Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: ( A ) Schematic of mouse whole brain in vivo Perturb-seq using 10x Genomics Flex Apex platform. ( B ) UMAP of whole brain in vivo Perturb-seq dataset encompassing 7.7 million sequenced nuclei, colored by developmental neighborhoods, anatomical region, and neurotransmitter type, inferred using MapMyCells . ( C ) Heatmap of the gene expression levels of 1,947 neurodevelopmental disease-associated risk genes in non-targeting control nuclei across different developmental neighborhoods. ( D ) Histogram of in vitro gRNA activity distribution of 45 selected gRNAs (15 genes, 3 gRNAs per gene) compared to safe-targeting controls by insertion-deletion analysis. ( E ) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally administered with 6e8 total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar, 1 mm), accompanied by zoomed in images to show representative MOI in each major brain region (scale bar, 50 μm), and stacked bar plot quantifying GFP and mScarlet viral labeling efficiency as well as double labeling rate. ( F ) UMAPs of whole brain in vivo Perturb-seq dataset separated by neighborhoods, colored by inferred cell subclass using MapMyCells . ( G ) Violin plots of number of genes and RNA UMIs recovered per nucleus from each developmental neighborhood. ( H ) Ranked bar plot showing proportion of sampled nuclei by brain region. ( I ) Stacked bar plot showing percentage of nuclei with no guide, single, double, or multiple guide assignment within each developmental neighborhood. ( J ) Histogram of total nuclei number distribution of nuclei recovered per perturbation. ( K ) Ranked dot plot of nuclei number in each perturbation and cell type pair and the minimum cell number cut off for perturbation and cell type pair for downstream analyses (dashed line). ( L ) Scatter plot of weighted mean log fold-changes of target genes across all cell types against their weighted mean expression levels in non-targeting control nuclei.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: In Vivo, Gene Expression, Control, In Vitro, Activity Assay, Immunofluorescence, Labeling, Expressing

Journal: bioRxiv

Article Title: Genome-scale functional mapping of the mammalian whole brain with in vivo Perturb-seq

doi: 10.64898/2026.03.16.711480

Figure Lengend Snippet: (A) Immunofluorescence image of sagittal section of a P37 mouse brain retro-orbitally injected at P16 with high (1e9), mid (6e8), or low (1.5e8) total vg per gram of body weight of AAV PHP.eB encoding either GFP or mScarlet (1:1 ratio) (scale bar = 1 mm). (B) Quantification of GFP and mScarlet viral labeling efficiency as well as double labeling rate in (A). (C) Representative FACS gating strategy to enrich transduced neuronal nuclei. (D) Bar plot of sex and weight at harvest of animals used in this study. (E) Animal tracking information showing the litter, age at harvest for each animal, as well as AAV-labeling rate by FACS and total nuclei number per hemisphere used for Flex hybridization. (F) Schematic of snRNA-seq data processing and quality control workflow.

Article Snippet: Briefly, HEK293T cells were transfected with above pooled plasmid library or plasmid encoding a PiggyBac transposase , along with AAV-PHP.eB capsid (Addgene #103005) and pHelper plasmids using PEI (Polysciences, #24765-1) at 80-90% confluency.

Techniques: Immunofluorescence, Injection, Labeling, Hybridization, Control

Journal: Nature Communications

Article Title: Molecular signatures of resilience to Alzheimer’s disease in neocortical layer 4 neurons

doi: 10.1038/s41467-026-68920-4

Figure Lengend Snippet: a Violin plots showing KCNIP4 gene expression across major cell types (left) and excitatory neuronal subtypes from BA9 and BA17 (right). b Violin plots showing KCNIP4 expression across AD disease groups in Ex2 and Ex5 neurons from BA9 and BA17. Log-normalized expression levels of KCNIP4 are shown. c Immunostaining for KCNIP4, EYA4, and NeuN in cryosections from low, intermediate, and high pathology stages illustrating increased expression of KCNIP4 in L4 EYA4 + neurons in BA17. d Quantification of KCNIP4 protein expression levels in L4 EYA4 + neurons, L4 EYA4 − neurons, and L2/3 neurons from BA17 across disease stages ( n = 6 donors per disease group). Data are shown as median ± IQR; whiskers represent minimum and maximum values. One-way ANOVA with two-sided Tukey’s test was used for multiple comparisons (* p -value < 0.05; *** p -value < 0.001; **** p -value < 0.0001; exact p -values are available in the Source Data file). Scale bars: 200 µm for low magnification images; 30 µm for high magnification images. Source data are provided as a Source Data file.

