LECTURE 8: Prep Lab 4
Investigating “Smooth muscles”.
BRING YOUR
DISSECTING KIT
IN THE
LAB
A- OBJECTIVES: Find info (Textbook - Google – your
Mom) and come up with an
hypothesis BEFORE COMING TO
THE LAB!!
What are the effects of the sympathetic and parasympathetic autonomic
systems on uterine contractions? Best guess? (=Hypothesis)
What are the effects of two hormones (oxytocin & epinephrin) on uterine
contractions? Best guess? (=Hypothesis)
What receptor type triggers the uterus and intestine response to acetylcholine?
Best guess? (=Hypothesis)
B- SETUP: PART I page 12 of lab manual
10ml
Label! Please…
C- THE UTERUS:
C1- EXCISION OF THE UTERUS:
PART II page 12 of lab manual
Before cutting the uterus out of the rat,
MEASURE THE LENGTH OF YOUR UTERINE HORN and WRITE IT ON YOUR
Graph in datastudio: Length of uterine horn in situ = ## mm
C2- MOUNTING THE UTERUS IN THE BATH:
PART III page 13 of lab manual
Uterus
Uterine horn
Ask your partner
to hold the rod!
Be careful:
1- Do not touch the uterus with your forceps; Hold it delicately by its extremities;
2- uterus is not
- caught under the loop of the steel tubing;
- wrapped around the steel tubing;
- sticking on the wall of the chamber or on the steel tubing
3- Always -> Bubbles and solution in bath
4- Thread (attaching uterus to the lever) -> vertical.
5- The arm of lever -> free range (should not bump on any part of the set-up);
6- When the muscle contracts or relaxes, the arm of the transducer should not be at the limit of its
mechanical range of motion;
7- Muscle contracts-> trace goes upward;
8- Watch the level of water in the water bath: no underwater experiment
9- Temperature of bath between 37 - 39 oC
10- After “washing” to remove the drugs from the bath, make sure that the clamps are closed properly;
D- MAKING YOUR TEMPLATE FILE & CALIBRATING YOUR
TRANSDUCER: PART IV page 13 of lab manual To do
REMEMBER YOUR VERY FIRST LAB??? before attaching
the uterus
onto the lever
What data acquisition rate will you choose?
a)- 1/min
b)- 1/sec
c)- 10/sec
d)- 100/sec
e)- 1000/sec
E- How to introduce drugs in the bath
MICROPIPETS
t he
are le
ew sib
B vi o
in er
z
Safety!
0
F- Pay attention -> how to make your life easy and happy:
ake up template and calibrate - save template - Call us
ount uterus - Call us
For all your recordings:
-Use event marker if relevant!
-Indicate what is it you are injecting and when;
INDICATE HOW MUCH;
-Indicate when you are washing the uterus to remove the
drugs from the bath;
- Give a decent length of pre-treatment trace.
G- Organization: Find info (Textbook - Google – your
Mom) and come up with an
hypothesis BEFORE COMING TO
THE LAB!!
What are the effects of the sympathetic and parasympathetic autonomic
systems on uterine contractions?
What are the effects of two hormones (oxytocin & epinephrin) on uterine
contractions? Best guess? (=Hypothesis)
What receptor type triggers the uterus and intestine response to acetylcholine?
Best guess? (=Hypothesis)
1 rat per bench
2 setups per bench
2 students/setup
1 student will man the computers
-> calibration
-> traces
-> labelling – timing – event marker
1 student will take care of the muscles + set-ups
-> dissection
-> mount the uterus/intestine in the muscle bath,
-> take care of the muscle preparation during the experiment
-> observe muscle (communicate observation to 2nd student)
-> introduce drugs in bath
PROTOCOLS
1- Response of Uterus to Norepinephrine and Acetylcholine
2- What receptor type triggers the uterus response to acetylcholine?
One pair of students One pair of students
1 nook 1 rat 1 rat
One pair of students One pair of students
1- Response of Uterus to epinephrine and oxytocin
2- What receptor type triggers the uterus response to acetylcholine?
H- Getting the data!
Response of Uterus to norepinephrine (or epinephrine)
- Start recording on datastudio and record the spontaneous activity for about 5 minutes or more.
- Add 0.2 ml of norepinephrine (or epinephrine) by inserting the drug under the surface of
the bath with a pipette. Mark the point (time) of addition on the recording.
- Record the response for 5 minutes
- Wash out the hormone/drug with fresh Thyrode’s by releasing the metal clamp close to the muscle bath.
Count slowly to ten before you stop the wash. You can wash more than once if needed!
- Wait 5 minutes following the wash and then stop recording.
NOTE: 5 minutes of wait after the wash is an approximate duration. Wait until the trace reaches a steady
pattern for at least 5 minutes before you stop recording.
After the wash do not expect to get a steady pattern identical to the one you had before adding the drug.
Save your file – Name it L##C##R##U##EP or L##C##R##U##NE
R## is the rat number; U## is the length of the uterus in mm
To be uploaded into your lab section data storage & on my memory card.
Response of Uterus to acetylcholine (or oxytocin)
- Start recording on datastudio and record the uterus activity for about 5 minutes or more.
- Add 0.2 ml of acetylcholine (or oxytocin) by inserting the drug under the surface of
the bath with a pipette. Mark the point (time) of addition on the recording.
- Record the response for 5 minutes
- Wash out the hormone/drug with fresh Thyrode’s by releasing the metal clamp close to the muscle bath.
Count slowly to ten before you stop the wash. You can wash more than once if needed!
- Wait 5 minutes following the wash and then stop recording.
NOTE: 5 minutes of wait after the wash is an approximate duration. Wait until the trace reaches a steady
pattern for at least 5 minutes before you stop recording.
After the wash do not expect to get a steady pattern identical to the one you had before adding the drug.
Save your file – Name it L##C##R##U##AC or L##C##R##U##OX
R## is the rat number; U## is the length of the uterus in mm
To be uploaded into your lab section data storage & on my memory card.
What receptor type triggers the uterus response to acetylcholine?
-The 4 students together design the protocol & state predicted results.
- You can use curare and atropine (find what they are used for!) in your textbook
or a quick “google”.
Save your file – Name it L##C##R##U##RE or L##C##R##U##RE
R## is the rat number; U## is the length of the uterus in mm
To be uploaded into your lab section data storage & on my memory card.
What is a steady pattern?
CLEANUP
- Glass pipets -> rince with tap water -> leave them
where you found them
Safety
- Disposable plastic containers -> empty them in the
container labeled chemical waste, rinse them and throw
them in the regular garbage bin
- Tips from micropipets -> throw them in the “sharps collector”
- Papers; kimwipes; -> throw them in the regular garbage bin.
- Rat bodies -> in the tray labeled rat bodies in the fridge.
- Animal tissues and fluids -> in the bag labeled animal waste.
