Protocolos de Laboratório de Bioquímica:Fisiologia Animal.pdf

7/29/2019 Protocolos de Laboratrio de Bioqumica:Fisiologia Animal.pdf

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LABORATRIOS DE

BIOQUMICA/FISIOLOGIA- Trabalhos de Fisiologia Animal

Licenciatura em Bioqumica

Lusa Maria Pinheiro Valente

[email protected]

ICBAS 2013-2014


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ndice

OBJETIVOS _1

PROGRAMA _2

CALENDARIZAO DOS TRABALHOS PRTICOS 3

BIBLIOGRAFIA E AVALIAO 4

PROTOCOLOS EXPERIMENTAIS _5

Cincia em animais de laboratrio__________________________________5

Fisiologia Virtual: estimulao nervosa______________________________7

Regulao hormonal:

o Regulao dos nveis plasmticos de cortisol 10

o Hormonas reguladoras da glicemia 18

Fisiologia Virtual: sistema muscular________________________________27

Identificao de diferentes tipos de fibras musculares 30

Fisiologia dos elementos figurados do sangue 36 Exame fsico e qumico da urina____________________ _______________49

Taxa de filtrao glomerular: clearance da inulina 58

Metabolismo. Determinao da taxa metablica 65


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2

PROGRAMA

1- Introduo. Cincia em animais de laboratrio

2- Fisiologia virtual: Fisiologia da estimulao nervosa

3- Regulao hormonal:

3.1- Regulao dos nveis plasmticos de cortisol.

3.2- Ao da insulina e adrenalina na glicmia.

4- Taxa de filtrao glomerular: clearance da inulina.

5- Exame fsico e qumico da urina.

6- Elementos figurados do sangue: Contagem de glbulos brancos e

vermelhos. Contagem diferencial dos glbulos brancos.

Determinao dos grupos sanguneos pelo sistema ABO.

7- Identificao de diferentes tipos de fibras musculares atravs de

tcnicas de imunohistoqumica (Actividade da succinato

desidrogenase SDH).

8- Fisiologia virtual: Fisiologia do msculo estriado esqueltico.

9- Metabolismo. Determinao da taxa metablica.

CONTENTS

1-Introduction. Animals in laboratory practices.

2- Virtual Physiology: Physiology of nerve stimulation

3- Hormonal control:

3.1- Insulin and adrenalin effect on glycemia.

3.2 Regulation of cortisol plasma levels.

4- Glomerular filtration rate: inulin clearance.

5- Physical and chemical testing of urine.

6- Blood cells: White and red blood cells counting and identification. Blood group

identification.

7- Muscle fiber types identification through immunohistochemistry (succinat

desidrogenase activity, SDH).

8- Virtual Physiology: Physiology of the striated musculature.9- Metabolism: Assessment of the metabolic rate of some animals.


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Laboratrios de Bioqumica e Fisiologia

4

Bibliografia/Bibliography

Randall, D., Burggren W. e French, K.. Eckert Animal Physiology.Mechanisms and adaptations. 5th Edition. W. H. Freeman and

Company, New York. 1988. 683 p.

Schmidt-Nielsen, K. Animal Physiology: Adaptation and Environment.

3rd Edition. Cambridge University Press, New York. 1989. 619 p.

Guytton, A. C. Texbook of Medical Physiology. 8th Edition. Saunders

Company

Mtodo de AvaliaoAvaliao contnua e Exame final.

Assessment method:In-course assessment and Final written exam.

Metodologia de ensinoAulas prticas

Teaching methodsPractical classes

Obteno de FrequnciaParticipao em 2/3 das aulas.

Eligibility for examsStudents must attend 2/3 of the classes.


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LICENCIATURA EM BIOQUMICA

Laboratrios de Bioqumica/Fisiologia

5

Cincia em animais de laboratrio

I. OBJETIVO

Pretende-se, nesta aula, explorar o tema cincia de animais de

laboratrio com os seguintes objetivos:

1) Conhecer os objectivos da cincia de animais de laboratrio

2) Identificar a politica dos 3 Rs

3) Conhecer os requisitos de Bem-estar animal em animais de

laboratrio

4) Encorajar a auto responsabilidade

5) Promover a discusso sobre alguns aspectos ticos

II. INTRODUO

A utilizao de animais em trabalhos experimentais de pesquisa

cientfica tem sido de importncia fundamental, no s pelos avanos que

permite no conhecimento dos mecanismos dos processos vitais, mas

tambm no aperfeioamento dos mtodos de preveno, diagnstico e

tratamento de doenas tanto na medicina humana como na prpria

medicina veterinria. Porm, durante muito tempo, os animais utilizados

em experimentao animal foram colocados em segundo plano. No

entanto, a evoluo do conhecimento sobre os animais e o aumento da

consciencializao da necessidade de tratar os animais com dignidade e

respeito alterou de forma drstica as relaes entre o ser humano e os

animais. Esta nova conscincia conduziu a um crescente questionamento

sobre o uso de animais pelo homem, nomeadamente o uso de animais em

experimentao cientfica, tanto pela sociedade como pela prpria

comunidade cientfica.


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Fisiologia Virtual 6

O desenvolvimento cientfico a favor do homem no pode, nem

deve, servir de argumento para o uso indiscriminado e para o desrespeito

para com os animais. necessria uma postura tica frente necessidade

do desenvolvimento da cincia e a adopo de medidas que diminuam o

sofrimento dos animais e favoream seu bem-estar. Para alm, dos

valores ticos, tem-se verificado que a credibilidade dos prprios

resultados experimentais est totalmente dependente do modelo animal

utilizado e do seu bem-estar. Desta forma surgiu a cincia em animais

de laboratrio, dedicada produo, cuidado e estudo de animais de

laboratrio utilizados na pesquisa biomdica e educao, que ao tratar de

temas relevantes como o bem-estar dos animais, mtodos alternativos,

biotica, engloba uma srie de reas incluindo: sanidade, gentica,maneio, bem-estar animal, anatomia, biologia comparada, engenharia,

cincia ambiental, tica, microbiologia, farmacologia, fisiologia, toxicologia

e comportamento animal.

A cincia de animais de laboratrio considera o bem-estar animal

como um dos principais factores que podem influenciar o resultado da

experincia e valoriza o uso tico dos animais retomando o princpio dos

trs R's

Refine Reduce and Replace. Nesta perspetiva os valores ticosesto envolvidos em todo o processo experimental, desde a justificativa

para a realizao da experincia, a escolha do modelo adequado para a

pesquisa, o nmero de animais e como estes sero alojados, a utilizao

de anestsicos e analgsicos quando necessrios.

Todas estas questes ticas conduzem necessidade de

regulamentao. Atualmente qualquer projeto de investigao tem que

ser avaliado pelas comisses de tica. Estes organismos no retiram

responsabilidade tica ao investigador obrigando-o, pelo contrrio, a

desenvolver as suas atividades de investigao de forma transparente e

guiadas por uma conduta tica.

III. Parte Prtica:

Descubra qual o seu perfil face experimentao animal em:

http://ae.imcode.com/en/1008
http://ae.imcode.com/en/1008http://ae.imcode.com/en/1008http://ae.imcode.com/en/1008


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FISIOLOGIA VIRTUAL

NEUROFISIOLOGIA E IMPULSOS NERVOSOS

I. OBJETIVO

Pretende-se, neste trabalho, fazer a simulao da estimulao

nervosa, utilizando o nervo citico de uma r atravs de um CD-ROM

interativo. O programa atravs de uma apresentao 3D do equipamentolaboratorial, permite que os alunos simular um conjunto de experincias,

testando diferentes condies experimentais e observar os resultados

obtidos. Com este trabalho pretende-se:

1) Definir os termos excitabilidade; condutibilidade; potencial demembrana de repouso, bomba de sdio-potssio; estmulo limiar;

despolarizao; potencial de ao; repolarizao; hiperpolarizao;

perodo refratrio absoluto; perodo refratrio relativo, impulso

nervoso; impulso nervoso composto e velocidade de conduo.

2) Descrever a relao entre o dimetro das fibras nervosas e avelocidade de conduo;

3) Descrever a relao entre a mielinizao das fibras nervosas e avelocidade de conduo.

De forma a estudar a fisiologia dos neurnios, nesta simulao

sero utilizados vrios aparelhos. O primeiro o estimulador eletrnico.

Este aparelho administra um choque eltrico que permite o controlo

preciso da durao, frequncia e voltagem do choque. O estimulador

possui 2 terminais: o positivo o vermelho e o negativo o preto. A

voltagem sai do estimulador pelo terminal vermelho, passa pelo nervo e

regressa ao estimulador pelo terminar preto de forma a completar o

circuito. O segundo aparelho, o osciloscpio, mede as alteraes da

voltagem ao longo do perodo de tempo. Ao aplicarmos um estmulo a um


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nervo, o ecr do osciloscpio apresentar um dos 3 resultados:

inexistncia de resposta; b) uma linha plana; ou c) um pico. Este ltimo

indicar que se gerou um potencial de ao.