Article Snippet: Neurons were transduced on day in vitro 7 (DIV7) with AAV-PHP.eB-CaMKIIa- Kcnip4 -P2A-EGFP or control AAV-PHP.eB-CaMKIIa-EGFP (Addgene #50469-PHPeB) at a multiplicity of infection (MOI) of 5000 viral genomes (vg)/cell.

Techniques: Gene Expression, Expressing, Immunostaining

Journal: Nature Communications

Article Title: Molecular signatures of resilience to Alzheimer’s disease in neocortical layer 4 neurons

doi: 10.1038/s41467-026-68920-4

Figure Lengend Snippet: a In vitro approach to evaluate AAV-mediated Kcnip4 overexpression on neural activity in primary excitatory cortical neurons using calcium imaging. b Representative neuronal Ca 2+ transients quantified as ΔF/F₀ at DIV 14 for each condition. c , Quantification of Ca 2+ transient frequency for each condition. Event frequency (events per minute) was averaged at the well level, with each well considered a biological replicate (4 wells per condition, 2 fields per well, 3 GFP-positive neurons per field). d In vivo approach to evaluate AAV-mediated Kcnip4 overexpression in App SAA and WT mice, and representative coronal section (50-µm thick) of a treated mouse illustrating transduction of cortical neurons. e Western blot representative image and quantification of KCNIP4 levels in cerebral cortex lysates following two different doses of Kcnip4 AAV ( n = 3 per group). f Representative images of cerebral cortex and hippocampus from Kcnip4 AAV-treated mice and quantification of transduction efficiency of the different AAVs in SSC in WT and App SAA mice. g − i Representative images and quantification of cortical amyloid beta, GFAP, and IBA1 immunostaining in App SAA mice treated with Kcnip4 AAV or control AAV (6−7 mice per group). j Representative immunofluorescence image through the SSC co-stained with GFP and c-Fos. k Percentage of c-Fos-positive cells in all cortical neurons across study groups. l − o Quantification of c-Fos in GFP + compared to GFP − neurons from App SAA and WT mice treated with Kcnip4 AAV or control AAV (5−7 mice per group). p Representative immunofluorescence image through the SSC co-stained with GFP and Arc. q Mean Arc staining intensity in all cortical neurons across groups; r−u , Quantification of Arc staining intensity in GFP + compared to GFP − neurons from App SAA and WT mice treated with Kcnip4 AAV or control AAV (5−7 mice per group). Data are shown as median ± IQR. A two-sided t-test was used for pairwise comparisons, and one-way ANOVA with two-sided Tukey’s test was used for multiple comparisons (* p -value < 0.05; ** p -value < 0.01, *** p -value < 0.001, **** p -value < 0.0001; exact p -values are available in the Source Data file). Scale bars: 200 µm ( f ); 50 µm ( j , p ). Source data are provided as a Source Data file.

Article Snippet: Neurons were transduced on day in vitro 7 (DIV7) with AAV-PHP.eB-CaMKIIa- Kcnip4 -P2A-EGFP or control AAV-PHP.eB-CaMKIIa-EGFP (Addgene #50469-PHPeB) at a multiplicity of infection (MOI) of 5000 viral genomes (vg)/cell.

