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June 3, 2008 BEHAVIORAL NEUROSCIENCE LAB I.
BASIC
MAINTENANCE..........................................................................................................
1
A. Animals........................................................................................................................
1
1.
HOUSING.................................................................................................
1
2. DISEASES.................................................................................................
2
B. Labs.............................................................................................................................
3
1.
CLEANING...............................................................................................
3
2. TRASH.......................................................................................................
3
3.
GLASS
AND NEEDLES...........................................................................
3
4.
CHEMICALS.............................................................................................
3 II.
GENERAL PROCEDURES......................................................................................................
4
A. Animals........................................................................................................................
4
1.
HANDLING...............................................................................................
4
2.
INJECTIONS.............................................................................................
4
a.
Intraperitoneal
(ip)........................................................................
4
b.
Subcutaneous
(sc).........................................................................
5
3.
RAT
BITES................................................................................................
5
4.
IDENTIFICATION....................................................................................
5
5.
EUTHANASIA..........................................................................................
6
6.
CARCASS
DISPOSAL.............................................................................
6
B. Labs.............................................................................................................................
6
1.
CONTROLLED
SUBSTANCES...............................................................
6
2.
CLOTHING...............................................................................................
6
3.
EATING/DRINKING................................................................................
7
4.
CONTACT
LENSES.................................................................................
7
5.
FUMES......................................................................................................
7
6.
GOGGLES
AND SPLASH SHIELDS.......................................................
7
7.
PARTICLE
AND VAPOR MASKS..........................................................
7
8.
GLOVES....................................................................................................
7
9.
HEARING
PROTECTION........................................................................
7
10. TOXIC
SUBSTANCES.............................................................................
8
11. GENERAL
CHEMICALS..........................................................................
8
12. CHEMICAL
SPILLS.................................................................................
8
13. GOING
HOME..........................................................................................
8
C. Stereotaxic
Surgery....................................................................................................
9
1.
ANESTHESIA...........................................................................................
9
2.
GENERAL
SURGERY..............................................................................
10
a.
Incision
and Closure....................................................................
10
3.
PLACEMENT
IN STEREOTAXIC..........................................................
11
4.
POST-ANESTHETIC
CARE....................................................................
11
5.
ASPIRATION...........................................................................................
11
6.
RESUSCITATION....................................................................................
12
D. Behavioral
Testing.....................................................................................................
13
1.
TAIL-FLICK.............................................................................................
13 III.
LAB
EXERCISES..................................................................................................................
14
A. Stimulation
of the Motor Cortex...............................................................................
14
1.
MATERIALS
.............................................................
14
2.
PROCEDURE...........................................................................................
14
3.
DATA
SUMMARY AND INTERPRETATION.......................................
16
B. Inhibition
of the Tail-Flick Response by
Stimulation of the
Periaqueductal Gray......................................
17
1.
MATERIALS
.............................................................
17
2.
PROCEDURE...........................................................................................
18
a.
Naloxone,
scopolamine or methscopolamine:................
19
b.
Chronaxie
Determination:...............................................
19
c.
Paired-Pulse
Assessment:...............................................
19
C. Behavioral
Effects of Periaqueductal Gray Lesions................................................
21
1.
MATERIALS............................................................................................
21
2.
METHODS...............................................................................................
21
a.
Fluid
Intake..................................................................................
23
b.
Open
Field Test...........................................................................
23
c.
Social
Cohesiveness....................................................................
23
d.
Morphine
Analgesia....................................................................
23 3.
DATA SUMMARY AND INTERPRETATION.......................................
24 All
animal use on campus is performed in compliance with the Guide for the
Care and
Use of Laboratory Animals published by the National Institutes of
Health. A copy of this guide can be found
over the
flammable liquids cabinet in the wet lab (204A). Students
are encouraged to consult this
guide. If you have any concerns about
compliance either with these guidelines or with the animal maintenance
and
research protocols described herein, report these immediately to the
lab
assistant and/or Dr. Cannon. The
institutional official who is responsible for overseeing all
instructional and
research use of animals on campus is Dr. Robert E. Powell, Dean of the
Graduate
School and Director of Research.