Nota:

Enquanto realiza as experincias que se seguem, no se esquea

que est a trabalhar com um nervo o qual formado por vrios

neurnios e no com um neurnio isolado. O potencial de ao que ver

no ecr do osciloscpio reflete o somatrio dos potenciais de ao de

todos os neurnios do nervo, denominado potencial de ao composto.

Embora um potencial de ao siga a lei do tudo-ou-nada num dado

neurnio, isto no significa necessariamente que o mesmo se passe num

nervo. Quando aplica um estmulo eltrico com determinada voltagem a

um nervo, este poder provocar a despolarizao da maior parte dos

neurnios, mas no necessariamente de todos eles. Para alcanar a

despolarizao de todos os neurnios poder ser necessrio aplicar um

estmulo de maior voltagem.

II. Protocolo Experimental

Nas simulaes seguintes ir utilizar o programa Physiol Ex para

investigar que tipos de estmulos desencadeiam um potencial de ao.

Proceder da seguinte forma:

1- Inserir o CD no computador e aceitar que se faam alteraes nocomputador

2- Selecionar o Physiol Ex seguido do ficheiro StartHere.html3- Aparecer o seguinte:


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1Site requi rements: Requisitos do programa

2Selecionar o Exerccio 3 e seguir as instrues


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Neurons have two major physiological properties: excitability, or the abil-ity to respond to stimuli and convert them into nerve impulses, and con-ductivity, the ability to transmit an impulse (in this case, to take the neural

impulse and pass it along the cell membrane). In the resting neuron (that is, a neu-ron that does not have any neural impulses), the exterior of the cell membrane ispositively charged and the interior is negatively charged relative to the outside.This difference in electrical charge across the plasma membrane is referred to asthe resting membrane potential, and the membrane is said to be polarized. Thesodium-potassium pump in the membrane maintains the difference in electricalcharge established by diffusion of ions. This active transport mechanism moves

3 sodium ions (Na

) out of the cell while moving in 2 potassium ions (K

).Therefore, the major cation (positively charged ion) outside the cell in the extra-cellular fluid is Na, and the major cation inside the cell is K. The inner surfaceof the cell membrane is more negative than the outer surface, mainly due to in-tracellular proteins, which, at body pH, tend to be negatively charged.

The resting membrane potential can be measured with a voltmeter by puttinga recording electrode just inside the cell membrane with a reference, or ground,

Neurophysiologyof Nerve Impulses

3E X E R C I S E

31

O B J E C T I V E S

1. To define the following terms:irritability, conductivity, resting membrane

potential, polarized, sodium-potassium pump, threshold stimulus, depo-

larization, action potential, repolarization, hyperpolarization, absolute

refractory period, relative refractory period, nerve impulse, compound

nerve action potential, and conduction velocity.

2. To list at least four different stimuli capable of generating an action

potential.

3. To list at least two agents capable of inhibiting an action potential.

4. To describe the relationship between nerve size and conduction velocity.

5. To describe the relationship between nerve myelination and conductionvelocity.

+ + + + +

+ + + + +

Voltmeter

Microelectrodeinside cell

Plasmamembrane

Microelectrodeoutside cell

70mV

Neuron

Axon

F I G U R E 3 . 1 Resting

membrane potential is

measured with a

voltmeter.


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electrode outside the membrane (see Figure 3.1). In the giantsquid axon (on which most early neural research was con-ducted), or in the frog axon that will be used in this exercise,the resting membrane potential is measured at70 millivolts(mV). (In humans, the resting membrane potential typicallymeasures between 40 mV and 90mV.)

The Nerve ImpulseWhen a neuron is activated by a stimulus of adequate inten-sity, known as a threshold stimulus, the membrane at itstrigger zone, typically the axon hillock, briefly becomes morepermeable to Na ions (sodium ion channels in the cell mem-brane open). Na rushes into the cell, increasing the numberof positive ions inside the cell and changing the membranepolarity. The interior surface of the membrane becomes less

32 Exercise 3

[Na+]

[K+][Na+]

[K+]

(a)

Na+

Stimulus

(b)

(c)

K+

(d)

(e)

Cell

exterior

K+

Diffus

ion

Na+Diffu

sio

n

PumpK

+

Na+

Plasmamembrane

Cell

interior

Repolarization

Actionpotential

Millivolts

RestingpotentialDepolarization

Time

Changes in the membrane charge duringdepolarization and repolarization(steps b through d).

(f)

+30

0

70

Since in the resting cell Na+ ions tend to diffuseinto the cell and K+ ions tend to diffuse out ofthe cell, the resting potential is maintained by theactive sodium-potassium pump.

+++++++++++++++++++++++++++++++++

+

+

+

+

+

+++++++++++++++++++++++++++++++++

++

+

+

+

+

+

+

+

+

++++++++++++++++++++++++++++

++++++++++++++++++++++++++++

+ + + + +

+ + + + +

++++++++++++++++++++++++++++

+ + + + +

++

+

+

+

++++++++++++++++++++++++++++++

+ + +

++++++++++++++++++++++++++++

+ + + + +

+ + +

++++++++++++++++++++++++++++++

F I G U R E 3 . 2 The nerve impulse. (a) Resting membrane potential (70 mV). There is an excess of

positive ions outside the cell, with Na the predominant ion in extracellular fluid and K the predominant

intracellular ion. The plasma membrane has a low permeability to Na. (b) Depolarizationreversal of the

resting potential. Application of a stimulus changes the membrane permeability, and Na ions are allowed to

diffuse rapidly into the cell. (c) Generation of the action potential or nerve impulse. If the stimulus is of adequate

intensity, the depolarization wave spreads rapidly along the entire length of the membrane. (d) Repolarization

reestablishment of the resting potential. The negative charge on the internal plasma membrane surface and

the positive charge on its external surface are reestablished by diffusion of K out of the cell, proceeding in

the same direction as in depolarization. (e) The original ionic concentrations of the resting state are restoredby the sodium-potassium pump. (f) A tracing of an action potential.


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negative and the exterior surface becomes less positive, aphenomenon called depolarization (see Figure 3.2b). Whendepolarization reaches a certain point called threshold, anaction potential is initiated (see Figure 3.2c) and the polarity ofthe membrane reverses.

When the membrane depolarizes, the resting membranepotential of70 mV becomes less negative. When themembrane potential reaches 0 mV, indicating there is no

charge difference across the membrane, the sodium ionchannels start to close and potassium ion channels open. Bythe time the sodium ion channels finally close, the mem-brane potential has reached 35 mV. The opening of thepotassium ion channels allows K to flow out of the celldown its electrochemical gradientremember, ions of likecharge are repelled from each other. The flow of K out ofthe cell causes the membrane potential to move in a nega-tive direction. This is referred to as repolarization (seeFigure 3.2d). This repolarization occurs within a millisec-ond of the initial sodium influx and reestablishes the restingmembrane potential. Actually, by the time the potassium ionchannels close, the cell membrane has undergone a hyperp-olarization, slipping to perhaps75 mV. With the channelsclosed, the membrane potential is quickly returned to thenormal resting membrane potential.

When the sodium ion channels are open, the membraneis totally insensitive to additional stimuli, regardless of theforce of stimulus. The cell is in what is called the absoluterefractory period. During repolarization, the membranemay be stimulated if a very strong stimulus is used. Thisperiod is called the relative refractory period.

The action potential, once started, is self-propagating,spreading rapidly along the neuron membrane. The actionpotential is aphenomenon, all-or-none, in which the neuronmembrane either depolarizes to threshold and the action po-tential is generated, or it does not. In neurons, the action po-tential is also called a nerve impulse. When it reaches theaxon terminal, it triggers the release of neurotransmittersinto the synaptic cleft. Depending on the situation, the neu-rotransmitter will either excite or inhibit the postsynapticneuron.

In order to study nerve physiology, we will use a frognerve and several electronic instruments. The first instrumentis the electronic stimulator. Nerves can be stimulated bychemicals, touch, or electric shock. The electronic stimulatoradministers an electric shock that is pure direct current (DC),and allows duration, frequency, and voltage of the shock tobe precisely controlled. The stimulator has two output termi-nals; the positive terminal is red and the negative terminal isblack. Voltage leaves the stimulator via the red terminal,

passes through the item to be stimulated (in this case, thenerve), and returns to the stimulator at the black terminal tocomplete the circuit.

The second instrument is the oscilloscope, an instrumentthat measures voltage changes over a period of time. The faceof the oscilloscope is similar to a black-and-white televisionscreen. The screen of the oscilloscope is the front of a tubewith a filament at the other end. The filament is heated andgives off a beam of electrons that passes to the front of the tube.Electronic circuitry allows the electron beam to be broughtacross the screen in preset time intervals. When the electronshit the phosphorescent material on the inside of the screen, aspot on the screen will glow. When we apply a stimulus to anerve, the oscilloscope screen will display one of the follow-

ing three results: no response, a flat line, or a graph with apeak. A graph with a peak indicates that an action potentialhas been generated.

