red): 0.90 ± 0.1 Hz, n = 8 mice, 32 cells, P = 0.007) with no difference in the amplitude of mEPSC (control: 15.72 ± 0.44 pA, MIA: 16.91 ± 0.51 pA, P = 0.097) was observed in the MIA offspring. (c) mIPSCs recorded from layer 2 pyramidal neurons in somatosensory cortex from control and MIA offspring. (d) No difference in mIPSCs frequency (control: 3.82 ± 0.26 Hz, n = 12 mice, 41 cells; MIA: 3.33 ± 0.29 Hz, n = 9 mice, 35 cells, P = 0.226) and increased mIPSC amplitude (control: 36.51 ± 1.82 pA, MIA: 42.25 ± 1.93 pA, P = 0.046) was observed in MIA offspring. Data are represented as mean ± SEM. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 5

Postnatal treatment with an anti-inflammatory drug ibudilast, prevents spine loss in young mice. (a) Layer 5 pyramidal neuron apical tuft dendrites from P17 control + vehicle, control + ibudilast, MIA + vehicle and MIA + ibudilast offspring mice treated during the first 2 postnatal weeks. (b) Two-way ANOVA was first used to test for the presence of interaction between drug treatment and experimental groups. This revealed an interaction consistent with the observation that ibudilast increased dendritic spine density in the MIA offspring but had no effect on the controls F(1, 20) = 6.296, P = 0.021. Post hoc two-way ANOVA with Bonferroni's correction confirm a reduction in spine density in MIA + vehicle offspring (control + vehicle: 0.89 ± 0.03 spines/micron, n = 6 mice; MIA + vehicle: 0.74 ± 0.01 spines/micron, n = 8, mice, P = 0.026) and that reduction is prevented by postnatal treatment with the anti-inflammatory drug ibudilast (MIA + ibudilast: 0.93 ± 0.05 spines/micron, n = 5 mice, MIA + vehicle: 0.74 ± 0.01 spines/micron, n = 8 mice, P = 0.007). No change in spine density in control + ibudilast offspring (control + vehicle: 0.89 ± 0.03 spines/micron, n = 6 mice; control + ibudilast: 0.90 ± 0.05 spines/micron, n = 5 mice, P > 0.9).

Fig. 6

Surface MHCI proteins are increased in vivo in MIA offspring. (a) Representative confocal images of cortical P17 control + vehicle, control + ibudilast, MIA + vehicle and MIA + ibudilast offspring, immunostained for sMHCI. (b) Quantification of surface MHCI. Two-way ANOVA was first used to test for the presence of interaction between drug treatment and experimental groups. This revealed an interaction consistent with the observation that ibudilast decreased sMHCI in the MIA offspring but had no effect on the controls (F(1, 28) = 6.779, P = 0.015). Post hoc two-way ANOVA with Bonferroni's correction confirms an increase in sMHCI in MIA + vehicle offspring (control + vehicle: 115 ± 12.2 a.u., n = 8; MIA + vehicle: 158.3 ± 9.3 a.u., n = 8, P = 0.01) but the increase is not completely prevented by the postnatal treatment with ibudilast (MIA + ibudilast: 130.9 ± 5.7 a.u., n = 8, MIA + vehicle: 158.3 ± 9.3 a.u., n = 8, P = 0.16).

Fig. 7

Dendritic spine impairments in MIA offspring persist into adulthood and are correlated with increased marble burying. (a) Layer 5 pyramidal neuron apical tuft dendrites from 3 month old control + vehicle, control + ibudilast, MIA + vehicle and MIA + ibudilast offspring mice treated during the first 2 postnatal weeks. (b) Two-way ANOVA was first used to test for the presence of interaction between drug treatment and experimental groups. This revealed an interaction consistent with the observation that ibudilast increased dendritic spine density in the MIA offspring but had no effect on the controls F(1, 19) = 4.62, P = 0.04. Post hoc two-way ANOVA with Bonferroni's correction indicates that a reduction in spine density in MIA + vehicle offspring persists into adulthood (control + vehicle: 0.85 ± 0.07 spines/micron, n = 6 mice; MIA + vehicle: 0.58 ± 0.05 spines/micron, n = 6 mice, P = 0.04) but is not prevented by postnatal treatment with the anti-inflammatory drug ibudilast (MIA + ibudilast: 0.77 ± 0.09 spines/micron, n = 6 mice, MIA + vehicle: 0.58 ± 0.05 spines/micron, n = 6 mice, P = 0.25). No changes in spines density in control + ibudilast offspring (control + vehicle: 0.85 ± 0.07 spines/micron, n = 6 mice; control + ibudilast: 0.77 ± 0.04 spines/micron, n = 5 mice, P > 0.9). (c) Marble burying is increased in MIA offspring and this increase is prevented by postnatal ibudilast treatment. There was a main effect of prenatal treatment, F(1,67) = 16.99, P = 0.0001, as well as a main effect of drug treatment, F(1,67) = 14.37, P = 0.0003 on marble burying but no significant interaction F(1,67) = 0.5, P = 0.48. Marble burying increased in MIA offspring (control + veh: 37.7 ± 3.3%, n = 18 mice from 4 litters; MIA + veh: 53.7 ± 2.7%, n = 18 mice from 4 litters). This increase is reduced in MIA offspring treated with ibudilast during the first 2 postnatal weeks (MIA + Ibud: 37 ± 2.9%, n = 17 from 4 litters). There was also a mild effect of ibudilast on control offspring (control + Ibud: 27.6 ± 3.8%, n = 17 from 4 litters). (d) The spine density observed at 3 months is inversely correlated with the percent of marbles buried at P60 (Pearson r = −0.49, n = 18, P = 0.048). Data are represented as mean ± SEM.