Enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits

Abstract

We present an enhancer AAV toolbox for accessing and perturbing striatal cell types and circuits. Best-in-class vectors were curated for accessing major striatal neuron populations including medium spiny neurons (MSNs), direct and indirect pathway MSNs, as well as Sst-Chodl, Pvalb-Pthlh, and cholinergic interneurons. Specificity was evaluated by multiple modes of molecular validation, three different routes of virus delivery, and with diverse transgene cargos. Importantly, we provide detailed information necessary to achieve reliable cell type specific labeling under different experimental contexts. We demonstrate direct pathway circuit-selective optogenetic perturbation of behavior and multiplex labeling of striatal interneuron types for targeted analysis of cellular features. Lastly, we show conserved in vivo activity for exemplary MSN enhancers in rat and macaque. This collection of striatal enhancer AAVs offers greater versatility compared to available transgenic lines and can readily be applied for cell type and circuit studies in diverse mammalian species beyond the mouse model.

Keywords: Addgene; Adeno-associated virus; Medium spiny neuron; Pvalb; Sst-chodl; basal ganglia; caudoputamen; cholinergic; macaque; spiny projection neuron; striatonigral; striatopallidal.

Figure 1.. Pipeline for the discovery, validation, and distribution of striatal enhancer AAVs.

A) Diagram of enhancer selection process. Isolated peaks are chosen based on proximity to marker genes from multiple different ATAC-seq datasets. B) Putative enhancer sequences were screened for cell type specific activity by retro-orbital injection using SYFP2 native fluorescence. Example shown is of plasmid AiP12237 that contains the D2 MSN enhancer AiE0452h driving expression of SYFP2. C) The most promisng “Best-in-Class” enhancers were further validated for on-target activity by comparing mRNA and protein expression across multiple techniques. D) Striatal enhancer AAV characterization data is publicly available through the Allen Institute for Brain Science Genetic Tools Atlas (https://portal.brain-map.org/genetic-tools/genetic-tools-atlas). Enhancer AAV plasmid DNA and select virus aliquots are available from Addgene for distribution to the research community.

Figure 2.. Putative enhancers can drive cell type specific expression in the striatum.

A) Heatmap of median transcript detection log2(CPM+1) of striatal cell type marker genes from CCN20230722 mouse whole brain taxonomy cluster matrix. Colored boxes group marker genes by corresponding cell type common name. B) Sankey diagram with three nodes indicating the sources for all putative enhancer peaks for each cell target, their predicted expression specificities, and their validated expression. Values indicate number of enhancers at each node. C-D) Putative enhancer peaks selected for striatal cell types from BOCA (C) and CATlas (D) datasets. Tracks are grouped by brain region for BOCA (PMC: primary motor cortex, PVC: primary visual cortex, AMY: amygdala, HIPP: hippocampus, MDT: medio -dorsal thalamus, NAC: nucleus accumbens, PUT: putamen) and by CCN20230722 whole mouse brain taxonomy designated cell subclass for CATlas. The marker gene each peak was found proximal to is noted above each peak (N/A indicates peak was not found near any known marker gene). Enhancer peaks are grouped by target (gold: D1 MSN, turquoise: D2 MSN, green: pan-MSN, pink: cholinergic interneurons, dark blue: Sst-Chodl interneurons. Maroon: Pvalb-Pthlh interneurons. E) Top: diagram of expression patterns indicative of striatal MSN cell populations. Middle: example sagittal sect ions of native SYFP2 expression from enhancer AAVs for each indicated MSN population. Bottom: zoomed in views of axonal projection patterns in basal ganglia target regions from the same enhancer AAV as middle panel (GPi: globus pallidus internal segment, GPe: globus pallidus external segment, SNr: substantia nigra pars reticulata). F) Left: diagram of expression patterns indicative of striatal interneuron populations. Right: example sagittal section of native SYFP2 from enhancer AAV with interneuron expression selectivity. G) Example of morphological differences for distinguishing striatal interneuron populations. Note large size of cholinergic interneuron somata and thick dendrite caliber, elongated or bipolar Sst-Chodl interneurons with thin dendrite caliber, and compact dendrites of multipolar Pvalb-Pthlh interneurons.

Figure 3.. Striatal enhancer AAVs demonstrate significant improvement in cell type specificity over existing tools.