Techniques: In Vitro, Over Expression, Activity Assay, Imaging, In Vivo, Transduction, Western Blot, Immunostaining, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Schematic showing the IV injection of the AAV-EGFP or AAV-sDLK1-T2A-GFP into the mice. B. Quantification of the protein levels of sDLK1 in the cortex of the mice, measured by mouse DLK1 ELISA. C. Distributions of the normalized numbers of genes up-regulated (top) and down-regulated (bottom) in different cell types of the mice injected with AAV-sDLK1-T2A-GFP, compared to mice injected with AAV-EGFP. The gene burden score is defined as the number of differentially expressed genes per 1000 UMI detected in each cell type. D. Scatter plot showing the positively correlated genes between DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected oligodendrocytes. E. Western blot of MBP (top) and MOBP (bottom). F–G. Quantification of the MBP (F) and MOBP (G) western blot. Data were analyzed by two-tailed unpaired t -test. ** p =0.0095 (MBP); *** p =0.0009 (MOBP). H. Ridge plot of the predicted chronological ages for oligodendrocytes in the mice injected with AAV-EGFP (top) and AAV-sDLK1-T2A-GFP (bottom). I. Quantification of the predicted age of oligodendrocytes. Data were reported as a box & whisker plot showing min to max and analyzed by t-test. Each dot represents a cell. **** p < 0.0001. J. UMAP plot showing the enrichment of OPC2 caused by increased sDLK1. K. Ratios of each OPC cluster. Data are reported as mean ± s.e.m. and analyzed by two-way ANOVA. * p =0.0432. L. Running enrichment score and pre-ranked list showing a negative enrichment of oligodendrocyte differentiation predicted by OPC3 markers

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: IV Injection, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Two Tailed Test, Whisker Assay

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. UMAP plots showing eight major cell types identified in the mouse hippocampus. B. Dot plot showing expression levels of canonical cell markers in each identified cell type. C. Proportion of each cell type within animals injected with AAV-GFP and AAV-sDLK1-T2A-GFP. D. Proportion of each cell type within individual samples. E. Violin plots showing the number of unique features (left); the number of total RNA count (middle), and the percentage of mitochondrial genes (right) detected in each identified cell type. F. Correlation between UMI counts and percentage of mitochondrial genes (left) or total gene counts (right) per nuclei for each individual sample.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Dot plot showing the expression level of DLK1 in different cell types in the hippocampus tissue. The size of each dot represents the percentage of cells with detected DLK1 mRNA. B. Chord diagram showing DLK signaling predicted by CellChat. The lengths of the segmented outer circle reflect the expression levels of ligand proteins in each cell type and of receptor proteins in the receiving cells, showing strong expression of DLK1 signaling originating from interneurons and astrocytes to oligodendrocytes and OPCs. C. Bubble plot showing the DLK1 interactions originating from interneurons and astrocytes to different receptors. D. Venn diagram showing the overlap of upregulated DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected oligodendrocyte(top) and the overlap of downregulated DEGs in G3 Terc-/-and AAV-sDLK1-T2A-GFP injected oligodendrocyte (bottom). E. Dot plot showing the change of expression levels of myelination proteins in oligodendrocytes caused by the increase of sDLK1. F. Cnet plot showing the network of genes associated with myelination-related Gene Ontology terms and myelin-related diseases, based on enrichment analysis of the top 500 differentially expressed genes in oligodendrocytes from mice with AAV-sDLK1-T2A-GFP versus mice with AAV-GFP. Nodes represent genes or GO terms; edge colors represent the pathways each node is involved in.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Expressing, Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A. Representative 20X images of Olig2 in the CA1 region of the hippocampus in C57BL/6 mice injected with either AAV-GFP or AAV-sDLK1-T2A-GFP. Scale bar represents 100 µm. B. Quantification of Olig2+ cell density in CA1. N = 5 mice injected with AAV-GFP and N = 4 mice injected with AAV-sDLK1-T2A-GFP. 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.9480. Data were analyzed by unpaired t-test. C. Quantification of Olig2+ cell density in CA3. N = 5 mice injected with AAV-GFP and N = 4 mice injected with AAV-sDLK1-T2A-GFP. 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.2008. Data were analyzed by unpaired t-test. D. Representative 20X images of PDGFRα in the CA1 region of the hippocampus in C57BL/6 mice injected with either AAV-GFP or AAV-sDLK1-T2A-GFP. Scale bar represents 100 µm. E. Quantification of PDGFRα+ cell density in CA1. N = 5 mice/treatment group and 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.3534. Data were analyzed by unpaired t-test. F. Quantification of PDGFRα+ cell density in CA3. N = 5 mice/treatment group and 2-3 hippocampal sections/mouse were imaged and analyzed. Data are reported as mean ± SEM. p = 0.6744. Data were analyzed by unpaired t-test.

