Sensory experience remodels genome architecture in neural circuit to drive motor learning

Abstract

Neuronal-activity-dependent transcription couples sensory experience to adaptive responses of the brain including learning and memory. Mechanisms of activity-dependent gene expression including alterations of the epigenome have been characterized^1-8. However, the fundamental question of whether sensory experience remodels chromatin architecture in the adult brain in vivo to induce neural code transformations and learning and memory remains to be addressed. Here we use in vivo calcium imaging, optogenetics and pharmacological approaches to show that granule neuron activation in the anterior dorsal cerebellar vermis has a crucial role in a delay tactile startle learning paradigm in mice. Of note, using large-scale transcriptome and chromatin profiling, we show that activation of the motor-learning-linked granule neuron circuit reorganizes neuronal chromatin including through long-distance enhancer-promoter and transcriptionally active compartment interactions to orchestrate distinct granule neuron gene expression modules. Conditional CRISPR knockout of the chromatin architecture regulator cohesin in anterior dorsal cerebellar vermis granule neurons in adult mice disrupts enhancer-promoter interactions, activity-dependent transcription and motor learning. These findings define how sensory experience patterns chromatin architecture and neural circuit coding in the brain to drive motor learning.

Extended Data Fig. 1.. Pharmacological inactivation of the ADCV and lobule VI in delay tactile startle conditioning

(a) Mice were subjected to tactile stimulation of the tail using an air puff (n=3 mice). (b) The delay tactile startle conditioning paradigm as in Fig. 1c–e using a 300 ms inter-stimulus interval (ISI) (left) and the percentage of trials with a conditioned response (CR) (right, day1 vs day7, P=1.8×10⁻⁵, two-tailed t-test, n=5 mice). (c, d) Cannula placement in the ADCV for drug delivery (c). Top: sagittal view, bottom: coronal view. Black or red/pink crosses indicate sites of saline or muscimol injections, respectively. Muscimol conjugated with the fluorophore BODIPY TMR-X injected one day after the last day of delay tactile startle conditioning was used to identify the location of the cannula tip. A representative injection site is shown (d, n=38 mice). (e) Mice injected with saline or muscimol in the ADCV were tested on the DigiGait system (n=5 mice). (f, g) Cannula placement in lobule VI for drug delivery (f). The percentage of conditioned responses during delay tactile startle conditioning (g, top) and unconditioned responses (g, bottom) upon muscimol-dependent neuronal inactivation in lobule VI (n=8 mice). In all panels, data show mean and shading or error bars denote standard error.

Extended Data Fig. 2.. Optogenetic inactivation and stimulation of the ADCV and lobule VI in delay tactile startle conditioning

(a) Day 5 backward conditioned responses of control mice subjected to delay tactile startle conditioning and optical stimulation in the ADCV as in Fig. 1i (Ai40 ctrl +ADCV laser, n=9 mice), Ai40-GC mice subjected to delay tactile startle conditioning in the absence of optical stimulation (Ai40-GC +no laser, n=6 mice), or Ai40-GC mice subjected to delay tactile startle conditioning and optogenetic silencing of lobule VI during the CS (Ai40-GC +L. VI laser, n=7 mice). (b) Acute sagittal cerebellar slices were prepared from mice expressing channelrhodopsin in granule neurons (Ai32-GC mice). Granule neuron action potentials were recorded in response to 100 ms optogenetic stimuli each composed of a 50 Hz train of 10 ms pulses (blue squares). A representative membrane potential trace for a granule neuron (left, n=2 neurons) and the relationship between the intensity of optostimulation of granule neurons and action potential firing (right, n=4 neurons). (c) Mice expressing channelrhodopsin in granule neurons (Ai32-GC mice) were subjected to optostimulation of the granule neuron pathway in the ADCV as the CS in the absence of the US (n=3 mice). (d) Control mice were subjected to optical stimulation in the ADCV as the CS together with the US (n=3 mice). (e) Mice expressing channelrhodopsin in granule neurons (Ai32-GC mice) were subjected to three days of delay tactile startle conditioning using optostimulation of lobule VI as the CS (backward conditioned responses in naïve vs day3, P=6.1×10⁻¹¹, two-tailed t-test, n=6 mice). In all panels, data show mean and error bars denote standard error.

