You will receive an anesthetized turtle with the plastron removed. The instructor will show you how to hook the turtle's ventricle to the myograph. You will also need to attach a sleeve electrode to the vagus nerve on the right side of the turtle's neck. When applying drugs put them directly on the sinus venosus. Measure the rate and force of contraction before and after any stimulation or drug application. Measure all periods of downtime in seconds, label all examples of Starling's law, and measure their force of contraction.
Part A: Comparison of the Effects of Vagal Stimulation and Acetylcholine on the Heart.
Test the vagal stimulus at the settings below and make sure that the heart stops contracting within 10 seconds. If the heart does not stop you will need to increase the voltage (try increments of 3 Volts) and try again. Once you have determined the appropriate voltage, start the record. First record 30 seconds of ventricular contractions then stimulate the vagus nerve just until the heart stops contracting. Record the subsequent "down-time" and allow the record to run for 30 seconds (after the heart begins to contract again) before adding 0.5 ml of Acetylcholine to the sinus venosus. Allow the record to run until you have 30 seconds of record after the effects of Acetylcholine are visible (decreased rate, increased force). Now wash off the Acetylcholine using amphibian saline and use cotton balls to soak up the excess fluid.
The recording criteria are as follows:
Transducer: F-60 Myograph
Paper Speed: 0.25 cm/second
Timer: 10 sec.
Stimulus: Electrical
Duration: 2msec
Intensity: 10 Volts
Frequency: 30 stimuli per second
Stimulus: Chemical
Drug administered: Acetylcholine (drug concentration)
Drug dosage: 0.5 ml
Part B: Effects of Physostigmine and Pilocarpine on the Heart
For the first record get a 30 second control period then stimulate the vagus until the heart stops contracting (should be less that 10 sec.). Record until the heart returns to normal then stop the record. Apply 1 drop of Physostigmine to the sinus venosus and wait 1 minute before recording again. Turn the record on and allow it to run for 30 seconds. You should be able to see a decrease in rate or increase in force. Now stimulate for the same amount of time as you did at the beginning of this record to stop the heart (i.e. if it took 6 seconds last time, stimulate for exactly 6 seconds this time). Continue to record for 30 seconds after the contractions begin again. Stop the record and quickly wash the Physostigmine off the tissue. Recording criteria:
Transducer: F-60 Myograph
Paper Speed: 0.25 cm/second
Timer: 1 sec.
Stimulus: Electrical
Duration: 2msec
Intensity: 10 Volts (or higher)
Frequency: 30 stimuli per second
Stimulus: Chemical
Drug administered: Physostigmine (conc.)
Drug dosage: 0.1 ml (1 drop)
For the second record, get a 30 second control period then add 3 drops of Pilocarpine to the sinus venosus. Wait until the effects are noticeable then record for 30 seconds. Recording criteria:
Transducer: F-60 Myograph
Paper Speed: 0.25 cm/second
Timer: 10 sec.
Stimulus: Chemical
Drug administered: Pilocarpine (conc.)
Drug dosage: 0.3 ml (3 drops)
Part C: The effects of Nicotine on the Heart (Biphasic action of Nicotine)
Record 30 seconds then add dilute nicotine to the sinus venosus. Allow the record to run until you have 30 seconds of record showing the slowing effects of Phase I, then add the concentrated nicotine. Record the effects until you are sure the heart is in Phase II then stimulate the vagus to prove you are in that stage. Now add the Ach to show that the muscarinic receptors were not the site of blockade. Record the effect of Ach for 30 seconds. Stop the record and wash the nicotine off the tissue. Recording criteria:
Transducer: F-60 Myograph
Paper Speed: 0.25 cm/second
Timer: 10 sec.
Stimulus: Electrical
Duration: 2msec
Intensity: 10 Volts (or higher)
Frequency: 30 stimuli per second
Stimulus: Chemical
Drug administered: Dilute Nicotine (conc.), Concentrated Nicotine (conc.), Acetylcholine (conc.)
Drug dosage: 0.4 ml (4 drops), 0.4 ml (4 drops), 1.0 ml, respectively
Part D: Effects of Atropine, followed by vagal stimulation and Acetylcholine.
ReturnRecord 30 seconds then add Atropine to the sinus venosus and turn off the record for about 1 minute. Begin recording again and you should see a slight increase in rate, now stimulate for the amount of time it took to stop the heart last time. Once you have shown that the Parasympathetic route is blocked, apply Ach to the sinus venosus and record for 30 seconds. This will indicate whether the muscarinic receptors are the site of the blockade. Recording criteria:
Transducer: F-60 Myograph
Paper Speed: 0.25 cm/second
Timer: 10 sec.
Stimulus: Electrical
Duration: 2msec
Intensity: 10 Volts (or higher)
Frequency: 30 stimuli per second
Stimulus: Chemical
Drug administered: Atropine (conc.), Acetylcholine (conc.)
Drug dosage: 0.3 ml (3 drops), 0.5 ml