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Injection

Journal: bioRxiv

Article Title: Soluble DLK1 secreted by telomere-shortening-induced senescent microglia impairs oligodendrocyte functions and alters neuronal activity

doi: 10.64898/2026.01.14.699608

Figure Lengend Snippet: A Venn diagram showing the overlap of upregulated (top) and downregulated (bottom) DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP -injected excitatory neurons. B. Dot plot of the top 10 Reactome pathways inferred by the upregulated overlapping DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected excitatory neurons. C–D. Running enrichment score and pre-ranked list showing a positive (C) and negative (D) enrichment of calcium ion transmembrane transport predicted by the upregulated overlapping DEGs in G3 Terc-/- and AAV-sDLK1-T2A-GFP injected excitatory neurons. E. Schematic illustrating the experiment setup to test the chronic effects of DLK1 on neuronal activities. F. Representative fluorescence image of human iPSC-derived neurons expressing GCaMP8f showing spontaneous activity. G. Representative spontaneous calcium traces. H–J. Quantification of synchronized firing rate (H). firing amplitude (I), or spontaneous firing rate (J). K. Representative averaged calcium traces from one KCl stimulation experiment in neurons treated with DLK1 (red) and the untreated control neurons (black). Recording 400 seconds. Mean± s.e.m. L–N. Quantification of peak amplitude (L), the delayed KCl stimulation-induced neuronal responses (M), the time each neuron spent to reach peak intensity (N), from KCl stimulation-induced neuronal responses. Data are presented as mean ± s.e.m. and analyzed by unpaired t -test. ** p =0.0055 (L) **** p <0.0001 (N).

Article Snippet: Intravenous injections of AAV particles encoding mouse sDLK1 were performed in C57BL/6 mice, while PHP.eB AAV encoding GFP alone (Addgene, 37825-PHP.eB) served as a negative control.

Techniques: Injection, Fluorescence, Derivative Assay, Expressing, Activity Assay, Control

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Top, Schematics of the intravenous injection. AAV PHP.eB particles were injected in the retro-orbital sinus of Emx1-Cre mice. Bottom, The viral genome contained insert gene coding for ChR2(H134R) fused to eYFP. Black and grey triangles indicate lox2272 and loxP sites. B Fluorescence image of a coronal section from an Emx1-Cre mouse brain showing expression of ChR2(H134R)-eYFP 12 weeks after AAV PHP.eB injection. The neocortex, hippocampus and white matter structures display high fluorescence. The whisker-associated primary somatosensory cortex (wS1) and the internal capsule (IC) are outlined in white boxes (see higher magnification in & ). RS: retro-splenial cortex; DG: dentate gyrus; CA2: subregion of the Cornus Ammonis (CA) in the hippocampus; M1: primary motor cortex; M2: secondary motor cortex; trS1: trunk region of S1. C Left , Section adjacent to the section displayed in panel B and treated with cytochrome C oxidase. Top right , Higher magnification image of wS1. The arrows show three barrels stained in layer 4. Bottom right , Corresponding fluorescence image of wS1 from the section displayed in panel B (white box 1). Arrows indicate low-density spots located in the regions corresponding to the barrels. Thick vertical fiber bundles cross all layers of wS1. D Inset, Fluorescence image of a tangential cortical section of an Emx1-Cre mouse brain 52 weeks after injection, centered on wS1 layer 4. Higher magnification shows low fluorescence in the barrels compared to septa. E Left , Fluorescence image of a coronal section of an Emx1-Cre x Ai32 mouse brain including the wS1 area. Right , Higher magnification shows high fluorescence in the barrels. F Same as D for an Emx1-Cre x Ai32 mouse brain. The higher magnification image shows high fluorescence in the barrels compared to septa. G Schematic of the three main hypotheses explaining the presence/absence of fluorescence in the neuropil of layer 4 in Emx1-Cre mice. The entities expressing or not the transgene could be: (1) thalamo-cortical neurons projections to L4; (2) projections of other neurons from the same column to the L4; (3) neurites of intra-barrel neurons. H,I Left , Transgene fluorescence (green) and NeuN immunostaining (red) from a tangential cortical section including layer 4 of wS1 from an Emx1-Cre mouse injected with PHP.eB mouse brain ( H ) and from an Emx1-Cre x Ai32 mouse ( I ). Middle , neuronal nuclei revealed by NeuN staining show a slightly higher concentration of somas at the barrel walls. Z-projection of 5 scan sections representing a total thickness of 20 µm. An example barrel is outlined in white. Right , Z-projection of transgene eYFP fluorescence from the same sections. wS1 barrels display low fluorescence in Emx1-Cre mouse injected with PHP.eB ( H ) and high fluorescence in Emx1-Cre x Ai32 mouse ( I ). Note that in panel F, the sparse bright eYFP spots do not colocalize with somatic NeuN spots, suggesting they are due to neuropil bundles (see also ). J Schematic representation of the estimated transgene expression level in the membranes of neurites and somata across cortical layers using the PHP.eB viral strategy ( Top ) and the transgenic mouse line strategy ( Bottom ).

