https://doi.org/10.5061/dryad.wh70rxwv4

Code and data for mouse hippocampal single-neuron projectome and spatial transcriptome analysis and visualization.

Description of the data and file structure

The neuronal data is represented as tree-structured swc files following specifications as: http://www.neuronland.org/NLMorphologyConverter/MorphologyFormats/SWC/Spec.html. All the swcs are registered to the Allen Template Brain CCF 2017 (Wang, Quanxin, et al. "The Allen mouse brain common coordinate framework: a 3D reference atlas." Cell 181.4 (2020): 936-953) with brain regions following Allen naming conventions, e.g. HPF: Hippocampal formation; HIP: Hippocampal region; CA1: Field CA1; CA3: Field CA3; DG: Dentate gyrus; SUB: Subiculum.

We organize the repository following research article figure groups; at the same time, we provide description for our data and scripts below for easy and independent access to our resources. The projectome data are mainly presented in: 1. readable tabular and JSON files, with description listed below; 2. pickle objects that can be read using Python pickle.load(open($file_path, 'rb')) function, stored within each figure with variable description in [note for pkl files.ipynb] under its designated directory. The spatial transcriptome data are stored as Seurat object that can be read using R readRDS($file_path) function.

The main structure for the repository:

• archive

  ###pre-requisites to run python files.
  ├─ requirements.txt

  ###data and scripts (*.ipynb, *.py, *.R) for analysis grouped by figures.
  ├─ figures

  ###Projectome subtype classifications of hippocampal neurons.
  │ ├─ fig1
  │ │ ├─ fig1_D
  │ │ ├─ fig1_E
  │ │ ├─ fig1_panelD.ipynb ###Code for heatmap showing the whole-brain projection strength of caudal part and rostral part for each single neuron
  │ │ └─ fig1_panelE.ipynb ###Code for hierarchical clustering based on the whole-brain projection patterns.
  │ │ └─ note for pkl files.ipynb

  ###projection target-dependent soma locations of hippocampal neurons.
  │ ├─ fig2
  │ │ ├─ fig2_A
  │ │ ├─ fig2_A.ipynb ###Code for the top 50 most frequent single-cell target patterns
  │ │ ├─ fig2_C
  │ │ ├─ fig2_C.ipynb ###Code for the distribution of LEC-MEC selectivity for each ENT projecting neurons and their distribution in CA1, SUB, ProS and SUBr neurons
  │ │ ├─ fig2_G
  │ │ ├─ fig2_G.ipynb ###Code for showing the neuron number and soma locations in given joint projection patterns and subtypes
  │ │ ├─ fig2_H
  │ │ ├─ fig2_H.ipynb ###Code for showing the soma locations for specified projection patterns among given target areas.
  │ │ └─ note for pkl files.ipynb

  ###Spatial correlation between transcriptome and projectome subtypes;
  │ ├─ fig3
  │ │ └─ figure_3.xlsx ###list of data and functions linked to spatialTranscriptome/R_data and spatialTranscriptome/python_data;

  ###Bi-hemispheric projecting neurons in hippocampus.
  │ ├─ fig4
  │ │ ├─ fig4_A
  │ │ ├─ fig4_A.ipynb ###Code for showing the distribution of bilaterally projecting neurons in different subtype
  │ │ ├─ fig4_E
  │ │ ├─ fig4_E.ipynb ###Code for showing the distribution of CA3 neurons and CA1 neurons that bilaterally projecting to HIP
  │ │ ├─ fig4_H
  │ │ ├─ fig4_H.ipynb ###Code for showing the projection strength in different layers of bilateral LEC and MEC for ProS,PAR,PRE,POST neurons that could bilaterally projecting to RHP
  │ │ ├─ fig4_I
  │ │ ├─ fig4_I.ipynb ###Code for showing intersection of neurons that bilaterally projecting to different areas.
  │ │ ├─ fig4_J
  │ │ ├─ fig4_J.ipynb ###Code for showing the neuron number, soma distribution in sub-hip regions and ipsilateral or contralateral projection strength in different target areas
  │ │ ├─ fig4_L
  │ │ ├─ fig4_L.ipynb ###Code for showing the ipsi-preference index of neurons having bilateral projections
  │ │ ├─ fig4_M
  │ │ ├─ fig4_M.ipynb ###Code for showing the ratio of ipsi-only,contra-only and bilateral projection neurons
  │ │ └─ note for pkls.ipynb

