Experimental Protocol
(craniotomy, neural stimulation and optical imaging)
Xylazine: 4mg/kg
solution:
.3g/ml
Cocktail dosage: .3ml/every 100g
of weight
Cocktail (for 10ml)
Ketamine: 5ml
Furosemide: 1ml/kg
Xylazine: 1.1ml
solution: 1mg/ml
Sterile water: 3.9ml
Cocktail dosage: .01ml/every100g of
weight
4-Aminopyridine: 0.5microliters
(25mM in 1% NaCl)
Ø Weigh rat
Ø Inject rat with
anesthetic (Ketamine 35-100 mg/kg and Xylazine 4 mg/kg). The Ketamine/Xylazine cocktail is faster
acting then Urethane. Urethane is used,
as the primary anesthetic after the rat has been put under by the
Ketamine/Xylazine cocktail. The
intraperitoneal injection should be given while holding the rat by the tail so
the organs are pushed towards the head by gravity. The point of the injection is towards the
middle left in the lower abdomen (the liver is located in the center right of
the abdomen so inject in center left to avoid damaging it); make certain that the
angle of the needle allows for the injection to be between the skin and
intestines.
Ø Warning!!!! Do not inject to deep avoid puncturing vital
organs.
Ø Warning!!!! Urethane is carcinogenic; when working with
it use gloves and store it in the vapor hood, avoid handling if you are
pregnant.
Ø Turn on
electronics, then do the first urethane injection
(.15g/100g); average time to take effect is around 7 –10 minutes, however some
cases may take longer and may take more then one injection. Three tests are can be done to check the consciousness
of rat. First, one can gently touch the
eyes with a dull instrument; if the rat does not blink, it is in deep
sleep. Second, you can pinch the rat
between toe and heel, use a good amount of force; if it does not pull its leg
away then it is another good indication of deep sleep. Finally, one can check the heart rate monitor
for sudden increase in heart rate. If
there is a large sudden increase or decrease it can mean the rat is either
waking up or dying.
Ø When rat is in
deep sleep, perhaps after more then just one injection, shave the head of the
rat from the top of eyes to the back of the head.
Ø Set heating pad
on the operating base of the stereotaxic frame, tape a folded diaper covering
the heating pad. {Approx time elapsed
around 20 minutes}
Ø Place rat in sterotaxic
frame. Have the bite of the rat over the
bottom bar and the upper clamp over the nose.
Next, lightly place the ear bars into the ears. Anesthetize rat at any point it seems to be
coming to. For quick acting anesthesia,
give 0.5ml Ketamine/Xylazine, Urethane is much slower acting. After, the head is in the correct position
push the ear bars into the ears till the point where you can feel it touching
the skull. If pushed in too hard the
skull will be crushed, if not hard enough the head will move during
surgery. There are small indentures in
the rat’s skull in which you will be trying to place the ear bars into. If done correctly then the head will not move
down when pushed on.
Ø Give an injection
of Furosemide (1ml/kg) in order reduce the blood and cerebral spinal fluid
pressure.
Ø Hook up the
capnograph (in this case it is both the CO2 detector and the heart monitor) to
the rat’s foot. Again check to see the
consciousness of rat with either the heel pinch test or eye test. (Approx time
elapsed 40 minutes; approximate amounts of anesthesia are around .18ml/100grams
of weight every half an hour, although this can very greatly with each
individual rodent. Make certain to use
the three tests mentioned above as a true indication of the depth of anesthetization.
Ø In order to get
the most accurate heart rate, hook up an ECG recording device. Pick a channel on spike two connect a needle
to wire and place just underneath rats skin in the bottom half of the rat
towards the rear below the heart. Connect
the other wire to an alligator clip and connect it to rats fur just above the
heart, towards the head, on the other side
Ø Lift skin on head
with tweezers and cut with scissors making a circular hole over the skull that
is around 1.5 inches in diameter. Use a
bone scraper over exposed area to clear away any connective tissue on the
skull. Area cleared with scalpel is
slightly smaller then the hole, around 1.25 inches in diameter. Use a swab to remove any excess blood. Use a cautery device to stop any points
around the cut area and on the skull that are bleeding. Rinse the area clean with 0.9%NaCl and clear
excess NaCl and blood with a suction device.
Ø When the skull is
exposed, cleaned and for the most part dry identify the points of bone
connection on the skull. The (Y)
connection at the back top of the skull is called Lamda and the (+) connection
at the front of the top of the skull is referred to as Bregma. These are the two junctures that mark the
part of the brain that is the area of interest you will be exposing.
Ø Using a dental
drill start at the Lambda position and go down to the Bregma then either to
left or to the right (depending on which side of the midline you are on) half
the distance just traveled. Make a
rectangle in an area as large as possible, over the entire hemisphere if
possible, to expose the area either on the right or the left side of the brain
you wish to gain access to. Begin to
thin the square you have just formed making small shaving motions with the
drill. Use 0.9%NaCl wash during drilling
to keep the brain from being damaged due to overheating and to clear away any
skull dust particles that may be limiting the view of your progress. Thin the skull as evenly as possible to avoid
rough spots and ridges. The drill can
bounce off these irregularities and puncture the skull. Use the tweezers to essentially pull out the
thinned square skull piece. If it does
not come out easily drill more. It is
possible to pull away the square of skull in rats because there is no connective
tissue between the brain and the dura.
Ø Warning: Be careful of the midline on the skull, blood
vessels directly underneath could be damaged and cause excess bleeding making
optical imaging impossible.
Ø Rinse the exposed
dura (the thin tissue covering the brain) with 0.9%NaCl wash, then use the suction device to remove and excess fluid and
blood. (Approx time elapsed 75 minutes)
Ø Note: 350 beats/minute is normal for the conscious rat;
experiments typically go well if the rat is stable around 280 beats/minute. This heart rate can have much variance based
on differences (size, age, inherent unknown characteristics) in each individual
rodent.
