Lab 1: Biomedical Signals Acquisition

Signals

• Signals are functions of one or more independent variable and typically contain information about the
behaviour/nature of some phenomenon.
• Waveform: the representation of a signal as a graph of amplitude vs time, the pattern of which gives us
information about the signal
o ex: EKG, Spirograph, EEG
• Deterministic signals: a signal is deterministic if it is exactly predictable for the time span of interest
(ex: sine wave can be described mathematically)
• Stochastic or random signals: a signal whose value has some element of chance associated to it, thus
cannot be predicted exactly. Statistical properties are probabilities are associated with it: mean, median,
mode, range (peak-to-peak)
o Biological signals often have both deterministic and stochastic signals
• Continuous (Analogue) Signals: the independent variable (time) is continuous, thus the signals are
defined for a continuum of values of time – continuously changing with time.
• Discrete time (digital) signals: signals are only defined at discrete times; the independent variable only
takes on a discrete set of values X(n).
• Desired signal: not interrupted by noise
o A discrete time signal/digital sequence can represent a pheneomon for which the independent
variable is already discrete (ex: # of calories ingested per day) or can be a digitized version of an
inherently continuous variable.
o In this lab: used ADC to convert analog (physiological) signal to digital so can be processed by
computer
• Signal:Noise Ratio: measurement of the amplitude of variance of the signal vs variance of the noise.
o Sources of noise include thermal (motion of atoms), interfence (unwanted signal from outside
source), and sampling noise (when you digitize a continuous signal with an A/D converter that
has a finite # of steps)
o The higher the signal:noise ratio, the better the signal can be distinguished from noise
• Sampling Frequency: frequency at which the ADC samples the analogue signal
• Sampling period: reciprocal of sampling f
 • Offset; a fluctuation in the baseline value of the signal; a constant and steady deviation of the measured
signal from the set point.

Signal Amplification

• Amplifier=device that tracks the amplitude of an incoming signal and proportionally increases the
voltage, current ,or power of the signal by using power from another source. Frequency is unchanged!
• Gain and amplification: factor by which you multiply your signal.
o Gain=1: signal stays the same
o Gain > 1: amplified
o Gain < 1: reduced
• Types of amplifiers:
o Single-ended amplifier --> uses a refernece input such as signal in skin to amplify the signal of
the EEG
o Differential amplifier --> uses 2 electrodes to get 2 signals from 2 diff. Sources. V1-V2
difference is amplified. Useful b/c if theres interfence, both electrodes pick it up and the only
thing amplified between the electrodes is both potentials while the common noise is substracted

• Spectral Analysis: any waveform can be decomposed into several (a sum of) waveforms
o This is what the so-called Fourier analysis does; it decomposes the waveform in different
components and measure the amplitude (power) of each frequency component. What is plotted is
a graph of power (amplitude) vs. frequency.
o Allows us to see what f’s and what amplitude make up the signal
• Powerlab contains A/D converter, amplifiers/ USB port
• Function generator generates sine wave which becomes the biological signal that goes to the powerlab
and then the computer
• The ADC does analog --> digital by sampling it --> computer system --> display

Sampling

• The process by which an analogue signal is converted to a signal signal in order to be sampled by the
computer. This is done by sampling the analogue signal at discrete points in time.
• The time between each sampling is called the sampling interval.
 • The electronic circuit that carries out the process of sampling the signal and A/D conversion is called an
analogue-to-digital converter (ADC). Being an electronic device, it requires an electrical signal at its
input. Thus the first step in the process of A/D conversion is to convert the analogue (non-voltage)
signal into an analogue voltage signal. The device that carries out this function is called a transducer.
For signals which are inherently voltages such as the electrocardiogram from the heart, the
electrooculogram from the eyes, or the electromyogram from muscle, transduction is of course not
necessary.
• Nyquist Interval: maximum time interval (period) between equally spaced samples of a signal that will
enable the signal waveform to be completely determined.
o The nyquist interval is equal to the reciprocal of 2x the highest f sampled (in reality, should use
more due to digitizing quantization error introduced by the digitizing process)
o Safety factor: 5-10x rather than 2x
• Nyquist Sampling Rate: the value of the sampling f equal to 2x highest frequency recorded
• Why not always sample at very high f?
o Cost, storage capacity, computing time

Resolution

• ADCs are described by bits.

