Abstract
Transcript labeling in intact tissues using in situ hybridization chain reaction has potential to provide vital spatiotemporal information for molecular characterization of heterogeneous neuronal populations. However, large tissue labeling in non-perfused or fresh-frozen rodent and postmortem human samples, which provide more flexible utilization than perfused tissues, is largely unexplored. In the present study, we optimized the combination of in situ hybridization chain reaction in fresh-frozen rodent brains and then evaluated the uniformity of neuronal labeling between two clearing methods, CLARITY and iDISCO+. We found that CLARITY yielded higher signal-to-noise ratios but more limited imaging depth and required longer clearing times, whereas, iDISCO+ resulted in better tissue clearing, greater imaging depth and a more uniform labeling of larger samples. Based on these results, we used iDISCO+-cleared fresh-frozen rodent brains to further validate this combination and map the expression of a few genes of interest pertaining to mood disorders. We then examined the potential of in situ hybridization chain reaction to label transcripts in cleared postmortem human brain tissues. The combination failed to produce adequate mRNA labeling in postmortem human cortical slices but produced visually adequate labeling in the cerebellum tissues. We next, investigated the multiplexing ability of in situ hybridization chain reaction in cleared tissues which revealed inconsistent fluorescence output depending upon the fluorophore conjugated to the hairpins. Finally, we applied our optimized protocol to assess the effect of glucocorticoid receptor overexpression on basal somatostatin expression in the mouse cortex. The constitutive glucocorticoid receptor overexpression resulted in lower number density of somatostatin-expressing neurons compared to wild type. Overall, the combination of in situ hybridization chain reaction with clearing methods, especially iDISCO+, may find broad application in the transcript analysis in rodent studies, but its limited use in postmortem human tissues can be improved by further optimizations.
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Acknowledgements
This work was supported by the Pritzker Neuropsychiatric Research Consortium, the Hope for Depression Research Foundation, National Institute of Health R01MH104261, Office of Naval Research Grant N00014-12-1-0366 and National Institute on Drug Abuse U01DA043098. The authors have no conflicts of interest to declare. The authors would like to thank Ms. Jennifer Fitzpatrick, Mr. Evan Hughes, Ms. Claire Barcelo, and Mr. Hui Li for their technical assistance.
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V.K., D.M.K., E.K.H., B.M., M.F., H.A., and S.J.W. are members of the Pritzker Neuropsychiatric Research Consortium, which is supported by the Pritzker Neuropsychiatric Disorders Research Fund, LLC (Fund). There exists a shared intellectual property agreement between the academic and philanthropic entities of the Consortium. The Fund has no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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“All animal care and procedures performed in studies involving animals were in accordance with the ethical standards of the guide for the Care and Use of Laboratory Animals: Eighth Edition (revised in 2011, published by the National Academy of Sciences), and approved by the University of Michigan Committee on the Use and Care of Animals.“This article does not contain any studies with human participants performed by any of the authors.”
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Supplementary file1 (M4V 11080 KB) Supplemental movie ESM_1. 3D visualization of differences in the HCR-FISH labeled Sst signal between CLARITY (left panel) and iDISCO+ (right panel) processed mouse cortical slices. First half of this movie shows a series of xy-plane images moving in z-direction of confocal acquired representative stacks, followed by maximum intensity projection (mip) 3D pre-quantitation volume and post-quantitation surface rendering of Sst neurons. Second half of the movie shows a series of xz-plane images moving in y-direction, followed by mip 3D pre-quantitation volume and post-quantitation surface renderings of Sst neurons. This animation highlights the higher number density of detected Sst neurons and uniform labeling across the z-plane in iDISCO+ compared to CLARITY.
Supplementary file2 (M4V 43589 KB) Supplemental movie ESM_2. 3D visualization of HCR FISH-labeled Sst expressing neurons in a rat forebrain hemi-slice cleared using iDISCO+. This movie first shows a series of xy-plane images acquired on a COLM followed by a mip 3D volume rendering. The characteristic layer-specific expression pattern of Sst in rat cortex can be observed in this movie.