  ###Projection patterns of CA1, SUB and SUBr neurons
  │ ├─ fig5
  │ │ ├─ fig5_A
  │ │ ├─ fig5_A.ipynb ###Code for showing enriched target patterns in CA1, SUB, ProS and SUBr neurons.
  │ │ ├─ fig5_B
  │ │ ├─ fig5_B.ipynb ###Code for showing the neuron number percentage which have different target number
  │ │ ├─ fig5_C
  │ │ ├─ fig5_C.ipynb ###Code for showing the collateral index of CA1,SUB,ProS and SUBr neurons projecting to different downstream areas.
  │ │ ├─ fig5_EFG
  │ │ ├─ fig5_EFG.ipynb ###Code for showing the correlation motif of downstream areas for CA1,SUB,ProS and SUBr neurons
  │ │ ├─ fig5_H
  │ │ ├─ fig5_H.ipynb ###Code for showing the intra-HPF projection patterns and extra-HPF projection patterns of neurons preferring different intra-HPF targets.
  │ │ ├─ fig5_I
  │ │ ├─ fig5_I.ipynb ###Code for showing the collateral index of neurons preferring different intra-HPF target
  │ │ ├─ fig5_K
  │ │ ├─ fig5_K.ipynb ###Code for showing the correlation of projection strength in intra-HPF tragets and extra-HPF targets
  │ │ └─ note for pkl files.ipynb

  ###Longitudinal spread of Schaffer collaterals and mossy fiber axons
  │ ├─ fig6
  │ │ ├─ fig6_C
  │ │ ├─ fig6_D
  │ │ ├─ fig6_F
  │ │ ├─ fig6_H
  │ │ ├─ fig6_I
  │ │ ├─ fig6_J
  │ │ ├─ fig6_K
  │ │ ├─ fig_6C.ipynb ###Code for showing the T-L index of axon projections in ipsilateral and contralateral CA3 from every single CA3 neuron
  │ │ ├─ fig_6D.ipynb ###Code for showing the T-L index of axon projections in ipsilateral and contralateral CA1 from every signle CA3 neuron
  │ │ ├─ fig_6F.ipynb ###Code for showing the T-L index of axon projections in DG and CA3 from every signle DG neuron
  │ │ ├─ fig_6H.ipynb ###Code for showing the statistic of overall T-L index for all CA3 neurons
  │ │ ├─ fig_6J.ipynb ###Code for showing the statistic of overall T-L index for all DG neurons
  │ │ ├─ fig_6i.ipynb ###Code for showing the statistic of overall span for all CA3 neurons
  │ │ └─ fig_6k.ipynb ###Code for showing the statistic of overall span for all DG neurons
  │ │ └─ note for pkl files.ipynb

  #Dependence of axon arbor distribution on the transverse location of soma.
  │ ├─ fig7
  │ │ ├─ HIP_CA1_CA3_proximodistal_analysis.py ###code for HPF transverse plane analysis
  │ │ ├─ allen_annotation
  │ │ │ └─ allen_struct_info.json ###json file that store the Allen CCFv3 structure information
  │ │ ├─ resource
  │ │ │ ├─ CA1_ipsi_targets_proximodistal_info.csv ###table of CA1 neuron's location on CA1 proximodistal and longitudinal position, mean terminal location in specific target regions.
  │ │ │ ├─ CA1_soma_info.csv ###table of CA1 neuron's soma information, for example, neuron id, neuron's soma location, neuron's laterality, neuron's depth in CA1.
  │ │ │ ├─ CA1_terminal_info.csv ### table of CA1 neuron's terminal information, for example, terminal location, laterality, CCFv3 structure id that the terminal located in.
  │ │ │ ├─ CA3_soma_info.csv ###table of CA3 neuron's soma information, for example, neuron id, neuron's longitudinal location in HIP, neuron's laterality, neuron's proximodistal location in CA3.
  │ │ │ └─ CA3_terminal_info.csv ### table of CA3 neuron's terminal information, for example, laterality, CCFv3 structure id that the terminal located in, terminal's longitudinal position and depth in CA1.
  │ │ └─ utils
  │ │ ├─ init.py
  │ │ └─ allen_struct_info.py ### map function between Allen CCFv3 structure informations, for example, map CCFv3 structure id to CCFv3 structure acronym.

