Laboratory 10

Mammalian Dive Response

Diving bradycardia in Homo sapiens

Objectives

Your objective today is to run a series of experiments that will uncover the proximal cues for diving bradycardia.

Introduction

When mammals dive, they must cope with the problem of being denied an external source of oxygen. Consummate divers such as the remarkable Weddell seal may remain submerged for up to two hours. To do so, they rely on anatomical features and physiologicalresponses that increase oxygen storage while reducing the use of oxygen for nonessential activities during a dive. Blood vessels supplying nonessential organs are constricted, redirecting blood to the oxygen-requiring brain and heart. Because it is supplying fewer organs with blood, the heart can beat more slowly (a condition known as bradycardia) while maintaining adequate blood pressure to the brain, the most metabolically sensitive organ; a further benefit of bradycardia is that the heart requires less oxygen as well. Diving bradycardia is an easily measured component of a group of reflexes that also include holding the breath (apnea) and peripheral vasoconstriction. Together these reflexes constitute the "diving response."

In comparison with diving mammals, humans are poorly adaptedto life in the water. In 2002, free-diving champion Mandy-Rae Cruikshank set a women's world record for static apnea of 6 minutes 13 seconds (the men's record, set in 2001by Scott Campbell, is 6 minutes 45 seconds), but most of us are comfortable holding our breaths for less than a minute.The first part of this laboratory exercise is designed to demonstrate that, despite our terrestrial nature, humans experience bradycardia when simulating a dive by holding the breath and immersing their face in cold water. The second part of this investigatory asks you to consider the cues that stimulate the bradycardia reflex. This question might be phrased "how does the body know that it is diving?" As biologists ,we might restate this question as "what are the proximate signs that trigger diving bradycardia?" The simulated dive holds several potential proximate cues: 1) apnea and 2) exposure of the face to cold water, which may be further broken down into several components including coldness, wetness, and pressure. The goal of the second part of todays exercise is to determine which of these cues triggers diving bradycardia in humans.

Proceedures

All tests are conducted in the same posture: leaning over the lab bench, headdownwith elbows resting on the lab bench.

You should work in groups of two or three but we will need the data from the entire class to tease out small effects with statistical analysis. Each student takes a turn being the experimental subjece, data collector or data recorder.

Each test lasts 30-60s. Most students will be able to hold your breath this long without too much trouble but should always stop anytime they experience discomfort. Test subjects will appreciate being tapped on the back every 10 s by the timer, particularly when being asked to hold their breath, because it is easy for subjects to lose track of time during the test.

A few points to remember

Equipment Setup

Note: This setup demonstrates basic Lead II ECG connections on a human subject. Student experiments may require variations.\

No Calibration is required but you should collect a bit of data to be certain that the gain for each channel is appropriate. Adjust as required.

Subject — Electrode Connections

Remember that diving bradycardia also includes reduction in blood flow to the extremitise in order to maximize flow to the brain.  Total blood flow to the extrematies can be recorded as the area under the curve plotted from the photoelectric plethismograph.  Set the plethismograph up on channel 2 and attach the sensor to a finger tip. Do not over tighten the strap.

An additional parameter that may chenge in response to diving is core heat conservation. Changes in the surface temperature of the skin can be monitored with surface thermisters.  Thermisters should be mounted with tape on the back of a hand or the top of a foot.

Running the Experiment

Hints for minimizing data error

Student-designed experiments:
pairs of test conditions for evaluating proximate cues.

After demonstrating that diving bradycardia exists in humans, You should perform a series of experiments that will uncover the proximate cues that stimulate the decrease in heart rate. Potential proximate cues can be evaluated with a series of paired experiments. Note that some variables can be investigated with more than one set of test conditions; each of these tests yields slightly different information, and consideration of several experiments togethercan yield further information not revealed by tests considered separately.

The data

Paired data for each experimental variable and its control allows the use of the very powerful paired t-test. You should record the data (HR) for the 15- to 30-s measurement period priorto each experiment followed immediatly by the experimental data on the same recording . Remember that each paired data set is to be treated as a separate experiment. Making more than one comparison within an experiment requires more advanced statistical tools—ANOVA or Bonferroni-corrected t-tests. In the event that the distribution of the data is significantly nonnormal, the nonparametric Wilcoxon matched pairs test, which makes no assumptions about the distribution of the data, should be substituted for the paired t-test.

To make use of paired t-tests, each student must complete both test conditions in each experiment, and data must be compiled so that pairs of results from each student are kept together (for instructions on how to format data, see instructions accompanying your statistical analysis software).
Order effects should be controlled for within each experiment. Use a random number generator to determine the order of the experiments. Flip a coin to determine which of the paired experiments is conducted first.

Data analysis: You need to calculate the frequency of the heart rate in bpm. This can be accomplished automatically by following the instructions in the BioPac manual but the set up is tedious and requires attention to detail. You have only a few data points so manual calculation may be more appropriate.

Set one of the calculations windows to frequency and one to integrate

  1. Frequency: Enlarge the output so you are looking at the recording for last 15 sec of the control or experimentl data and using the the I beam tool select the interval between each beat for about 10 beats. Record the frequency for each interval and calculate the mean. Remember that BioPac will calculate Htz and you need to convert to BPM. Repeat this for the last 15 sec of each dive oe wexperimen.
  2. Blood flow: Again enlarge the output so you are looking at about 15 sec of data (control or experimental). Using the I beam tool select the start and end of this 15 second interval. set one of the calculation windows to area and record this number as representitive of flow. This value is in mV/sec and is relative to the set up.Because of this you may need to convert the data to percent change to evaluate the results of the class data.
  3. The frequency and relative blood flow data can then be analysed statistically. Note: one approach to eliminating the varience in induividuals is to calculate the % change from control to dive. If you do this there is no need to run paired T tests as yo will have only one number for each student for each experiment ( and the entire data set can be analysed woth an ANOVA.

Report

A table presenting the data and its statistical significance and a graphic representation of the results are required along with a paragraph describing the proximate cause of the dive response. If you move through the experiments quickly and everyone enters data the analysis should warent a report. In the event that our n is to low I will add students from previous years.

References

Walter I. Hatch
wihatch@smcm.edu

November 18, 2012