While performing the following experiments, keep inmind that you are working with a nerve, which consists ofmany neuronsyou are not working with just a singleneuron. The action potential you will see on the oscilloscopescreen reflects the cumulative action potentials of all the

neurons in the nerve, called a compound nerve action po-tential. Although an action potential follows the all-or-nonelaw within a single neuron, it does not necessarily follow thislaw within an entire nerve. When you electrically stimulate anerve at a given voltage, the stimulus may result in the depo-larization of most of the neurons but not necessarily all ofthem. To achieve depolarization of all of the neurons, ahigher stimulus voltage may be needed.

Eliciting (Generating)a Nerve Impulse

In the following experiments, you will investigate what kindsof stimuli trigger an action potential. To begin, select Exercise3: Neurophysiology of Nerve Impulses from the drop-downmenu and click GO. Before you perform the activities, watchthe Nerve Impulses video to see how ambient activitiy ismeasured. Then click Eliciting a Nerve Impulse. The open-ing screen will appear in a few seconds (see Figure 3.3). Notethat a sciatic nerve from a frog has been placed into the nervechamber. Leads go from the stimulator output to the nervechamber, the vertical box on the left side. Leads also go fromthe nerve chamber to the oscilloscope. Notice that these leadsare red and black. The current travels along the red lead to thenerve. When the nerve depolarizes, it will generate an electri-cal current that will travel along the red wire to the oscillo-

scope and back to the nerve along the black wire.

A C T I V I T Y 1

Electrical Stimulation

1. Set the voltage at 1.0 V by clicking the () button nextto the Voltage display.

2. Click Single Stimulus.

Do you see any kind of response on the oscilloscope

screen? ____________

If you saw no response, or a flat line indicating no actionpotential, click the Clear button on the oscilloscope, increasethe voltage, and click Single Stimulus again until you see atrace (deflection of the line) that indicates an action potential.

What was the threshold voltage, that is, the voltage at which

you first saw an action potential? _______ V

Click Record Data on the data collection box to record yourresults.

3. If you wish to print your graph, click Tools and thenPrint Graph. You may do this each time you generate a

graph on the oscilloscope screen.

Neurophysiology of Nerve Impulses 33


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4. Increase the voltage by 0.5 V, and click Single Stimulus.

How does this tracing compare to the one trace that was gen-erated at the threshold voltage? (Hint: Look very carefully atthe tracings.)

What reason can you give for the change?

Click Record Data on the data collection box to record yourresults.

5. Continue to increase the voltage by 0.5 V and to clickSingle Stimulus until you find the point beyond which nofurther increase occurs in the peak of the action potentialtrace.

Record this maximal voltage here: __________ V

Click Record Data to record your results.

Now that we have seen that an electrical impulse cancause an action potential, lets try some other methods ofstimulating a nerve.

A C T I V I T Y 2

Mechanical Stimulation

1. Click the Clear button on the oscilloscope.

2. Using the mouse, click the glass rod located on the bot-tom shelf on the left side of the screen, and drag it over to thenerve. When the glass rod is over the nerve, release the

34 Exercise 3

F I G U R E 3 . 3 Opening screen of the Eliciting a Nerve Impulse experiment.


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mouse button to indicate that the rod is now touching thenerve. What do you see on the oscilloscope screen?

How does this tracing compare with the other tracings thatyou have generated?

Click Record Data to record your results. Leave the graph onthe screen so that you can compare it to the graph you willgenerate in the next activity.

A C T I V I T Y 3

Thermal Stimulation

Click on the glass rod and drag it to the heater, releasing

the mouse button. Click on the Heat button. When the rodturns red, indicating that it has been heated, click and dragthe rod over the nerve and release the mouse button. Whathappens?

How does this trace compare to the trace that was generatedwith the unheated glass rod?

What explanation can you provide for this?

Click Record Data to record your results. Then click Clearto clear the oscilloscope screen for the next activity.

A C T I V I T Y 4

Chemical Stimulation

1. Click and drag the dropper from the bottle of sodiumchloride (salt solution) over to the nerve in the chamber andthen release the mouse button to dispense drops.

Does this generate an action potential? _________________

2. Look back at Activity 1 for the voltage you determined.Set the voltage at that level, and click Single Stimulus tostimulate the nerve.

Does this tracing differ from the original threshold stimulus

tracing?__________


3. Click the Clean button on top of the nerve chamber. Thiswill return the nerve to its original (nonsalted) state. ClickClear to clear the oscilloscope screen.

4. Click and drag the dropper from the bottle of hydrochlo-ric acid over to the nerve, and release the mouse button to dis-pense drops.

Does this generate an action potential?__________________

Does this tracing differ from the one generated by the original

threshold stimulus? _____________________


5. Click on the Clean button on the nerve chamber to cleanthe chamber and return the nerve to its untouched state.

6. Click Tools Print Data to print the data you haverecorded for this experiment.

To summarize your experimental results, what kinds of stimulican elicit an action potential?

Inhibiting a Nerve ImpulseNumerous physical factors and chemical agents can impairthe ability of nerve fibers to function. For example, deeppressure and cold temperature both block nerve impulsetransmission by preventing local blood supply from reachingthe nerve fibers. Local anesthetics, alcohol, and numerousother chemicals are also effective in blocking nerve transmis-sion. In this experiment, we will study the effects of variousagents on nerve transmission.

To begin, click the Experiment menu and select In-hibiting a Nerve Impulse. The display screen for this activ-ity (Figure 3.4) is similar to the screen in the first activity. Tothe left are bottles of several agents that we will test on thenerve. Keep the tracings you printed out from the first activ-ity close at hand for comparison.



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A C T I V I T Y 5

Testing the Effects of Ether

1. Using the mouse, click and drag the dropper from thebottle of ether over to the nerve in between the stimulatingelectrodes and recording electrodes. Release the mouse but-ton to dispense drops.

2. Look back at Activity 1 for the voltage you determined.

Set the voltage at that level, and click Single Stimulus tostimulate the nerve. What sort of trace do you see?

What has happened to the nerve? ______________________


3. Click on the Time (min) button on the oscilloscope. Thisbutton toggles the time scale between minutes and millisec-onds. The screen will now display activity over the course of10 minutes (the space between each vertical line representing1 minute). Because of the change in time scale, an actionpotential will look like a sharp vertical spike on the screen.

4. Click the () button underInterval between Stimuli onthe stimulator until the timer is set for 2.0 minutes. This willset the stimulus to stimulate the nerve every two minutes.

Click on Stimulate to start the stimulations. Watch theElapsed Time display. With the change in time scale, theaction potential will look like a straight vertical line.

How long does it take for the nerve to return to normal?

5. Click on the Stop button to stop this action and to returnthe Elapsed Time to 0.0.

36 Exercise 3

F I G U R E 3 . 4 Opening screen of the Inhibiting a Nerve Impulse experiment.


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6. Click the Time (msec) button on the oscilloscope to re-turn it to its normal millisecond display.

7. Click Clear to clear the oscilloscope for the next activity.

8. Click the () button under Interval between Stimuliuntil it is reset to 0.00.

A C T I V I T Y 6

Testing the Effects of Curare

Curare is a well-known plant extract that South American In-dians used to paralyze their prey. It is an alpha-toxin thatbinds to acetylcholine binding sites on the postsynaptic cellmembrane, which will prevent the acetylcholine from acting.Curare blocks synaptic transmission by preventing the flowof neural impulses from neuron to neuron.

1. Click and drag the dropper from the bottle of curare andposition the dropper on the nerve in between the stimulatingand recording electrodes. Release the mouse button to dis-pense drops.

2. Look back at Activity 1 for the voltage you determined.Set the voltage at that level, and click Single Stimulus tostimulate the nerve. Does this generate an action potential?

What explains this effect?____________________________

What do you think would be the overall effect of curare onthe organism?


3. Click on the Clean button on the nerve chamber to re-move the curare and return the nerve to its original untouchedstate.

4. Click Clear to clear the oscilloscope screen for the nextactivity.

A C T I V I T Y 7

Testing the Effects of Lidocaine

Note: Lidocaine is a sodium-channel antagonist that preventssodium channels from opening.

1. Click and drag the dropper from the bottle of lidocaineand position it over the nerve between the stimulating andrecording electrodes. Release the mouse button to dispensedrops. Does this generate a trace?

2. Look back at Activity 1 for the voltage you determined.Set the voltage at that level, and click Single Stimulus tostimulate the nerve. What sort of tracing is seen?

Why does lidocaine have this effect on nerve fiber transmis-

sion?

Click Record Data to record your results. Click Tools Print Data if you wish to print your data.

3. Click on the Clean button on the nerve chamber to re-move the lidocaine and return the nerve to its original un-touched state.

Nerve Conduction VelocityAs has been pointed out, one of the major physiological prop-erties of neurons is conductivity: the ability to transmit thenerve impulse to other neurons, muscles, or glands. Thenerve impulse, or propagated action potential, occurs whenNa floods into the neuron, causing the membrane to depo-larize. Although this event is spoken of in electrical terms,and is measured using instruments that measure electricalevents, the conduction velocity, that is, the velocity of theaction potential along a neural membrane, does not occur atthe speed of light. Rather, this event is much slower. In cer-tain nerves in the human, the velocity of an action potentialmay be as fast as 120 meters per second. In other nerves, con-duction speed is much slower, occurring at a speed of lessthan 3 meters per second.