• The ADC used in the lab is 16-bit and thus can take on 2^16 values=65536, representing the integers 0
to 65535. Our ADC also has an input range of -10 000 millivolts (mV) to +10 000 millivolts (mV). This
means that the binary value 0000000000000000, which is equivalent to the decimal number 0, will be
returned by the ADC to the computer when a voltage of -10 000 mV is present at its input, and the
binary number 1111111111111111, whose decimal equivalent is 65535, will be returned when the input
voltage is +10 000 mV. Thus, the input voltage range from -10 000 mV to +10 000 mV (=20 000 mV) is
divided into 65536 levels, with each level being 20 000 mV/65536 = 0.305 mV wide. This value will
determine the resolution of the sampled signal, i.e. This system alows us to measure 0.305 mV.
• In the example above, the resolution is 0.305 millivolts. An input voltage lying within one of these
0.305mV wide ranges is converted into a specific binary number: for example, any voltage lying in the
range from -10 000 to -9 999.695 will be converted to the binary number: 0000000000000000, while
any voltage in the range between +9 999.695 and +10 000 mV will be converted to the decimal number
65535.
• Resolution = Voltage Range / Digital Range (= 2 ^# bits)
• Q: How would the resolution for an 8-bit converter compare to the 12-bit case?
 • Resolution Answer The resolution of an 8-bit converter is not as good as that of a 12-bit one. The
resolution for the 8-bit converter is 0.078 volts versus 0.0049 volts for the 12-bit. The 8-bit converter
will not be able to resolve the smaller, finer changes in the input signal; therefore, the sampled signal
will not be as accurate a representation of the true signal. ---> The smaller the resolution, the better!
o Ex: 300 uV is higher resolution (smaller #) than 78 mV which is low resolution

Aliasing

• Aliasing: phenomenon which occurs whenever a signal is not sampled at greater than 2x maximum
bandwidth. Can cause a high f signal to appear low f, because don’t sample enough points, the f is
wrong.
• Artifactual result due to improper choice of sampling rte. Should always be 2x max, if not more (usualy
3-4x)

Saturation

• Occurs when the intensity of signal exceeds values of sampling range (ex: input 20 V but range is 10 V
= +/- 5 --> output will be 5 mV which is wrong) --> the graph will appear “cut”
• The input signal must always remain within the input voltage range (-10 000 to 10 000 mV).
o If signal is > 10 000 mV: will be read as 10 000 mV --> saturation
o If signal is < - 10 000 mV: should be amplified
o The signal input should span as much of the range as possible
• For example, if the signal to be recorded is much smaller than +/- 10 000 mV, say +/- 5 000 mV, then
the range over which the board operates should be decreased. By changing the hardware gain from 10
000 mV (10 V) to 5 V, the operating range of the board is changed from +/- 10 000mV to +/-5 000 mV.
This allows the experimenter to record a +/- 2V signal with a significant improvement in signal
resolution ( 2 times greater). This occurs because the minimum resolvable voltage would be 2x5000= 10
000 mV/65536 or 0.152 mV versus 0.305 mV when the board's operating range was set to +/-10 volts.
o Lowering the voltage range decreases the resolution, which is better quality
o Spanning the sampling to the most accurate range possible means better resolution
o Overall: the input signal should span as much of the ADC input voltage range as possible
(because this increases resolution), without saturating the signal

Filters
• A biological signal can be broken down into fundamental frequencies – usually only interested in a
certain range of f’s
• Filters permit certain f components of a signal to pass easily while inhibiting or preventing others -->
used o separate desired signal from noise
• 4 types of filter:
o High Pass (Low f): filters low f’s by attenuating signals above a given threshold
o Low pass. (High f): filters high f’s by removing low and middle range f’s
o Notch filter: filters one f, usually 60 Hz from power lines
o Band pass filter: allows only signals within the specified range to pass through the filter. Useful
when want to retain only specific waves from an EEG record (ex: if only want alpha, set the
band pass filter to 8-13 Hz)
• Real filters/hardware filters alter the f composition of the signal, and so after filtering, we cannot recover
the f’s that have been filtered
• Digital filters change the f of the signal by performing calculations on the data, eliminates unwanted f’s
but can still restore old f’s if desired

Electroencephalogram (EEG)

