last update 28 October 2016 at 3:15 pm
Have a set of dissecting tools near the videopresenter for the demonstration surgery.
One large tank that can be filled to a depth of about 5 inches with tap water to test for swimming.
Input A = Ch 3,
Input B = Off,
Timebase = 50 ms,
Samples = 2560 (40KHz)
BioAmp range 10 mV or 5 mV or as necessary to see Compound Action Potential
Display: Overlay stimulator to Input A,
Set up: Sampling = Multiple= Superimpose, Samples 10 or 20 or 30 as desired. Source= User, 0.5 sec delay,
Set up Stimulator: check Isolated, mode = pulse, delay = 10 ms, duration = 100 us, Amplitude = 20 mA.
MS Word File to print with above information: Click here.
Measure time from stim to a) first deviation from basedline or b) first peak/trough of Compound Action Potential.
Stimulate and elbow and then at wrist, recording distances and subtracting times to calculate APCV, using analogy of car and highway and garage entry.
ADInstruments Human Motor Nerve Conduction Instructor's Note
Erik STÅLBERG , Hacer ERDEM, Nerve conduction studies. NOROL BIL D 17:
2 , 2000 http://www.med.ege.edu.tr/norolbil/2000/NBD10900.html
Journal of Neurological Sciences (Turkish)
Abstract: A review of the principles of motor and sensory conduction studies is presented in this article. These studies are important in the neurophysiological evaluation of patients with nerve muscle disorders. A thorough knowledge of pathophysiology, methodology and analysis of pertinent parameters together define the quality of such an investigation. The review deals with these aspects and contains updated knowledge of the parameters that may be useful in routine work. Examples of the findings in some diseases are also given.
This paper has a photograph indicating multiple stimulation sites for the median nerve which could allow us to eliminate the need to account for synaptic delay.
LaFratta CW, Smith OH. A study of the relationship
of motor conduction velocity in the adult to handedness and sex. Arch Phys
Med Rehabil 1964; 54: 475-477.
Stetson DS, Albers JW, Silverstein BA, Wolfe RA. Effects
of age, sex and antropomethric factors on nerve conduction measures. Muscle
Nerve 1992; 15: 1095-1104.
Have students determine what should be recorded in the spreadsheet. Test both arms in each subject, record any nerve/elbow damage/conditions.
Initials, sex, distance, athletic/not so, latency from stimulus artefact to onset (or optionally, the peak) of CAP are entered into a Excel spreadsheet at the front desk.
Spreadsheet with graphs showing the effects of changing sampling intervals: sampling_effect.xls
Blindspot cards/brickwall color print with + and empty circle.
Rulers and meter stick for near point of accommodation study
Data sheets for NPA numbers
Desk lamps for photobleaching
Laser pointer (beam and bar) for Hubel and Wiesel experiments
If necessary, lab assistants should calibrate von Frey hairs using fine balance.
Front table should have rubber stamp (grid pattern) and ink pad.
Extra Sharpie markers
Ice bath for charging cold probes
Hot water bath at 65 centigrade for charging warm probes.
Digital thermometer for monitoring hot bath
Computers with Excel software to enter data,
Calipers with von Frey bristles glued to jaws.
Felt tip marking pens (ultra-fine point Sharpies)
calibrated von Frey hair on wooden handle
Thermal probe (modified syringe with aluminum rod) (check in advance for leaks)
Sharp metal probe with wooden handle for "pain" stimulus
Data sheets for
_____receptor density in palm and calf and cheek (takes quite a while to complete)
_____point localization palm and calf (easily completed) with 3 trials: write at least 4 hypotheses on back of indivisual data sheet for lab quiz grade.
_____two point discrimination (slow process using grid stamped on back of hand and fingertip, cheek, or calf.
Instructions and demonstration on how to conduct 3 cutaneous receptor tests requires about 35 minutes. Afterwards, students work at their own (rapid) pace to complete as many experiments as possible until one hour before the end of lab, at which time the Auditory System powerpoint is done lecture style supplemented with the freqency generator attached to the audio input on the front teaching table.
Alternatively, instead of the Auditory System lecture, the case studies on spinal cord injuries can be done.
60-70 minutes at end of lab: diagram ascending pathways for pain & temperature
and touch & proprioception.
