Maternal inflammation has a profound effect on cortical interneuron development in a stage and subtype-specific manner

Abstract

Severe infections during pregnancy are one of the major risk factors for cognitive impairment in the offspring. It has been suggested that maternal inflammation leads to dysfunction of cortical GABAergic interneurons that in turn underlies cognitive impairment of the affected offspring. However, the evidence comes largely from studies of adult or mature brains and how the impairment of inhibitory circuits arises upon maternal inflammation is unknown. Here we show that maternal inflammation affects multiple steps of cortical GABAergic interneuron development, i.e., proliferation of precursor cells, migration and positioning of neuroblasts, as well as neuronal maturation. Importantly, the development of distinct subtypes of cortical GABAergic interneurons was discretely impaired as a result of maternal inflammation. This translated into a reduction in cell numbers, redistribution across cortical regions and layers, and changes in morphology and cellular properties. Furthermore, selective vulnerability of GABAergic interneuron subtypes was associated with the stage of brain development. Thus, we propose that maternally derived insults have developmental stage-dependent effects, which contribute to the complex etiology of cognitive impairment in the affected offspring.

Fig. 1

Poly I:C induces rapid upregulation of IL-6 in maternal plasma and behavioral impairments in maternal inflammation-affected offspring. a Upregulation of IL-6 protein in maternal plasma 3 h after i.v. injection of poly I:C (n = 8 and 9 for PBS and poly I:C respectively, 5 wild-type and 3 and 4 GAD67-EGFP mice, respectively, labeled by filled and green circles/squares, respectively). Prenatal exposure to maternal inflammation results in impaired social zone preference (b) (n = 12 each), as well as a decrease in time spent in social zone (c) (F = 3.23). Mice exposed to maternal inflammation during gestation present signs of hyperactivity as seen by total distance traveled (d) (n = 12 each) and speed of running over a 10 min period (e) (n = 12 each). f Maternal inflammation during early gestation induces schizotypal effects in offspring that can be gauged by a decreased prepulse inhibition (at 72 dB) (n = 15 and 9 for PBS and poly I:C, respectively). Comparison of means by Student’s t-test in a, d, and f, t-test with Holm-Sidak correction for multiple comparison in b and two-way ANOVA in c and, e (mean ± SEM are shown, *p < 0.05, **p < 0.005)

Fig. 2

Early migratory deficits of GAD-EGFP+ neuroblasts due to maternal inflammation. a A schematic overview of the migratory routes taken by GAD+ neuroblasts during embryonic development [57, 58]. b, c Reduction in the density of EGFP+ neuroblasts migrating into the cortical intermediate zone at E14.5 as a result of maternal inflammation. Panel b shows somatosensory region of the developing cortex. Dotted box denotes the region of counting. Quantifications from the dotted boxes are shown in c (motor cortex: n = 8 each; somatosensory cortex: n = 8 each). d, e Superficial regions of the cortical column show a reduction in density of EGFP+ neuroblasts due to maternal inflammation. Cortical columns from developing motor (d) and somatosensory (d’) cortical regions were divided into ten equal-sized bins and the number of EGFP+ cells counted in each (with the exception of bin 1). Bin 10 represents the upper margin of the IZ. Densities in the graphs (e) show a reduction in bins 3–4. f Embryos exposed to maternal inflammation showed a decreased total density of migratory neuroblasts at E17.5. EGFP+ cells were counted in the cortical plate and intermediate zone across bins 2–10 (n = 10 each). g Radar plot showing that a greater percentage of neuroblasts in the cortical plate migrate laterally due to maternal inflammation as assessed by the angle of leading process. Angles were grouped into 30° bins and relative percentages were plotted. Each spoke represents 10%. Comparison of means by Student’s t-test in c, and f, t-test with Holm–Sidak correction for multiple comparison in e (mean ± SEM are shown, *p < 0.05, **p < 0.005). Scale bars: 50 µm