Supplementary file3 (M4V 8041 KB) Supplemental movie ESM_3. Volume rendering of a rat hippocampus showing Sst expression pattern in the iDISCO+ cleared intact tissue and labeled using HCR FISH method. This movie shows a series of xy-plane images acquired on a COLM followed by 3D mip volume rendering.
Supplementary file4 (M4V 38281 KB) Supplemental movie ESM_4. 3D visualization of a mouse left hemisphere showing Sst expressing neurons across the entire rostro-caudal extent of the brain. Post HCR FISH labeling, brain tissue was cleared using the iDISCO+ method and imaging was performed on a COLM.
Supplementary file5 (MP4 108109 KB) Supplemental movie ESM_5. Sst expression in an iDISCO+ cleared sample of a mouse left hemi-brainstem. COLM-acquired images are first rendered as a series of xy-planes progressing in the medio-lateral direction and subsequently as mip 3D volume rendering.
Supplementary file6 (M4V 79471 KB) Supplemental movie ESM_6. Volume rendering of HCR FISH-labeled Pvalb expressing neurons in a rat forebrain hemi-slice. This representative COLM-acquired image stack of an iDISCO+-cleared brain shows the predominant cortical expression of Pvalb.
Supplementary file7 (MP4 94155 KB) Supplemental movie ESM_7. 3D visualization of HCR FISH-labeled Pvalb expressing neurons in a rat brainstem hemi-slice showing the expression pattern in cerebellum and several nuclei. iDISCO+-cleared and COLM-acquired image stacks are first projected as a series of xy-planes moving in a rostral-caudal-rostral direction, then as a mip 3D volume rendering.
Supplementary file8 (MP4 18243 KB) Supplemental movie ESM_8. 3D visualization of an iDISCO+ cleared rat mid-brain slice showing the expression of Th mRNA in the SNr/c, VTA, PBP and PAG regions. This movie first shows the rostro-caudal progression of a series of xy-plane images acquired on the COLM followed by a mip 3D volume rendering.
Supplementary file9 (M4V 22808 KB) Supplemental movie ESM_9. Dbh expressing neurons in the rat locus coeruleus are shown here first as a series of xy-plane images acquired on a confocal microscope followed by a mip 3D volume. This representative brainstem hemi-slice was processed through HCR FISH and cleared using the iDISCO+ method.
Supplementary file10 (M4V 6137 KB) Supplemental movie ESM_10. 3D visualization of HCR FISH-labeled CALB expressing neurons in the postmortem human cerebellum block. Confocal microscope-acquired image stacks are rendered first as a series of xy-plane images followed by a mip 3D volume rendering.
Supplementary file11 (M4V 13962 KB) Supplemental movie ESM_11. PVALB expressing neurons in the postmortem human cerebellum block are visualized in this movie as a series of xy-plane images then by a mip 3D volume. This representative block was cleared using iDISCO+ and images were acquired on a confocal microscope.
Supplementary file12 (MP4 10459 KB) Supplemental movie ESM_12. 3D visualization of COLM acquired dual HCR FISH labeling in iDISCO+ cleared mouse hemisphere. Movie shows labeling of Pvalb (green, AF-647) and Vglut1 (red, AF-594) mRNAs in a series of dorso-ventral planes from a down-sampled image volume. Left pane shows progression of z-planes from an intact hemisphere whereas right pane shows a zoomed-in view of ventral hippocampus and cortex (regions of lateral entorhinal cortex) whose z-plane progression is synced anatomically with the whole hemisphere in the left pane.
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Kumar, V., Krolewski, D.M., Hebda-Bauer, E.K. et al. Optimization and evaluation of fluorescence in situ hybridization chain reaction in cleared fresh-frozen brain tissues. Brain Struct Funct 226, 481–499 (2021). https://doi.org/10.1007/s00429-020-02194-4
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DOI: https://doi.org/10.1007/s00429-020-02194-4