Ø Set up the
micromanipulators with the bipolar electrode and the field potential electrode.
As a local field potential (LFP) electrode we usually using glass electrode
(diameter of tips is 20 – 60 microns, impedance 0.5 – 2. 0 Megohms). But we
also can use tungsten electrode (nonexpendable). Use the knobs on the stereotaxic device to
adjust the rat’s head to the right height for the most proper placement for the
size of your rat. Make certain to check
whether your rat is in need of more anesthesia by
doing the toe or eye test. Again make
certain to clean the brain with 0.9% NaCl, suction out excess and use tweezers
and small sponge to dry and clean surgical area.
Ø If the dura has
not been removed at this point, which is not necessary except for fluorescent
dye experiments, make a small slit in the durra matter in order to put both the
bipolar electrode and the field potential electrode into the cortex.
Ø Note: If using 4-AminoPyridine (injected into
layers two and three) to induce neural stimulation the bipolar electrode will
not be used.
Ø Begin to insert
the bipolar electrode into the brain, you will wish to put it 0.2-0.3
millimeters below the dura matter at an angle of 30-35 degrees. The reason for the shallow angle is so that a
cover slip can be placed over the two electrodes almost horizontal to fit in
the small working distance between the brain and cameral lens. If the position were extremely important the
bipolar electrode and the field potential electrode would be at 90 degrees and
we would use the Lamda, Bregma and coordinates on the stereotaxic frame for
precision. The field potential electrode
(made by stretching a glass pipette, breaking the tip, running a wire through
it and filling the pipette with an NaCl solution) is to be placed around
1millimeter or slightly less from the bipolar electrode and should go into
layers four-five of the cortex (.5-.6millmeters below surface). A reference electrode will be attached to the
rat by means of a metal clip that is attached by clipping onto ledge of fur and
exposed tissue at the back of the head, almost on the neck area. The ground electrode is attached to the
table. {Approx time elapsed 100 minutes}
Ø Electrodes are
tested to see if the spontaneous recording is being picked up and displayed on
the computer. Any issues with
electronics are adjusted. If you are not
getting a reading from the field potential electrode perhaps you will need to
break the tip due to it being too fine.
Our amplifiers are designed for low impedance recording (field
potential). A high impedance amplifier
will allow one to use finer electrodes to get single neuron recordings.
Ø Filters used
(Green 546nm, Red 694nm, Blue 394nm). It is not necessary to remove the durra
matter for the IOS (intrinsic optical signals) a thinned skull will
suffice. Opti-lite filter is referred to
as the excitation filter and the camera filter is referred to as the emission
filter. Only the excitation filter is
used for the IOS imaging.
Ø Warning!!!! The next three steps are done quickly, so to
avoid the agar drying out. If the agar
dries out, place it back into the microwave for a few seconds. Have the small glass piece ready, mentioned
in the steps below, before you make the agar
Ø Agar is made
next. Mix around .2 grams of 1.0% Agar
with 25ml of 0.9%NaCl, or is better to use 0.3 g of agar and 35 ml soline. (if amount of solution is small it’s getting very viscous
even if ratio agar / soline is the same).
Swirl solution and put into microwave watching closely for two or three
eight-second intervals until it forms a viscous
transparent goo. Let it sit and cool
down for a minute. Once again check to
see if rat is in need of more anesthesia. The agar helps to prevent movement artifacts
due to the brain pulsation, prevents brain swelling and prevents specular
reflection of saline from the cortex {Approx time elapsed 110 minutes}
Ø Warning!!!! If the rat begins to awake at any time while
the electrodes are in, quickly remove them.
Once they are removed give another shot of anesthesia and wait till the
rat is again under. Clean the area with
the 0.9% NaCl solution, suction clean and again use tweezers and sponge. If the electrodes break off in the brain of
the rat then you will have a ruined experiment, a bloody rat brain and a dead
rat! If you do the toe pinch test
regularly hopefully this will not happen.
Ø A small
transparent glass piece is roughly broken to fit size of exposed brain. The agar mix is put into a syringe without a
needle. Put a small amount of agar mix
on your wrist to make certain the temperature is around body temperature i.e.,
not feeling cold or hot when put on your wrist.
Next put the agar onto the exposed area of the rat’s tissue, almost
filling the entire 1.45-inch diameter hole you made in your initial cuts. Take the piece of broken glass and lay it
atop the electrodes and the open area of brain, making certain not to let the
broken glass cut the brain. Fill any
open areas under the cover slip with more Agar. This has to be done quickly so that the agar
is all in place before it starts to harden.
Avoid air bubbles at all costs they will negatively affect the imaging
causing specular reflection and can move around and grow during the course of
the experiment. When set properly the
glass should cover the two ends of the electrodes that are in the brain and
will be almost flat, only at a small angle of inclination.
Ø CCD Camera is now
positioned over rat’s brain and turned on.
Attempt to get a clear picture on the computer screen. Focus the camera
on the surface of the cortex and take the green image of the blood vessel
pattern. When the picture is clear
adjust the viewing size to show the two electrodes and the brain. The new view will be about 25% of the initial
view. Next defocus image on the surface
so that it is approximately focused 500 micrometers below the cortical
surface. The light that will be
reflected will then be reflected from a lot of different layers i.e., the six
layers that make up the rat neocortex that includes approximately 1mm below the
cortical surface {Approx time elapsed 124 minutes}
Ø WHEN EXPERIMENT
IS COMPLETE REMEMBER TO TURN OFF THE CCD CAMERA!!!
Ø Sacrifice animal
by injection of Sleepaway (2-5 ml).