Case studies for spinal cord injuries (6 cases, each lab table solves two cases and presents their findings to the class.)
Do Case # 1 as a demonstration and have teams of students solve remaining cases
Group 1 does Case 2 & 3
Group 2 does Case 3 & 4
Group 3 does Case 4 & 5
Group 4 does Case 5 & 6
Group 5 does Case 6 & 2
For this lab students will design experiments to demonstrate
Wave summation .
To expedite two-point calibration of the force transducer, weigh the standard lab clamps (~41.0 g) in advance so that students don't have to spend time doing so. Key point: Use 4 decimal places in the units conversion; otherwise the scale is displayed in kg.
Provide each group of 4 students with a
Calibrate with lead fishing weights hung from paper clip.
Consider pre-stressing each muscle with 2 grams of tension in an attempt to standardize sarcomere lengths from the onset of the experiments to minimize the variation in tension observed.
Make sure pin electrodes are thoroughly cleaned each day.
For Fatigue experiments, make three measurements: Time at which tension is reduced by 1/3, 1/2, and 2/3 of peak tetanic tension.
Include peak tension to muscle mass ratio calculation in the spreadsheet.
New notes from Fall 2013:
Showed dissection via video recorded from new presenter.
Used only gastroc muscles, stimulated one gastroc from each frog via nerve, the other directly to the muscle to compare latent periods.
Frogs were smaller than usual so we didn't attempt wrist extensors. Compare nerve and muscle stim sites was good.
For next lab: list everthing that must be measured early on (TD, CT, latent period, Max Twitch Tension, etc.) and make the first four measurements only from the twitch obtained with the 10 V (max) stimulus.
Consider measuring cross-sectional area of each muscle and including that data. Could use a razor to slice through the thickest portion of the muscle, dip the cut face in ink, and blot to a paper. Then measure with Image J.
Consider using small fish hooks instead of string to attach mucles.
Major potential improvement: Stimulate sciatic nerves for gastroc muscle and direct stimulation of wrist extensors to 1) equalize degree of difficult for setting up apparatus, 2) more complete recruitment of gastroc myofibers, and 3) comparing latent period for direct and nerve stimulation.
Number the frogs consectively throughout the week, instead of frog 1 and 2 for each lab day. This avoids confusion.
At the beginning, do a series of stimulations for which the "stretch" of the muscle is systematically altered for the purpose of approximating the "optimal length" for which all subsequent experiments will be done. With the 10 V stim, keep incrementally tighting the apparatus by 3 turns for gastrocs, and 1/2 turn for wrist extensors until the peak twitch tension plateaus, or if it diminishes, release some of the stretch to return to the flat portion of the Length/tension.
Once threshold has been established, use two units above threshold for all subsequent stimulations. (i.e. if threshold was 0.3 V, use 1.2V for subsequent recordings.)
Eliminate the "fatigue" component from this lab since fatigue is covered in next week's human forearm muscle fatigue lab. This eliminates the confusion about converting from tetanic interval to tetanic frequency, and eliminates the need to rush to switch to the Chart program. However, once experiments are concluded, have students deliver a tetanic stim for the full second of the recording and monitor tetanic tension. We saw fatigue in the nerve-stimulated gastroc preps in less that a second, and no evidence of fatigue in the wrist extensors after a second of contraction. Cool!
After the complete tetanus experiment, have student record a second maximal twitch and record this in the spreadsheet to allow a better comparison between peak twitch and peak tetanic tension.
Nerve stim preps often show some spontaneous contractions, presumably as the nerve fails. This was eliminated in one prep by crushing the nerve where it entered the gastroc at the knee, and converting this prep to direct stimulation of the muscle.
New definition of endpoint for twitch duration: time at which tension is reduced to 10% of peak tension. (i.e. if peak twitch tension was 39.2 g, the endpoint of twitch duration is the time at which the tenion was 3.9 grams.) This results is a more consistent estimate of twitch duration.
Once all recordings are complete, reset timescale to 5 seconds, and deliver a tetanic stimulus for the full five seconds to look for fatige. Measure by what percentage the tension drops over 5 seconds and, based on that, estimate how long it would take at that same rate to fully fatigue the muscle.
Consider using vaseline to keep the nerve moist.
Time permitting: Have the lab tables show their prep and records to other lab tables so that all are familiar with the differences in muscle sizes and peak tensions.