• Recorded waveforms reflect cortical electrical activity

• Some recoded activity is generated by APs but most is generated by EPSPs and IPSPs.
• Signal intensity: EEG activity is small, measured in mV
• Signal frequency: the main frequencies of the EEG waves are:
o Delta: <= 3 Hz, high amplitude/slow wave, dominant in infants and stage 3 + 4 of sleep,
usually most prominent frontally in adults and posteriorly in children, found in local regions
when patient has encephalitis, subcortical lesions, hydrocephalus, deep midline lesions
o Theta: 3.5 – 7.5 Hz, slow activity, abnormal in awake adults but ok in teens, can be seen in
encephalopathy and hydrocephalus
o Alpha: 7.5-13 Hz, best seen in posterior regions of the head, appears when closing eyes and
relaxing, and disappears when eyes open or when alerting mechanisms of thought and
consciousness, present after age 13
o Beta: 14-20 Hz, fast activity, seen on both side and most evident frontally, accentuated by
sedatives and benzodiazepines/barbiturates, may be absent in areas of cortical damage,
dominant in patients who are anxious, alert, eyes open
 • EEG activity can be rhythmic (normal), arrhythmic (no stable rhythm), dysrhythmic (associated with
disease)
• EEG voltage activity can be described via attenuation (amplitude dampens), hypersynchrony
(increase in voltage and rhythmic activity), paroxysmal (rapid onset to high voltage and culminating
down to lower voltage)
• EEG electrode placement: letter=lobe, even number=right side of head, odd=left. The midline = 0.
o ex: F7=frontal lobe on left side
• EEG montage: placement of electrodes
o Bipolar: the reference electrode for one channel is he active electrode for the next channel
o Referential: reference for each electrode is in common with the other electrodes

Experiments

1. Waveform Acquisition
• Must acquire, amplify and transform an analogue signal into digital signal.

2. Artifacts contaminating EEG recordings

• Activity of cortical neurons is synchronized in regular firing rhytms --> brain waves. Electrodes pick up
these waves.
• EEG is affected by state of arousal of the cortex and can show stages of sleep. EEG is used to diagnose
epilepsy and brain death.
• Signals are small because the recording electrodes are separated from the brain surface via hair, scalp,
skull, and CSF. Need a specially designed amplifier. Even so, may get artifacts.
• Blinking artifacts (EOG=electro-oculographic signals=potentials arising from eye movement)
• EMG (not EEG!) artifacts (facial movements causing EMG activity in muscles)
• Mechanical movement of electrodes (especially occipital, insecured by the hair)
• In this experiment: frontal electrode on forehead, occipital on call, and a third ground electrode to reduce
electrical inference

3. Alpha waves in the EEG

• Examining the effects of visual activity on alpha waves from different electrode arrangements
o Eyes shut, eyes open
o Alpha waves naturally occur when eyes closed/relax
 Lab 2: Blood Laboratory Exercises

Measurement of Erythrocyte Fragility via Osmotic Hemolysis

• Aim: determine the relationship between the extent of hemolysis and osmolarity of the medium.
• Cell membranes are semipermeable; the amount of osmotic pressure depends upon the difference
between non-diffusable ions (osmosis/L) on each side of the membrane.
• 1 osmotic=one mol of dissolved, non-diffusible, non-ionized substance
• If the substance ionizes: one mol of dissolved substance yields 2 osmols
• Osmolarity:
o Convert % into g/L (1%=1g/100 mL)
o Determine molarity (M/L)
o Determine osmolarity (Osm/L)
o Osmotic pressure = osmolarity x 22.4 x 760
o Ex: Determine osmolarity and osmotic pressure of a 0.9% NaCl solution: 0.9%=0.9g/100 mL = 9
g/L --> 9g/L divided by 58 g/mol = 0.155 M solution --> 0.155 M = 0.31 osmols (2 x 0.155
because NaCl) (=310 mOsm). --> 0.31 x 22.4 x 760=570 mmHg
• 0.9% NaCl --> isotonic, no net influx or efflux of water
• Hypertonic: osmotic efflux of water causes cells to lose their normal biconcave disc shape, undergoing
collapse, leading to crenation.
o Ex: 1.8%
• Hypotonic: influx of water occurs; cells swell and membrane is disrupted, allowing escape of
hemoglobin, causing hemolysis.
o ex: 0.4%
• In this experiment, we make use of the fact that the number of cells lysing is proprtional is to the
hypotonicity of the ECF. The [c] of liberated hemoglobin in each medium is an index of the extent of
osmotic hemolysis.
o More Hb in medium, more hemolysis
• Estimation of the [Hb] is done via optical density, which increases linearly with concentration. The OD
is proprtional to the # of solute particles (Hb) in the solution. Amount of Hb depends on how many
RBCs have listed.
• The zero value for the calibration curve is 0.9% NaCl solution --> no hemolysis.
• Pure distilled water provides the value for 100% hemolysis.
Erythrocyte Sedimentation Rate (ESR)

