TITLE: Protocols for stereotaxic injections into mouse brain and ex-vivo electrophysiology
AUTHORS: Jyoti Gupta, Michael J. Higley

[DESCRIPTION]
This protocol describes the method for injection of α-Synuclin PFF and monomer into the mouse brain. The second part of the protocol describes preparation of acute slices from these mice and whole-cell patch clamp recordings.

[STEPS]
SECTION: Protocol for stereotaxic injections
1. Anesthetize the mouse with isoflurane by placing in the anesthesia chamber.
SECTION: Protocol for stereotaxic injections
2. Place the anesthetized mouse in the stereotaxic frame and continually maintain isoflurane anesthesia using a vaporizer (Harvard Apparatus).  
SECTION: Protocol for stereotaxic injections
3. Locate the injection sites relative to bregma and make holes into the skull using a drill.
SECTION: Protocol for stereotaxic injections
4. Lower the injection pipette into the brain at the predetermined coordinates. Perform the injection at a rate of 20 nL/s using a Nanoject III microinjector (Drummond Scientific).
SECTION: Protocol for stereotaxic injections
5. Following the injection, leave the pipette in place for 10 minutes to prevent backflow while withdrawing. 
SECTION: Protocol for stereotaxic injections
6. Seal the incisions with sutures and allow the mice to recover on a heating pad until conscious. 
SECTION: Protocol for stereotaxic injections
7. Continue to monitor the mice and administer the post-operative analgesia. 
SECTION: Protocol for acute slice preparation and electrophysiology
8. Anesthetize the mouse with isoflurane.
SECTION: Protocol for acute slice preparation and electrophysiology
9. Transcardially perfuse the anesthetized mouse with oxygenated (95% O2/5% CO2) ice-cold choline artificial cerebrospinal fluid (Choline ACSF) containing (in mM):  

 

SECTION: Protocol for acute slice preparation and electrophysiology
10. Decapitated the mouse and quickly remove the brain.
SECTION: Protocol for acute slice preparation and electrophysiology
11. Mount the brain in the vibratome and cut 300 μm thick slices in the coronal plane.
SECTION: Protocol for acute slice preparation and electrophysiology
12. Transfer the slices to warm ACSF (32 °C) containing (in mM): 

 


SECTION: Protocol for acute slice preparation and electrophysiology
13. After incubating at 32 °C for 30 min, transfer the slices to Room temperature (RT).

SECTION: Protocol for acute slice preparation and electrophysiology
14. Transfer the slices to the slice chamber that is perfused with oxygenated ACSF.
SECTION: Protocol for acute slice preparation and electrophysiology
15. Identify layer 2/3 pyramidal cells in the primary somatosensory cortex.
SECTION: Protocol for acute slice preparation and electrophysiology
16. Backfill glass electrodes (3-3.5 MΩ) with an internal solution containing (in mM): 

 

SECTION: Protocol for acute slice preparation and electrophysiology
17. Add 1 micromolar (µM) tetrodotoxin to the bath to record miniature postsynaptic currents. 

SECTION: Protocol for acute slice preparation and electrophysiology
18. Voltage-clamp the cells at -70mV to record miniature excitatory postsynaptic currents (mEPSCs) and at 0mV to record miniature inhibitory postsynaptic currents (mIPSCs).