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Injection, Fluorescence, Expressing, Whisker Assay, Staining, Immunostaining, Concentration Assay, Transgenic Assay

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Left Fluorescence image of a sagittal section from an Emx1-Cre mouse brain showing expression of ChR2(H134R)-eYFP 4 weeks after AAV PHP.eB injection. The neocortex, hippocampus and white matter structures are regions of high fluorescence. The frontal pole (3) and the hippocampus (4) are outlined in white boxes. As expected with the Emx1 dependency, no fluorescence was observed in the cerebellum (delineated in white). Cerebellar peduncles display a high level of fluorescence as they convey cortico-cerebellar projections. AC: Anterior commissure; CbP: cerebellar peduncles. Middle Higher magnification of inset 3. Cortical layers are delimited, layer 5 displays the highest fluorescence level. L: layer; CC: corpus callosum. Right Higher magnification of inset 4. Among subregions of the Cornus Ammonis (CA), CA2 displays the highest fluorescence level. Fluorescence drops sharply at its boundaries. DG: Dentate gyrus. B Fluorescence image of the transgene superimposed with DAPI staining of a coronal section through the dentate gyrus from an Emx1-Cre mouse brain 4 weeks after AAV PHP.eB injection. Nuclei are densely packed in the granule cell layer and do not co-localize with the transgene. This illustrates that most of the fluorescence from the transgene comes from the neurites and that the opsin is expressed at the neuronal membrane. C Left Fluorescence image of a coronal section through the internal capsule from an Emx1-Cre mouse brain 12 weeks after AAV PHP.eB injection. Higher magnification of inset 2 from . The internal capsule is a pure white matter structure (only axons). Several bundles of fluorescent axons are visible. Right Higher magnification of the inset 5 showing the axon-like morphology of the fluorescent fibers. D Fluorescence image of a coronal section from an Emx1-Cre mouse brain showing expression of ChR2(H134R)-eYFP 12 weeks after AAV PHP.eB injection. As expected, no fluorescence is observed from the midbrain, the hindbrain and the cerebellum due to the Emx1 dependency. Pyramids are fluorescent since they contain cortical axons projecting down to the medulla. IC: Inferior colliculus; Cb: Cerebellum; V4: fourth ventricle; Pyr: Pyramids. E Fluorescence image of a sagittal section from an Emx1-Cre x Ai32 mouse brain. In agreement with , the whole neocortex is fluorescent and the barrels are brighter than the neighboring layers. S1: primary somatosensory cortex; ulS1: upper limb S1; wS1: whisker S1; Vis. & V1: Visual areas and primary visual cortex. F Fluorescence image of a coronal section from a Scnn1a-Cre mouse brain 7 weeks after PHP.eB injection. Layer 4 of the PPC and auditory areas also show the highest fluorescence among their cortical layers. RSP: retro-splenial cortex; PPC: posterior parietal cortex; A1/AUD: primary auditory cortex and auditory areas. G,H Transgene fluorescence (green) and NeuN immunostaining (red) from a tangential cortical section including layer 4 of wS1 from an Emx1-Cre x Ai32 mouse brain ( G ) and from an Emx1-Cre mouse injected with PHP.eB ( H ). These images are higher magnifications of images in . Neuronal nuclei are circled ( Left ) and match with low-fluorescence areas of the eYFP pattern ( Right ), illustrating that the transgene is expressed at the neuronal membrane and not in the cytoplasm.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Fluorescence, Expressing, Injection, Staining, Membrane, Whisker Assay, Immunostaining

