Lab 18: Modulation of Lumbriculus
heart rate
I.
Layout of the oligochaete circulatory system (Fig. 17-12)
A.
The
circulatory system of oligochaetes is a closed system
1.
The
blood plasma contains a hemoglobin-like respiratory pigment (for carrying
oxygen) called erythrocruorin
2.
Erythrocruorin
gives oligochaete blood a bright red color
3.
The
blood of most invertebrates is only slightly pigmented (colors range from
yellow to blue!)
B.
The
dorsal blood vessel is pulsatile & is the functional heart of the worm
(Fig. 17-12A)
1.
The
aortic arches (sometimes called hearts) are also pulsatile & assist in
movement of the blood
C.
Blood
is propelled forward by waves of muscular contractions that start at the rear
of the dorsal vessel
1.
Blood
moves from the dorsal blood vessel to the ventral blood vessel via a system of
smaller vessels (including capillaries)
D.
The
ventral vessel is not pulsatile & blood flows in it posteriorly
1.
Blood
eventually returns to the dorsal vessel (through other vessels) & the cycle
starts over
II.
Patterns of dorsal blood vessel contraction
A.
While
the contractions of the dorsal vessel start at the rear, not all of the
contractions proceed the entire length of the worm
1.
Some
of the contractions die out
B.
This
means that pulsation rates may be higher in the rear of the worm than at more
anterior sites
C.
Practical
consequence for today is that you want to monitor pulsation rates at the same
site before & after drug treatments
III.
Pulsation rate can be altered by a number of substances
A.
Acetylcholine
is a common neurotransmitter involved in the regulation of heart rate in many
animals
1.
In
earthworms, acetylcholine increases heart rate
a)
But
the effects of acetylcholine are complex and depend on concentration
2.
Nicotine
is an acetylcholine “agonist”
a)
An
agonist is a substance that mimics a natural neurotransmitter
b)
Thus,
nicotine produces the same action as would application of acetylcholine
c)
Many,
but not all, drugs we take recreationally are agonists for different types of
neurotransmitters
B.
Caffeine
is a complex drug that has several types of actions
1.
The
most likely effect in today’s experiments has to do with the ability of
caffeine to inhibit an enzyme known as phosphodiesterase
2.
To
see the significance of phosphodiesterase inhibition, we have to back up a
little
a)
In
lecture, we talked about the activation of chemically-gated channels by
acetylcholine during synaptic transmission
b)
There’s
a whole other class of neurotransmitter receptor that does not open an
ion channel when activated
(1)
When
some of these channels are activated, they produce a chemical called cyclic AMP
(cAMP) inside the postsynaptic cell
(2)
cAMP
than produces a cellular effect (like changing heart rate) via a complex
biochemical pathway
(3)
The
actions of cAMP are terminated by phosphodiesterase
(a)
Inhibition
of phosphodiesterase by caffeine will stop this breakdown of cAMP &cAMP
levels will go up (producing a greater cAMP-induced effect)
IV.Procedures
A.
Catch
a worm & put in a small plastic beaker
B.
Place
the worm on a slide & wick off most of the water using a Kimwipe
C.
Using
a scalpel, cut off the front & rear thirds of the worm
1.
Cutting
off the front third reduces the amount the worm will crawl around & cutting
off the rear third makes the pulsation rate more consistant
2.
Save
the middle third & put the other two parts of the worm back into the
aquarium
a)
They’ll
each regenerate the missing parts, so we’ll have three worms when we started
with one
D.
Fill
the slot on the observation slide with pond water & place the worm in the
slot
1.
Wick
off the excess water so that it is level with the top
2.
Place
the slide under either a dissection microscope (at fairly high power) or a
compound (at lowest power)
3.
Worms
can be manipulated by lightly touching them with a hair attached to an
applicator stick
E.
Count
the number of pulsations that pass one point on the body over 1 minute (record
on your data sheet)
1.
Repeat
this twice more (three total readings) & calculate the average
F.
Add
extra water to the slot & transfer the worm to a beaker containing a
particular concentration of drug (in pond water)
1.
Let
the worm sit in the drug for 15 minutes
a)
While
the worm is sitting in drug, you can do two other worms (see Fig. 2 in the
handout)
2.
Remove
the worm & briefly rinse the worm in clean pond water
G.
Return
worm to the slot & take three more readings of its heart rate
H.
Repeat
for the other concentrations of both drugs
V.
Data analysis
A.
We
will examine four concentrations of each drug & also a control group (that
will be used to compare to the effects of both drugs)
1.
Nicotine
concentrations = 0.1 mM, 0.25 mM, 0.5 mM, & 1 mM
2.
Caffeine
concentrations = 1 mM, 3 mM, 5 mM, & 10 mM
3.
Control
worms sit in untreated pond water for 15 minutes
a)
This
group will allow us to see if there is any effect of handling the worms
B.
Between
individual worms there is a fair amount of variability in heart rate
1.
We
can reduce this variability by looking only at the change in heart rate (rather
than the rate directly
a)
For
example, say one worm had a pretreatment mean heart rate of 12 beats per minute
(bpm) & a posttreatment mean rate of 17 bpm – the change in heart rate is 5
bpm
C.
Determine
the mean change in heart rate for each of your worms & write it in the
appropriate section of the table on the front blackboard
1.
Make
two graphs (one for each drug) that plot the class average change in heart rate
versus the drug concentration
a)
Plot
the data for the control worms as a concentration of “0 mM” on each graph
D.
For
your lab report, hand in your data sheets, the graphs, and a short description
of the effects of each drug on the heart rate of the worms