Videos before lab were useful. However, many students were disappointed they
didn't get to do the dissection for themselves. Perhaps we could provide the
legs with skin stripped and let them go from there. They ask for a video before
lab showing how to set up the muscle on the force transducer.
They were really impressed by the establishment of the optimal length by adjusting the stretch of the muscle at the outset to get a length at which tension was maximal.
They were also impressed that muscle tension persists after the last of a tetanic stimulus because it takes some time to return Ca++ to the SR.
They also clearly appreciated the advantages of stimulating the nerve to activate myofibers throughout the muscle, whereas direct stimulation activates only those myofibers beneath and near the electrodes.
For the Monday lab, we didn't finish in time to talk about the data in the spreadsheet. Many students would appreciate questions that show them what aspects of the data they should be attentive to. For example, many were amazed that a single muscle can lift more than the mass of the frog. Questions such as "Why is peak tetanic tension greater than peak twitch tension?" would get them to address the important issue.
We do get to a set of questions in a future lab when data from the entire week is in the spreadsheet. We should emphasize that we'll return to the full data set later.
One student recommended that near the end of lab that those who worked on gastrocs move over to the wrist extensor preps and vice versa. Seems like a good idea to me. They could actually deliver a few stimuli. Have the host table go through their recordings that demonstrate twitch, threshold, recruitment, etc. That way all groups could be progressing through this simultaneously.
One problem that arose was the stretching of the string over the lab period. Let's think of a better way to attach the muscle to the transducer.
In the following lab, review all frog muscle data using the Review Questions posted to the website.
Same set-up as for Frog Muscle I.
Announce for students who want to pith to arrive in lab 20 minutes early.
Once 3 frogs are pithed, have early-arriving students remove the skin from the hindlimbs.
Sever the hindlegs at the hips and distribute one leg to each of 6 lab tables.
At beginning of lab, tell students that they are to use previous experience
with Scope and Chart to test the hypothesis as stated on the website.
With a diagram on the board, describe the dissection to free the sciatic nerve and to isolate the gastrocnemius muscle from the lower leg.
Remind them to calibrated the force transducer using the two-point calibration technique (standard clamps weigh 41.0 grams).
Some groups will need help recalling the stimulus parameters.
In an attempt to have each muscle prep stretched to the same degree prior to collecting data, leave the muscle completely slack, zero the bridge pod. Then tighten the muscle/string combination until the bridge pod reads 10.0 grams. Then, re-zero the bridge pod. Doing this for all muscles means that each one has been stretched to the same extent (10 g) and that initial tension has be zeroed out.
To prevent tensions that read in kg instead of grams, changes the decimal place to 4 instead of 2 when doing the two-point calibration.
We tried: having students determine what threshold voltage is for stimulating the sciatic nerve, then use that same voltage for direct stimulation of the muscle. In many cases, the threshold voltage for the nerve was subthreshold to generate twitches in the muscle by direct stimulation. Therefore, it is advisable to use 10V stim for both the nerve and the muscle. For 2009, Monday lab used 10 V, Tuesday used, 5 V and Thursday lab used 2.5 V. Twitch tensiosn did not follow consistently follow the prediction that sciatic stim produces greater tension that direct stimulation of the muscle.
Warn about overstretching the nerve and letting the nerve dry out. Also, don't let a drop of Ringer's span the two electrode tips; the current will pass through the Ringer's and not through the tissue.
Warn about no changes during data collection except the site of stimulation.
Demonstrate how to set up and conduct a t-test on JMP using the computer. Have students take notes.
Have a spreadsheet on the front computer into which each group can enter all data for each of the ten trials for direct and indirect stimulation.
Students are to design experiments to collect data that will allow them to
address the following relationships here expressed as hypotheses:
H1. Increasing the load on a muscle increases the twitch duration.
H2. Increasing the load on a muscle increases the latent period.
H3. Increasing the load on a muscle increases the distance shortened.
H4. Increasing the load on a muscle increases the velocity of shortening.
Provide each group of 3 students with a
A balance should be set up to measure the loads and muscle masses.
Three double-pithed frogs provide 6 gastrocnemius muscles. Weigh frogs immediately
Time may not permit all groups to determine maximum load lifted.
Lab jacks are not stable when extended.
