mapseq sindbis virus library  (Cold Spring Harbor Laboratory Meetings)

Journal: bioRxiv

Article Title: Topographic alignment of auditory inputs to the visual cortex

doi: 10.1101/2025.07.29.667237

Figure Lengend Snippet: a, Two hypothetical modes of information transfer from auditory cortex (AC) to the visual cortex. Auditory information (arrows) could be uniformly distributed across visual cortical areas (left) or selectively broadcast to specific visual areas (right). b, Experimental schematic for mapping single neuron projections using MAPseq. The barcoded Sindbis virus library is injected into the primary auditory cortex (A1), labelling neurons with unique RNA barcodes (BC) that are transported into axon terminals. Brain regions of interest are dissected into cubelets via laser capture microdissection (LCM) and barcodes present in each cubelet are sequenced. c, Total bulk MAPseq counts in individual cortical target cubelets recapitulate conventional anterograde tracing projection patterns (Pearson correlation coefficient r = 0.65, p =7×10 −42 ; 338 cubelets from 3 mice, mean z-scored bulk GFP signal from 3 mice). d, MAPseq recapitulates brain-wide projection patterns of pyramidal tract (PT), corticothalamic (CT) and intratelencephalic (IT) neurons (4141 neurons across 3 mice). e, Layer distribution of IT, CT and PT neurons. CT and PT neurons are predominantly found in deep layer cubelets (885 and 1910 superficial and deep layer neurons). f, Mean area-level corticocortical normalised projection density estimated by MAPseq recapitulates conventional anterograde tracing (25 areas, N=3 mice for A1 MAPseq and N=3 mice for bulk anterograde tracing from A1; error bars are standard deviation across mice in log space back-transformed to linear scale; Pearson correlation coefficient r=0.85, p =7×10 −8 ) . g, Example projection patterns of single neurons across cortical cubelets visualized on a cortical flatmap (top) and their projection densities across cortical areas (bottom) normalized to the maximum for each neuron. Colormap indicates normalised projection density for the sampled areas. Areas in white were not sampled. h, Proportions of neurons projecting only to majority auditory cortex cubelets and outside of the auditory cortex ( N=3 mice). i, Proportions of neurons targeting different cortical areas. Black bars group neurons projecting to any of the cortical areas specified on top of the panel. Purple bars show proportions for individual areas (N=3 mice).

Article Snippet: The MAPseq Sindbis virus library (JK100L2, MAPseq/BARseq Core facility, Cold Spring Harbor Laboratory) was injected through a small craniotomy into the auditory cortex.

Techniques: Virus, Injection, Laser Capture Microdissection, Anterograde Tracing, Standard Deviation, Transformation Assay

Journal: bioRxiv

Article Title: Topographic alignment of auditory inputs to the visual cortex

doi: 10.1101/2025.07.29.667237

Figure Lengend Snippet: a, Cortical flatmaps showing distribution of bulk MAPseq barcode counts across cubelets for individual mice. b, Flatmaps of summed projection strength of bulk anterograde GFP tracing for three different experiments from the Allen Connectivity Atlas. c, Z-scored bulk projection density for individual Allen Connectivity Atlas anterograde tracing experiments plotted against each other. d, Comparison of mean area-specific normalised projection density between mice using the “homogeneous across cubelet” (where barcodes are assumed to be homogeneously distributed across cubelets) e, “ homogeneous across area,” in which barcode distribution is assumed to be homogeneous across brain areas, and f, “ area is main” where barcode counts assigned to brain regions with largest representation within each cubelet (see methods).

Article Snippet: The MAPseq Sindbis virus library (JK100L2, MAPseq/BARseq Core facility, Cold Spring Harbor Laboratory) was injected through a small craniotomy into the auditory cortex.

Techniques: Anterograde Tracing, Comparison

Journal: bioRxiv

Article Title: Topographic alignment of auditory inputs to the visual cortex

doi: 10.1101/2025.07.29.667237

Figure Lengend Snippet: a, Heatmap showing mean Spearman correlation coefficient between cortical brain areas in LCM samples across all 3 mice. In addition to biases in observed co-projections based on cubelet structure, axon projection labelling efficiency by MAPseq Sindbis virus can vary between neurons. Neurons with lower labelling efficiency would have fewer barcode counts in their target areas, which would lead to some projection targets being missed due to limits in sensitivity. b, To test whether differences in labelling efficiency affected observation of co-projection motifs, we simulated observed/expected number of co-projections with either equal efficiency of labelling (left) or variation in neuron labelling efficiency (right). In the simulation, 100,000 neurons were sampled with p=0.2 of targeting each of the 10 areas and given either a constant or variable labelling efficiency. Expected co-projections are the product of individual marginal probabilities (independence assumption). We observed that simulated differences in axon barcode labelling efficiency can lead to artificial over-representation of co-projection motifs. c, To correct for spurious over-represented co-projection motifs that arise when two target areas fall within the same cubelet, we compared observed data to the same dataset after shuffling neuron barcode identifies within cubelets. However, normalising by the log₂ fold change between observed and shuffled simulations revealed that simple random shuffling (left) does not correct for the overrepresentation bias due to variable efficiency. In contrast, shuffling using the curveball algorithm (right) which preserves individual neuron labelling efficiencies was able to correct for artificial overrepresentation due to variable labelling efficiencies in the simulation. d, As predicted by simulations, applying this same principle to our MAPseq dataset showed that correcting for both variable labelling efficiencies and shared areas within cubelets using the curveball shuffle approach (right) decreased co-projection motif effect sizes compared to the random shuffle approach (which only corrects for shared areas within cubelets, left). e, Heatmaps showing conditional probabilities of A1 neurons targeting cortical co-projection targets ‘Co-projection target’ given neurons already target a given cortical area (‘Cortical area’). e, shows actual data conditional probabilities. To determine statistical significance from chance, we used a shuffle population of 100,000 shuffles, maintaining differences in labelling efficiency and marginal probabilities (see Methods). For visualisation, the e’ heatmap shows the mean of 100,000 shuffles, and e’’, shows the effect size (log2(observed/shuffled conditional probabilities) red and blue circle colours indicate increased and decreased effect sizes respectively, shading indicates strength of p -value (as indicated on the colour-bar), black outline indicates p ≤0.05. P -values are calculated from the null shuffled population distribution after Bonferroni correction.

