Recent success in identifying gene-regulatory elements in the context of recombinant adeno-associated virus vectors has enabled cell-type-restricted gene expression. However, within the cerebral cortex these tools are largely limited to broad classes of neurons. To overcome this limitation, we developed a strategy that led to the identification of multiple new enhancers to target functionally distinct neuronal subtypes. By investigating the regulatory landscape of the disease gene Scn1a, we discovered enhancers selective for parvalbumin (PV) and vasoactive intestinal peptide-expressing interneurons. Demonstrating the functional utility of these elements, we show that the PV-specific enhancer allowed for the selective targeting and manipulation of these neurons across vertebrate species, including humans. Finally, we demonstrate that our selection method is generalizable and characterizes additional PV-specific enhancers with exquisite specificity within distinct brain regions. Altogether, these viral tools can be used for cell-type-specific circuit manipulation and hold considerable promise for use in therapeutic interventions.
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The data that support the findings of this study are available from the corresponding author upon reasonable request. The scATAC datasets presented in the present study are available on GEO with the accession no. GSE152449. All AAV plasmids and their corresponding sequences are available from Addgene (pAAV-S5E2-dTom-nls-dTom (Addgene, no. 135630), pAAV-S5E2-GFP-fGFP (Addgene, no. 135631), pAAV-S5E2-GCaMP6f (Addgene, no. 135632), pAAV-S5E2-C1V1–eYFP (Addgene, no. 135633), pAAV-S5E2-ChR2-mCherry (Addgene, no. 135634), pAAV-S5E2-Gq-P2A-dTomato-short (Addgene, no. 135635), pAAV-S5E1-dTom-nlsdTom (Addgene, no. 135637), pAAV-S5E3-dTom-nlsdTom (Addgene, no. 135638), pAAV-S5E4-dTom-nls-dTom (Addgene, no. 135639), pAAV-S5E5-dTom-nlsdTom (Addgene, no. 135640), pAAV-S5E6-dTom-nlsdTom (Addgene, no. 135641), pAAV-S5E7-dTom-nls-dTom (Addgene, no. 135642), pAAV-S5E8-dTom-nlsdTom (Addgene, no. 135643), 018_pAAV-S5E9-dTom-nls-dTom (Addgene, no. 135644), pAAV-S5E10-dTom-nlsdTom (Addgene, no. 135645), pAAV-E11-ChR2GFP2x (Addgene, no. 153434), pAAV-E14-ChR2GFP2x (Addgene, no. 153435), pAAV-E22-ChR2GFP2x (Addgene, no. 153436) and pAAV-E29-ChR2GFP2x (Addgene, no. 153437)).
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We thank S. Gerard, P. Delvenne and C. Parotte for their useful comments on the manuscript. J.D. is supported by NIH grants (nos. R01-MH111529 and UG3MH120096) as well as the Simons Foundation Award (no. 566615) and a gift from the Friends-Of-FACES foundation. T.P.F. is supported by fellowships from the Belgian American Educational Foundation and the George E. Hewitt Foundation for Medical Research, an NARSAD Young Investigator grant from the Brain and Behavior Research Foundation, and a Kavli Institute for Brain and Mind Innovative research grant. C.J.M. was awarded an Intramural Research Program of NINDS and a Eunice Kennedy Shriver NICHD Intramural grant. J. Smith is supported by the NINDS (grant no. K99 NS106528). G. Fishell is supported by the NINDS (grant nos. NS081297, NS074972), NIMH (grant no. MH071679) and NIH (grant no. UG3MH120096), and the Harvard’s Dean Initiative, as well as support from the Simons Foundation award (no. 566615). We thank the individuals who selflessly volunteered to participate in this study.
The Broad Institute of MIT and Harvard has filed patent applications related to this work with G. Fishell and J.D. listed as inventors.
Peer review information Nature Neuroscience thanks Z. Josh Huang, Botond Roska and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, UMAP plot of 3500 neuronal nuclei collected from 4 Dlx6a-cre::Sun1-eGFP mice showing promoter accessibility of the indicated canonical interneuron markers. b, Fluorescent images of sagittal sections from adult mice that were injected systemically with the indicated rAAV-E[x]-dTom and analyzed 3 weeks post-injection with IHC for the viral reporter. Scale bar for left panels represents 500um; Scale bar for right panels represents 100um. See method section for details on the reproducibility of the representative images presented in panel b.