A) Diagram of triple modality enhancer AAV validation process including scRNA -seq (Smart-seq V4, SSv4), immunohistochemistry (IHC), and mFISH (RNAscope). B) Enhancer specificities determined by SSv4 scRNA -seq (n=1–2 mice and on average 50 cells per enhancer). Sequenced transcriptomes were mapped against the 10X Whole Mouse Brain taxonomy CCN20230722 using hierarchical mapping algorithm in MapMyCells (Allen Brain Map). Cell types in dendrogram encompass all neuronal basal ganglia subclasses from the taxonomy , with the six cell types targeted in this manuscript highlighted in color. C) Quantification of on-target enhancer AAV specificities across all three validation methods. Black boxes indicate no data and stars indicate 3 of the top enhancers. D -E) Top: example sagittal sections of hChATp (D) or AiE0743m_3xC2 (E) driven native SYFP2 fluorescence. Bottom: Identification of striatal cholinergic interneurons by IHC using ChAT subclass marker antibody. Each panel is a representative ROI from three different brain regions. F) RNAscope using probes against SYFP2 for three versions of the AiE0779m D1 MSN enhancer (FL=full length enhancer, 3xC2=optimized enhancer with 3x core region, and 6×C2=optimized enhancer with 6x core region). G) Quantification of completeness of labeling D1 MSNs from RNAscope image. Fisher’s exact test revealed differences in proportions of SYFP2-positive cells between AiE0779m enhancer versions (p < 0.0001). Post-hoc pairwise comparisons with Bonferroni correction showed significant differences between all enhancers (***p <0.001). H) SYFP2 signal intensity per cell from RNAscope images. The Kruskal-Wallis test revealed a significant difference in SYFP2 intensity across enhancers (χ² = 424.39, df = 2, p < 0.0001). Post-hoc Dunn’s test with Bonferroni correction showed that all pairwise comparisons were significant: 3xC2 vs. 6xC2 (p < 0.0001), 3xC2 vs. FL (p < 0.0001), and 6xC2 vs. FL (p < 0.0001). n=1–2 mice and 1 ROI per enhancer version for RNAscope quantification.

Figure 4.. Specificity of labeling for striatal enhancer AAVs across different delivery routes and cargos.

A) Diagram of enhancer AAV vector build and range of recommended AAV dosages across stereotaxic (STX), intracerebroventricular (ICV), and retro-orbital (RO) routes of administration in mice. Note ICV delivery is in P1-P2 pups while STX and RO are in adult mice. B-E) Representative sagittal sections of native SYFP2 fluorescence across all three delivery routes for four different enhancers targeting B-C) D1 MSNs, D) D2 MSNs, and E) cholinergic interneurons. F) Summary of D1 and D2 MSN enhancer and interneuron enhancer specificities by route of administration. Each point represents a single enhancer. A Kruskal-Wallis test revealed no significant difference in specificity across routes for the MSN enhancers (p = 0.7963), while a significant difference was observed for interneuron enhancers (p = 0.04383). Post-hoc Dunn’s test with Bonferroni correction indicated that the RO route differed significantly from the STX route (p < 0.05), but no significant difference was found between ICV and other routes. G) Heatmap of on-target specificities for striatal enhancers for the three routes of administration determined by scRNA-seq and mapping (D1 and D2 MSN enhancers) or IHC with cell type marker antibodies (interneuron enhancers). H) Images of IHC comparing two cholinergic enhancers AiE0873m_3xC2 and AiE0743m_3xC2 by STX injection. White arrows show colocalization of SYFP2 and Chat. White circles in the AiE0873m_3xC2 images demonstrate off-target SYFP2 expression in MSNs. I) Diagram of enhancer AAV vector build for delivery of functional transgenes by STX injection into dorsal striatum. J) Representative sagittal images of native fluorescence for enhancer AAVs driving functional transgenes. Left: D1 MSN enhancer AiE0779m_3xC2 driving ChR2(CRC)-EYFP (AiP13278). Middle: D2 MSN enhancer AiE0452h_3xC2 driving CoChR-EGFP (AiP14035). Right: D2 MSN enhancer AiE0452h_3xC2 driving jGCaMP8m (AiP14134).