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Top Fluorescence image of a coronal section from two Emx1-Cre mouse brains showing expression of GCamP8m 4 weeks after AAV PHP.eB injection. Apart from the gene of interest (coding for GCamP8m and not ChR2-H134R) the construct is the same as before: Cre dependency and CAG promoter. Part of the whisker S1 area (wS1) is outlined a white box. llS1: lower limb S1; ulS1: upper limb S1; trS1: trunk S1. Bottom Higher magnification of the insets. Fluorescent somas are mostly located in layers 2/3 (L2/3) and layer 5 (L5). Neurons of layer 5 appear brighter than neurons of layer 2/3, probably because of their larger cytoplasm and consequently the higher amount of GCamP8m. There is almost no visible fluorescent soma in layer 4. The background noise and autofluorescence on the right image (mouse n°13) are probably due to the fact that the mouse has not been perfused.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Fluorescence, Expressing, Injection, Construct, Whisker Assay

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Left Fluorescence image of a coronal section from a Scnn1a-Cre mouse brain 7 weeks after AAV PHP.eB injection, centered on wS1 (white box). Right Higher magnification image of wS1. White arrows indicate layer 4 barrels. B Left Fluorescence image of a tangential section from another Scnn1a-Cre mouse 7 weeks after injection. Right Higher magnification image of wS1. C Same as A from a Scnn1a-Cre x Ai32 mouse brain. Only layer 4 barrels are labelled in wS1. llS1: lower limb S1 area; ulS1: upper limb S1 area; S2: secondary somatosensory area. D Same as C for a tangential section from a Scnn1a-Cre x Ai32 mouse brain, centered on wS1 (white box).

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Fluorescence, Injection

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Left , Schematics of the setup. An Emx1-Cre mouse injected with AAV PhP.eB particles was head-fixed under anaesthesia. A 32-channel silicon probe was inserted below the cranial window through a small opening. We recorded neuronal activity while projecting light spots with a digital projector (DLP) through the intact part of the window. Fifteen different spots were projected in pseudo-random sequences, according to a 3×5 stimulation matrix centered above the inserted probe. Right , image of the cranial window with the electrode penetrating the cortex from the side. A sketch of the light spots and of the electrode shank are overlaid. B Mean firing rates of all 5 single units before and during the photostimulation protocol (not specifically after a given spot projection). All units showed an increased activity (Wilcoxon test, * p < 0.05). C Raster plots of action potentials for one single unit, centered on the beginning of the first flash of spot 1 (Top) and spot 11 (Bottom). Four to five phase-locked peaks of response were observed in response to spot 11 while spot 1 didn’t trigger any activity. D Average increase in the number of action potentials per second during the 80 ms following a light spot onset compared to the baseline period activity, for each of the 15 light spots. Same neuron as panel C. E Detection task. Scnn1a-Cre mice injected with AAV.PHP.eB (carrying the gene coding for ChR2-H134R) or control mice were head-fixated under the same DLP system as in A. Mice were awake and water-restricted. For each trial, an optogenetic spot (300 µm diameter) was projected onto the window, either targeting the C2 barrel or targeting the dental cement (sham catch trial). The mouse had to lick a spout to obtain a water reward in the two seconds following the stimulus start. A masking blue light was directed towards the eyes to mask the projection of spots. F Timeline of a single trial. Each trial started with a No Lick Period of random duration (between 3 and 5 s). This period was reset by each lick to force the mice to stop licking and focus on detecting the stimulation. Then, the stimulus was projected during 500 ms: 50 flashes of 5 ms separated by 5 ms (same duty cycle as in ). The trials in which the spot was projected onto the cement accounted for 10% of the total, with one catch trial randomly placed per group of 10 trials. From the first flash, the mouse had 2 seconds to lick the spout twice in order to receive a reward (Reaction period). Licks to both cortical and cement spots were rewarded by a water droplet. Finally, an intertrial period (6 to 8 s long) allowed the mouse to drink its reward before the start of the next trial. G Licking activity for the Scnn1a-Cre mouse injected with the virus (“only L4”) during session 9 of the detection task. Top , Raster plots of licks during all trials of the session. The type of trial (spot onto cortex or cement) is shown by a colored tick on the left (cortex black, cement red) and by the matching color of the licks. Bottom , Mean lick distribution computed over all trials of the session, for trials in which the light spot was projected onto the cortex (black) or cement (red). Time 0 corresponds to the beginning of the first optogenetic flash.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Injection, Activity Assay, Control, Virus