Adult mice were injected systemically with rAAV-E2-dTomato. a, b, Slice recording of the intrinsic properties of virally labeled neurons in S1 cortex and PFC. The left panels show plots of recorded cells with the indicated intrinsic properties. The blue dots represent cells with stereotypical fast-spiking properties. The right panels indicate the proportion of fast spiking cells recorded. c, Representative slice recording traces of cells indicated in (b). (d) Representative image of virally labeled chandelier cells. (e) Coronal and sagittal sections were analyzed with IHC for the viral reporter and PV, and the specificity to PV was reported across brain regions. (f) The native viral expression was analyzed from the indicated organs. Scale bars represent 50um (d), 100um (e) and 250um (f). On the graphs, dots represent individual measurements and the lines represent average + /- s.e.m. Values for specificity are listed in the supplementary table 2. See method section for details on the reproducibility of the representative images presented in panel d and f.
Mice were injected locally with the following constructs (a - P14 injection with rAAV-E2-GCaMP6f; b – rAAV-E2-C1V1-eYFP; c – rAAV-E2-GqDREADD). (a) Mice were analyzed 1-week post-injection. The left panel shows widefield images of two representative peaks shown by the pound signs in Fig. 3. The right panel shows a fluorescent image taken after GCaMP recordings. (b) Slice electrophysiology current clamp recordings were performed 1-week post-injection. Cells expressing the viral reporter were targeted with either 10 Hz or 40 Hz laser stimulation (550 nm) while the voltage was recorded over 3 seconds. (c) Slice electrophysiology current clamp recordings were performed 1-week post-injection. The voltage was recorded before and after bath application of CNO. Scale bars represent 500um. The red bars represent laser stimulation. On the graphs, dots represent individual measurements. c right panel: p-value = 0.0039; t = 3.859; df = 9; c right panel: p-value = 0.0258; t = 3.135; df = 5); *, ** and *** correspond to a p-value < 0.01, <0,001 and < 0,0001 respectively. See method section for details on the reproducibility of the representative images presented in panel a.
a, Human brain tissue obtained from surgical resection that was exposed to rAAV-E2-dTomato and maintained in culture for 7–14 days. Left - Representative image of the dendrites of virally labeled cells filled with Biocytin during the recording session. Right - Slice recording of the intrinsic properties of virally labeled neurons. The quantifications show the indicated parameters. Scale bars represent 100um for the left images and 2um for the right images. b, Adult mice were injected with the indicated modified rAAV-E2-dTomato construct and analyzed 3 weeks post-injection with IHC for the viral reporter and PV. The corresponding specificity is shown in the right panel. Scale bars represent 100um. On the graphs, dots represent individual measurements and the lines represent average + /- s.e.m. Values for specificity are listed in the supplementary table 2.
Adult macaques were injected locally in the prefrontal or S1 cortex with the indicated rAAV-E[x]-eGFP and analyzed 8 weeks post-injection with immunohistochemistry for the reporter and indicated markers. The right panels display the injection sites (above), and the boxed quantified regions (below). The corresponding sensitivity is shown in the bottom left. Scale bars represent 25um (lower panels) and 50um (upper panels). On the graphs, dots represent individual measurements and the lines represent average + /- s.e.m. Values for sensitivity are listed in the supplementary table 2.
Each panel shows the indicated enhancer sequence displayed on a fixed region of 750 bp, where each block of the lower part of the left graph shows conserved (dark gray) and non-conserved (light gray) regions of the enhancer. On the upper part of the left graph, each trace shows an individual transcription factor binding site mapped using CiiiDER (see methods). The blue traces represent TFBS found only in mice and the orange traces represent the TFBS found both in mice and humans. The two bar charts show the proportion of the TFBS found either on conserved or non-conserved regions of the enhancer, for all TFBS and for the subset of conserved TFBS, respectively.
Supplementary Table 1. Enhancer selection: table listing all the candidate enhancers tested in this study and their corresponding features. Associated gene: gene closest to the candidate enhancer; Target population: neuronal population targeted by the candidate enhancer. Specificity for target population: percentage of virally labeled cells co-expressing a marker for the corresponding target population. Location: genomic location of the candidate enhancer in the mouse reference genome (mm10) expressed as chromosome/start/stop; Presence of ATAC peaks: presence of an ATAC-seq peak in any one of the five neuronal populations included in the study (derived from the scATAC-seq data obtained for PV, SST, VIP and ID2 and bulk ATAC-seq for excitatory neurons from the Mo et al.21); Conservation with human sequence: percentage of identical base-pairs between the mouse sequence and the corresponding human sequence identified by local alignment using the EMBOSS Needle pairwise sequence alignment. See Methods for further details on each feature. Supplementary Table 2. Quantification and statistics: table containing the metadata associated with each of the quantification plot presented in the figures.
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Vormstein-Schneider, D., Lin, J.D., Pelkey, K.A. et al. Viral manipulation of functionally distinct interneurons in mice, non-human primates and humans. Nat Neurosci 23, 1629–1636 (2020). https://doi.org/10.1038/s41593-020-0692-9
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