Extended Data Fig. 3.. In vivo imaging of granule neuron coding during delay tactile startle conditioning

(a) Sagittal sections from the cerebellum of mice expressing GCaMP6f in granule neurons (Ai95-GC) were subjected to immunohistochemistry using the GFP and Calbindin antibodies and the DNA dye Bisbenzimide (Hoechst) (n=2 mice). Scale bar: 200 μm, 10x magnification (top); 50 μm, 40x magnification (bottom). Molecular layer: ML, Purkinje cell layer: PCL, and internal granule layer: IGL. (b) In vivo two-photon calcium imaging of Ai95-GC mice subjected to delay tactile startle conditioning, followed by image registration and auto cell-segmentation. In a representative imaging session, the average mouse locomotion during ten training trials (top), segmented granule neuron somas (middle, left) and their calcium responses (middle, right), and population granule neuron responsivity (bottom) (n=6 mice). (c) Locomotion of Ai95-GC mice during delay tactile startle conditioning on day 1 and after training for 6–9 days (n=6 mice). (d) The percentage of granule neurons that are active in the ADCV during the CS period in tactile startle conditioning (*P=0.031,***P=7.3×10⁻⁵, one-way ANOVA with Dunnett’s post-hoc test, n=5,4,4 mice for day1,day6–9CR+,day6–9CR-). (e) Population responsivity of granule neurons in lobule VI during the CS period in tactile startle conditioning (**P<0.01,***P<0.001, two-way repeated measures ANOVA with Dunnett’s post-hoc test, n=5 mice). In all panels, data show mean and shading or error bars denote standard error

Extended Data Fig. 4.. In vivo imaging of Purkinje cell dendrite coding during delay tactile startle conditioning

(a, b) The AAV delivery approach to label Purkinje cells in the ADCV and lobule VI (a). Sagittal sections from the cerebellum of Pcp2-Cre mice injected with AAV9-FLEx-GCaMP6f (AAV9-GCaMP6f-PC) were subjected to immunohistochemistry using the GFP antibody and Hoechst (b, n=2 mice). Scale bars: 500 μm, 4x magnification (top); 50 μm, 40x magnification (bottom). (c) In vivo two-photon calcium imaging of AAV9-GCaMP6f-PC mice subjected to 60 seconds of free wheel locomotion and 10 trials of delay tactile startle conditioning with randomized inter-trial intervals. In a representative imaging session, Purkinje cell dendrite calcium responses were analyzed as in Extended Data Fig. 3b (n=10 mice). (d) Locomotion of AAV9-GCaMP6f-PC mice during delay tactile startle conditioning on day 1 and after training for 5–8 days (n=10 mice). Data show mean and shading denotes standard error

Extended Data Fig. 5.. Identification of cell type-enriched and sensorimotor experience-dependent gene modules in the cerebellum in mice

(a) Gene modules identified in Fig. 3b were intersected with cerebellar cell type-specific TRAP-Seq data and analyzed as in Fig. 3c. (b) Heatmaps of gene module expression induced by sensorimotor stimulation as in Fig. 3a (n=52 samples, log2 mean centered). (c) Comparison of the log2 fold change in gene expression induced by free wheel locomotion compared to homecage control and the log2 fold change in gene expression upon treatment with the GABA(A) receptor agonist muscimol compared to saline control during free wheel locomotion in the ADCV of mice. Genes in the lightcyan module induced by locomotion that were restored to homecage control expression levels upon silencing of the ADCV cortical activity with muscimol (reversal) fall on the dotted line. (d) Sensorimotor stimulation-dependent gene modules analyzed as in (c). (e) The average velocity of mice subjected to delay tactile startle conditioning (US+CS) or control (US) condition during inter-trial intervals (n=4 mice). (f, g) Gene expression in the ADCV or lobule VI of mice subjected to delay tactile startle conditioning (US+CS) or control (US) condition (n=4 mice). In all panels, data show mean and error bars denote standard error

Extended Data Fig. 6.. Optostimulation of granule neurons potentiates CS pathway-regulated gene modules

(a) UCSC genome browser tracks of chromatin-bound (Chrom-Seq) and nucleocytoplasmic (NucCyto-Seq) RNA at the fosl2 locus upon optostimulation of granule neurons in the ADCV in mice. The chromatin-bound fraction contained immature unspliced RNA and the nucleocytoplasmic fraction contained spliced mature RNA. (b-d) Comparisons of the log2 fold change in chromatin-bound RNA and the log2 fold change in nucleocytoplasmic RNA upon optostimulation of granule neurons together with the log2 fold change in total RNA upon sensorimotor stimulation in the ADCV in mice (n=4,4,18) mice for chromatin, nucleocytoplasmic, total RNA). Data show mean ± standard error. (e) Pulse chase analyses were performed by optogenetically stimulating granule neurons in the ADCV for 10 minutes and returning mice to their homecage for 10, 50, or 140 minutes. The ADCV of optostimulated or unstimulated control mice was then subjected to RNA-Seq using the chromatin-bound (nascent) or nucleocytoplasmic (mature) fractions. (f-i) Time course of chromatin-bound (nascent) or nucleocytoplasmic (mature) RNA expression following optostimulation of granule neurons as in (e) (n=2 mice).