To see the setup for this experiment, click the Experimentmenu and select Nerve Conduction Velocity (Figure 3.5). Inthis exercise, the oscilloscope and stimulator will be used alongwith a third instrument, the bio-amplifier. The bio-amplifieris used to amplify any membrane depolarization so that theoscilloscope can easily record the event. Normally, when amembrane depolarization sufficient to initiate an actionpotential is looked at, the interior of the cell membrane goesfrom70 mV to about40 mV. This is easily registered andviewable on an oscilloscope without the aid of an amplifier.However, in this experiment, it is the change in the mem-brane potential on the outside of the nerve that is beingobserved. The change that occurs here during depolarizationwill be so minuscule that it must be amplified in order to bevisible on the oscilloscope.

A nerve chamber (similar to the one used in the previ-ous two experiments) will be used. The design is basically aplastic box with platinum electrodes running across it. Thenerve will be laid on these electrodes. Two electrodes willbe used to bring the impulse from the stimulator to the nerveand three will be used for recording the membrane depolar-ization.



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In this experiment, we will determine and compare theconduction velocities of different types of nerves. We willexamine four nerves: an earthworm nerve, a frog nerve, andtwo rat nerves. The earthworm nerve is the smallest of thefour. The frog nerve is a medium-sized myelinated nerve. Ratnerve 1 is a medium-sized unmyelinated nerve. Rat nerve 2 isa large, myelinated nervethe largest nerve in this group.We will observe the effects of size and myelination on nerveconductivity.

The basic layout of the materials is shown in Figure18B.5. The two wires (red and black) from the stimulatorconnect with the top right side of the nerve chamber. Threerecording wires (red, black, and a bare wire cable) are at-tached to connectors on the other end of the nerve chamberand go to the bio-amplifier. The bare cable serves as aground reference for the electrical circuit and provides thereference for comparison of any change in membrane poten-tial. The bio-amplifier is connected to the oscilloscope sothat any amplified membrane changes can be observed. The

stimulator output, called thepulse, has been connected to theoscilloscope so that when the nerve is stimulated, the tracingwill start across the oscilloscope screen. Thus, the time fromthe start of the trace on the left side of the screen (when thenerve was stimulated) to the actual nerve deflection (fromthe recording electrodes) can be accurately measured. Thisamount of time, usually in milliseconds, is critical for deter-mining conduction velocity.

A C T I V I T Y 8

Measuring Nerve Conduction Velocity

1. On the stimulator, click the Pulse button.

2. Turn the bio-amplifier on by clicking the horizontal baron the bio-amplifier and dragging it to the On setting.

On the left side of the screen are the four nerves that willbe studied. The nerves included are the earthworm, a frognerve, and two rat nerves of different sizes. The earthworm as

38 Exercise 3

F I G U R E 3 . 5 Opening screen of the Nerve Conduction Velocity experiment.


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a whole is used because it has a nerve running down its ven-tral surface. Afrog nerve is used as the frog has long been theanimal of choice in many physiology laboratories. The ratnerves are used so that you may compare (a) the conductionvelocity of different sized nerves and (b) the conduction ve-locity of a myelinated versus unmyelinated nerve. Rememberthat the frog nerve is myelinated and that rat nerve 1 is thesame size as the frog nerve but unmyelinated. Rat nerve 2,

the largest nerve of the bunch, is myelinated.

3. Using the mouse, click and drag the dropper from thebottle of ethanol over the earthworm and release the mousebutton to dispense drops of ethanol. This will narcotize theworm so it does not move around during the experiment butwill not affect nerve conduction velocity. The alcohol is at alow enough percentage that the worm will be fine and back tonormal within 15 minutes.

4. Click and drag the earthworm into the nerve chamber. Besure the worm is over both of the stimulating electrodes andall three of the recording electrodes.

5. Using the () button next to the Voltage display, set the

voltage to 1.0 V. Then click Stimulate to stimulate the nerve.Do you see an action potential? If not, increase the voltage byincrements of 1.0 V until a trace is obtained.

At what threshold voltage do you first see an action potential

generated? _______ V

6. Next, click on the Measure button located on the stimu-lator. You will see a vertical yellow line appear on the far left

edge of the oscilloscope screen. Now click the () button un-der the Measure button. This will move the yellow line to theright. This line lets you measure how much time has elapsedon the graph at the point that the line is crossing the graph.You will see the elapsed time appear on the Time (msec) dis-play on the stimulator. Keep clicking () until the yellow lineis exactly at the point in the graph where the graph ceases be-

ing a flat line and first starts to rise.7. Once you have the yellow line positioned at the start ofthe graphs ascent, note the time elapsed at this point. ClickRecord Data to record the elapsed time on the data collec-tion graph. PhysioEx will automatically compute the conduc-tion velocity based on this data. Note that the data collectionbox includes a Distance (mm) column and that the distanceis always 43 mm. This is the distance from the red stimulat-ing wire to the red recording wire. In a wet lab, you wouldhave to measure the distance yourself before you couldproceed with calculating the conduction velocity.

It is important that you have the yellow vertical measur-ing line positioned at the start of the graphs rise before youclick Record Dataotherwise, the conduction velocity cal-

culated for the nerve will be inaccurate.

8. Fill in the data in the Earthworm column on Chart 1.

9. Click and drag the earthworm to its original place. ClickClear to clear the oscilloscope screen.

10. Repeat steps 4 through 9 for the remaining nerves. Re-member to click Record Data after each experimental runand to fill in the chart for question 8.

11. Click Tools Print Data to print your data.


Frog Rat nerve 1 Rat nerve 2Earthworm (medium nerve, (medium nerve, (large nerve,

Nerve (small nerve) myelinated) unmyelinated) myelinated)

Thresholdvoltage

Elapsed timefrom stimulationto action potential

Conduction

velocity

CHART 1


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Which nerve in the group has the slowest conduction velocity?

What was the speed of the nerve? ____________________

Which nerve in the group of four has the fastest conduction

velocity?

What was the speed of the nerve? _____________________

What is the relationship between nerve size and conduction

velocity? ________________________________________

Based on the results, what is your conclusion regarding con-duction velocity and whether the nerve is myelinated or not?

What is the major reason for the differences seen in con-duction velocity between the myelinated nerves and the

unmyelinated nerves? _______________________________

Histology Review Supplement

For a review of nervous tissue, go to Exercise H: HistologyAtlas and Review on the PhysioEx website to print out theNervous Tissue Review worksheet.

40 Exercise 3


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41

3R E V I E W S H E E T

Eliciting (Generating) a Nerve Impulse1. Why dont the terms depolarization and action potential mean the same thing?

2. What was the threshold voltage in Activity 1? ______________________________________________________________

3. What was the effect of increasing the voltage? How does this change correlate to changes in the nerve?________________

4. How did the action potential generated with the unheated rod compare to that generated with the heated rod? ____________

5. Describe the types of stimuli that generated an action potential.________________________________________________

6. If you were to spend a lot of time studying nerve physiology in the laboratory, what type of stimulus would you use and why?

7. Why does the addition of sodium chloride elicit an action potential? Hint: Think about the sodium permeability of the neuron

(Figure 3.2e)._____________________________________________________________________________________

Neurophysiologyof Nerve Impulses

E X E R C I S E

NAME ____________________________________

LAB TIME/DATE ________________________


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Inhibiting a Nerve Impulse8. What was the effect of ether on eliciting an action potential? __________________________________________________

9. Does the addition of ether to the nerve cause any permanent alteration in neural response?

10. What was the effect of curare on eliciting an action potential?

11. Explain the reason for your answer to question 10 above.

12. What was the effect of lidocaine on eliciting an action potential?

Nerve Conduction Velocity13. What is the relationship between size of the nerve and conduction velocity? ______________________________________

14. Keeping your answer to question 13 in mind, how might you draw an analogy between the nerves in the human body andelectrical wires?

15. How does myelination affect nerve conduction velocity? Explain, using your data from Chart 1. ______________________

16. If any of the nerves used were reversed in their placement on the stimulating and recording electrodes, would any differencesbe seen in conduction velocity? Explain.

42 Review Sheet 3


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10

REGULAO DOS NVEIS PLASMTICOS DE CORTISOL

I. OBJETIVO

Pretende-se com este trabalho fazer a determinao dos nveisplasmticos de cortisol em animais submetidos a diferentes condies, e

relacionar os valores obtidos com o estado fisiolgico de cada um.

II. INTRODUO TERICA

O crtex das glndulas supra-renais quando estimulado pela

hormona adrenocorticotrpica (ACTH) sintetiza e secreta uma famlia de

hormonas esteroides derivadas do colesterol. Estas hormonas dividem-se

em 3 categorias: hormonas reprodutivas, mineralocorticides, que

regulam a funo renal e glucocorticides com uma ao vasta, incluindo

a mobilizao de aminocidos e glucose e aes anti-inflamatrias. Dentro

das hormonas com atividade glucocorticide, o cortisol a mais

importante.

