Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro

Application of Airy beam light sheet microscopy to examine early neurodevelopmental structures in 3D hiPSC-derived human cortical spheroids.

BACKGROUND: The inability to observe relevant biological processes in vivo significantly restricts human neurodevelopmental research. Advances in appropriate in vitro model systems, including patient-specific human brain organoids and human cortical spheroids (hCSs), offer a pragmatic solution to this issue. In particular, hCSs are an accessible method for generating homogenous organoids of dorsal telencephalic fate, which recapitulate key aspects of human corticogenesis, including the formation of neural rosettes-in vitro correlates of the neural tube. These neurogenic niches give rise to neural progenitors that subsequently differentiate into neurons. Studies differentiating induced pluripotent stem cells (hiPSCs) in 2D have linked atypical formation of neural rosettes with neurodevelopmental disorders such as autism spectrum conditions. Thus far, however, conventional methods of tissue preparation in this field limit the ability to image these structures in three-dimensions within intact hCS or other 3D preparations. To overcome this limitation, we have sought to optimise a methodological approach to process hCSs to maximise the utility of a novel Airy-beam light sheet microscope (ALSM) to acquire high resolution volumetric images of internal structures within hCS representative of early developmental time points. RESULTS: Conventional approaches to imaging hCS by confocal microscopy were limited in their ability to image effectively into intact spheroids. Conversely, volumetric acquisition by ALSM offered superior imaging through intact, non-clarified, in vitro tissues, in both speed and resolution when compared to conventional confocal imaging systems. Furthermore, optimised immunohistochemistry and optical clearing of hCSs afforded improved imaging at depth. This permitted visualization of the morphology of the inner lumen of neural rosettes. CONCLUSION: We present an optimized methodology that takes advantage of an ALSM system that can rapidly image intact 3D brain organoids at high resolution while retaining a large field of view. This imaging modality can be applied to both non-cleared and cleared in vitro human brain spheroids derived from hiPSCs for precise examination of their internal 3D structures. This process represents a rapid, highly efficient method to examine and quantify in 3D the formation of key structures required for the coordination of neurodevelopmental processes in both health and disease states. We posit that this approach would facilitate investigation of human neurodevelopmental processes in vitro

An Autism-Associated Variant of Epac2 Reveals a Role for Ras/Epac2 Signaling in Controlling Basal Dendrite Maintenance in Mice

The architecture of dendritic arbors determines circuit connectivity, receptive fields, and computational properties of neurons, and dendritic structure is impaired in several psychiatric disorders. While apical and basal dendritic compartments of pyramidal neurons are functionally specialized and differentially regulated, little is known about mechanisms that selectively maintain basal dendrites. Here we identified a role for the Ras/Epac2 pathway in maintaining basal dendrite complexity of cortical neurons. Epac2 is a guanine nucleotide exchange factor (GEF) for the Ras-like small GTPase Rap, and it is highly enriched in the adult mouse brain. We found that in vivo Epac2 knockdown in layer 2/3 cortical neurons via in utero electroporation reduced basal dendritic architecture, and that Epac2 knockdown in mature cortical neurons in vitro mimicked this effect. Overexpression of an Epac2 rare coding variant, found in human subjects diagnosed with autism, also impaired basal dendritic morphology. This mutation disrupted Epac2's interaction with Ras, and inhibition of Ras selectively interfered with basal dendrite maintenance. Finally, we observed that components of the Ras/Epac2/Rap pathway exhibited differential abundance in the basal versus apical dendritic compartments. These findings define a role for Epac2 in enabling crosstalk between Ras and Rap signaling in maintaining basal dendrite complexity, and exemplify how rare coding variants, in addition to their disease relevance, can provide insight into cellular mechanisms relevant for brain connectivity

Asymmetric distribution of Ras/Epac2/Rap/p-BRaf in cortical neuron dendrites.

<p>(A) Distribution of Epac2 in apical and basal dendrites. Endogenous MAP2 indicates dendrites. (B) Epac2 fluorescence intensity (normalized to unit area, µm², and indicated by pseudocoloring) is reduced in secondary basal dendrites. (C–F) Analysis of Ras (C–D) or Rap (E–F) distribution revealed that Ras and Rap are asymmetrically distributed over apical and basal dendrites. GFP outlines dendrite morphology. (G) Distribution of phosphorylated (active) BRaf in apical and basal dendrites. Endogenous MAP2 indicates dendrites. (H) p-BRaf levels are reduced in basal dendrites compared to apical dendrites. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; Scale bars, 35 µm (A, C, E, G), 5 µm (A, C, E, G insets).</p

Epac2 is required for selective maintenance of basal dendrite complexity and length.

<p>(A) Binary images of cultured cortical neurons (DIV 23–28) expressing GFP, GFP+Epac2-RNAi, or GFP+Epac2-RNAi+Epac2-rescue. (B) Epac2-RNAi selectively reduces complexity of basal arbors. Expression of RNAi-insensitive Epac2-rescue recovers Epac2-RNAi-induced reduction of basal dendrite complexity to control levels. (C) Epac2-RNAi reduces basal dendritic length, while Epac2-rescue prevents this effect. ‡, difference between Epac2-rescue and Epac2-RNAi, <i>p</i><0.05, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; scale bars, 100 µm.</p

Disruption of Epac2 interaction with Ras by point mutation impairs basal dendritic maintenance.

