TITLE: Determining the effects of mecamylamine in the mouse striatum using a Conditioned Preference Place (CPP) paradigm 
AUTHORS: Yan-Feng Zhang, Stephanie J Cragg

[DESCRIPTION]
This protocol is to determine the effects of administrating mecamylamine in the mouse brain striatum using a Conditioned Preference Place (CPP) paradigm. This model measures the increased preference for an environment associated with the administration of a drug. The goal is to create a conditioned stimulus able to evoke a specific behaviour based on the nature of the drug tested.

[BEFORE_START]
We performed these experiments in male C57BL/6N mice (42-50 days-old).


[STEPS]
SECTION: Preparing the mouse for surgery
1. Anesthetize the mouse in an induction chamber with isoflurane (5% induction; 1.5 - 2% maintenance).
SECTION: Conditioned Place Preference Testing
7. Place mice in a 40 cm × 40 cm transparent plexiglass arena that is divided into two equal chambers separated by doorway. 
 

SECTION: Conditioned Place Preference Testing
8. On day 1, allow mice to freely shuttle between two chambers to assess place preference at baseline, expressed as % time spent in right chamber. 
 

SECTION: Conditioned Place Preference Testing
9. On days 2 and 3, administer alternating bilateral striatal injection with either mecamylamine (10 µg/side) or saline vehicle (0.9%) in a volume of 0.5 µl over 1 min in AM and PM. Constrain animals respectively in the right or left chamber for 20 min. 
 

SECTION: Conditioned Place Preference Testing
10. On day 4, calculate the post-conditioning chamber preference as the % of time spent in the right mecamylamine-associated chamber compared to on pre-conditioning day 1. 
SECTION: Conditioned Place Preference Testing
11. For the next two days (days 5-6), administer bilateral saline injections and allow animals to explore both chambers for 20 min.
 

SECTION: Conditioned Place Preference Testing
12. Repeat the conditioning procedure with bilateral saline for both chambers, with a pre-conditioning test on day 7, two days of conditioning on days 8-9, and a post-conditioning test on day 10. 

 

SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
18. Dehydrate samples using 30% sucrose solution for 24 hours.

SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
17. Dissect brains and fixed them overnight in 4% PFA.
SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
16. Perfuse animals transcardially with a saline solution followed by 4% paraformaldehyde (PFA) to clear blood and preserve brain for immunocytochemistry. 
SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
19. Freeze brains and section it to 50 µm slices using a vibratome. 
SECTION: Preparing the mouse for surgery
2. Mount the mouse on the warmed stereotaxic apparatus for surgery.
SECTION: Surgical Procedure
3. Using the stereotaxic arm, localize the cannula to the landmark of interest (bregma or dura).
SECTION: Surgical Procedure
4. Once localized, raise the cannula tip 1-2 mm above the skull surface and move to the desired coordinates. For dorsal striatum injections, we use the coordinates AP +1.0 mm; ML +/- 1.6 mm to bregma; DV -2.4 mm (from dura). 
SECTION: Surgical Procedure
5. Using bilateral injection needles (O.D. 0.21 mm, I.D. 0.11 mm, RWD, China), drill through the skull at the identified coordinates to create a hole for the cannula to be inserted.
SECTION: Surgical Procedure
6. Once the procedure is complete, mice recovered for three days in a warmed home cage. 
SECTION: Open Field Testing
13. Administer bilateral striatal injection of either saline vehicle or mecamylamine (10 µg/side).
SECTION: Open Field Testing
14. Place animals into the open field chamber and assess total running distance and average velocity within 20 minutes.
SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
20. Follow steps 2-4, 10-18 in Protocol: Immunocytochemistry of acute brain slices used in ex vivo voltammetry recordings. 
SECTION: Verification of intrastriatal cannulae location in the dorsal striatum
15. Anesthetise mice with intraperitoneal injection of sterile Avertin (250 mg/kg body weight).