A secreo de glucocorticides e, por isso, o seu efeito nos tecidos

regulado por estmulos nervosos atravs de um mecanismo de retroao

negativo. Estes estmulos induzem a secreo da hormona libertadora de

corticotropina (CRH) pelas clulas neurosecretoras do hipotlamo. A

consequente libertao da hormona adreno-corticotrpica (ACTH) pelas

clulas anteriores da hipfise estimula a secreo de glucocorticides pelo

crtex das suprarrenais. Estes esterides levam a um aumento da glucose

plasmtica e do glicognio heptico estimulando a converso de


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Cortisol 11

aminocidos e gorduras em glicose. Um mecanismo de retroao

negativo, tanto a nvel da hipfise como do hipotlamo, pode limitar a

secreo de ACTH, reduzindo a concentrao plasmtica de

glucocorticides.

O nvel basal de secreo de glucocorticides apresenta um ritmo

circadiano, resultante da variao cclica na secreo de CRH, que parece

ser influenciado por um relgio biolgico endgeno. Os nveis basais nos

humanos so mximos durante as primeiras horas da manh, antes de

qualquer atividade. Estes ciclos so uma forma de adaptao muito til

dada a sua ao mobilizadora das fontes energticas. Alm da regulao

endgena de secreo, o crtex das suprarrenais estimulado para

secretar glucocorticides como resposta a vrios tipos de stress (incluindo


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Cortisol 12

jejum). O stress atravs do sistema nervoso leva a uma elevao da ACTH

e consequentemente a um estmulo do crtex das suprarrenais.

Os glucocorticides atuam a nvel heptico, aumentando a sntese

de enzimas que estimulam a gluconeognese (sntese de glucose a partir

de aminocidos e cidos gordos). Alguma desta glucose armazenada no

fgado como glicognio, mas a maioria libertada levando a um aumento

dos nveis de glicose sanguneos. Contudo, a utilizao desta glicose

restrita uma vez que os glucocorticoides, ao contrrio da insulina,

reduzem a sua absoro nos tecidos perifricos, como o msculo, que por

sua vez levado a liberar aminocidos para o plasma. O resultado final

a degradao heptica das protenas tecidulares para manter adequados

os nveis plasmticos de glicose e a produo de energia para tecidos

crticos como o crebro. A aco do cortisol pode ser resumida nos

seguintes passos:

1. Aumento da gluconeognese hepatica2. Aumento do catabolismo proteico tecidular (msculo, tec. adiposo)3. Aumento da glicognese heptica4. Inibio da secreo de ACTH (mecanismo de retroao)

A meia-vida do cortisol na circulao sangunea de 70-90

minutos; o local principal em que metabolizado o fgado. Os valores

normais de cortisol plasmtico pelas 8 da manh esto compreendidos no

seguinte intervalo: 60-230 ng/ml. Nveis mais elevados de cortisol e falta

de variao diurna podem indicar sndroma de Cushing (hipersecreo de

ACTH) ou tumor suprarrenal. Nveis mais baixos so encontrados em

casos de insuficincia renal primria (hipoplasia adrenal ou doena de

Addison) e deficincia de ACTH. Na maioria dos casos, medies isoladas

do valor basal no so de nenhuma utilidade, sendo geralmente

necessrio testar vrias amostras para determinar oscilaes ao longo do

dia.

Hoje em dia a sade e bem-estar animal uma das preocupaes

em todas as produes de bovinos, ovinos, sunos, aves, cavalos e peixes


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Cortisol 13

de aquicultura. Muito recentemente a Autoridade Europeia para a

Segurana dos Alimentos (AESA/EFSA) emitiu um parecer relativo aos

aspetos respeitantes ao bem-estar das principais espcies animais que

so submetidas a insensibilizao e preparao para a morte no quadro

das prticas de abate, tanto em matadouro comercial como na

explorao. O Painel cientfico recomenda que devem ser selecionadas,

para cada espcie, as condies e os mtodos mais apropriados em

termos do bem-estar animal. O texto integral do parecer est disponvel

em http://www.efsa.eu.int.

Dentro dos indicadores credveis do bem-estar animal os nveis de

cortisol tm sido bastante utilizados dada a sua correlao com fatores

que induzem stress, nomeadamente as condies ambientais

(temperatura, arejamento, qualidade de gua) o maneio, o confinamento

espacial e densidade populacional, o transporte e o acesso a alimento

entre outros. Para que o cortisol seja utilizado como indicador

necessrio conhecer os nveis normais para cada uma das espcies a

estudar, no sendo, no entanto a sua determinao simples. Trata-se de

um mtodo invasivo podendo a amostragem interferir nos resultadosobtidos.

III. FUNDAMENTO DO MTODO

Neste trabalho vai-se utilizar um mtodo competitivo

imunoenzimtico colorimtrico (ELISA - Enzyme Linked Immuno Sorbent

Assay) para a determinao quantitativa da concentrao de cortisol no

plasma de dois animais submetidos a duas situaes de stress.

As paredes das microplacas esto revestidas com um anticorpo

anti-cortisol (fase slida). O cortisol (antignio) presente na amostra vai

competir com um antignio marcado com uma enzima (cortisol conjugado

com peroxidase) para se ligar ao anticorpo adsorvido na microplaca. Aps

incubao, todo o cortisol que ficou livre (no ligado ao anticorpo) rejeitado mediante uma simples lavagem da fase slida. O complexo
http://www.efsa.eu.int/http://www.efsa.eu.int/


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Cortisol 14

formado pelo antignio marcado enzimaticamente, presente na frao

ligada, vai ento ser visualizado atravs da reao com o substrato H2O2

(perxido de hidrognio) - TMB (tetrametilbenzidina). Esta reao leva ao

aparecimento de um produtode colorao azul que passa a amarelo aps

a adio da soluo de paragem de cido sulfrico (H2SO4). A intensidade

da cor desenvolvida proporcional concentrao de antignio marcado

que ficou ligado placa e, logo, inversamente proporcional concentrao

de cortisol presente na amostra. A absorvncia determinada a 450 nm

num leitor de microplacas.

IV. PROTOCOLO EXPERIMENTAL

Recolheu-se sangue de um coelho, antes de uma interveno

cirrgica (T0) e 45 minutos aps o incio da cirurgia (T45), e de um peixe,

antes e 45 minutos depois de ter sido submetido a um maneio indutor de

stress (forte agitao da gua com redes de pesca). O sangue foi

centrifugado a 3000 rpm, 15 minutos, e o plasma congelado (-20 C) at

ser analisado.

Material:

- Leitor de microplacas (: 450 nm);

- teste ELISApara cortisol;

- Pipetas multicanais.

Reagentes:

Presentes no kit(EIAgen CORTISOL):

Padres de cortisol (C0-C4);Conjugado (cortisol conjugado com peroxidase);Substrato TMB (H2O2-TMB; 0,25 g/l));Soluo de paragem (cido sulfrico; 0,15 M);Microplaca revestida (anti-cortisol) adsorvido na

microplaca.


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Cortisol 15

Preparao dos reagentes:

Todos os reagentes, amostras e padres devem ser colocados

temperatura ambiente (20-250C) antes de iniciar o ensaio.

1) Preparao dos Padres: C0-C1-C2-C3-C4

Antes da utilizao dos vrios padres a utilizar para obter uma

curva de calibrao, deixar num agitador rotativo durante pelo menos 5

minutos. Os padres possuem as seguintes concentraes de cortisol:

Padres C0 C1 C2 C3 C4

ng/ml 0 10 50 150 500

Estveis durante 6 meses a 2-8 C aps a abertura

2) Preparao da amostra:

A determinao de cortisol pode ser efetuada em plasma (heparina

ou EDTA) ou em soro. Se o ensaio no for efetuado no mesmo dia da

colheita, conservar a amostra a -20 C.

Advertncias:

- Evitar a utilizao de amostras hemolisadas;

- Fazer a reconstituio e distribuio dos reagentes com a mxima

preciso;

- No utilizar reagentes de lotes diferentes;

- O presente mtodo permite determinar concentraes de cortisol

de 10 ng/ml a 500 ng/ml;

- A administrao de esteroides naturais ou sintticos pode alterar

os nveis hemticos de cortisol.


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Cortisol 16

Procedimento:

Uma vez que necessrio fazer uma operao em duplicado,

preparam-se dois poos para cada um dos pontos da curva de calibrao

(C0-C4), dois para cada amostra e um para o branco.

1) Aps agitao, distribuir as vrias solues pelos poos da microplaca

de acordo com a seguinte tabela:

Padres Amostra Branco

Padres C0-C4 20 l - -

Amostra 20 l -

Conjugado 200 l 200 l -

2) Misturar bem durante 10 segundos e incubar 1 h a 37 C.

3) Rejeitar a mistura de reao e lavar adicionando a cada poo 0.3 ml

de gua destilada: repetir a lavagem removendo completamente a gua

(inverter as placas sobre papel absorvente).

4)Adicionar o substrato por todos os poos:

Calibrador Amostra Branco

Substrato TMB 100 l 100 l 100 l

5)Incubar exatamente durante 15 min temperatura ambiente (22

C-28 C) ao abrigo da luz.