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Top, Detection task, same as . Bottom , Rationale of the task design. We wanted to figure out if the detection of the optogenetic flash was due to the activity it induced or by sensing it in any other way. By rewarding both spots, the mouse had every reason to lick as soon as it detected a spot, whether optogenetically or visually. Thus, the absence of licking for the spot on the cement indicates that it was not detected. B Licking activity for the Scnn1a-Cre mouse injected with the virus (“only L4”) during session 9 of the detection task. Top, Middle Same as . Bottom, For each trial, we quantified the delay for ‘lick onset’ as the time of the first lick after the start of stimulation (reaction time). The mean lick onset distribution was computed over all trials of the session, same color convention as in . The distribution of lick onset delays showed a single narrow peak (mean +/- s.d. = 689 +/- 189 ms). As the duration of the No Lick Period was random, this fixed reaction time to the stimulation further indicated that the mouse was indeed guided by the cue and not exploiting a timing strategy. C Left, Same representations as in B for a Scnn1a-Cre control mouse (no virus injected) during session 9 of the detection task. Left Top , Raster plots of licks. Left Middle, The lick distributions for both spots, through the window and on the cement, were similar, suggesting that there was no activation of neurons or retinal photoreceptors by light from the projector entering through the cranial window. Left Bottom, The distribution of lick onsets was not concentrated around one reaction time but as very broad (mean +/- s.d. = 540 +/- 455 ms), suggesting random initiation of licking bursts after the necessary still No-lick period. See also panel D. Right, Same analysis as Left for session 10 in the same control mouse, during which a spot was presented on wS1 or no stimulation was presented (no light or any cue at all) (purple curve and ticks). Top and Middle, In the absence of any stimulation, the animal still licked in the same way as it did for the spot on the cortex. Bottom, Reaction times to both spots were also similar. Overall, this suggests that the apparent detection of both spots by the control mouse was merely the effect of recurrent and sustained licking, i.e., a timing strategy that the mouse developed to take advantage of the temporal structure of the task. Due to technical issues, two consecutive partial sessions were run and pooled for each of sessions 9 ( Left ) and 10 ( Right ) of the control mouse. D Distributions of trial durations during session 9 for the virus-injected (green) and the control mice (orange). The duration of a trial was computed as the time spent in the No Lick Period (including all the resets) and the reaction period. The best theoretical mean trial duration (without any reset of the No Lick Period) would be 6 s. Arrows indicate the mean trial duration. Trial durations were much longer for the control mouse (up to 98 s, mean +/- s.d. = 25.6 +/-20.0 s) compared to the PhP.eB injected mouse (mean +/- s.d. = 6.6 +/- 1.7 s), mostly because of repeated resetting of the No Lick period. This difference confirmed the use of a timing strategy in the absence of opsin expression.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Activity Assay, Injection, Virus, Control, Activation Assay, Expressing