Additional emergency numbers are posted on the colony door. There
are individuals who, with a religious fervor, wish to suppress all
research and
educational uses of animals. These
individuals use both legal and illegal means to attain their goals. Do not bring anyone into the lab without the
permission of Dr. Cannon. If a stranger
appears in the lab, direct them to Dr. Cannon's office or to the
departmental
secretary. If they refuse to leave the
lab, call security (7888 for a non-emergency, 7777 if you have an
emergency of
any kind). Feel
free to discuss this lab and any of its activities with whomever you
wish. When talking with someone outside of
the lab,
however, be careful not to engage in embellishments and/or "black
humor" regarding the procedures that involve animals.
As you can see, we take every precaution to
ensure that our subjects are treated humanely.
Irresponsible portrayals of laboratory activities will require
your
dismissal from the lab.
Your
rats will be housed singly in wire hanging cages. Except
when required by research protocols,
the animals should have an ample supply of fresh water and lab chow. Food and water should be checked daily.
General
feeding and watering occurs on Monday, Wednesday, and Friday and is
performed
by Mr. Manning, our animal care technician.
Mats under the cages are changed on the same days.
If you should need to refill a water bottle,
empty it completely and give it a rinse.
Be certain that you never switch water bottles between rats. When in doubt, get a new bottle.
Food that has fallen on the floor is never
given to animals--throw it out.
Cages
and bottles are sterilized by Mr. Manning at least every two weeks. Sterilization also must occur before an
animal is given either a new cage or water bottle.
The
temperature in the animal colony is maintained at 72o F and
the
humidity is kept between 40 and 60 percent.
There are temperature alarms for each of the two animal housing
rooms as
well as one for an animal running room.
When triggered, the alarms sound from a box outside of Dr.
Cannon's
office, as well as outside the building.
You can silence the alarm by pushing the reset button on the
alarm box
outside of Dr. Cannon's office. Having
done this, either report the situation directly to Dr. Cannon, or call
one of
the emergency numbers found on the alarm box.
Do not simply silence the alarm and forget about it; little
lives are
counting on you!
The
animals are maintained on a 12 hr/12 hr day/night schedule with the
animal
colony fluorescent lights being off during most of the daylight hours. Red lights are on during the "dark"
cycle to allow us to work in the room.
The times of lights on and lights off are posted in the animal
room. Report any perceived malfunctions
of the environmental control systems immediately.
Rats
that make chattering or wheezing sounds typically have upper
respiratory
infections. Infected animals will be
euthanized and their cages and bottles sterilized.
Rats
that display a marked twisting of the body, particularly the head may
have an
ear infection that will also required that they be euthanized and their
cages
and bottle sterilized.
Report
the preceding conditions immediately.
Similarly, if you detect any signs of disease or insect
infestation in
the animal colony, report this immediately.
Always
leave the lab clean--no exceptions. This
includes all surgical instruments, equipment, lab tables, and floors.
Change
trash bags before they become too full.
Tie them closed and leave them along the curved wall outside the
emergency exit in the dry lab (204B).
Always place a new trash bag in the vacated receptacle. When necessary, feel free to double bag. Trash bags are located under the counters in
the main animal work room.
Never
place sharp objects in the regular trash where they would pose a hazard
to the
maintenance staff. There are special
receptacles for broken glass and used needles in the wet lab.
Some
chemicals require special disposal procedures (e.g., formal/saline,
hemisol). These disposal instructions
are posted in the wet lab--always follow them to the letter. When required, storage containers for used
chemicals also can be found in the wet lab.
In
general, the rats in our lab are tame, gentle and friendly. When treated with proper respect, they remain
that way. Do not handle rats with
gloves. It is unnecessary and frightens
them.
Rats
can generally be picked up by the base of the tail, but DO NOT grab
near the
tip. Tip-grabbing may result in broken
or skinless tails. Always handle the rat
from the 5 cm nearest the body. Once a
rat has become accustomed to being handled, it can be safely picked up
for injection
by first placing it calmly into the crook of your arm (wear a lab coat). Pick the rat up with your thumb and index
fingers just under the shoulders and support the animal's midsection
gently,
but firmly.