Use short strings
Sample rate on Scope must be set to highest value so that 1ms stim pulse can be selected.
Calibrate displacement transducers at Range = 500 mV or 200 mV
Pith and weigh 3 frogs
Have early arriving students begin dissection to remove leg skin
Diagram leg surgery on board, emphasizing being careful with sciatic nerve
Use videopresenter to read calipers (measure diameter of a coins)
Have member with least experience on MacLab run the computer this week
Allow groups 1 min each to answer questions on minimzing fatigue and establishing optimal length
Consider how load might affect fatigue
Consider otpimal number of trials
Review photos of previous setups
Remind students to change range to see twitches better
Most important: emphasize NO CHANGES other than load between two sets of trials!
Diagram typical recording and agree to marking and measuring conventions, especially for contraction velocity
Provide data sheet only after all instructions provided and questions have been answered.
Quickly take through set-up of Scope and stimulator.
Some groups finish 30 minute early.
Have lab assistants collect data sheets and enter values in master spreadsheet.
Each lab set up should have
Make sure EKG Diagnoses files are on each computer.
Do blood pressure measurements in a quiet room.
Lab Assistants will oversee the measurment of blood pressure.
Provide each group of 3 students with a
For general use
For Fall 2010, drugs made at 7:30 am worked well in the 8:00 am morning lab, but did not work at all for the 2:30 pm lab.
Epinephrine and Acetylcholine are very reliable. Atropine often gives unexpected results (expect small increase or no change in HR.)
Pith frogs before lab time. Have student do the surgery to expose the heart quickly.
Topics to mention:
Smooth muscle contraction in gut in response to ACh and EPI.
Skeletal muscle contraction in response to ACh.
Strategies to maximize the survival of in vitro tissue (students write ideas on note cards.)
Definition of death (last use of ATP or cessation of breathing or brainwaves or heartbeat?)
Describe degrees of heart block.
4 labs of 15 students each will consume about 500 ml of 0.171 N silver nitrate for each lab and about 50 ml of Potassium chromate for the entire week.
Unused Silver nitrate from burettes can be returned to the stock bottle for use the subsequent year. (This is a very expensive chemical, and our titrations need only be approximations.)
To make 0.171 Normal Silver nitrate, use 2.90 g silver nitrate to make a final volume of 1 liter or 11.6 to make 4 liters.
Have computers booted to Internet Explorer (for searching on-line Merck Manual.)
Provide each lab group with a hardcopy of the Merk Manual and a medical dictionary and texts from library.
(Medical dictionaries can be borrowed from the Biology Seminar Room.)
Each lab group will solve four cases, and be responsible for presenting one during a ten-minute span at the end of lab.
Each lab group will draw a card from the cookie jar with about 1 hour remaining in the lab period. The card indicated the case they are to present.
Tips based on some things that worked in 2011:
Have 7 groups and tell each group which case they should start with:
Group 1 starts with Case 2 ,then 3, 4, 5.
Group 2 starts with Case 3, then 4,5,2
Group 3 starts with Case 4 .
Group 4 starts with Case 5
Group 6 starts with Case 2
Group 7 starts with Case 3.
This way, not all groups are working simultaneously on the same case and, all groups aren't rushed to finish case 5 since some of them do it as a first or second case.
At 30 minute intervals, announce the time, and indicated that they should allocate their time so as to be able to finish all 4 cases in 2 hours or slightly less.
Some groups finished all 4 cases a little early so get them started on the survey. A couple of groups may nott quite finish all questions for their final case, but overall, the timing is about right.
At precisely 2 hours into the lab, ring the bell and announcee the drawing of assignments and how the presentations will to begin promptly with 40 minutes remaining in the lab, with each presenting group allocated no more than 10 minutes for their presentation. Three groups will serve as consultants to specific cases and the lab assistant and you can serve as the consultants for the remaining case.
Began the presentations right on time with 40 minutes left to go in lab and finsh right at the end of the period.