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Left, Sensory-guided licking task. A rotating light bar was projected on wS1 through a cranial window by a digital light processor (DLP). A water tube detected licks and delivered rewards when adequate. Right, An example image of the cortical surface during optogenetic stimulation (the white bar corresponds to blue light in grey scale). B Position of the stimulation space (blue circle) on the cortical functional map (grey) in S1. The space is centered on the C2 barrel and is restricted to the wS1 area. The map contours were adapted from , and , and positioned according to intrinsic imaging of several barrels (panel C). C Example mapping of wS1 barrels under anesthesia prior to training. Left, Contours of intrinsic imaging responses to single whisker stimulations (Alpha, Gamma, C2 and Delta whiskers) superimposed on an image of the cortical surface. Right , Temporal light fluctuations (ΔI/I0) of the corresponding regions of interest during trials. The stimulation period is indicated by the grey shading. D Left, Different zones of the stimulation space. When the bar was in the Rewardable zone (green), licks were rewarded. In the No-lick zone (red), a lick ended the trial. In the Neutral zones (white), licks were ignored. Right, Snapshots of an example trial showing the displacement of the light bar on the barrel cortex. E Spatial distribution of the optogenetic bar angle at all lick timestamps for one example Emx1-Cre mouse trained 5 weeks after AAV PHP.eB injection, for the 1st and the 10th (Last) sessions. F Average proportions (mean ± s.e.m) of all licks for which the optogenetic bar was in the Rewardable (green), No-lick (red), and Neutral zones (black) during training of Emx1-Cre mice injected with PHP.eB of the “all layers except L4” group (n = 5). The “Last” session consists of the 10th session for two mice and the 15th for three mice. Wilcoxon test, * p < 0.05. G Average proportions (mean ± s.e.m) of all licks during training (same quantification as F ). Left, Mean values from the first and last sessions for Emx1-Cre mice injected with PHP.eB (n = 5). Middle, Same for two Scnn1a-Cre mice (A and B) injected with PHP.eB (n = 2). Right, Same for Emx1-Cre x ChR2-tdT mice (n = 8). The “Last” session differs between groups, see panel H. Wilcoxon test * p < 0.05. H Left, Schematics of expression profile for the three groups: ‘all layers except L4’ Emx1-Cre + virus (purple), ‘only L4’ Scnn1a-Cre + virus (pink) and ‘all layers’ Emx1-Cre x Ai27 (green) mice. Blue light intensity was adapted to the cortical depth of opsin-expressing cells (see Methods). Right, Individual learning curves, quantified by the percentage of rewarded trials normalized by subtracting the chance level (see Methods). Same color code. I Learning speed measurement. Number of training sessions (one per day) to reach at least 10% of rewarded trials after normalization (corresponding to around 30% of raw percentage, ). Each circle represents a mouse. Same color code as panel H . The empty circle (n.a) represents an animal that never exceeded the chance level.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Functional Assay, Imaging, Whisker Assay, Injection, Expressing, Virus

Journal: bioRxiv

Article Title: Specific targeting of layer 4 for direct cortical stimulation is not necessary to induce reliable perception

doi: 10.64898/2026.01.08.698338

Figure Lengend Snippet: A Raw (Left) and normalized (Right) learning curves quantified by the percentage of rewarded trials for Emx1-Cre mice injected with PHP.eB (n = 5, A ); Scnn1a-Cre mice injected with PHP.eB (n = 2, B ); and Emx1-Cre x ChR2-tdT mice (n = 8, C ). The normalized rewarded trial percentage is obtained by subtracting the chance level (see Methods). For the three mouse groups, the raw learning curves have similar shapes and dynamics to the corresponding normalized ones. The “Last” session differs between groups. For the group Emx1-Cre + virus, 2 mice were trained up to day 10 and three up to day 15. Scnn1a-Cre + virus mice were trained up to day 15 and 19. For the Emx1-Cre x Ai27 group, five mice were trained up to day 5, two mice up to day 7 and one up to day 10. Curves on the right are the same as but truncated. Wilcoxon tests, * p < 0.05., ** p < 0.01.

Article Snippet: We injected AAV PHP.eB particles (Addgene #127090) to obtain a Cre-dependent expression of ChR2-H134R fused to eYFP in excitatory neurons in all cortical layers (10 Emx1-Cre mice) or in L4 (4 Scnn1a-Cre mice).

Techniques: Injection, Virus