6) Adicionar 100 l da soluo de paragem (cido sulfrico 0,15M) a

todos os poos:

7)Ler a absorvncia (A) num leitor de microplacas a 450 nm, repondoa zero com o branco.

C0 C0C1 C1C2 C2C3 C3C4 C4CLH0 CLH0 PX0 PX0CLH45 CLH45 PX45 PX45BRA BRA


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Cortisol 17

IV. APRESENTAO DOS RESULTADOS

a) Calcule a absorvncia (A) mdia de cada ponto da curva decalibrao (C1-C4) e de cada amostra. Subtrair a cada uma das

mdias de absorvncia o valor de absorvncia do branco.

b) Calcule a percentagem de ligao mxima (%B/B0) dividindo aabsorvncia de cada padro pela absorvncia do padro C0 (possui

mxima ligao) e multiplique por 100. Transforme esta razo

numa funo exponencial ou logit onde:

Logit= ln (%B/B0/(100-%B-B0)).

c) Construa uma curva de calibrao para o cortisol a partir dosvalores obtidos para cada um dos padres (C1-C4).

d) Indique na curva de calibrao os valores percentuais de B/B0relativos a cada amostra e extrapole a concentrao de cortisol em

ng/mL correspondente.

e) De acordo com os valores obtidos avalie a utilizao do cortisolcomo indicador do bem-estar animal.

Bibliografia:

Guyton, A. C., 1991. Textbook of Medical Physiology, 8 th edition . W. B. Saunders

Company. London, Toronto.

Randall, D., Burggren W. e French, K. Eckert Animal Physiology. 1988. Mechanisms and

adaptations. 5th Edition. W. H. Freeman and Company, New York.

Animais Cortisol (ng/ml)

Coelho (tempo 0)

Coelho (aps 45 min)

Peixe (tempo 0)

Peixe (aps 45 min)


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18

ACO DA INSULINA E ADRENALINA NA GLICMIA

I. OBJETIVO

O objetivo deste trabalho estudar a ao reguladora de duashormonas, a insulina e a adrenalina, na glicemia, usando o coelho como

animal de experincia.

II. INTRODUO TERICA

O valor da glicemia no Homem controlado dentro de uma faixamuito estreita, oscilando entre 80 e 100 mg/dl pela manh e em jejum.

Considera-se como limite superior da normalidade em jejum o valor de

110 mg/dl. Esta concentrao aumenta imediatamente aps as refeies

para 120-140 mg/dl e cai novamente para valores inferiores aos normais

dentro de aproximadamente 2 horas.

A insulina o regulador hormonal mais importante do

metabolismo energtico. sintetizada e secretada pelas clulas do

pncreas quando a glicemia aumenta, e favorece o armazenamento dos

metabolitos energticos bem como as snteses proteicas.

A ligao da insulina aos recetores das membranas das clulas

musculares, adiposas e de outras clulas do corpo, constituindo cerca de

80% da totalidade das clulas, torna as membranas altamente

permeveis glucose (fig. 1a e b). Uma vez dentro das clulas a glucose mediatamente fosforilada.


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Hormonas Reguladoras da Glicmia 19

a)

b)

Fig. 1 a) Ligao da insulina aos recetores b) Formao da ligao covalente entre

os grupos os grupos aminocidos livres na insulina e a protena do recetor.

Alm do aumento da permeabilidade da membrana para a glucose,ela fica mais permevel para os aminocidos e para os ies potssio,

magnsio e fosfato. A insulina promove ainda um aumento da atividade

das enzimas metablicas celulares. Aps a ingesto de uma refeio rica

em hidratos de carbono, a entrada de glucose para o sangue resulta numa

secreo rpida de insulina, que pode aumentar a taxa de transporte de

glucose para as clulas cerca de 10 a 20 vezes.


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A ao da insulina ocorre aos seguintes nveis:

1. A insulina inibe a fosforilase do fgado, evitando a hidrlise doglicognio.

2.

A insulina facilita a entrada de glucose para a clula heptica,aumentando a atividade que promove a fosforilao da glucose.

3. A insulina aumenta a atividade do fosfofrutocinase e daglicogenosintetase, a enzima interveniente na sntese doglicognio.

O efeito global desta hormona , portanto, uma diminuio da

glicemia. Um nvel baixo de insulina entre as refeies inverte estes

efeitos, ativando a fosforilase que hidroliza o glicognio a glucose. Se omsculo est em repouso, a glucose armazenada sob a forma de

glicognio at um limite de 2% (no fgado a insulina promove tambm a

formao de glicognio). Assim, nos intervalos das refeies, quando a

glucose no est disponvel, e o nvel sanguneo desta comea a cair, o

glicognio heptico mobilizado impedindo que os nveis sanguneos de

glucose baixem.

Quadro 1

FgadoTecido

adiposo

Msculo

esqueltico

Transporte de glucose + +

Transporte de aminocidos + +

Sntese de glicognio +

Glicogenlise -Gliconeognese -

Cetognese -

Lipognese + +

Liplise - -

Sntese proteica + + +

Protelise - - -


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O crebro diferente dos outros tecidos, no que respeita entrada

de glucose para as clulas, que feita sem a interveno da insulina. Os

efeitos da insulina nos principais tecidos implicados no metabolismo

energtico esto resumidos no quadro 1.

A manuteno dos nveis sanguneos de glucose indispensvel,

uma vez que o crebro, a retina e o epitlio germinativo das gnadas

dependem exclusivamente deste nutriente.

Papel da adrenalina

A ativao do Sistema Simptico conduz a descargas de

noradrenalina (nas terminaes nervosas e da suprarrenal) e de

adrenalina (s na suprarrenal) que provocam, entre numerosos outros

efeitos no organismo, uma hiperglicemia transitria.

A hiperglicemia pode ser assim explicada:

1. Estimulao da glicogenlise (pela variao da concentrao de

AMPC, de Ca2+ e de calmodulina, segundo os tipos de receptores a ou

p recrutados) e da gluconeognese heptica (fig 2).

Cell membrane

Blood

glucose

Glycogen

Uridine diphosphate glucose (Phosphorilase)

Glucose 1-phosphate

Glucose 6-phosphate

Glycolisis

(glucokinase)

(phosphatase)

Cell membrane

Blood

glucose

Glycogen

Uridine diphosphate glucose (Phosphorilase)

Glucose 1-phosphate

Glucose 6-phosphate

Glycolisis

(glucokinase)

(phosphatase)

Fig. 2 As reaces qumicas da

glucognese e da glicognolise,

mostrando tambm as intervenes

entre a glucose sangunea e o

glicognio do fgado.


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2. Estimulao da glicogenlise muscular com baixa utilizao da

glucose plasmtica.

3. Aumento da liplise no tecido adiposo, pela ao sobre a lipase

homono-sensvel.

O mecanismo de ao da adrenalina via AMPc est representado na

Fig.3. O AMPc inicia uma cascata de reaes qumicas que ativam a

fosforilase, segundo a seguinte sequncia:

A adrenalina ativa a adenilciclase na membrana das clulas;

A qual ativa a protena reguladora da protena cinase; Que ativa a protena cinase; Que ativa a fosforilase b cinase; Que converte a fosforilase b em fosforilase a; Que promove a degradao do glicognio a glucose-I-fosfato; Que desfosforilada e libertada para o sangue.

Fig. 3 O mecanismo do AMPcatravs do qual muitas hormonas

exercem o controlo na funo celular.


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III. FUNDAMENTO DO MTODO

A ao da insulina e adrenalina na glicemia ser verificado em

coelhos, aos quais sero feitas colheitas de sangue em tempos

determinados, aps a administrao das respectivas hormonas. O

doseamento da glucose baseia-se na seguinte reao:

IV. Protocolo Experimental

a) Administrao das hormonas e recolha de sangue

1. Anestesiar dois coelhos com uma mistura de Imalgne 1000 e Rompun

2% (1:1) 1ml da mistura/kg do coelho;

2. Cateterizar a jugular direita e a cartida esquerda de cada coelho;

3. Injetar na jugular 1,5 ml da soluo de heparina, esperar 2 min;

4. No tempo 0 (zero) recolher da cartida 5 ml de sangue (amostras A0 e

B0) para tubos heparinizados; recolher mais 5 ml de sangue para um

outro tubo e recuperar o plasma para doseamento do cortisol (trabalhoprtico seguinte).

5. Injetar na jugular de cada um dos coelhos 1,5 ml da soluo de

Hormona A e Hormona B, respetivamente (T0) e cronometrar;

6. Recolher 5 ml de sangue aos 3, 7, 15, 30 e 45 minutos, em cada um

dos coelhos, para tubos previamente heparinizados e conservar a 5oC

at sua utilizao. Para o trabalho do cortisol (aula seguinte), aos 45

minutos, colher 5 ml extra de sangue, recuperar o plasma e congelar.