Extended Data Fig. 7.. Optostimulation of the CS pathway regulates uaRNA and eRNA expression

(a, b) Heatmaps of significantly differentially expressed (DE) uaRNA and eRNA from Chrom-Seq or NucCyto-Seq (two-sided P-value from negative binomial distribution with Benjamini-Hochberg post-hoc test, n=4 mice, false discovery rate (FDR) < 0.05, log2 mean centered). (c) Comparison of the log2 fold change in mRNA expression and the log2 fold change in uaRNA and eRNA expression at lightcyan, midnightblue, and brown gene modules upon optostimulation of granule neurons (n=83,67,342 transcript-uaRNA pairs and n=66,63,366 transcript-eRNA pairs for lightcyan, midnightblue, brown). The Pearson correlation coefficient (corr) is shown.

Extended Data Fig. 8.. Optostimulation of the CS pathway regulates histone modification and histone variant abundance at gene promoters

and enhancers (a-c) Comparison of the log2 fold change in gene expression and the log2 fold change in H3K27ac (a), H2A.z (b), or H3K4me3 (c) read density at the TSSs of granule neuron-enriched gene modules upon optostimulation of granule neurons in the ADCV in mice (n=85,355 transcripts for midnightblue, brown). The Pearson correlation coefficient (corr) is shown. (d) The profile of the histone marks H3K27ac, H2A.z, H3K4me3, and H3K27me3 surrounding the TSSs of genes whose expression was upregulated (left, mRNA fold change>1, log2), not changed (middle, −0.05<mRNA fold change<0.05, log2), or downregulated (right, mRNA fold change<−1, log2) upon optostimulation of granule neurons (n=2,3,2,2 biological replicates for H3K27ac,H2A.z,H3K4me3,H3K27me3). (e, f) Comparison of the log2 fold change in mRNA or eRNA and the log2 fold change in H3K27ac (top) or H2A.z (bottom) levels at the enhancers of granule neuron-enriched CS-regulated gene modules (e, n=266,256,1510 enhancers for lightcyan, midnightblue, brown). The Pearson correlation coefficient (corr) is shown. The profile of H3K27ac and H2A.z surrounding enhancers with eRNA levels that were upregulated (left, mRNA fold change>1, log2), not changed (middle, −0.05<mRNA fold change<0.05, log2), or downregulated (right, mRNA fold change<−1, log2) upon optostimulation of granule neurons (f, n=2,3 biological replicates for H3K27ac,H2A.z). In all panels, data show mean.

Extended Data Fig. 9.. Transcription factors enriched at CS pathway regulated genes

(a) Transcription factor binding motifs enriched at enhancers or promoters with upregulated (fold change>1 or >0.585, log2) or downregulated (fold change<−1 or <−0.585, log2) H3K27ac levels upon optostimulation of granule neurons (n=1379,419 enhancers for FC>1,FC<−1; n=290,166 promoters for FC>0.585,FC<−0.585). (b) Transcription factor binding motifs enriched at enhancers or promoters of granule neuron CS pathway activated gene modules (n=364,288,1139 enhancers and n=37,38,69 promoters for lightcyan, midnightblue, brown). In all panels, Fisher’s exact test with Benjamini post-hoc test. NS indicates no significant motifs identified.

