General Information

This readme file was generated on 2024-12-05 by Saroj Sah

These datasets involve the analysis of synaptic loci in the M2 prefrontal cortex of mice at 1 month, 3 months, and 6 months post-PFF injection into the dorsal striatum. 
Alpha-synuclein monomer served as the control. 
Brain sections were stained with antibodies against pre-synaptic proteins, post-synaptic proteins, and p-synuclein S129. 
The total number of pre- and post-synaptic puncta was determined using Imaris, based on puncta intensity and size.

Pre- and post-synaptic puncta with the top 20% intensity were analyzed to generate a Gaussian distribution (frequency distribution) of synaptic loci formed by the nearest neighboring pre- and post-synaptic proteins. 
For the VGluT2/Homer1 synaptic loci analysis, the top 60% intense pre- and post-synaptic puncta was considered. 

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List of Dataset analyzed for synapse loss at time point 1 month post inject (1MPI), 3 month post injection (3MPI) and 6 month post injection (6MPI IS(Ipsilateral) and CS(Contralateral)):
SEQUIN Dataset 1MPI_VGlut1_Homer1_Synaptic_Loci_Frequency_Distribution_Density.xlsx
SEQUIN Dataset 3MPI_VGlut1_Homer1_Synaptic_Loci_Frequency_Distribution_Density.xlsx
SEQUIN Dataset 3MPI_VGlut2_Homer1_Synaptic_Loci_Frequency_Distribution_Density.xlsx
SEQUIN Dataset 3MPI_VGAT_Gephyrin_Synaptic_Loci_Frequency_Distribution_Density.xlsx
SEQUIN Dataset 6MPI_VGAT_Gephyrin_Synaptic_Loci_Frequency_Distribution_Density.xlsx
SEQUIN Dataset 6MPI_VGlut1_HomerSynaptic_Loci_Frequency_IS.xlsx
SEQUIN Dataset 6MPI_VGlut1_HomerSynaptic_Loci_Frequency_CS.xlsx

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File Overview
The analyses in the various worksheets of of an excile file come from the same imaging dataset. Analyses were performed for excitatory and inhibitory synaptic loci: VGluT1-Homer1 (1MPI, 3MPI, 6MPI), VGAT-Gephyrin (3MPI, 6MPI), and VGluT2-Homer1 (3MPI).

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Data-Specific Information in each excel file for the imaging dataset is shown in different worksheets:

1. SynapticLoci_Freq_Distribut
- This sheet contains the frequency distribution of synaptic loci formed by the nearest pre- and post-synaptic puncta (NN) pairs, presented on an image-wise basis for each mouse.
  - Column headings:
    - HistogramBin: NN pairs are distributed along the column for each image against a geometric series of separations: 10 nm, 30 nm, 50 nm, etc.
    - Monomer: Mice injected with monomer.
    - PFF: Mice injected with PFF.
  - Raw headings:
    - Injection: Injected material.
    - MouseNo.: Animal ID.
    - ImageNo.: The Image analyzed for the given mouse.
    - SynapticLoci_Frequency: Number of NN pairs for each image against a particular geometric series of separations.

2. Synaptic_Loci_Density
- This sheet contains cumulative NN pairs that fall within the Gaussian distribution for each image and the density calculated from the image volume and cumulative NN pairs.
  - Column headings:
    - Group: Mouse group injected with either PFF or Monomer.
  - Raw headings:
    - MouseNo.: Animal ID.
    - ImageNo.: The Image analyzed for the given mouse.
    - Synaptic_Loci#: Cumulative NN pairs within the Gaussian distribution for each image.
    - Density_# / µm³: Synaptic_Loci# per µm³ (density) for each image.

3. Pre_Post_Synaptic_Puncta_Density
- This sheet contains details on the number of pre- and post-synaptic puncta analyzed using Imaris, and the density was calculated.
  - Column headings:
    - Group: Mouse group injected with either PFF or Monomer.
  - Raw headings:
    - MouseNo.: Animal ID.
    - ImageNo.: The Image analyzed for the given mouse.
    - Pre-synaptic_puncta_#: Number of pre-synaptic puncta.
    - Post-synaptic_puncta_#: Number of post-synaptic puncta.
    - Pre-synaptic_puncta_Density_# / µm³: Number of pre-synaptic puncta per µm³ (Density) for each image.
    - Post-synaptic_puncta_Density_# / µm³: Number of post-synaptic puncta per µm³ (Density) for each image.

4. NN_Separation
- This sheet (only for VGlut1-Homer and VGluT2-Homer) contains details on the mean separation between the nearest pre- and post-synaptic puncta (NN pairs) for synaptic loci, analyzed from each image using Gaussian distribution. The peak of the Gaussian distribution gives the NN-Separation distance.
  - Column headings:
    - Group: Mouse group injected with either PFF or Monomer.
  - Raw headings:
    - MouseNo.: Animal ID.
    - ImageNo.: The Image analyzed for the given mouse.
    - NN_Separation (nm): Mean separation between the nearest pre- and post-synaptic proteins of synaptic loci.

Methodology
Data was collected using a Zeiss LSM 900 Airyscan microscope with a 63x 1.4 NA objective. 
Images were pre-processed in Zeiss Zen Blue using a strength setting between 6 to 7.5. 
The same strength was applied across all images analyzed for a particular set of analyses.
Airyscan-processed images were analyzed using IMARIS and MATLAB codes (Reitz, S.J., Sauerbeck, A.D., Kummer, T.T., 2021).

Data Access and Sharing
Use of this data must reference Saroj Sah and Thomas Biederer.

Funding
This research was funded in whole or in part by Aligning Science Across Parkinson’s [ASAP‐020616] through the Michael J. Fox Foundation for Parkinson’s Research (MJFF).