Glucose + O2 cido glucnico + H2O2

Glucoseoxidase

Glucose + O2 cido glucnico + H2O2

Glucoseoxidase

H2O2 + Fenol + Amino-4-antipirina Quinoneimina corada + 4 H2OH2O2 + Fenol + Amino-4-antipirina Quinoneimina corada + 4 H2O


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b)Doseamento da glucose

1. Pipetar para tubos de centrfuga:

Reagentes (ml)TUBOS

Amostra Padro Branco

Sangue total 0,1 - -

Glucose (100 mg/dl) - 0,1 -

gua destilada - - 0,1

cido perclrico 0,9 0,9 0,9

2. Misturar cuidadosamente;

3. Centrifugar as amostras a 3.000 r.p.m, durante 15 minutos (excepto o

padro e o branco);

4. Com uma pipeta Pasteur passar cuidadosamente os sobrenadantes

obtidos em 3 para outros tubos;

5. Pipetar para novos tubos de ensaio:

Reagentes (ml)TUBOS

Amostra Padro Branco

Sobrenadante obtido em 4. 0,15 0,15 0,15

Reagente de colorao (*) 3,0 3,0 3,0

6. Misturar cuidadosamente;

7. Incubar a 37C, durante 10 min, evitando a luz intensa;

8. Ler no espetrofotmetro, a 500 nm, as absorvncias das amostras e do

padro, contra o branco.

* O reagente de colorao contm glicose oxidase, fenol, amino-4-

antipirina e peroxidase.


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V. APRESENTAO E DISCUSSO DOS RESULTADOS

a) Sabendo que a concentrao do padro de glucose 100 mg/dl, calcule

a concentrao da glucose nas amostras de sangue.

Hormona A D.O Concentraes

Padro 100 mg/dl

A0

T3

T7

T15

T30

T45

Hormona B D.O Concentraes

Padro 100 mg/dl

A0

T3

T7

T15

T30

T45

b) Com os valores obtidos construa um grfico representando as

concentraes de glucose em funo do tempo e interprete os

resultados com base no conhecimento da atuao destas hormonas.


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c) De acordo com os valores da glicemia obtidos, identifique a hormona

que foi administrada a cada um dos coelhos. Justifique.

d) Interprete cada grfico referindo-se forma de atuao de cada

hormona (velocidade e tempo de atuao), bem como aos possveismecanismos de retroao verificados.

Bibliografia

GUYTON. Textbook of Medical Physiology. 12nd edition. W.B. Saunders Company. London,Toronto.


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27

FISIOLOGIA VIRTUAL

FISIOLOGIA DO MSCULO ESQUELTICO

I.OBJETIVO

Pretende-se neste trabalho fazer a simulao da fisiologia do

msculo, atravs de um CD-ROM interativo, utilizando o msculo da perna

de uma r. O msculo retirado da r juntamente com o nervo e

estimulado eletricamente num laboratrio de eletrofisiologia virtual.

Quando um msculo esqueltico, retirado de um animal experimental,

estimulado eletricamente, comporta-se da mesma forma que um msculo

estimulado in vivo, portanto este tipo de experincias proporciona um

meio valioso para o estudo do comportamento do msculo permitindo

compreender importantes conceitos sobre a contrao muscular:

1.Definir os seguintes termos utilizados na descrio da fisiologia domsculo: somatao de mltiplas unidades motoras; estmulos

mximos; gradao em escada, somao de ondas, tetania;

2.Desenhar um grfico que relacione a fora do estmulo com a forade uma contrao simples, ilustrando a resposta gradual do

msculo.

3. Explicar como proceder para diminuir, suavizar ou suster acontrao do msculo-esqueltico.

4.Identificar as condies que levam a que a contrao muscular sejaisomtrica ou isotnica.

5.Descrever, em termos de comprimento e fora, as transies entreos estados isomtricos ou isotnicos durante uma mesma contrao

muscular simples.


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6.Explicar porque permanece constante a fora muscular durante aretrao isotnica.

7.Explicar os resultados obtidos na execuo deste trabalho emtermos de estrutura muscular.

II.PROTOCOLOEXPERIMENTAL

Nas simulaes seguintes ir utilizar o programa physiol Ex para

investigar que tipos de estmulos desencadeiam um potencial de ao.

Proceder da seguinte forma:1- Inserir o CD no computador e aceitar que se faam alteraes no

computador

2- Selecionar o Physiol Ex seguido do ficheiro StartHere.html3- Aparecer o seguinte:


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1Site Requi rements: Requisitos do programa

2Selecionar o Exerccio 2 e segui r as instr ues


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S

keletal muscles are composed of hundreds to thousands of individual cells,each doing their share of work in the production of force. As their name sug-

gests, skeletal muscles move the skeleton. Skeletal muscles are remarkablemachines; while allowing us the manual dexterity to create magnificent works of art,they are also capable of generating the brute force needed to lift a 100-lb. sack ofconcrete. When a skeletal muscle from an experimental animal is electrically stim-ulated, it behaves in the same way as a stimulated muscle in the intact body, that is,in vivo. Hence, such an experiment gives us valuable insight into muscle behavior.

This set of computer simulations demonstrates many important physiologi-cal concepts of skeletal muscle contraction. The program graphically provides allthe equipment and materials necessary for you, the investigator, to set up experi-mental conditions and observe the results. In student-conducted laboratory in-vestigations, there are many ways to approach a problem, and the same is true ofthese simulations. The instructions will guide you in your investigation, but youshould also try out alternate approaches to gain insight into the logical methodsused in scientific experimentation.

Try this approach: As you work through the simulations for the first time, fol-low the instructions closely and answer the questions posed as you go. Then tryasking What if . . . ? questions to test the validity of your hypotheses. The majoradvantages of these computer simulations are that the muscle cannot be acciden-tally damaged, lab equipment will not break down at the worst possible time, andyou will have ample time to think critically about the processes being investigated.

Because you will be working with a simulated muscle and an oscilloscopedisplay, you need to watch both carefully during the experiments. Think aboutwhat is happening in each situation. You need to understand how you are exper-imentally manipulating the muscle in order to understand your results.

Skeletal Muscle Physiology

2E X E R C I S E

17

O B J E C T I V E S

1. To define these terms used in describing muscle physiology:multiple

motor unit summation, maximal stimulus, treppe, wave summation, and

tetanus.

2. To identify two ways that the mode of stimulation can affect muscle

force production.

3. To plot a graph relating stimulus strength and twitch force to illustrate

graded muscle response.

4. To explain how slow, smooth, sustained contraction is possible in a

skeletal muscle.

5. To graphically understand the relationships between passive, active,

and total forces.

6. To identify the conditions under which muscle contraction is isometricor isotonic.

7. To describe in terms of length and force the transitions between isomet-

ric and isotonic conditions during a single muscle twitch.

8. To describe the effects of resistance and starting length on the initial

velocity of shortening.

9. To explain why muscle force remains constant during isotonic

shortening.

10. To explain experimental results in terms of muscle structure.


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Electrical StimulationA contracting skeletal muscle will produce force and/or short-ening when nervous or electrical stimulation is applied. Theforce generated by a whole muscle reflects the number of mo-tor units firing at a given time. Strong muscle contraction im-plies that many motor units are activated and each unit hasmaximally contracted. Weak contraction means that few motorunits are active; however, the activated units are maximallycontracted. By increasing the number of motor units firing, wecan produce a steady increase in muscle force, a process calledrecruitment ormultiple motor unit summation.

Regardless of the number of motor units activated, a sin-gle contraction of skeletal muscle is called a muscle twitch(Figure 2.1b). A tracing of a muscle twitch is divided intothree phases: latent, contraction, and relaxation. The latentperiod is a short period between the time of stimulation andthe beginning of contraction. Although no force is generatedduring this interval, chemical changes occur intracellularly inpreparation for contraction, such as the release of calciumfrom the sarcoplasmic reticulum. During contraction, themyofilaments are sliding past each other, and the muscleshortens. Relaxation takes place when contraction has endedand the muscle returns to its normal resting state and length.

The first activity you will conduct simulates an isometric,or fixed length, contraction of an isolated skeletal muscle.This activity allows you to investigate how the strength and fre-quency of an electrical stimulus affect whole muscle function.Note that these simulations involve indirect stimulation by anelectrode placed on the surface of the muscle. This differs fromthe situation in vivo where each fiber in the muscle receives di-rect stimulation via a nerve ending. In other words, increasingthe intensity of the electrical stimulation mimics how the ner-vous system increases the number of motor units activated.

Single StimulusChoose Exercise 2: Skeletal Muscle Physiology from thedrop-down menu and click GO. Before you perform the ac-tivities, watch the Skeletal Muscle video to gain an appreci-ation for the preparation required for these experiments. Thenclick Single Stimulus. The opening screen will appear in afew seconds (Figure 2.1a). The oscilloscope display, the gridat the top of the screen, is the most important part of thescreen because it graphically displays the contraction datafor analysis. Time is displayed on the horizontal axis. A fullsweep is initially set at 200 msec. However, you can adjustthe sweep time from 200 msec to 1000 msec by clicking anddragging the 200 msec button at the lower right corner of theoscilloscope display to the left to a new position on the time

axis. The force (in grams) produced by muscle contraction isdisplayed on the vertical axis. Clicking the Clear Tracingsbutton at the bottom right of the oscilloscope erases all mus-cle twitch tracings from the oscilloscope display.