• The ESR is a test which indirectly measures the presence of inflammation in the body.
• When anticoagulated whole blood is put in a tube, the RBCs will settle out from the plasma at the
bototm of the tube because the density of the RBCs is > density plasma. The rate at which they settle is
measured as the mm of clear plasma present at the top of the column after one hour (mm/hr). The rate at
which they settle depends on fibrinogen and globulin [c]’s, which make RBCs stick to form rouleauxs.
o Men: ~15 mm/hr avg
o Women: ~20 mm/hr avg
 Values are higher in older people, for both genders
• When the RBCs settle, they undergo rouleaux formation (form large aggregates as there is an alteration
in the distribtion of charges on the surface of the RBC)
• Rouleax formation -> increased levels of plasma fibrinogen and globulins, which increase in
inflammation, tumors, neurodegernative diseases (as [F] or [Glob] increases during inflammation, ESR
increases)
o Thus ESR reflects mainly changes in plasma proteins that accompany acute and chronic
infections. In these cases, ESR is much > 20 mm/hr. Chronic inflammatory diseases increase
ESR.
o ESR is used to follow the progress of the diseases state or monitor effectiveness of treatment.
o Ex: the ESR of a male with lupus would be > 15 mm/hr. Lupus=autoimmune disorder, lots of
inflammation, ESR increases.
o ESR is decreased when RBCs are abnormally shaped (sickle cell, spherocytosis) or during
polycythemia (increased RBC).

Hemostasis

• Hemostasis=blood halting. Depends on:

o Vasoconstiction and formation of platelet plug
o Blood clotting
o Clot retraction
• 1. Clotting Time Test: the time taken for blood to clot mainly reflects the time required for generation of
thrombin. Lack of fibrinogen is very rare and is unlikely to be a cause of prolonged clotting time! If
prohombin or some other factor is low, clotting time is prolonged.
o Prothrombin --> Thrombin --> fibrinogen --> fibrin --> clotting
 o Decrease prothrombin, increase clotting time
• II. Bleeding Time Test: measures the time taken for vasoconstriction and platelet plug formation to
occur. No clot is allowed to form, such that the arrest i of bleeding depends solely on vessel constriction
and platelet action. Put filter paper on blood --> the bleeding time is the first half minute when no blood
is sucked up by the filter paper.
• III. Prothrombin Time Test: addition of Ca2+ and other thrombin factors to a sample of blood.
o Measures the integrity of the extrinsic system (factor VII) as well as factors common to both
extrinsic + intrinsic system (factors X, V, prothrombin, fibrinogen)
• IV. Partial Thromboplastin Time: to a sample of blood, add kaolin, cephalin, and ionized Ca2+.
o Measures integrity of intrinsic system (factors XII, XI, VIII, IX) and the common clotting
pathways (X, Y, prothrombin, fibrinogen)
o Liver disease increases PTT by decreasing level of necessary factors

Blood Cell Indices

• Tests that involve blood cells:

o RBC count: using a hemocytometer, precise grids allow for the counting of RBCs in a diluted
blood sample. Must count cells within a counting chamber and obtain an average number. That
number is multiplied by a factor that compensates for the amount of dilution. The final result
expresses the # of RBCs per cubic mm of the original blood sample.
o Hematocrit: = the ratio of volume of packed RBCs to the total blood volume. Determine ratio
using scale on the centrifuge.
o Male: 40-52%
o Female: 36-48%
o Hemoglobin content determination: using an optical measuring cartridge --> the restraints in it
function to hemolyze the blood and mediate a modified azide methemoglobin rxn. Hb-Iron is
converted from the ferrous to the ferric state to form methemglobin via sodium nitrite.
Methemoglobin combines with azide to form azide methemoglobin, causing a color change on
the cartridge. The reflectance measured by the cartridge at the end point is directly proportional
to the hemoglobin [c].
 Male: 13.5-17.5 g/dL (=g/100 mL), Female=11.5-15.5
o Total WBC count: mammalian blood contains 5 different types of WBCs
o Differential WBC count: % of each type of WBC in a sample of blood
 Neutrophil: multilobed nucleus, pale red/blue granules
  Eosinophil: two lobed nucleus, granules in cytoplasm
 Basophils: purple/black, 2 lobed nucleus, granules
 Lymphocyte: large spherical nucleus, thin rim of pale blue cytoplasm
 Monocytes: kidney-shaped nucleus, abundant pale blue cytoplasm
• The first 3 tests allow for the calculation of the Mean Corpuscular Volume (MCV) and Mean
Corpuscular Hb Concentration (MCHC)
• Low values of RBC count, hematocrit, or Hb --> anemia (decreased oxygen-carrying capacity of the
blood). There are many causes of anemia: loss of blood via hemorrhage, bone marrow disease, iron
deficiency, vitamin B12 or folic acid deficiency, low RBC production or high destruction, small RBC
size)
o Iron deficiency anemia: microcytic and hypochromic
o B12 deficiency anemia: macrocyclic and normochromic
• High values of RBC count: polycythemia --> due to excess RBC production or larger RBC size
• MCV = hematocrit (%) x 10 / RBC count (millions/mm3 blood)
o High MCV: macrocytic
o Low MCV: microcytic
o Normal: normochromic
• MCHC = hemoglobin (g/100ml) x 100 / hematocrit (%)
o High: hyperchromic
o Low: hypochromic
• Deviations from normal values often indicate a diseased state:

Expected Ranges
Neutrophil (%) 50-70
Eosinophil (%) 1-4
Basophil (%) 0.1
Monocyte (%) 2-8
Lymphocyte (%)
•
o Neutrophilia (high neutrophil count): localized infections such as appendicitis
o Neutoropenia (decrease in neutrophil count): typhoid fever, infectious diseases, measles
o Eosinophilia: allergic rxns or parasites such as Trichinella
o Eosinopenia: elevated corticosteroids (state of stress)
o Lymphocytosis: viral infections
Blood Group Typing

• The blood group refers to the presence of certain antigens on human RBCs.
o Ex: ABO blood group: one, two, or none of the 2 blood group antigens A and B --> A, B, AB, O.
o The corresponding antigen and antibody are never found in the same individual since, when
mixed, they would form antigen-antibody complexes and agglutinate in blood.
o Ex: Type A --> antigen=A, antibody=B. Type AB --> antigens A and B, no antibodies. O --> no
antigen, antibodies A and B. Someone with type A blood contains anti-B antibodies, which will
attack type B surface antigens.
o Antigens=cell surface proteins, agglutination=antibodies against RBC antigens
• In lab: add anti-A antiserum to the drop of blood marked A and anti-B antiserum to end marked B (same
blood).
o The subject is type A if agglutination occurred at the A end, and B if agglutination occurred at B
end, AB if agglutination occurred at both ends, and
• Rh system: Rh antigens are also antigens expresed on RBCs. RBCs expressing Rh antigen are called
Rh+.
• Rh system becomes important if Rh is incompatible between mother and fetus. During birth, leakage of
baby’s RBCs into mother’s circulation often occurs. If baby is Rh+ from the father and mother is Rh-,
these Rh+ RBCs will cause mother to form antibodies against Rh antigen. These IgG class antibodies do
not cause problem for first baby, but if second baby is born, the Rh+ antibodies can attack the Rh+ fetus.
The RBCs are destroyed --> anemia, jaundice. Leads to erthyroblastosis fetalis or hemolytic disease of
the new born – may be fatal.
o If mother is Rh+ then it does not matter what the baby is
• In lab: add drop of blood to anti-Rh. If agglutination occurs, blood is Rh+.
• Ex: Mother is group A, father is AB: Mother’s genotype can be AA or AO. Father is AB. The baby can
be BO, aka type B.
 Lab 3: Immunology