Extended Data Fig. 10.. Chromatin architecture in the cerebellum

(a) Features of genomic loci interacting with promoters in the adult cerebellum. MAPS analyses of PLAC-Seq data identified promoter-centric interactions enriched for regulatory regions of the genome marked by occupancy of CTCF or H3K27ac. (b, c) PLAC-Seq analyses of the number of home-specific and opto-specific interactions (b, P=4.7×10⁻⁵, Chi-square test, n=2 biological replicates) or changes in interaction frequency (c, **P=0.0045,***P=0.00026, two-sided Wilcoxon signed rank test, n=83,49 enhancer-promoter pairs for FC>0.585,FC<−0.585) between promoters and enhancers harboring upregulated (blue, fold change>0.585 or >1, log2) or downregulated (green, fold change<−0.585, log2) H3K27ac levels upon optostimulation of granule neurons. Box plots show median, quartiles (box) and range (whiskers). (d) Features of frequently interacting regions (FIREs) in the adult cerebellum. (e) The number of home-specific and opto-specific FIREs within 200Kb of TSSs harboring upregulated (blue, fold change>0.585 or >1, log2) or downregulated (green, fold change<−0.585, log2) H3K27ac levels upon optostimulation of granule neurons (P=8.8×10⁻⁷, Chi-square test, n=3 biological replicates). (f) The profile of H3K27ac surrounding the distal (left) or proximal (right) enhancers of genes indicated in Fig. 4d (n=2 biological replicates). Data show mean. (g, h) A representative image of a granule neuron labeled with DNA FISH probes targeting the nr4a3 (green) or gapdh (red) gene together with Hoechst (g, n=416 nuclei). The dotted line indicates the nucleus. The co-localization of the nr4a3 and gapdh genes upon optostimulation (h, n=2 mice). Data show mean. (i) Comparison of the change in compartment strength (λ) and the change in H3K27ac levels in 100Kb bins along chromosomes upon optostimulation of granule neurons (n=24046 bins). The Pearson correlation coefficient (corr) is shown. (j) PLAC-Seq analyses of inter-chromosomal normalized interaction frequency between genomic loci at 100Kb resolution harboring upregulated (blue, fold change>0.585, log2) or downregulated (green, fold change<−0.585, log2) H3K27ac levels as in Fig. 4j. (k) Sagittal sections from the cerebellum of Cas9EGFP-GC mice infected with AAV9-gRNA-mCherry as in Fig. 5f and subjected to immunohistochemistry using the GFP and DsRed antibodies and DAPI (n=4 mice). Scale bar: 100 μm. (l) The ADCV of conditional CRISPR Rad21 knockout or control mice analyzed as in Fig. 5j (n=2,2,5,5 mice for home-ctrl,home-Rad21cKO,US+CS-ctrl,US+CS-Rad21cKO). Data show mean and error bars denote standard error.

Fig. 1.. The ADCV plays a crucial role in delay tactile startle conditioning

(a) Schematic of the tactile stimulus apparatus. (b) Mouse locomotion in response to tactile stimulation of the nose (n=20 mice). (c) The delay tactile startle conditioning paradigm using an LED light as conditioned stimulus (CS) and tactile stimulation as unconditioned stimulus (US). ISI: inter-stimulus interval. (d, e) Mouse locomotion during delay tactile startle conditioning (d, left and middle), and the maximum negative velocity of responses during catch trials (d, right, ***P=0.0006,0.0002 for day1-day10,day5-day10, one-way ANOVA with Dunnett’s post-hoc test, n=10,10,9 mice for day1,5,10) or percentage of trials with a conditioned response (CR) (e, backward CR on day1 vs day10, P=1.5×10⁻²³, two-tailed t-test, n=25,12 mice for day1,10). UR: unconditioned response. (f) Mice injected daily with muscimol or the saline vehicle control in the ADCV. The percentage of CR with training (left) and UR (right) upon muscimol-dependent neuronal inactivation during delay tactile startle conditioning (**P=0.0019, one-way ANOVA with Dunnett’s post-hoc test, n=8,7,7 mice for saline,musimol-rostral,muscimol-caudal). (g, h) Head-fixed mice expressing archaerhodopsin in granule neurons (Ai40-GC), channelrhodopsin in granule neurons (Ai32-GC), or channelrhodopsin in Purkinje cells (Ai32-PC) were optogenetically silenced or stimulated using fiber optic cannulae overlying the ADCV or lobule IX (L. IX). (i) Optogenetic silencing of the granule neuron pathway during the CS in delay tactile startle conditioning (*P=0.033, two-tailed t-test, n=9 mice). (j) Optostimulation of granule neurons in the ADCV or lobule IX as the CS in delay tactile startle conditioning (***P=0.00048, two-tailed t-test, n=5 mice). (k) Optostimulation of Purkinje cells in the ADCV or lobule IX as the US in delay tactile startle conditioning (*P=0.033, two-tailed t-test, n=7,5 mice for ADCV,lobule IX). In all panels, data show mean and shading or error bars denote standard error.

