Dan Notes

Day Before Injection

Prepare:

- 20mL of tramadol solution in a falcon tube by mixing 10mL of Ora Sweet (F), 10mL of Ora Plus (G) and crushing a tramadol pill

- 30mL of KMR solution in a falcon tube by mixing one scoop of KMR (E) with water

- Vial of liquid green dye by obtaining less than a fruit fly size of powder green dye (B) with the end of a Q tip (H) and tapping it into a plastic vial (D) and mixing it with ringers solution (C). You can obtain ringers via a 1mL Luer Lock syringe (L) and an 23 or 18 Gauge needle (M)

- Cut two pieces of parafilm (S) big enough to cover the bottom of a 500mL glass beaker (T)

- Physio suite (5) and position appropriately. Tape (R) a paper towel onto the heating pad (6). Connect animal sensor and heating pad to physio suite.

- EKG cable (4) and plug into EKG (11)

- Head fixing apparatus (3) with animal holding apparatus (8)

- Calibrate microscope (10) by putting a ruler on where the animal will be. Make sure you can see the ruler markings clearly.

- Ensure that drill (9) is in place and the pedal is facing where the surgeon will be sitting

- Nanoinjector (1) and nanoinjector holder (7). Put nanoinjector through the holder.

- Glass pipettes (2) that have been pulled and grinded. If this has not been done, do it now!

- Containers (W and X) holding gauze (V) filled with ethanol (BB) and iodine (AA). Each container should have at least 5 gauzes.

- Micropipette size 20 microliter tips (FF) and micropipetter (EE),

- Virus injection paper card (O), write in virus information

- Neosporin (Z) and Vaseline (Y)

- Surgical sutures (CC)

- Bag of 1mL easy touch disposable syringes (Q)

- Electric shaver (P)

- Penicillin and other drugs (Ketamine/Xylazine, Atropine, Ketoprofen, Bupivacaine)

- Sterile scalpel blade (N)

- Cauterizer (U)

- Sterile gloves (DD), surgical tools and gown

- Dry ice containers: one large Styrofoam container, a medium size plastic container and a small size plastic container (Russian Doll Style)

- Survival log

- Plastic cup (I) with bleach (K)

- Pen and notepad

- Large plastic trash bag (dispose in hazardous waste bin in foster)

Day of Virus Injection

- Obtain dry ice with dry ice containers and obtain virus from Bassine

- Administer tramadol and ketoprofen to animals before ketamine/xylazine administration

- Prepare drugs and syringes

- Detailed protocol on how to inject virus's can be found here Virus

Post Operative Care

- Coordinate with personnel on who will do post op care at night and who will cover morning

Tips

- You can air out the 1mL easy touch syringes and label them with tape beforehand so they're ready to go

- You can have the glass pipettes filled with mineral oil

- Use a 1mL Luer Lock syringe with a 23 Gauge needle for penicillin administration

2-photon Headplate Surgery

Day before 2-photon Headplate Surgery

Prepare:

10mL ringers in a 10mL luer syringe and 18 gauge needle.

10mL ringers with dextrose in a 10mL luer lock syringe and 18 gauge needle.

Cut intramedic polyethylene (IP)

Change settings on IP administrative device (correlate flowrate with animal weight)

Tape paper towel onto heating pad

Ensure isoflurane mask is properly connected

Ensure isoflurane dispenser is full

Ensure Oxygen, Nitrous Oxide and Nitrogen tanks are full

Prepare 1mL easy touch syringes for Ketamine, Bupivacaine, Dexamethasone and Atropine

Prepare 1mL Luer Lock syringe with 18 Gauge needle for euthasol

Day of 2-photon Headplate Surgery

1. Administer ketamine injection in leg of animal, wait 5 minutes

2. Turn on vacuum pump and dispense isoflurane. Turn on Nitrous Oxide and Oxygen tanks, set ratio to 2:1 Nitrous oxide: Oxygen

3. Gently put nose of animal on top of isoflurane mask

4. After animal becomes drowsy, push isoflurane mask deeper into nose. Adjust as needed.

5. After animal falls asleep, full cover nose with isoflurane mask.

6. Shave head, neck, left side of stomach and tail

7. Bring ferret over to operating area of table.

8. Obtain heart rate and body temperature.

9. Inject .1mL of Bupivacaine under skin of throat. Administer Dexamethasone and Atropine at this time into the leg muscle.

