Psychology 111L Laboratory in Biopsychology Instruction for the lab involving: Stereotaxic Surgeries Materials needed - Scale for

r weighing the rats one for the whole lab - Heat sterilizer Steri - one for the whole lab - Heating pads Six, one for each stereotaxic & two for two recovery cages - One atlas of a the rat brain G. Paxinos and C. Watson, The rat brain in stereotaxic coordinates, second ed., Academic Press Inc., San Diego, 1986. - Hot water bath to warm the sterile saline - 4 Stoelting Stereotaxic Apparatuses - 4 bilateral guide cannulae - Plastic One, 2mm apart, 5 mm beyond the pedestal, 22GA sterilized - 4 dummies to fit these cannula sterilized - 20 jewel screws (5 per station) sterilized - 4 Bone Curate - 4 Scalpel Handle - 4 Hemostatic Forceps sterilized - 16 bulldog clamps (4 per station) sterilized - 4 Tissue Forceps sterilized - 4 Screw driver (will be heat sterilized) - 4 Scalpel Blade #10 - 4 Small Drill bit sterilized - 4 Large Drill bit sterilized - 4 bottles of dental cement - 4 bottles of dental acrylic Gloves (sterile & non-sterile) Sterilized Q-Tips Sterilized cotton swabs Blue pads calculators Saline (sterile & bacteriostatic) Banamine Xylazine Ketamine HCl Timentin Wound powder Betadine Cleanning Alcohol Puralube Vet ointment 15 ml centrifuge tubes Soy milk 27 gauge needles 25 gauge needles 1 ml syringes 3 ml syringes 4 surgery record pages 4 surgery plan pages

Procedure Preparing for the surgery drugs, assigning roles and attaching cannula: Weight your rat and record its weight on the surgery record page. Prepare your Banamine syringe - Connect a 1 ml syringe to a 25 gauge needle; aspirate the right amount of Banamine from the bottle; You will inject 2 mg/kg of Banamine. The bottle contains 50 mg/ml, if your rat weighs 300 grams, you will need about 0.012 ml of Banamine (0.3kg X 2 = 0.6mg). Since you have 50 mg in 1 ml you need to inject 0.6/50 ml (i.e. 0.012 ml), which will contain 0.6 mg of Banamine. The following will most likely be done by your T.A.!

Sterilize your cannulae by holding the plastic part with forceps in the bead sterilizer for 20 seconds. Place your cannulae on the carrier-arm, using the plastic part only (and not the metal tubes) to hold it. Make sure that the cannulae is held straight by moving one of its metal tubes up and down in front of one line on one of the ear-bars and making sure its aligned with that line in all three dimensions. Designate the anesthesiologist (that will monitor the rats anesthesia throughout the surgery), the surgeon (which will make the incision, clean the skull, and make all the measurements), and the surgeon assistant (which will drill the holes). Prepare your sterile surface. Surgeon put on sterile gloves and place your sterile instruments on the sterile surface. Surgeon assistant place the blade on the blade holder and place it on the sterile surface Your instructor will inject your animal IM with a Ketamine/Xylazine mix as follows: 56.25 mg/kg mg/kg Ketamine + 7.5 mg/kg Xylazine. Since the Ketamine is in concentration of 100 mg/ml we will need 0.5625 ml/kg + since the xylazine is in 100 mg/ml solution we will need 0.075 ml/kg of its solution so total volume of 0.6375ml/kg (Your instructor will mix 3ml of keyamine with 0.4 ml of the xylazine). If a rat then, weigh 300g it will be injected with 0.3 * 0.6375 ml of the solution or 0.19125 ml solution. As soon as the animal down we will shave it quickly clean it and put it in the stereotaxic.

