TITLE: EtOH Self-Administration
AUTHORS: Brent Boomhower, Lani Tieu, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines in detail the procedure for conducting ethanol self-administration studies (e.g., Pharm) including the use of passive vapor to induce dependence. </div></div>

[STEPS]
?. [Begin front lever training (Water only)]
Run 24 animals in an overnight session with only the front lever connected to water and a handful of food added directly into the chamber (see "Operant Chamber Set-up"  below for specific session instructions).
?. [Begin front lever training (Water only)]
Run the next 24 animals the following day for their overnight session in the same manner.
?. [Operant Chamber Set-up]
Fill syringes with water to maximum capacity (~33 mL).
?. [Operant Chamber Set-up]
Connect syringes to the EtOH pump and tubing, which operates via the front lever and fills the front well, respectively.
?. [Operant Chamber Set-up]
Push through 1-2 ml water to flush out air bubbles from the line.
?. [Operant Chamber Set-up]
Start a test session on both computers to test the levers and flush out any remaining air bubbles from the lines. Do so by repeatedly pressing the lever to activate the pump.
?. [Operant Chamber Set-up]
Open ETOHi12 application to set up session on each computer (switch between computers by pressing Scroll Lock twice).
?. [Operant Chamber Set-up]
Set levers used from 'Both Levers' to 'Front Lever'.
?. [Operant Chamber Set-up]
Start test session for each chamber by pressing 'Start Group'.
?. [Operant Chamber Set-up]
Use paper towels to soak up all the flushed water.
?. [Operant Chamber Set-up]
End the test session.
?. [Operant Chamber Set-up]
Now, set-up the computers for the overnight session.
?. [Operant Chamber Set-up]
Set Max Rewards to 300 to prevent animals from pressing to an empty syringe.
?. [Operant Chamber Set-up]
Set Levers Used from 'Both Levers' to 'Front Lever'.
?. [Operant Chamber Set-up]
Load subject names to proper animal IDs under Group Settings tab based on chamber placement for data collection.
?. [Operant Chamber Set-up]
Set session length to 1140 minutes to allow the program to run all night.
In future sessions, the session length will be much shorter and progressively shorten until reaching 30 minutes.
?. [Operant Chamber Set-up]
Change File Name.
File names should be simple yet descriptive (e.g., “pharm7.30min2F1” for the first female 2-lever, 30 min session of the 7th cohort for the pharm grant).
?. [Operant Chamber Set-up]
Place each animal into their respective chamber and start the overnight session.
?. [Operant Chamber Set-up]
Before the session ends the next day, check if any animals have responded to the maximum rewards. Take out all animals that have pressed to the maximum rewards.
?. [Operant Chamber Set-up]
Allow all other animals to continue until they reach the maximum rewards OR the session ends.
?. [Operant Chamber Set-up]
Clean the bedding trays and operant chambers.
?. [Operant Chamber Set-up]
Only animals that did not reach at least 150 lever responses need to be run in a joint session after both 24 animal groups have done one overnight session. Use same parameters as outlined above.
Typically, there will be less than 24 animals that have significantly low pressing, therefore one joint session is possible.
?. [Front lever training (EtOH only)]
Prepare a solution of  ethanol (EtOH) in the wetlab area.
?. [Front lever training (EtOH only)]
For 1 liter volume solution, pipet  190 Proof EtOH  (95%) into a 1000 mL capacity graduated cylinder.
105 mL
?. [Front lever training (EtOH only)]
Add  water from the sink to the graduated cylinder.
895 mL
?. [Front lever training (EtOH only)]
Pour contents of the graduated cylinder into a 1000 mL capacity glass bottle with a screwable cap and shake.
?. [Front lever training (EtOH only)]
Label contents as "10% EtOH" including the date and your initials.
?. [Front lever training (EtOH only)]
Run the first group of 24 animals in a 3-hour session with  EtOH in the front lever. *Begin this first session earlier in the day so the next 3-hour session will not run too late.*
All session parameters will remain the same as the overnight sessions, except the session length (180 min) and the file name.