If
a rat tries to get away, tries to bite, or is generally uncooperative,
do not
become angry at the rat and DO NOT be mean to it. You
probably will end up getting what you
deserve--bit. Get someone else to help
you or find a towel or lab coat in which to hold the rat.
IMPORTANT: All
injections should be recorded in your lab
books. All injections involving
controlled substances should be recorded in the log book kept in the
first
drawer on the left along the long wall in the wet lab.
This drawer also contains syringes, needles,
and most substances for injection.
Wipe
the top of the drug bottle with 70% alcohol.
Similarly, the abdominal skin of the rat should be swabbed with
a cotton
ball soaked in 70% alcohol. Always use a
new needle tip.
Invert
the rat and expose the abdomen. From the
rat's perspective, the injection site should be just to the left of the
midline
at a point midway between the diaphragm and the penis.
Take care not to puncture or inject into
hollow organs (bladder) or solid organs (spleen, liver) by using a
short needle
(1/2 inch). Always push the needle
through the abdominal wall quickly and at an angle about 40o slightly
off of perpendicular to the abdomen.
Always pull the plunger out slightly (aspirate) after insertion
and
prior to injection to check for blood or urine in the needle. In survival surgery, if either are present
you should discard your needle and start over.
In an acute experiment, reposition the needle and check again. If your needle is clear, inject quickly and
withdraw. When placing the animal back
in its cage, drop the rat crisply and cleanly from a height of 1-2
inches (this
will not hurt the rat and will get your hand out of the way just in
case the
animal wants to direct a bit of displeasure your way).
There
is rarely an excuse for getting bit.
Rats are cute and like to be petted, but don't invite rats to
sniff or
taste your fingers as they may bite.
Watch the rat when you go to pick it up and make sure the rat
knows you
are there so that you do not frighten it.
If
you do receive a bite, make the wound bleed as much as possible and
wash well
with soap and warm water. Rinse with
BACDOWN antimicrobial soap and then Betadyne, which are available in
the lab. If possible, do not bandage. If you have had a tetanus shot within the
last five years, no additional booster shots are necessary unless the
wound is
unusually deep. If you have any doubts
concerning how serious your injury is and/or the desirability of
obtaining a tetanus
shot, contact the school nurse at extension 7667.
Each
cage has an identification card on it.
In addition to the age, sex, and supplier of the rat, you should
record
any surgical, experimental, and/or pharmacological interventions that
an animal
has received on these cards.
Additionally,
during any time period when an animal's liquid or solid diet is
modified, a
second tag should be prominently taped to the front of the cage
describing this
modification and the length of time it will be in effect.
Without this tag, an animal will be placed
back on free feed and water. Be certain
to return your animal to free food and water as soon as your research
protocol
requires it. This is particularly
important if an animal is being completely deprived of food or water
for some
interval of time.
Should
you need to mark individual animals directly, use an indelible
felt-tipped pen
to stain the fur on the rat's back and/or place bands on the upper part
of the
rat's tail.
Care
should be taken during the course of research that animals are not
subjected to
unnecessary pain or discomfort. Rats
that undergo surgical procedures must be rendered incapable of feeling
pain via
suitable anesthesia. The same principle
applies to animals that are to be sacrificed or euthanized at the end
of a
study.
The
term euthanasia means "good death".
Methods of euthanasia should result in rapid, painless and
humane death
for the subject. For this lab, you will
administer at least 55 mg/kg pentobarbital intraperitoneally (see # 2). Again, don't forget to record the injection. The animals will then either be perfused
transcardially or decapitated.
Death
should be verified (absence of heart beat, cold body and rigidity) in
any
euthanized animal prior to its disposal.
If in doubt, consult the instructor or the lab assistant. Carcasses should first be wrapped in a
garbage bag and then placed in an orange biohazard bag that is tied and
identified with the tags available in the wet lab.
The bags should then be placed in the freezer
of the refrigerator in the wet lab (be certain that the freezer
door is
closed). When this freezer is full, the
carcasses will be transported to the large freezer near the main stairs
in the
basement of Loyola Hall by Mr. Manning.
You should NEVER take an animal carcass from the lab. |