The Immune System

• Primary lymphoid organs:

o Thymus and bone marrow
o Function: to produce a large reserve of cells able to respond to foreign (non-self) cells, to
eliminate self-reactive cells
o Major site of lymphopoiesis (production of lymphocytes)
• Secondary lymphoid organs:
o Spleen, lymph nodes, mucosal-associated and bronchial-associated and gut-associated lymphoid
tissue, tonsils, adenoids, Peyer’s patches.
o Function: provide an environment for immune cell proliferation and maturation (of cells
involved in adaptive immune response), filter and trap antigens, provide an environment for cell-
cell interaction and cell-cytokine interaction.
• The innate response uses phagocytize cells (neutrophils, monocytes, macrophages) which release
inflammatory mediators (basophils, mast cells, eosinophils) and natural killer cells.
• The acquired (humoral) response involves the proliferation of antigen-specific B and T cells.
• Both responses usually work together.
• Major Histocompatibility Complex (MCH): all nucleated cells of the body express MHC Class I,
whereas only cells that help present the antigen to T cells express MHC class II (thus smaller in #)
o These antigen presenting cells include macrophages, dendritic cells, and B cells
o Mononuclear phagocytes: most important group of long-lived phagocytic cells, they are bone
marrow-derived and their function is to engulf particles, internalize them and destroy them
o Dendritic cells are found in T-cell areas and lymph node and spleen. Most important for the
initial activiation of naïve T cells.
o B cells are bone marrow-derived. Upon exposure to antigen, B cells multiply and become plasma
cells, which produce large amounts of antibody.

• T cells originate from bone marrow stem cells and undergo further differentiation in the thymus where
they migrate.
• T cells express in an orderly fashion certain markers or cell surface proteins --> nomenclature=CD
numbering.
• CD4+ T cells=cytokine-secreting helper cells can be divided into 2 major types:
 o Type 1 helper T cells --> secrete interleukin 2 and interferon
o Type II helper T cells secrete interleukin 4 and 5
• The production of cytokines by TH1 helps with cell-mediated immunity (activation of macrophages and
T-cell mediated cytotoxicity)
• TH2 helps B cells produce antibodies

• B cells mature in bone marrow, T cells in thymus

• When a B cell encounters its matching antigen, it will engulf the antigen and digest it into single amino
acids or fragments/chains of a.a.. It will then display these antigen fragments on its surface to attract a T
cell. Cytokines secreted by the T cell help the B cell to multiply and mature into antibody-producing
plasma cells OR can become a memory cell. The antibodies are now ready for action if you should be
exposed to this antigen a second time. The antibody-antigen complexes produced go into the blood and
lock onto matching antigens.
• Serum: the fluid component of uncoagulated blood – uncoaggulated because lacks fibrinogen.
• Antiserum: the fluid component of uncoaggulated blood, contains antibodies --> serum from an
immunized person
• Immunoglobulin: large glycoproteins molecules that have specific structure --> no immunization
• Antibody: an immunoglobulin produced in response to and can bind to a specific antigen.
o Making an antibody requires immunization
• Antibody structure:
• 2 heavy 2 light variable chains=antigen-binding chains
 • The constant region serves as a handle from which variable regions can interact with regions, involved
in Fc receptor binding. It is involved in starting complement cascade,
binds Fc receptors on NK cells, dendritic cells, and neutrophils. Involved
in opsonization (enhancement of phagocytic process). Fc portion also
determines immunoglobulin class.
• Types of Ig classes: IgD, IgE, IgG, IgG, IgA, IgM
• IgD, IgE, IgG, IgA = monomers
• IgA=monomer or dimer
• IgM=pentamer – does not cross the placenta thus it is the
only immunoglobulin a baby does not receive at birth

• Immune response: takes a few days for antibodies to form after

initial contact with antigen A. Once levels of antibodies are
formed, if cell comes into contact with antigen A again, there is a
quicker response and more antibodies formed. Why? --> memory
cells, larger reserve of cells ready to proliferate in case antigen is
introduced again
o Rationale for vaccination

Mechanism of Action

• Neutralization
o Neutralizes toxins, binds to pathogens and prevents it infecting new cells (like in HIV)
• Natural Killer Cells – antibody dependent cellular cytotoxicity will kill pathogen
• Macrophages, dendritic cells, neutrophils engulf antibody-bound pathoge
• Antibody bound to antigen can start complement cascade by binding C1q, first component of cascade