Fig. 2.. Transformation of mossy fiber/granule neuron and climbing fiber/Purkinje cell neural coding in the anterior cerebellum during motor learning in mice

(a) Schematic of glutamatergic brainstem projections to the cerebellum. (b, c) Head-fixed mice subjected to in vivo two-photon calcium imaging through a cranial window implanted over the anterior cerebellum (b, n=16 mice) during delay tactile startle conditioning (c). Scale bar: 500 μm, IC: inferior colliculus. (d-g) In vivo imaging of mossy fiber-driven granule neuron calcium activity in the anterior cerebellum of mice expressing GCaMP6f in granule neurons (Ai95-GC) (d, n=6 mice, scale bar: 20 μm). Population responsivity traces (e) and maximum CS-activated population responses (f, **P=0.0016,***P=1.3×10⁻⁵, two-tailed t-test) of granule neurons in the ADCV or lobule VI during delay tactile startle conditioning. The motor behavior of mice (g, left) and CS-activated population responsivity of granule neurons in the ADCV (g, right, *P<0.05,**P<0.01,***P<0.001, two-way repeated measures ANOVA with Dunnett’s post-hoc test) during trials with conditioned responses (CR+) or absent conditioned responses (CR-) after motor learning (in e-g, n=6,10 fields of view for ADCV,lobule VI in 6 mice). (h-k) In vivo imaging of climbing fiber-driven Purkinje cell dendrite calcium activity in anterior cerebellum of mice expressing GCaMP6f in Purkinje cells (AAV9-GCaMP6f-PC) (h, n=10 mice, scale bar: 100 μm). Population responsivity traces (i) and maximum CS-activated population responses (j, *P=0.042, two-tailed t-test) of Purkinje cell dendrites in the ADCV or lobule VI during delay tactile startle conditioning. The motor behavior of mice (k, left) and CS-activated population responsivity of Purkinje cell dendrites in the ADCV (k, right, *P<0.05, two-way repeated measures ANOVA with Dunnett’s post-hoc test) during trials with CR+ or CR- after motor learning (in i-k, n=11 fields of view in 10 mice). In all panels, data show mean and shading or error bars denote standard error.

Fig. 3.. Sensorimotor stimulation triggers epigenetic regulation of cell type-enriched gene modules in the cerebellum in vivo

(a) RNA from the cerebellum (total), ADCV, or lobule VI (L. VI) of adult mice undergoing forced rotarod locomotion, free wheel locomotion, delay tactile startle conditioning (DTSC), or homecage condition was subjected to RNA-Seq. A heatmap of significant differential gene expression induced by sensorimotor stimulation versus homecage condition (two-sided P-value from negative binomial distribution with Benjamini-Hochberg post-hoc test, n=52 biological samples, false discovery rate (FDR)<0.05, log2 mean centered). (b) Weighted gene co-expression network analysis (WGCNA) of the gene expression profiles in (a) and the heatmap of gene interconnectivity between modules. (c) Interrogation of gene modules identified in (b) using cell type-specific TRAP microarray data. Molecular layer interneurons: MLI, progenitor: pro, mature: mat. (d) Correlation of the first principal component of gene modules (eigengene) with locomotion (n=40,12 biological samples for locomotion,rest). (e) Annotation of gene modules using DAVID (Fisher’s Exact test with Benjamini post-hoc test, n=118,93,98,452 transcripts for purple,lightcyan,salmon,brown). (f) Correlation of the eigengene with CS-activation in the delay tactile startle conditioning task (n=8 biological samples). (g) Optostimulation of ADCV granule neurons, followed by RNA-Seq of the chromatin bound fraction (nascent RNA) or nucleocytoplasmic fraction (mature RNA). (h) Effects of optostimulation of granule neurons compared with free wheel locomotion on gene module expression. (i) Transcripts and epigenetic regulation at enhancers and gene promoters. (j) A UCSC genome browser track at the fos locus. (k) Comparison of the log2 fold change of mRNA expression with the log2 fold change of enrichment of H3K27ac (left) or H2A.z (right) at TSSs of lightcyan module genes upon optostimulation of granule neurons. Pearson correlation coefficient (corr) is shown (n=79 transcripts). In d, f, correlation P-value with Storey post-hoc test.