Student feedback from 2005:
Solve only 3 cases to avoid rushing
Groups of no more than 3 people so that everyone has plenty to do
Mix up groups so that students interact with people other than their usual lab partners
Allow students to solve an additional case as a take-home assignment for extra credit
Include a case on a hormone which has not been addressed in class
Have students to some advance preparation
Make sure each student has a print-out of the cases
Students want more computers (could allow students to bring their own wireless laptops)
students omit hematocrit information, BUN, & creatinine, units are bothersome
for some cases (mEq/L vs. mg/L)
eliminate specific gravity from Case 1 to reduce time necessary
For demonstration purposes, have the Bell Spirometer and other small spirometers on display before introducing the PowerLab system.
Explain/demonstrate how the Bell Spirometer generates recordings.
Have woodshop supplies available (hidden) to emphasize hazards of inhalants to lung health.
Each lab table should have
PowerLab with Spirometry pod and Pneumotachometer
Disposable mouthpiece, filter, and noseclip for each subject. (
Physiology reference values sheets (two per table)
Data sheet for recording lung volumes and capacities
Spare copies of Respiration Case Study # 1
Instructions for Spirometry (this version omits the background information on the first two pages of the full document: Full Instructions with Background)
Important: Position each Pneumotachometer away from heat sources and let it "warm up" attached to the PowerLab for at least 5 minutes (preferabley more) prior to the beginning of the experiments.
Tips and Notes:
Time permits only on subject in each group to get mechanical lung function data while completing Case Study # 1: COPD Emphysema. Completing spirometry and analysis on one subject per lab table requires about 45 minutes.
Do the spirometry experiments just before the Lung Function portion of Case Study # 1 to allow students to make the same measurments on themselves as were made on the patient in CS #1.
Position Pneumotachometer (PT) such that tubing is pointing upward during recordings.
Follow printed instructions carefully for calibrating PT. Important: don't take a deep breath before fully exhaling during the set-up portion.
Emphasize that subjects must inhale as deeply as possible and then quickly exhale maximally to obtain a good recording for FEV and FEV 1.0.
Inform students that "Spirometry Report" allows them to compare their values with those computed by the software. The values should be comparable and student should not rely on computed values.
Have lab assistant collect data sheets and enter values into master spreadsheet.
MLA145 Student Disposalbe Respiratory Kit from ADInstruments $5.00 each, box of 75 = $375.00 plus shipping.
Supplies: Hand ergometer, colored pencils and rulers for graphing Andro muscle biopsy data, graph paper,
handouts: Frog Muscle Data sheets, Review Questions for Frog Muscle Spreadsheets, King Muscle Biopsy Methods.
Links to on-line case studies: http://www.mhhe.com/biosci/ap/ap_casestudies/cases/ap_case08.html and http://www.mhhe.com/biosci/ap/ap_casestudies/cases/ap_case07.html.
Experiment # 1 for recording fatigue with hand ergometer. (Which should be attached to the Powerlab 30 minutes before the experiment to allow the electronics inside the ergometer to reach stable operating output voltage. Otherwise, it well be necessary to perform multiple recalibrations for 100% contraction since the zero value drifts downward as the ergometer warms up. Range will be 20 or 50 mV.
Use printouts of PowerLab instructions after quickly reviewing the use of the Ergometer.
Have pairs of students do the fatigue experiments with the other pair at each table creates graphs from muscle biopsy data from the King et al. 1999 paper on Andro. Provide colored pencils for graphing.
Have pairs of students look at the Frog Muscle Data spreadsheets using the review questions. Tell them in advance that names will be drawn randomly so be prepared to answer any question when called upon.
Ergometer experiments, graphing Andro data, and reviewing frog muscle spreadsheet takes about 2.5 hours. Leave 30 minutes at end of lab for summarizing and discussing everything. Case studies can be done after lab.
For fun, do one calibration recording for somebody and have other students add their own 100% attempts to the same recording to determine who's the strongest!
It takes about an hour or a little more to do the fatigue experiments on both subjects in each pair. They really enjoyed this and the experiment worked well, providing clear, predictable, consistent results. We talked about their observations for a few minutes, nothing the various causes of fatigue and reviewing that the grip contractions are isometric and that brief 0.5 sec pauses acutally deliver some blood to the contracting muscles, which have been depeleting their creatine phosphate, glycogen, and glucose stores, and at some point will turn to blood-borne glucose and fatty acids and ketones for energy.
Also review corticospinal tracts, role of cerebellum and basal ganglia in producing the contractions, and role of spindle afferents and golgi tendon organs during the contractions.