  ###Dependence of axonal arbor distribution on the longitudinal location of soma
  │ ├─ fig8
  │ │ ├─ fig8_C
  │ │ ├─ fig8_D
  │ │ ├─ fig8_EF
  │ │ ├─ fig8_G
  │ │ ├─ fig_8C.ipynb ###Code for showing the topographic projection correlation between the somalocations and the axon terminal locations for all ENT projecting neurons
  │ │ ├─ fig_8D.ipynb ###Code for showing the whole-brain topographic projection correlation between the somalocations and the axon terminan locations
  │ │ ├─ fig_8EF.ipynb ###Code for showing the correlation between somalocations and the target number,the correlation between the longitudinal span and the target number
  │ │ └─ fig_8G.ipynb ###Code for showing the whole brain topographic projection patterns for all the neurons
  │ │ └─ note for pklfiles.ipynb

  ###dendrite analysis
  │ ├─ fig_s10
  │ │ ├─ CA1_to_MSC_dendritic_analysis.py ### Code for dendritic morphological feature analysis of MSC projecting CA1 neurons.
  │ │ └─ resource
  │ │ ├─ CA1_dendritic_cluster.csv ### Dendritic cluster of all CA1 neurons.
  │ │ ├─ CA1_projection_strength.csv ### MSC projecting CA1 neurons' projection strength in different target regions.
  │ │ ├─ CA1_to_MSC_dendritic_cluster.csv ###Dendritic clusters of MSC projecting CA1 neurons
  │ │ ├─ CA1_to_MSC_dendritic_features.csv ###Dendritic mophological features of MSC projecting CA1 neurons
  │ │ ├─ CA1_to_MSC_feature_importance.csv ###Dendritic feature importance for dendritic classicication.
  │ │ ├─ CA1_to_MSC_soma_info.csv ###Location information of MSC projecting CA1 neurons
  │ │ └─ MSC_projecting_axon_length.csv ### CA1 neurons' axon length in MSC

  ###Intra-HPF target-specific preference in hippocampal axon projections.
  │ ├─ fig_s11
  │ │ ├─ figS11_A.ipynb ###Code for clustermap of intra-HPF projection patterns.
  │ │ ├─ figS11_B.ipynb ###Code for intra-HPF projection subtype distribution in CA1, SUB, ProS, and SUBr neuron groups.
  │ │ ├─ figS11_DEFG.ipynb ###Code for intersection among neurons having different target numbers, neurons projecting different targets and intra-HPF projection subtype
  │ │ ├─ figS11_H.ipynb ###Code for co-projecting neuron numbers of intra-HPF areas and extra-HPF areas
  │ │ ├─ figS11_I.ipynb ###Code for the fraction of SUBr neurons in each intra-HPF prjectiono subtype
  │ │ ├─ fig_s11A
  │ │ ├─ fig_s11B
  │ │ ├─ fig_s11DEFG
  │ │ ├─ fig_s11H
  │ │ ├─ fig_s11I
  │ │ └─ note for pklfiles.ipynb

  ###superficial/deep CA1 neuron analysis
  │ ├─ fig_s12
  │ │ ├─ S12.py ### Code for demostrate the projection pattern difference of superficial CA1 neurons and deep CA1 neurons.
  │ │ └─ resource
  │ │ ├─ S12_deep_superficial_projection_features.csv ### Projection strength in different target regions of deep superficial CA1 neurons.
  │ │ └─ S12_dorsal_CA1_soma_depth_and_projection_features.csv ### Table of neurons' location and projection strength in target regions.

  ###Morphological features of neurons in hippocampal subregions.
  │ ├─ fig_s2
  │ │ ├─ fig_s2_morphology_parameters.ipynb ###Code for box plot for morphology features quantification.
  │ │ └─ morphology_parameters.xlsx ###Morphology features quantification.