Article Snippet: The MAPseq Sindbis virus library (JK100L2, MAPseq/BARseq Core facility, Cold Spring Harbor Laboratory) was injected through a small craniotomy into the auditory cortex.

Techniques: Virus

Journal: bioRxiv

Article Title: Topographic alignment of auditory inputs to the visual cortex

doi: 10.1101/2025.07.29.667237

Figure Lengend Snippet: a, Summary of different second strand synthesis approaches compared in optimisation of MAPseq sequencing library preparation. In the non-targeted approach, the nicked mRNA is used as primers for second strand synthesis, while in the targeted approach, RNA is degraded and a targeted primer anneals to the single stranded DNA and used for second strand synthesis. b, Comparison of non-targeted and targeted second strand synthesis approaches in MAPseq library preparation shows greater yield of final PCR product using the targeted approach. N = 4, p =0.017, paired two-tailed t- test. c, Diagram illustrating template switching during PCR amplification in MAPseq library preparation. d, 2D histogram showing the unique molecular identifier (UMI) count for neuron barcodes with the same UMI, where 1 st max is the UMI count for the neuron-barcode/UMI combination that is most abundant, and 2 nd max is UMI count for the neuron-barcode/UMI combination that is second most abundant. At 1 st max /2 nd max ratios of less than 10 we observe a decrease in entropy, as well as d’, an increase in AT content in the UMI sequence, suggesting that shared UMIs with neuron barcodes with similar UMI counts are resultant of sequencing artifacts or biases in random nucleotide synthesis during oligo synthesis. Ratios with 1 st max /2 nd max ≥10 abundance were therefore considered template switching events and only the most abundant neuron-barcode/UMI combination kept, while below this ratio all reads were discarded. e, UMI count distribution for negative control (olfactory bulb) LCM samples in MAPseq dataset. Setting a UMI count threshold of ≥2 removes 95% of barcodes from negative control.

Article Snippet: The MAPseq Sindbis virus library (JK100L2, MAPseq/BARseq Core facility, Cold Spring Harbor Laboratory) was injected through a small craniotomy into the auditory cortex.

Techniques: Sequencing, Comparison, Two Tailed Test, Amplification, Oligo Synthesis, Negative Control

Journal: Science Advances

Article Title: Networks of habenula-projecting cortical neurons regulate cocaine seeking

doi: 10.1126/sciadv.abj2225

Figure Lengend Snippet: ( A ) Summary of MAPseq procedure. ( B ) Percentage of barcodes detected in the mPFC also detected in brain sites listed on y axis. Data were collected from n = 2 mice. ( C ) Heatmap representation of mPFC barcodes (% total number) detected in brain regions shown on y axis and in regions shown on x axis. ( D ) Histogram of connectome of mPFC ➔PVT , mPFC ➔VTA , and mPFC ➔LHb neurons. Shown is the distribution of barcodes (%) for mPFC cells that project to the PVT, VTA, and LHb. ( E ) Summary of injection procedure to label cortical-habenular neurons that project to VTA and fluorescence image of mPFC from mice injected with CTb retrograde tracers in the LHb and VTA. Shown are CTb-594 (red; mPFC ➔LHb neurons)–labeled and CTb-488 (green; mPFC ➔VTA neurons)–labeled cells. White arrows identify dual-labeled cells (yellow; mPFC ➔LHb VTA neurons) in the mPFC. A total of n = 5 mice were imaged. ( F ) Summary of injection procedure to label cortical-habenular neurons that project to the LC and associated fluorescence image of mPFC from injected mice (red; mPFC ➔LHb neurons) and GFP (green; mPFC ➔VTA neurons). White arrows identify dual-labeled cells (yellow; mPFC ➔LHb LC neurons) in the mPFC. A total of n = 5 mice were imaged. ( G ) Injection procedure (the tracing the relationship between input and output method, also known as the TRIO method) to label cortico-habenular neurons that project to VTA. Shown are mCherry + cells in the mPFC, which are cortico-habenular neurons that synapse into LHb neurons that project to VTA. A total of n = 4 mice were imaged. ( H ) Injection procedure to label cortico-habenular neurons that project to the LC. mCherry + cells were detected in the mPFC (right), which are cortico-habenular neurons that synapse into LHb neurons that project to the LC. A total of n = 3 mice were imaged. ( I ) Graphical representation of the cortico-habenular connectome. A population of mPFC neurons (shown in blue) projects concurrently to the LHb and then to the same monoaminergic brain centers to which LHb neurons also project (shown in red).

Article Snippet: This was accomplished by injecting the MAPseq virus (Sindbis virus, Cold Spring Harbor Laboratories) into the mPFC.

Techniques: Injection, Fluorescence, Labeling