In the last 20 minutes or so, we reviewed the numbers and trends in the gastroc and wrist extensor spreadsheet with the Review Questions. We'd also expect peak tetanic tension to be about 3x twitch tension but in many cases this was not so. I asked them why and they generated several responses (fatigue, muscle not replenished with Ringers, movement of muscle underneath the pin electrodes, etc.) We examined how many times its own weight a muscle can lift and compared that to our own gastroc muscles which can easily lift our own body weight plus more. Finally, we looked at our data points for fatigue and related that to myofiber type proportions in the frog muscles and what we would expect in human wrist extensors (not so much for postural in humans) and human gastrocs (more for postural in humans).
Optional but not recommended because it is a replication of the frog muscle twitch lab where threshold, twitch, summation and tetanus are recorded:
Experiment # 2 for stimulating median nerve and recording recruitment, summation, and wave summation: Need piezoelectric pulse transducers and bar stimulating electrode with electrode paste.
Takes about 1:45 to do the recruitment/summation experiments and then summarize the expected results with the diagrams on the board.
Each lab table has: Powerlab with Hand Ergometer, two sleeved photocopies of frog muscle spreadsheets, 2 photocopies of questions for from muscle data analysis, 2 photocopies of questions for APCV and Ascending Pathways, sleeved copies of the summary data from Two Point Discrimination experiments, and sleeved pink photocopies of the keys to seven spinal cord injury case diagnoses previously distributed in lecture.
Upon arrival, the lab is very quickly shown how to set up Chart to record from the Hand Ergometers (which have been warming up for several hours prior to lab.) Students perform the two point calibration right along with the instructor who projects the set-up on the big screen and takes the student up to Page 3 Procedural Step 2 on the Photocopied Handouts placed at each PowerLab set-up. It takes about 40 minutes for a pair of students to complete the fatigue experiments on each other. Meanwhile, the other pair of students at each lab table are working on the Frog Muscle data.
For lab groups of 3 students, combine lab tables so that pairs are working on the data analysis simultaneously with the pair that is doing the fatigue experiment.
In the previous lab students studied skeletal muscle physiology from gastroc muscles of frogs stimulated directly or via the sciatic nerve. Group data was entered in a spreadsheet but there was no time during that lab to discuss the results. Two sets of plastic-sleeved photocopies of the 2013 frog muscle physiology data and 2012 data from gastrocs and wrist extensors was placed on each lab table along with a set of questions to guide pairs of students through the data analysis. Idea for human muscle fatigue lab: : measure time to fatige (by some criterion) and record in a spreadsheet for all human subjects. Then analyze with JMP to see if "athletes" are more fatigue resistant.
Once the first pair have completed their fatigue experiments, they swap locations with the pair who have been working on the Frog Muscle handouts. The pair who have just finished fatigue recordings are instructed to begin work on the APCV and Ascending Pathways worksheet.
Upon completion of the Fatigue experiments, instruct all students at a table to work together on JMP Analysis of a question based on data in the APCV spreadsheet that can be downloaded from the website. This serves as an excellent opportunity for students to review the use of JMP and t-testing. Have each table write up their results on a colored sheet of paper to share orally with the entire lab. It is useful to remind students to create two columns in JMP, the left containing a label (such as DOM or NONDOM or ATHLETIC or NA, etc) and a scodnd column with an APCV value for that subject. Remind them of the conditions in which one-and two-tailed t-tests are to be used. Have the lab TAs type the results of APCV analysis into a document to be send to the instructor.
Allocate time to review some of the questions on the worksheets, and if necessary, to finish any PPT lecture on Auditory or Vestibular Systems.
This very busy lab seems particularly well-suited as a last lab before Test 2 in that students are intensely reviewing muscle physiology and fatigue and ascending pathways. It is particulary timely to have them do JMP analysis in the week prior to the due date for their abstracts. This alone probably greatly facilitates independent statistical analysis for abstracts or executive summaries that will be due shortly.
Have student watch Education Portal Videos in advance of lab (Recommendations below with links)
Select 6 cases from Merck Manual On-line version, so that editing can occur in pairs of groups.
Intro for 5-10 minutes, 60 minutes to create cases, 20 minutes to peer edit cases, 30 minutes to revise and post cases and answers, 20 minutes to solve a new case, 35 minutes to present the case created.