Complement Cascade
 • First step=antigen and antibody complex binds C1q
o Can also be activated by antibody-antibody or an alternate pathway that does not involve
antibody binding at all
• The complement cascade can kill microbes
o C5-C9 = Membrane Attack Complex --> makes holes in cell wall
• Some activated complement components cause, indirectly, vasodilation, increased capillary
permeability, and chemotaxis
• C3b is an opsonin (helps allow pathogens to be engulfed)
o Antibodies themselves are also opsonins – phagocytes have membrane receptors for Fc portion
of IgG, which leads to phagocytosis --> antibodies link phagocyte to bacterium

• When a foreign antigen is introduced into an animal, the animal will respond to it: response can be cell-
mediated or humoral.
o Humoral: the effector molecule is immunoglobulin, which is secreted by antibody-forming cells
present in the secondary lymphoid organs (spleen, lymph nodes)

• Antibody by itself is rather ineffectual in eliminating foreign organisms. However, IgG and IgM
antibodies can activate the complement system resulting in stimulation of different effector functions
such as phagocytosis and lysis of foreign antigens.

Enzyme-Linked Immunosorbent Assay (ELISA)

• Used to detect the presence of antibodies in a serum sample --> used to test for disease due to pathogen
(HIV, influenza). If antibodies are present, person has been affected.
• Horse radish peroxidase is the second antibody – it catalyzes blue rxn if bound to primary antibody.
Thus is rxn turns blue, there is the presence of primary antibodies. If not, they are washed off.
• The antigen binding site of the secondary antibody has to recognize the primary antibody, i.e. be specific
for human immunoglobulin.
• The Fc region is conjugated with an enzyme Horseradish Peroxidase (HRP). The secondary antibody is
usually prepared in a species other than the one the primary antibody comes from i.e., If the primary
antibody came from a human the secondary antibody comes from a non-human animal source
• HRP –horseradish peroxidase is an enzyme. It enzymatically cleaves the substrate TMB. When this
occurs a blue color develops --> the intensity of the blue color is a measure of the amount of enzymatic
activity.
Hemagglutination

• Measures the [c] of antibody in an antiserum relative to a particle, such as a RBC

o Can measure the [antibody] in blood and see how much is needed to agglutinate the RBCs -->
“at what concentration do we get agglutination? At what [c] does it fall off (b/c too diluted)?”
• Expressed as a titer: titer = inverse of last dilution of the antibody sample that is positive for
hemagglutination
• Used in blood banks to make sure a blood recipient does not have pre-formed antibodies to the donor
blood cells (or vice versa)
• Serial dilutions: diluting a stock solution where the [c] decreases by the same quantity in each successive
step
o 2-fold serial dilution: 1:1 antibody/solution --> 1:2 --> 1:4 --> 1:8..
o 10-fold serial dilution: 1:1 – 1:10 --> 1:100
 • In the zone of equivalence, the correct proportion of antibody to antigen occurs, resulting in a visible
mat formed by Ag-Ab complex crosslinking.

Complement-Mediated Lysis

• Formation of MAC to induce cell lysis

• Used to test for the presence of an antibody to cell surface antigens
• Formerly used for HLA typing
• Antibody alone does not start the complement cascade
• Antibody alone does not cause cell lysis --> Antibody binding to antigen activates complement
• Cascade of reactions terminates with insertion of a hydrophobic pore into the lipid bilayer – causes lysis.
o Our experiment will demonstrate that you need an antibody-antigen complex to activate SRBC
(Sheep Red Blood Cell) lysis
 Lab 4: Resting Membrane Potential