The electrical stimulatoris the equipment seen just be-neath the oscilloscope display. Clicking Stimulate deliversthe electrical shock to the muscle through the electrodes lyingon the surface of the muscle. Stimulus voltage is set byclicking the (+) or () buttons next to the voltage window.Three small windows to the right of the Stimulate button dis-play the force measurements.Active force is produced duringmuscle contraction, whilepassive force results from the mus-cle being stretched (much like a rubber band). The total forceis the sum of active and passive forces. The red arrow at the

left of the oscilloscope display is an indicator of passiveforce. After the muscle is stimulated, the Measure button atthe right edge of the electrical stimulator becomes active.When the Measure button is clicked, a vertical orange linewill be displayed at the left edge of the oscilloscope display.Clicking the arrow buttons below the Measure button movesthe orange line horizontally across the screen. The Time win-dow displays the difference in time between the zero point on

the X-axis and the intersection between the orange measureline and the muscle twitch tracing.

The muscle is suspended in the support stand to the leftof the oscilloscope display. The hook through the upper ten-don of the muscle is part of the force transducer, which mea-sures the force produced by the muscle. The hook through thelower tendon secures the muscle in place. The weight cabinetjust below the muscle support stand is not active in this ex-periment; it contains weights you will use in the isotonic con-traction part of the simulation. You can adjust the startinglength of the muscle by clicking the (+) or () buttons lo-cated next to the Muscle Length display window.

When you click the Record Data button in the data col-lection unit below the electrical stimulator, your data isrecorded in the computers memory and is displayed in thedata grid at the bottom of the screen. Data displayed in the datagrid include the voltage, muscle length, and active, passive,and total force measurements. If you are not satisfied with asingle run, you can click Delete Line to erase a single line ofdata. Clicking the Clear Table button will remove all accumu-lated data in the experiment and allow you to start over.

A C T I V I T Y 1

Practicing Generating a Tracing

1. Click the Stimulate button once. Because the beginningvoltage is set to zero, no muscle activity should result. You

will see a blue line moving across the bottom of the oscillo-scope display. This blue line will indicate muscle force in theexperiments. If the tracings move too slowly across thescreen, click and hold the 200 button at the lower right cornerof the oscilloscope and drag it to the left to the 40 msec markand release it. This action resets the total sweep time to 1000msec to speed up the display time.

2. Click and hold the () button beneath the Stimulate but-ton until the voltage window reads 3.0 volts. Click Stimulateonce. You will see the muscle react, and a contraction tracingwill appear on the screen. Notice that the muscle tracing coloralternates between blue and yellow each time the Stimulatebutton is clicked to enhance the visual difference between

twitch tracings. You can click the Clear Tracings button asneeded to clean up the oscilloscope display. To retain yourdata, click the Record Data button at the end of each stimulus.

3. Change the voltage to 5.0 volts, and click Stimulateagain. Notice how the force of contraction also changes.Identify the latent, contraction, and relaxation phases in thetracings.

4. You may print your data by clicking Tools PrintData. You may also print out hard copies of the graphs yougenerate by clicking on Tools Print Graph.

Feel free to experiment with anything that comes to mind toget a sense of how whole muscle responds to an electrical

stimulus.

18 Exercise 2


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Skeletal Muscle Physiology 19

F I G U R E 2 . 1 Single stimulus and muscle twitch.

(a) Opening screen of the Single Stimulus experiment. (b) The

muscle twitch: myogram of an isometric twitch contraction.

(a)

0 5 10 15 20 25 30 35

Muscletension

Stimulus Time (msec)

Maximum tensiondevelopment

Relaxationphase

Contractionphase

Latentperiod

(b)


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A C T I V I T Y 2

Determining the Latent Period

1. Click Clear Tracings to erase the oscilloscope display.The voltage should be set to 5.0 volts.

2. Drag the 200 msec button to the right edge of the oscil-loscope.

3. Click the Stimulate button once, and allow the tracing tocomplete.

4. When you measure the length of the latent period from aprinted graph, you measure the time between the applicationof the stimulus and the beginning of the first observable re-sponse (increase in force). The computer cant look ahead,anticipating the change in active force. To measure the lengthof the latent period using the computer, click the Measurebutton. Then click the right arrow button next to the Timewindow repeatedly until you notice the first increase in theActive Force window. This takes you beyond the actuallength of the latent period. Now click the left arrow buttonnext to the Time window until the Active Force windowagain reads zero. At this point the computer is measuring thetime between the application of the stimulus and the lastpoint where the active force is zero (just prior to contraction).

How long is the latent period? ________________ msec

What occurs in the muscle during this apparent lack ofactivity?

The Graded Muscle Responseto Increased Stimulus Intensity

As the stimulus to a muscle is increased, the amount of forceproduced by the muscle also increases. As more voltage is de-livered to the whole muscle, more muscle fibers are activatedand the total force produced by the muscle is increased. Max-imal contraction occurs when all the muscle cells have beenactivated. Any stimulation beyond this voltage will not in-crease the force of contraction. This experiment mimics mus-cle activity in vivo where the recruitment of additional motorunits increases the total force produced. This phenomenon iscalled multiple motor unit summation orrecruitment.

A C T I V I T Y 3

Investigating Graded Muscle Response

to Increased Stimulus Intensity

1. Click Clear Tracings if there are tracings on your screen.

2. Set the voltage to 0.0, and click Stimulate.

3. Click Record Data. If you decide to redo a single stim-ulus, choose the data line in the grid and click Delete Line toerase that single line of data. If you want to repeat the entireexperiment, click the Clear Table button to erase all datarecorded to that point.

4. Repeat steps 2 and 3, increasing the voltage by 0.5 eachtime until you reach the maximum voltage of 10.0. Be sure toselect Record Data each time.

5. Observe the twitch tracings. Click on the Tools menuand then choose Plot Data.

6. Use the slider bars to display Active Force on the Y-axisand Voltage on the X-axis.

7. Use your graph to answer the following questions:

What is the minimal, or threshold, stimulus? __________ V

What is the maximal stimulus? __________ V

How can you explain the increase in force that you observe?

8. Click Print Plot at the top left corner of the Plot Datawindow to print a hard copy of the graph. When finished,click the X at the top right of the plot window.

9. Click Tools Print Data to print your data.

Multiple Stimulus

Choose Multiple Stimulus from the Experiment menu. Theopening screen will appear in a few seconds (Figure 2.2).

The only significant change to the on-screen equipmentis found in the electrical stimulator. The measuring equip-ment has been removed and other controls have been added:The Multiple Stimulus button is a toggle that allows you toalternately start and stop the electrical stimulator. When Mul-

tiple Stimulus is first clicked, its name changes to Stop Stim-ulus, and electrical stimuli are delivered to the muscle at therate specified in the Stimuli/sec window until the musclecompletely fatigues or the stimulator is turned off. The stim-ulator is turned off by clicking the Stop Stimulus button. Thestimulus rate is adjusted by clicking the () or () buttonsnext to the Stimuli/sec window.

A C T I V I T Y 4

Investigating Treppe

When a muscle first contracts, the force it is able to produceis less than the force it is able to produce in subsequent con-

tractions within a relatively narrow time span. A myogram, arecording of a muscle twitch, reveals this phenomenon as thetreppe, or staircase, effect. For the first few twitches, eachsuccessive stimulation produces slightly more force than theprevious contraction as long as the muscle is allowed to fullyrelax between stimuli, and the stimuli are delivered relativelyclose together. Treppe is thought to be caused by increasedefficiency of the enzyme systems within the cell and in-creased availability of intracellular calcium.

1. The voltage should be set to 8.2 volts, and the musclelength should be 75 mm.

2. Drag the 200 msec button to the center of the X-axis timerange.

20 Exercise 2


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3. Be sure that you fully understand the following threesteps before you proceed.

Click Single Stimulus. Watch the twitch tracing carefully. After the tracing shows that the muscle has completely

relaxed, immediately click Single Stimulus again.

When the second twitch completes, click Single Stimu-lus once more and allow the tracing to complete.

4. Click Tools Print Graph.

What happens to force production with each subsequentstimulus?

________________________________________________

_____________________________________________

A C T I V I T Y 5

Investigating Wave Summation

As demonstrated in Activity 3 with single stimuli, multiplemotor unit summation is one way to increase the amount offorce produced by muscle. Multiple motor unit summation re-lied on increased stimulus intensity in that simulation. An-other way to increase force is by wave, or temporal, summa-tion. Wave summation is achieved by increasing the stimulus

frequency, or rate of stimulus delivery to the muscle. Wavesummation occurs because the muscle is already in a partiallycontracted state when subsequent stimuli are delivered.

Tetanus can be considered an extreme form of wavesummation that results in a steady, sustained contraction. Ineffect, the muscle does not have any chance to relax becauseit is be

Documents

Protocolos de Laboratório de Bioquímica:Fisiologia Animal.pdf