Preparing the rat for surgery: Shave the upper surface of your rats head from the middle of its eyes to the start of its neck. Clean the shaved area thoroughly with alcohol, betadine, and repeat. Place your rat in the Sterotaxic Apparatus (first placing the ear bars in the ears start with the left ear bar that is tighten already and you never move, find the left ear canal and insert the left ear bar into it, move the ear around the ear bar gently until you feel that you are in a hole in the bone, hold the head in this position and now move the right ear bar until it is too in the hole in the bone, tighten the right ear bar, check that there is no movement of the head sideways only up and down; place the teeth in the teeth holder and push the teeth holder back until the teeth are tightly against it and, tighten it; fix the nose in place well in the mask and tighten it) Inject the Banamine SC (see calculation on the Surgery Plan sheet that was given to your group). When needed ask your instructor to supplement the anesthesia for your rat with 0.15 ml Ketamine straight from the bottle. Starting the surgery: Make an incision starting half a centimeter behind the eye line and stopping half a centimeter before the rear end of the skull. Push the skin and the fascia to the side and hold it with the four bulldog clamps Clean the skull from remaining fascia with the Bone Curate and then with saline Finding the Nucleus Accumbens: Find Lamda, which is the point junction between the two occipital and the two parietal bones. Place one of the cannulas metal tubes on it and read its dorsal-

ventral (DV) coordinates. This will be on the ruler that is on the arm perpendicular to the base of the stereotaxic. This ruler has a smaller ruler attached to it, one that goes only from zero to one. Find one is the line on the bigger ruler that is just below the zero on the smaller ruler. If this line is, for example the forth line from 2. Your reading is 24mm. (If this line is, for example the seventh line from 3. Your reading is 37mm.). Then find which of the lines on the smaller ruler completely aligns with a line on the bigger ruler. If this line (on the smaller ruler!) is for example the sixth from zero you add .6 mm to your previous reading and that together will be the DV coordinate for Lamda. Find Bregma, which is the point junction between the two parietal and the two frontal bones. Place one of the cannulas metal tubes on it and read its dorsal-ventral coordinates using the same procedure that was described for Lamda. Make sure that Bregmas and Lamdas ventral-dorsal coordinates are not more than 0.5 mm. If they are, call your T.A. or your instructor to adjust the nose bar and redo the measurements. Record the Anterior-Posterior (AP) and Medial-Lateral (ML) coordinates of Bregma, using the same procedure that was described for finding the DV coordinates with the exception that the ruler for AP is on the base of the stereotaxic and parallel to it, and the ruler for the ML coordinates is attached to the DV arm/ruler and is parallel to the base of the stereotaxic. Calculate your coordinates for the Nucleus Accumbens as follows: Record your AP coordinates for bregma on your Surgery Plan sheet. Add to it 1.0 mm as is written in the formula, this is the AP coordinate for the Nucleus Accumbens. Record your ML coordinates for bregma on your Surgery Plan sheet. If you recorded the AP coordinate for Bregma, using the right metal tube of the cannulae subtract from it 1.0mm, if you recorded the AP coordinate for Bregma, using the left metal tube of the cannulae add to it 1.0mm, this is the ML coordinate for the Nucleus Accumbens. Move your carrier-arm to the right AP and ML coordinates and mark the spots (one on each side of the midline) of entry to the nucleus accumbens. In an appropriate distance, not to interfere with the final position of the cannula on the skull, mark four spots for entry of the jewel screws. Move the carrier-arm to the side

Placing the cannula and screws: Drill four holes for the screws with the small drill bit. Lean the hand that holds the drill on your other hand that will rest of the stereotaxic. Drill a little and take the drill up do this until you fill that went drill throughout the bone, you wont feel any more resistance. Drill two holes for the cannulae with the big drill bit, using the same technique, as described above. Clean the skull with saline Screw the screws in, using the forceps designed to hold the screws. Place the carrier-arm back in place. Measure the coordinates for Bregma again. Make sure your cannula-holes are in the right spots.

Measure the dorsal-ventral coordinate next to the cannulae-hole and subtract 6.0 mm from them as in the formula. Insert the cannula

Finishing up: Mix (in the hood, in the last moment), and apply the dental cement After the dental cement is dry, disconnect the carrier arm from the cannula, by holding the cannulae with forceps and pushing down on it while raising the carrier arm Insert the dummy in the cannula Remove the bulldogs, and clean the head around the dental cement. Apply wound powder around the dental cement Inject 1ml of saline per 100 gram of body weight SC Place the rat on a heating pad, and watch it until its awake Place the rat back into its cage Post-Surgery Care Weight the rat every day and record it, if the rat loses more than 10% of its body weight inform your T.A., or your instructor Watch your rat for other signs of sickness (some of which are: blood around nose, redness around eyes, secretions around the dental cement, failure to groom, drink or eat), again if you notice any inform your T.A.