Fig. 4.. Activation of granule neuron CS pathway promotes enhancer-promoter interactions and compartmentalization in vivo

(a) Schematic of proximity ligation-assisted ChIP-Seq (PLAC-Seq) (left). A MAPS-normalized contact map at the pax6 gene locus in the ADCV (right). (b) Promoter-centric interactions at the nr4a1 locus at 10Kb resolution upon optostimulation of ADCV granule neurons. Glasses indicate viewpoint. (c, d) CS-regulated promoter interactions with enhancers (c, *P=0.014,**P=0.0035,***P=0.00043, two-sided Wilcoxon signed rank test, n=77,39,174 enhancer-promoters for lightcyan,midnightblue,brown) or activated promoter interactions with distal or proximal enhancers (d, ***P=0.00057, two-sided Wilcoxon signed rank test, n=22,38 multienhancer-promoters for proximal,distal) upon optostimulation of granule neurons. (e, f) Promoter-centric interactions at the nr4a3 locus and DNA FISH probes recognizing the distal nr4a3 enhancers and nr4a3 gene together with the DNA dye Hoechst (e, f, left, n=169 nuclei). Distance between the nr4a3 enhancers and gene upon optostimulation of granule neurons (f, right, ***P=0.00060, two-sided Mann-Whitney-Wilcoxon test, n=85,84 nuclei for homecage,optostim). (g) Genome organization of A/B compartments in chromosome 1 using the Pearson correlation matrix or first eigenvector (λ) of Hi-C contacts. (h) Change in compartment strength (λ) and H3K27ac levels along chromosome 1 upon optostimulation of granule neurons. (i) Compartment strength at genomic loci with changes in H3K27ac levels upon optostimulation of granule neurons (**P=0.0018,0.0023 for up,down, one-way ANOVA with Bonferroni post-hoc test). (j) Inter-chromosomal normalized interaction frequency between genomic loci with changes in H3K27ac levels (**P=0.0056, one-way ANOVA with Bonferroni post-hoc test). (k) A model of activity-dependent regulation of chromatin architecture at activated (blue) or repressed (green) genomic loci. In a, b, e, one-sided P-value from zero-truncated Poisson distribution with Benjamini-Hochberg post-hoc test. In a-f, n=2 biological replicates. In c, d, f, box plots show median, quartiles (box) and range (whiskers). In g-j, n=3 biological replicates. In i, j, data show mean ± standard error.

Fig. 5.. The core Cohesin subunit Rad21 is required for activity-dependent transcription and motor learning in mice

(a) Cohesin is a chromatin architectural protein that regulates enhancer-promoter interactions. (b) UCSC genome browser tracks at the fos or kcnh1 locus. (c, d) Profiles of Rad21 density at promoters (left), enhancers (middle) or flanking CTCF-bound insulators (right) of genes with increased H3K27ac (c) or reduced H3K27ac (d) upon optostimulation of granule neurons. (e, f) Schematic of AAV delivery approach to knockout Rad21 in ADCV granule neurons (e); an mCherry labeled cerebellum from adult mice expressing Cas9 in granule neurons (f, n=25 mice). Scale bar: 200 μm. (g) Conditional CRISPR knockout of Rad21 in ADCV granule neurons in adult mice significantly downregulated Rad21 mRNA levels in the ADCV, normalized to levels in lobule IX (***P=4.7×10⁻⁶, two-tailed t-test, n=4,6 mice for ctrl,Rad21cKO). (h, i) Aggregate peak analysis of enhancer-promoter interactions identified using MAPS analyses of PLAC-Seq data. HiC interactions are normalized to the mean interactions in the lower-left (LL) corner. Heatmaps of the 200Kb surrounding region (h) and barplots of the peak 10Kb bin normalized to the LL bins (i) upon conditional CRISPR knockout of Rad21 in ADCV granule neurons (n=2 biological replicates). (j) The ADCV of conditional CRISPR Rad21 knockout or control mice undergoing delay tactile startle conditioning or the homecage control condition was dissected and subjected to RNA-Seq analyses (Mann-Whitney-Wilcoxon test, n=2,2,5,5 mice for home-ctrl,home-Rad21cKO,US+CS-ctrl,US+CS-Rad21cKO). (k) Performance of conditional CRISPR Rad21 knockout or control animals in delay tactile startle conditioning (*P=0.025,0.040 for day3,day4, two-way repeated measures ANOVA with Sidak’s post-hoc test, n=10,11 mice for ctrl,Rad21cKO). In all panels, data show mean and shading or error bars denote standard error.