Figure 5.. Comparison of D1 MSN and D2 MSN enhancer AAVs to GENSAT Cre driver lines.

A) Diagram of breeding cross for generating conditional reporter transgenic mice by Cre/loxP recombination. B) Diagram of strategy for generating conditional reporter transgene expression using enhancer AAVs. C-D) ISH from www.gensat.org of GENSAT EY217 Drd1a-Cre driver line crossed to Rosa26-EGFP reporter line with C) cell body expression in striatum (STR) and islands of calleja in olfactory tubercle (OT) and D) projections to substantia nigra pars reticulata (SNr) with extra-striatal expression observed in cortex. E-F) STPT images of D1 MSN enhancer AiE0779m driving iCre recombinase point mutant R297T (AiP15260) injected into Ai14 reporter mouse line with E) cell body expression in STR but not islands of calleja of the olfactory tubercle (OT) and F) projections to SNr. G-H) ISH from www.gensat.org of GENSAT ER44 Drd2-Cre driver line crossed to Rosa26-EGFP reporter line with G) cell body expression in STR but not islands of calleja in OT and H) extra-striatal dopamine neuron expression in ventral tegmental area (VTA) and SNr. I-J) STPT images of D2 MSN enhancer AiE0452h driving iCre recombinase point mutant R297T (AiP13781) injected into Ai14 reporter mouse line with I) cell body expression in STR but not islands of calleja in OT and J) no off-target midbrain dopamine expression. K-P) Example sagittal sections of enhancer AAVs driving iCre(R297T) (AiP14825, AiP15578, and AiP13781) in Ai14 reporter mouse line either through STX (K-M) or RO (N-P) injection. Images are of native tdTomato fluorescence. Q) Comparison of on-target cell type specificity using scRNA-seq between RO and STX injection methods. Bars represent mean ± standard error (SE). Individual data points indicate values for each enhancer. A Wilcoxon rank sum test was performed to assess statistical differences between groups (W = 5, p = 1), indicating no significant difference between injection methods.

Figure 6.. Optogenetic stimulation with viral vector targeting D1 MSNs is sufficient to induce locomotion and contralateral rotations.

A-B) Experimental design with ICV injection of AAV vectors (AiP13278 for D1 ChR2 and AiP13044 for matched D1-SYFP2 control, 3.0E+10 vg each) into postnatal day 2 (P2) mouse pups. Optogenetic stimulation and behavioral tracking is performed at least 1 week after stereotaxic implantation of optical fibers in adult mice. C) Exemplar tracks of mouse locomotion before (light gray; 10 sec), during (blue, orange; 10 sec), and after (gray; 10 sec) optogenetic stimulation in a 30 cm circular arena. D-E) Unilateral light delivery through the optical fiber (450 nm, 0.3 mW, 10 sec) increased the locomotion speed in D1-ChR2 virus injected mice (*** P≤0.001 by paired t-test; n=8 mice) but not D1-SYFP2 controls (ns, not significant; n=9 mice). F-G) Unilateral light delivery increased rotation bias towards contralateral rotations in D1-ChR2 injected mice (***P≤0.001 by paired t-test; n=8 mice) but not D1-SYFP2 control mice (ns, not significant; n=9 mice). H-I) Optogenetic stimulation induced steady contralateral rotations within ~200 ms of the onset of light stimulation in D1-ChR2 virus injected mice but not D1-SYFP2 only controls. Rotations persisted throughout the stimulation.

Figure 7.. Concurrent striatal cell type identification and targeting by multiplexed enhancer AAV delivery in C57 mice.