Fig. 3

Maternal inflammation causes large-scale impairment in the distribution of GAD-EGFP+ neuroblasts and mature interneurons in the cortex. a–c Graphs showing a decrease in density of EGFP+ cells measured by counting EGFP+ cells in ten equally sized bins in the cortical plate of the presumptive motor and somatosensory cortices. Dotted lines represent the margins of IZ, CP, and MZ, respectively (P3 motor: F = 21.85, P3 somatosensory: F = 24.1, n = 8, 7 for PBS and poly I:C respectively; P6 motor: F = 21.72, P6 somatosensory: F = 19.59, n = 8, 8 for PBS and poly I:C respectively; P9 motor: F = 12.22, and P9 somatosensory: F = 13.77, n = 8, 10 for PBS and poly I:C respectively). d–h Significant reduction in the density of interneurons in the somatosensory but not in the motor cortex at P15. EGFP+ cells were quantified in the total cortical thickness as well as in each cortical layer. Panels in d show representative images of regions used for quantification. Graphs show the total (e, f) (n = 8 and 7 for PBS and poly I:C, respectively) as well as the layer-wise (g, h) reduction in EGFP+ cell density. Comparison of means by two-way ANOVA in a–c, Student’s t-test in e and f, and t-test with Holm–Sidak correction for multiple comparison in g and h) (mean ± SEM are shown, ***p < 0.0005). Scale bars: 50 µm (d)

Fig. 4

Interneuron subtype-specific effects of maternal inflammation. a–f Maternal inflammation resulted in a decreased density of PV and SST expressing interneurons at P15 but not at P60. Panels show representative images from P15 (a) and P60 (b). A significant decrease in PV+ interneurons could be seen in L4, while the difference was more pronounced in L2-4 for SST+ interneurons (c, d) (n = 8 and 7 for PBS and poly I:C, respectively). An appreciable but not statistically significant decrease could be seen at P60 (e, f). g–j Reciprocal changes in PV expression between P15 and P60 due to maternal inflammation. At P15, PV+ interneurons in both motor, and somatosensory cortical regions in maternal inflammation-exposed animals showed an increased expression of PV (h) (n = 8 and 7 for PBS and poly I:C, respectively) measured by the signal intensity in immunolabeled images (g). By P60 (i), this effect had reversed and instead a decreased expression was observed in both motor and somatosensory cortical regions (j) (n = 6 each). Statistical analysis by t-test with Holm–Sidak correction for multiple comparison in c–f, h, and j (mean ± SEM are shown, *p < 0.05, **p < 0.005, ***p < 0.0005). Scale bars: 50 µm (a, b)

Fig. 5

Acute and developmental stage-dependent impairment of progenitor proliferation due to maternal inflammation. a–c Maternal inflammation had acute effect on proliferation of MGE-derived Nkx2.1+ progenitors. Dams were injected with poly I:C at E9.5 and embryos assessed at E10.5 after a 2-h BrdU pulse-chase (a, b). Quantification of proliferating Nkx2.1+ BrdU+ cells showed that maternal inflammation resulted in reduction in Nkx2.1+ proliferating cells at E10.5 (b) (n = 9 and 8 for PBS and poly I:C, respectively) that did not sustain until E14.5 (c). COUP-TFII+ progenitors remained unaffected when studied at E14.5 (d). e–g Dams were injected with poly I:C at E12.5 and embryos assessed at E14.5 after a 2-h BrdU pulse-chase. Proliferation of CGE-derived COUP-TFII+ progenitors was reduced after maternal inflammation at E12.5 (e, g) (n = 9 and 8 for PBS and poly I:C, respectively) that correlated with the later neurogenic window of CGE-derived progenitors. In contrast, proliferation of Nkx2.1+ MGE-derived progenitors was unaffected at E14.5 (f). h–j Late maternal inflammation at E16.5 led to an increase in proliferation of both COUP-TFII+ (h, j) and Nkx2.1+ (i) progenitors (n = 8–9 for each PBS and Poly I:C). k Schematic representation of the findings in this manuscript. Maternal inflammation affects interneuron development at multiple stages such as proliferation, migration, differentiation and maturation leading to increased vulnerability to mental disorders. Central artwork was modified from [89]. Comparison of means by Student’s t-test in b–d, f, g, I, and j)(mean ± SEM are shown, *p < 0.05, **p < 0.005, ***p < 0.0005). Scale bars: 50 µm (a, e, h)