10. Perform Tracheostomy

Dan's Tips

- Patience is key. Before making a cut, press your finger on the neck and feel the trachea. Do not cut if you can't feel

- You should feel the trachea rolling left and right as you push your finger against it, like a straw under a plastic wrap

- Pull the throat skin from both sides with one hand to make the skin tight as you cut it.

- Feel and cut. Feel and cut. Do not guess where the trachea is, always feel and cut.

- Once you see the ridges of the trachea, using sharp forceps, pull upward the plastic-like wrap around the trachea and carefully cut around the plastic-like wrap.

- As you cut around the plastic-like wrap, the trachea should expose itself naturally and very clearly

11. Put IP tube into the animal. Place animal on its stomach

12. Fixate head onto the apparatus.

13. Inject .1mL Bupivacaine under the skin of the head

14. Expose skull and virus injection site

15. Perform headplate procedures (two people are needed)

- Ensure that the skull is dry. You may use a air blower (device next to drill) to gently air dry the skull

- Locate where the headplate will go.

- Apply zap gel around the base of the headplate. Apply some zap gel on skull (if needed)

- Remove both ear bars and have the bars that connect with the headplate ready to go

- One person will firmly press the headplate against the skull while the other person applies a solution to quickly dry the zap gel

- One person will continue to apply a continuous pressure such that the headplate does not come off the skull

- The second person will mix dental cement with curating solution to create a liquidy dental cement mixture. While the mixture is liquidy, pour some into the hole of the headplate

- When the dental cement mixture becomes more of a slushie, apply generously underneath on the left, rear and right sides. Also apply on top via the front and back.

- Carefully connect the headplate to the bars and screw the connections down but not completely

- Apply vetbond over the dental cement

- wait 25 min for dental cement to dry

16. After dental cement dries, perform craniotomy and durotomy.

17. Check for expression using portable dinolight microscope

Dark Chamber & Rearing

Dark Chamber Construction Schematics can be found here Dark Rearing Schematics

Behavioral Training

Retrieving and transporting animals

The kit will be placed into the carrier inside the dark chamber!

Ensure that the carrier has a diaper cloth and a solid container (i.e. sharp disposable container)

Identify which kit you will need to bring up (i.e. left shave, no shave, both shave, right shave, etc.)

Carefully place the kit in the center of the carrier and cover it with the container. Close the carrier lid.

Exit the dark chamber and gently bring the carrier to Bassine 419

Positioning animal in front of stimulus monitor

Remove the kit from the carrier and place into animal holder

The kit may squeak and move around when inside. This is okay. Wait for a few minutes for the kit to settle down.

Training stimulus

Turn on PC (master) and Mac (stimulus) computers

Click on MATLAB for PC (it takes a while for MATLAB to open on PC)

While you're waiting for MATLAB to open on the PC, switch over to Mac and open MATLAB, type initstims and hit enter. The Mac is now listening

Go back to the PC. On MATLAB, type RunExperiment and hit enter.

Now wait and a figure window will open.

Then a window will open asking you about the default path. Click "No - choose another"

Select the entire folder miketheheadless Z drive

Click select folder and wait for additional windows to pop up

Under the "RunExperiment window", under "Active Sheets", choose "Ferret Training" Wait for a window to pop up.

Click "edit base"

If the animal is a "slow" animal, set _tFrequency_ to 1 and _nCycles_ to 5; if the animal is "fast", set _tFrequency_ at 8 and _nCycles_ at 10

Set _contrast_ to 1"

Press "OK" to close the "edit base" window

Go to "Training control sheet" matlab window

Set preferred direction; if direction is up/down, leave as 0. If direction is left/right, set to 90

Press run

Verify that the stimuli of the correct speed and direction are running. There should be 5 seconds of the stimulus followed by 5 seconds of a blank screen

Overall, stimuli will run for 20 minutes followed by 10 minute "breaks" of gray screens. This is one interval and there will be four intervals.

You should see the intervals that were run from the PC MATLAB

Training Stimulus Additional Info.

On mac, holding down the space bar aborts but lets mac continue "listening." ctrl c aborts everything.

Step 1 can be found in https://sites.google.com/site/vhlabinternalsite/rigs/rig-4-multichannel-electrophysiology

To switch between MAC and PC, simply press the physical buttons above that say 1 and 2. 1 is for PC and 2 is for MAC. A light will flash from the computer currently chosen