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cm_plasticity

This repository contains the analysis code used in Lu et al, "Rapid, activity-dependent intrinsic plasticity in the developing zebra finch auditory cortex". The manuscript is currently under review but is available as a preprint on bioRxiv.

Setup

You will need Python 3.9-3.11 and R 4.2.1 or later.

Create a virtual environment and install dependencies:

python3 -m venv venv
source venv/bin/activate
python -m pip install --upgrade pip setuptools wheel
python -m pip install -r requirements.txt

Get the data

The first step in the analysis extracts information about current injections and spike times from the raw intracellular recording data, which was generated in Axon Binary Format. Once the paper is published, the output of this step can be downloaded from figshare as a zip archive here. Unpack the zip file, making sure that the files go into a build subdirectory under the top level of the repository.

All of the other data and metadata for the analysis can be found in the inputs subdirectory:

  • immuno_counts.csv: counts of immunopositive neurons in confocal images of CM sections (blinded)
  • em_counts.csv: counts of immunopositive clusters in electron microscopy images of CM sections (blinded)
  • immuno_birds.csv: information to unblind rearing conditions for fluorescence and EM data
  • biocytin_cells.csv: manual classification of biocytin- and Kv1.1-labeled cells as Kv1.1-positive/negative (blinded)
  • kv11_puncta.csv: automated (CellProfiler) counts of Kv1.1 puncta in biocytin-labeled neurons (blinded)
  • colocalization_epochs.csv: metadata for neurons/epochs examining Kv1.1 expression in biocytin-labeled cells
  • plasticity_epochs.csv: metadata for neurons/epochs examining plasticity under various conditions
  • reversal_epochs.csv: metadata for neurons/epochs examining pharamcological reversal of plasticity

Analysis

  1. On our local cluster, we ran batch/process-abfs.sh < inputs/spkstep_epochs.tbl to extract spike times and other metadata from the raw ABF files and store them in pprox files. Skip this step if you're using the pprox files from figshare.
  2. Run venv/bin/python scripts/response-stats.py --output-dir build build/*.pprox to collate data from all the pprox files.
  3. scripts/immuno.R has the analysis code for the immunohistochemistry (Fig 1)
  4. scripts/colocalization.R has the analysis code comparing Kv1.1 expression to intrinsic dynamics (Fig 2)
  5. scripts/plasticity.R quantifies plasticity in CR, PR, minimal injection, and BAPTA-AM conditions (Figs 3, 4, 6, 7)
  6. scripts/reversal.R tests whether 4-AP and/or alpha-dendrotoxin reverse intrinsic plasticity