?. [Front lever training (EtOH only)]
Run the second group in the same manner as above.
?. [Front lever training (EtOH only)]
After each session ends, record lever presses (physically and digitally).
?. [Front lever training (EtOH only)]
Thoroughly clean chambers after both sessions are finished.
?. [Overnight training sessions, continued]
Run animals with water connected to the front lever and following all other guidelines as in the first overnight sessions.
?. [Overnight training sessions, continued]
Take out animals from their overnight session the next day.
All animals should reach significantly high lever responses during this overnight session. If a few animals do not respond significantly, you should likely move forward in the protocol despite this, but first ask for further guidance from your mentor.
?. [Front lever training (EtOH only), continued]
After the overnight session finishes, run each set of animals in another 3-hour session with  EtOH connected to the front lever as outlined previously.
?. [Front lever training (EtOH only), continued]
After the session, check the average lever responses for each group. The average responses should be close to 50 or more for these 3-hour sessions.
If this is not the case, once again ask for further guidance from your mentor before continuing in the protocol.
?. [Front lever training (EtOH only), continued]
Continue to run animals each day as described above, except decrease the session lengths to 2 hours.Run 2-hour sessions for 2-3 days.
?. [Front lever training (EtOH only), continued]
Next, run animals in 1-hour sessions for 2-3 days.
Always keep an eye on average lever responses and decrease subsequent session lengths as lever responses stabilize.
?. [Front lever training (EtOH only), continued]
Finally, run animals in 30-minute sessions for at least 2-3 days.These sessions should start close to the beginning of the animals' dark cycle.*Consult your mentor before moving forward in the protocol to the 2-lever paradigm.*
?. [2-lever paradigm (EtOH & water)]
Upon reaching stable responses for  EtOH, begin 30-minute sessions with 2-lever access.
These sessions will be run under the same parameters as the previous 30-minute sessions, except the lever option will be left as 'Both Levers', rather than 'Front Lever'.
?. [2-lever paradigm (EtOH & water)]
Attach the  EtOH syringe to the front lever pump/line.
?. [2-lever paradigm (EtOH & water)]
Attach the water syringe to the back lever pump/line.
?. [2-lever paradigm (EtOH & water)]
Run animals with 2-lever access for 2-3 weeks.
At any point, if there are days that are overwhelmingly busy, talk to your mentor and up to 1-2 consecutive days may be skipped.
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
*Consult your mentor before proceeding.*Split animals into two groups (dependent & nondependent), with the dependent group defined as the group to be housed in the passive EtOH vapor chambers.
Dependent and nondependent groups should be split so each group has 1/2 the male and 1/2 the female animals.
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
For at least 2-3 weeks, all animals will not undergo self-administration sessions.The next few weeks are designated for the modulation of the dependent group's blood alcohol levels (BALs).See "Blood collection for BALs" protocol in "Biological Sampling" folder to begin the process of reaching animal BALs of ~150-250 mg/dL blood alcohol for the dependent group.
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
Upon reaching or nearing the desired BAL range for the dependent animals, begin 2-lever sessions again.These sessions will be run ONLY on Monday, Wednesday, and Friday for both nondependent and dependent groups.
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
Run the nondependent group within 1-2 hours of their light cycle switching to dark.
One operant chamber rack should be designated for males and the other females, since males and females will now be run simultaneously.
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
Next, run the dependent group 4-5 hours into passive vapor withdrawal (i.e., the pump turning OFF and ethanol beakers empty).
?. [Passive vapor, or chronic intermittent ethanol (CIE) exposure]
Continue to run both groups of animals and begin pharmacological treatments or another experimental paradigm.
?. [Treatment groups]
Both the nondependent and dependent groups will be split into a vehicle group and a treatment group.
?. [Treatment groups]
These groups will be formed by analyzing the last several sessions.High, moderate, and low responders must be distributed evenly into each group (see example below).
?. [Treatment groups]
Continue 2-lever sessions with applied treatments.