  ###Neuron clustering.
  │ ├─ fig_s4
  │ │ ├─ fig_s4D
  │ │ ├─ fig_s4E
  │ │ ├─ figs4_D.ipynb ###Code for dot plot of downstream target patterns along axon trajectories of axon arbor templates.
  │ │ ├─ figs4_E_caudal.ipynb ###Code for hierarchical clustering of caudal projection patterns
  │ │ ├─ figs4_E_combine.ipynb ###Code for generate whole-brain projection patterns by combining caudal projection patterns and rostral projection patterns
  │ │ └─ figs4_E_rostral.ipynb ###Code for hierarchical clustering of rostral projection patterns
  │ │ └─ note for pklfiles.ipynb

  ###Discrete longitudinal distribution of projectome subtypes.
  │ ├─ fig_s7
  │ │ ├─ fig_s7_plot_distribution.ipynb ###Code for distribution plot for projectome subtypes.
  │ │ ├─ neuronInfo.csv ###Neuron sample information.
  │ │ ├─ scatter.csv ###Downstream projection patterns for subtype5 and subtype21 neurons.
  │ │ ├─ slopes.csv ###Distribution quantification for each projectome subtypes.
  │ │ └─ slopes_region.csv ###Distribution quantification for each projectome subtypes per region.

  ###The long-range projection from neurons in DG, CA2 and CA3.
  │ ├─ fig_s8
  │ │ ├─ figS8_B.ipynb ###Code for pie plot and distribution plot for DG long-range projection neurons of subtype 5, 34, 35, 39.
  │ │ ├─ figS8_C.ipynb ###Code for violin plot of axon lengths in target areas (HPF, LSX and MSC) for DG long-range projection neurons of subtype 5, 34, 35, 39.
  │ │ ├─ figS8_E.ipynb ###Code for stacked bar plot of DG-mo, DG-po, and DG-sg neuron numbers in local and long-range projection DG neurons.
  │ │ ├─ figS8_I.ipynb ###Code for pie plot and distribution plot for CA3 neurons of subtype 5, 34, 35, 39, and 43.
  │ │ ├─ figS8_J.ipynb ###Code for violin plot of axon lengths in target areas (HPF, LSX and MSC) for CA3 neurons of subtype 5, 34, 35, 39, and 43.
  │ │ ├─ figS8_LMN.ipynb ###Code for neuron distribution and downstream projection patterns for CA2 neurons of subtype 34, 39, and 43.
  │ │ ├─ fig_s8B
  │ │ ├─ fig_s8C
  │ │ ├─ fig_s8E
  │ │ ├─ fig_s8I
  │ │ ├─ fig_s8J
  │ │ ├─ fig_s8LMN
  │ │ └─ note for pklfiles.ipynb

  ###Spatial transcriptome analysis for CA1 sections.
  │ └─ fig_s9
  │ └─ figure_s9.xlsx ###list of data and functions linked to spatialTranscriptome/R_data and spatialTranscriptome/python_data.

  ###data and scripts (*.R, *.ipynb) to analyze spatial transcriptome and its correlation to projectome, including Seurat objects for mouse CA1 spatial transcriptome analysis and documents on how to use them, mainly used for fig3 and fig_s9.
  └─ spatialTranscriptome
  ├─ How to process transcriptomic data.pptx

  ├─ R_data
  │ ├─ bregma.txt ###chip bregma coordinates.
  │ ├─ cluster
  │ │ └─ cluster.rds ###Seurat object of CA1 spatial bin-resolution cells
  │ ├─ cluster_pipeline.R ###code for CA1 cells processing and clustering
  │ ├─ mask_data ###Allen brain region mask.
  │ ├─ subcluster
  │ │ ├─ CA_subcluster_transcriptomic_proportion.csv ###transcriptomic proportion in CA1 subcluster C1.
  │ │ ├─ CA_subtype_projectomic_proportion.csv ###projectome proportion in bregma.
  │ │ └─ subcluster.rds ###Seurat object of CA1 subcluster C1 spatial bin-resolution cells
  │ └─ subcluster_pipeline.R ###code for CA1 subcluster C1 cells processing and clustering

  └─ python_data
  ├─ fig3_GI_figS9_J.ipynb ###code to plot cell distribution in coronal slice
  ├─ for_qc_heatmap_and_cor.csv ###sample information
  └─ mask.csv ###Allen brain region mask.

Sharing/Access information

This data is also available at:

• [https://doi.org/10.12412/BSDC.1697181607.20002] (Brain Science Data Center, Chinese Academy of Sciences)

Code/Software

All data can be used independently. All scripts can be run seperately using desired functions and data with pre-requisite libraries installed properly.

The pre-requisites to run the Python code are listed in requirements.txt, the Python version used is Python 3.8.18.

The pre-requisites to run the R code are listed below with R 4.3.1:

• Seurat 4.4.0
• tidyverse 2.0.0
• pheatmap 1.0.12
• reshape2 1.4.4
• harmony 1.0.3