TITLE: Immunohistochemical fluorescent labelling and activity readout of striatal cholinergic interneuron activity
AUTHORS: Emanuel F Lopes, Stephanie J Cragg

[DESCRIPTION]
This protocol delineates a method to identify choline acetyltransferase (ChAT)-expressing neurons and co-label these with a marker for global neuronal activity (phosphorylated-S6 Ribosomal Protein – pS6RP) in PFA perfusion-fixed 40 µm-thick mouse brain slices. Changes in the fluorescence of the pS6RP label are linked to changes in neuronal activity (Bertran-Gonzalez, J. et al. (2012)). Therefore, this immunohistochemical approach allows for the identification of cholinergic neurons alongside a readout of their activity. This protocol will focus on the use of this dual-labelling approach to target striatal cholinergic interneurons (ChIs) but may be applied to other brain regions or neurons of interest.

[BEFORE_START]
Note 1: pS6RP is a marker for neuronal activity, and will brightly fluoresce in tonically active neurons (e.g. striatal cholinergic interneurons). However, all neurons will express this marker and the stress induced by the perfusion-fixation process may lead to a confounding increase in expression in many neuron types. In terms of the tonically active ChIs, reducing stress in the perfusion step is ideal and lowers pS6RP signals from medium spiny neurons and other striatal neuronal populations. 

Note 2: Fixation with PFA does not prevent Ser/Thr phosphatases from dephosphorylating proteins. Perfusion, fixation, slicing and staining should use cold solutions and equipment to prevent degradation of the pS6RP signal. Slicing and staining should occur as soon as possible to prevent signal loss. If this is not possible consider maintaining slices in antifreeze in a -20°C freezer. Maintaining slices in -4°C during the staining process as much as possible is ideal for maximal signal-to-noise. The inclusion of sodium fluoride (NaF) in solutions can help inhibit phosphatase activity, maintaining pS6RP phosphorylation states throughout the staining protocol.

[STEPS]
SECTION: Perfusion-Fixation
1. Habituate animal for 3 days prior to perfusion. Handle mouse, and simulate performing an intraperitoneal injection once a day (scruff and poke belly with sheathed needle & syringe). 
SECTION: Perfusion-Fixation
2. On the day of the perfusion, anesthetize the mouse with an i.p. injection of pentobarbital (0.1 mL, 200 mg/ml). Perform a transcardial perfusion as normal, perfuse with ~5 mL PBS followed by ~5mL 4% cold PFA. Extract brain into chilled container with 4% PFA. 
 

SECTION: Perfusion-Fixation
3. Maintain brain in 4% PFA for 24 hours at -4°C.
SECTION: Perfusion-Fixation
4. Slice brain into 40 μm-thick slices.
 

SECTION: Perfusion-Fixation
5. Process slices for staining immediately after slicing, or store in anti-freeze at -20°C.
 

SECTION: Primary Antibody Incubation
6. Wash sections 5 times in PBS-NaF, each time for 5 minutes.
SECTION: Primary Antibody Incubation
7. Incubate for 5 min in PBS-NaF containing 3% H2O2 and 10% methanol (vol/vol). 
SECTION: Primary Antibody Incubation
8. Rinse 5 x 5 min in PBS-NaF.
SECTION: Primary Antibody Incubation
9. Incubate for 20 minutes in 0.2 % Triton in PBS-NaF + 10% Donkey serum (DS).
SECTION: Primary Antibody Incubation
10. Rinse 5 x 5 min in PBS-NaF.
SECTION: Primary Antibody Incubation
11. Incubate overnight at 4°C in PBS-NaF containing 0.2% Triton, 3% DS, 1:300 rabbit anti phosphorylated ribosomal protein S6, 1:200 goat anti-ChAT.
SECTION: Secondary Antibody Incubation
12. Wash sections in PBS-NaF 5 x 5 min.
SECTION: Secondary Antibody Incubation
13. Incubate for 60 minutes in 1:1000 Alexa Fluor 568 donkey anti-goat, 1:400 donkey anti-rabbit Alexa-488-coupled secondary antibody diluted in PBS-NaF + 3% DS. 
SECTION: Secondary Antibody Incubation
14. Wash sections in PBS 5 x 5 min.
SECTION: Secondary Antibody Incubation
15. Mount slices with Vectashield.