<p>(A) Domain structure of Epac2: cAMP-binding (cAMP); domain found in Dishevelled, Egl-10, and Pleckstrin (DEP); N-terminus of Ras-exchanger motif (REM); Ras-association (RA); Rap-Guanine Exchange Factor (Rap-GEF). Asterisk indicates position of Epac2-G706R rare variant. (B) Representative binary images of cortical neurons expressing GFP+Epac2-WT or GFP+Epac2-G706R. (C) Sholl analysis of apical and basal dendrites reveals a significant decrease in basal complexity in Epac2-G706R-expressing cells. (D) Quantification of apical and basal dendrite length. (E) Ras activation by GTPγs results in stronger coimmunoprecipitation of Epac2 with Ras in neurons, while inactivation of Ras by GDP reduces Ras-Epac2 interaction. (F) Quantification of (E). (G) Epac2-G706R point mutation exhibits impaired interaction with Ras in hEK293 cells. (H) Quantification of G. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; scale bar, 100 µm.</p

Comparison of paired cell morphology following in vivo knockdown of Epac2 by in utero electroporation reveals reduced basal arbors of layer 2/3 cortical neurons.

<p>(A) Left, image of paired cells, one positive for Epac2-RNAi, filled with biocytin and imaged by 2PLSM in 300 µm-thick cortical slices. Right, binary images of biocytin-filled paired layer 2/3 neurons to the left. Animals were electroporated at E16.5; cortical slices (300 µm) were made at P28. (B) Quantification of total dendrite numbers separated into apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites compared to paired control cells. (C) Epac2 knockdown in vivo selectively reduced basal dendrite length when compared to paired control cells. (D) “Skeleton outline” of basal and apical arbors, separated into primary, secondary, and tertiary branches, of neurons shown in (A). (E) Quantification of basal dendrite numbers reveals that Epac2 knockdown in vivo induces a loss of higher order branches compared to paired control; blue square, control; red circles, Epac2-RNAi; black squares with error bars, mean, SEM. (F) Analysis of basal dendrite length demonstrates a selective reduction in higher order basal length. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001; scale bars, 50 µm.</p

Epac2 regulates basal dendrite complexity in vivo.

<p>(A) Low magnification images of in utero electroporated layer 2/3 cortical neurons, expressing either control or Epac2-RNAi. Animals were electroporated at E16.5; coronal slices (50 µm) were made at P28. Red dashed rectangles indicate images represented in (B–D). (B) High magnification images of layer 2/3 cortical neurons outlined in (A). (C–D) Binary images (C) and “skeleton outline” of basal and apical arbors (D), separated into primary, secondary, and tertiary branches, of neurons shown in (B). (E) Quantification of dendrite numbers separated into total apical and basal dendrite branches. Epac2 knockdown in vivo induced a loss of basal dendrites. (F) Epac2 knockdown in vivo induced a selective reduction in basal dendrite length. (G) Quantification of dendrite branch numbers separated into basal/apical, primary/secondary/tertiary order branches. Epac2 knockdown in vivo induced a loss of basal dendrites, specifically driven by a loss of secondary and tertiary basal dendrites. (H) Epac2 knockdown in vivo selectively reduced secondary and tertiary basal dendrite lengths. *<i>p</i><0.05, ***<i>p</i><0.001; scale bars, 100 µm (A); 50 µm (B–D).</p

Asymmetric regulation of BRaf phosphorylation in cortical neuron dendrites.

<p>(A–C) Inhibition of Ras signaling by FTaseII (30 min, 200 nM) reduced p-BRaf immunofluorescence in basal dendrites compared to apical dendrites. Yellow boxes in (A) indicate areas in high magnification in (B). Endogenous MAP2 indicated dendrites. p-BRaf fluorescence intensity was normalized to unit area (µm²) and is indicated by pseudocoloring. White dashed lines outline dendrite. (C) Quantification of p-BRaf immunofluorescence in basal versus apical dendrites after 30 min FTaseII treatment relative to control apical levels (dashed gray line). (D–F) Inhibition of Epac2 function by Epac2-RNAi resulted in selective and robust reduction of p-BRaf immunofluorescence in basal dendrites compared to apical dendrites. Yellow dashed boxes in (D) indicate areas in high magnification in (E). Overexpressed GFP outlined dendrite morphology. p-BRaf fluorescence intensity was normalized to unit area (µm²) and is indicated by pseudocoloring. White dashed lines outline dendrite. (F) Quantification of p-BRaf immunofluorescence in basal versus apical dendrites in Epac2-RNAi-expressing cells relative to control apical levels (dashed gray line). **<i>p</i><0.01, ***<i>p</i><0.001 compared to control levels; #<i>p</i><0.001 compared to apical p-BRaf levels after treatment (C, F); scale bars, 35 µm (A, D), 5 µm (B, E).</p