A) Enhancer AAVs were individually packaged, pooled, and injected RO into adult C57Bl6/J mice. B) Example sagittal sections of triple labeling using D1 MSN enhancer AiE0779m driving SYFP2 (AiP12609), D2 MSN enhancer AiE0452h driving mTFP1 (AiP12700), and cholinergic interneuron enhancer (AiE0743m_3xC2) driving tdTomato (AiP13738). Zoomed in views of dorsal striatum (STRd, middle) and cortex (right). C) Example sagittal sections of triple labeling using striatal Sst-Chodl enhancer (AiE0682h) driving SYFP2 (AiP12689), Pvalb-Pthlh enhancer (AiE0140h_3xC2) driving mTFP1 (AiP13808), and cholinergic enhancer (AiE0743m_3xC2) driving tdTomato (AiP13738). Zoomed in views of dorsal striatum (STRd, middle) and cortex (right). Note off-target tdTomato expression in cortex from AiE0743m_3xC2. D-E) Quantification of both multiplex injection combinations in dorsal striatum (D) and cortex (E). Numbers in Venn diagrams represent cell counts for each enhancer driven fluorescent reporter. Bamard’s test was used to evaluate enhancer expression overlap in cortex vs STRd (n=1 mouse and ROI per region). For D1 MSN+D2 MSN+Cholinergic enhancers, no significant difference was found (two-sided p = 0.8882) while for Sst-Chodl+Pvalb-Pthlh+Cholinergic enhancers, a significant difference in enhancer expression overlap between cortex vs STRd was observed (two-sided p <0.0001). F-H) Single cell patch-clamp recordings from coronal brain slices derived from a mouse with multiplex injection of Sst-Chodl enhancer AiE1426m-SYFP2 (AiP15050), Pvalb-Pthlh enhancer AiE0140h_3xC2-mTFP1 (AiP13808), and Cholinergic enhancer AiE0743m_3xC2-tdTomato (AiP13738). F-H): Representative voltage responses to hyperpolarizing and depolarizing current steps (top) with example single action potential traces highlighting distinctive AP halfwidths (middle). Current steps used to generate recordings are shown (bottom). I-J) 3D plots comparing electrophysiological features that distinguish the three striatal interneuron types. Individual neurons are shown as shaded circles, whereas darker diamonds represent the mean values for each interneuron type. Circles with black outline correspond to the exemplary neuron traces shown in panels F-H. PERMANOVA statistical testing using all five electrophysiological features found significant differences across the three cell type populations (F= 51.071, p= 0.001). A pairwise PERMANOVA post-hoc analysis indicated significant differences between all cell type pairwise comparisons (p<0.01). I) Comparison of the slope of the FI curve (firing frequency vs. current injection), the steady state input resistance, and action potential upstroke to downstroke ratio. J) Comparison of the slope of the FI curve with the resting membrane potential and the action potential half-width. Sample sizes: Pvalb-Pthlh, n=13; Sst-Chodl, n=16; Cholinergic, n=8. A total of 7 mice were used for triple RO injection patch clamp recording experiments. Example voltage traces in panel F, G and H panels (top) were recorded from the same mouse.

Figure 8.. Cross-species conservation of striatal enhancer open chromatin and enhancer AAV activity in vivo.

A) Assessment of evolutionary conservation of open chromatin activity. Bulk ATAC-seq analysis showing open chromatin regions in the cortex and striatum of human, macaque, rat, and mouse at conserved orthologs of five selected enhancer loci. These enhancers include three pan-MSN enhancers, one D1 MSN enhancer, and one D2 MSN enhancer. ATAC-seq traces demonstrate conserved higher open chromatin activity in the striatum compared to the cortex for all enhancers except the ortholog of AiE0441h in rat, which shows no chromatin accessibility in either cortex or striatum. B-H) Conservation of striatal enhancer specificity in vivo. B) Enhancer AAVs corresponding to the set of enhancers shown in (A), including an additional optimized variant (AiE0441h_3xC2), were injected RO into mouse and ICV into rat. Sagittal brain sections show conserved patterns of enhancer activity in mouse versus rat brain for native SYFP2 fluorescence (green), with DAPI counterstain (blue). Images were not acquired under matched conditions and have been adjusted to optimally highlight brain wide expression patterns for enhancer specificity comparison purposes. C-H) Stereotaxic injection of AiE0780m driving SYFP2 (AiP12610) in macaque caudate and putamen using the Brainsight veterinary surgical robot C) Coronal section of striatum injection site with red=propidium iodide, blue=DAPI, green=native SYFP2. Insets of caudate (cd) and putamen (put) show native SYFP2 only. D) Example images of RNAscope from Putamen injection site. Yellow circles indicate SYFP⁺DRD1⁺PPP1R1B⁺ cells. E-H) Coronal sections posterior to the injection site showing axon projections through the GPi and SNr indicative of D1 MSNs with red = propidium iodide, blue=DAPI, green = native SYFP2.