TITLE: Imaging of cholinergic interneurons in post-mortem rodent tissue to identify striatal satellite astrocytes
AUTHORS: Shinil Raina, Stephanie J Cragg

[DESCRIPTION]
This protocol details confocal image acquisition steps and the analysis pipeline used to calculate inter-soma distance between cholinergic interneurons/pMSNs and their nearest astrocyte. The partner Protocol: Immunofluorescent Labelling of Post-Mortem Rodent Brain Tissue describes how to label cholinergic interneurons/pMSNs along with striatal astrocytes in PFA perfusion-fixed, 50-µm thick, post-mortem rodent brain tissue. 




[BEFORE_START]
The tissue used in the steps below were obtained after processing as described in Protocol: Immunofluorescent Labelling of Post-Mortem Rodent Brain Tissue. 

[STEPS]
SECTION: Confocal Imaging Acquisition
1. Turn on the Leica LSM980 confocal microscope and Zen Blue imaging acquisition software.
SECTION: Confocal Imaging Acquisition
3. Using a 10x objective, identify anatomical landmarks using the DAPI or ChAT/NeuN signal in the region of interest. This would include the corpus callosum for the dorsal striatum, and the anterior commissure for the ventral striatum.
 

SECTION: Confocal Imaging Acquisition
4. Switch to a 63x/1.4 NA (oil immersion) objective and identify randomly selected ChAT/NeuN positive somata within the region of interest. Center the objective over this particular neuron. 

 

SECTION: Confocal Imaging Acquisition
5. Image the identified cell across the different channels using the following lasers:
ChAT/NeuN - 568 nm wavelength laser
S100β - 488 nm wavelength laser
DAPI - 358 nm wavelength laser 

The laser wavelength can also be altered as appropriate for the secondary antibody that is used.

 

SECTION: Confocal Imaging Acquisition
6. Save the images with the appropriate details of the sample and imaging settings. A .czi format is recommended but other formats could also work.
SECTION: Image Analysis
8. Open the images in FIJI (ImageJ) and convert to a maximum projection across the z axis.
SECTION: Image Analysis
9. While remaining blind to the S100β (green) channel to prevent bias on astrocyte location, draw a region of interest (ROI) around the cell in the centre of the image with the polygon selection tool. This would either be the ChAT/NeuN positive cell, depending on the sample.
SECTION: Image Analysis
10. Open the S100β (green) channel. Draw a ROI around the nearest astrocyte by identifying a signal in the S100β (green) channel (>5 µm in diameter) featuring a DAPI-positive nucleus residing closest to centre of the image.
SECTION: Image Analysis
11. From the ROIs, measure the surface area of the cells and the x and y coordinates of the centre point to calculate the intersoma distance.
SECTION: Image Analysis
12. Using the x and y coordinates obtained earlier, calculate the Euclidean distance between the centre point of the ChAT/NeuN cell and their nearest astrocyte.
SECTION: Confocal Imaging Acquisition
2. Secure a glass slide containing one or more fluorescently-labelled brain section into the stage of the microscope. Take care at this step to ensure the coverslip does not break or dislodge. 
SECTION: Confocal Imaging Acquisition
7. Move to the next cell in the region of interest. Repeat image acquisition across the desired number of cells. Sample an equal number of cells from both hemispheres.
SECTION: Image Analysis
13. Repeat analysis steps outlined above for all imaged cells.