• All living cells under resting conditions have an electrical potential difference.
• By convention: inside is -, outside is 0 (the polarity of the charge - + or - - is stated in terms of the sign
of the excess charge on the inside of the cell)
• Excess ions are attracted to each other and collect along a thin shell on the inner and outer surface of the
plasma membrane, whereas the bulk of the ECF and ICF are electrically neutral.
• RMP ranges from -60 to -90 mV. It is a function of:
o Ratio of ion [c] in cell and ECF
o Permeability of membrane to these ions
 Relevant ions: Na+, K+, Cl-
 Ionic asymmetry and selective membrane permeability lead inevitably to a RMP
• Ionic asymmetry: K+ high in cell and low in ECF, Na+ high in ECF and low in ECF. This asymmetry is
maintained by ion pumps.
• Forces acting on movement of ions:
o Chemical: due to [c] gradient
o Electrical: due to potential difference (electrical gradient)
o Equilibrium potential: chemical force=electrical force, in opposite direction, thus no net
movement of ions.
 Thus, membrane potential is determined by electrochemical gradient and relative
membrane permeability to different ions.
• The value of the equilbirum poitential for any ion depends upon the [c] gradient for that ion across the
membrane. If [c] across both sides is equal, the force of the gradient would be zero, and eq potential
would be zero. Thus, increase in [c] gradient = increase in eq potential.
• The equilbirum potential for any ion can be calculated using the Nernst equation:
• Ek=58log10 ([K+]o=ECF/[K]i=ICF)
• Ek is a function of [c] ratio ONLY
• Idea: at rest, the muscle membrane is permeable only to K+. If this is so, the resting membrane potential
should be equal to the eq potential Ek. A graph of Ek to vs. Log10 [K+]o should be linear with
slope=58. As [K+]o increases, Ek becomes less negative. This is not entirely the case because Na+
permeability, although small, is not zero. The smaller the [K+]o, the stronger the Na+ influx.
o If [K+]o=ecf increases 10x --> Ek is depolarized by 58 mV
o If [K+]o=ecf increases 100x --> Ek is depolarized by 115 mV
 • The influence of other ions is best determined using the Goldman Equation, which is similar in form to
the Nernst Equation, but incorporates permeability to Na and Cl. (In fact, the inclusion of Cl does not
appreciably affect the solution of the equation.)
o Goldman Equation:

• Tetraethylammonium: blocker of K+ conductance, but not completely, even at very high [c] – there are
different K+ channels, not all are targeted.
o A complete block of K+ conductance would depolarize the membrane more, bring the RMP
closer to Na+, because relative permeability of Na+ and K+ changes, Na+ becomes more
important. This produces a change (decrease) in slope.
o A partial block of K+ conductance would alter in the same way but smaller effect.
 “The slope of the points is considerably less (smaller) than what we obtained without
TEA above. TEA is a compound that blocks potassium channels. If potassium
permeability has been reduced, we expect the membrane potential to move more towards
sodium's equilibrium potential (Ena), and this is exactly what has happened.”

Crayfish Muscle Resting Membrane Potential

• Aim: to measure the RMP of crayfish muscle cells (in superior extensor muscle), study the effect of
varying the extracellular K+ ion concentration on the membrane potential, and study the effects of
solutions containing TEA on membrane potential.

• Vt = E – Eb
o Vt=tip potential
o E=glass microelectrode
o Eb=Ag/AgCl

• When the glass electrode penetrates a muscle cell, a new potential exists between the two electrodes
(Vx) – this is equal to the tip potential + membrane potential --> Vx = Vt + Vm. Rearranging, we get:
o Vm = RMP = Vx – Vt
• Sample data collected for a given solution might appear as follows: 5 deflections of approximately equal
amplitude. The membrane potential for each penetration is calculated by subtracting the Tip Potential
from the Tip + Membrane Potential

• To prevent damage to muscle cell, the glass microelectromechanical must have a small tip. However, the
resistance associated with the fine tip impedes most current flow. The tip size can be assessed from the
electrode resistance.

• A high input impedance pre-amplifier recording instrument is needed:

o To reduce current flow through recording electrode
 During the recording, current will flow through both the high resistance of the recording
electrode and low impedance of the recording system. Since these two are attached in
series, their resistances add and their current can be substantial. This is bad because it
will alter the ionic environment of the cell.
o To accurately measure RMP
 If we don’t use this high input recording, a large fraction of the total Em will appear as a
voltage drop across the electrode, and only a small fraction will occur as a voltage drop
across the recording system. Thus the recorded RMP would only be a small fraction
(~1/10) of the actual Em. On the other hand, if the electrode is connected to a high
impedance (high resistance) amplifier, the current flow will be much smaller because
current and resistance are inversely proportional. In addition, most of the voltage drop
will occur across the amplifier and not across the electrode.
 In this experiment: a high impedance source (membrane potential and microelectrode)
has been coupled to a low impedance measuring device via a high impedance pre-
amplifier.
o To make measurements of RMP insensitive to changes of electrode resistance – you change
resistance of electrode by accident throughout experiment, but will not affect results.

• Why aspirate the old solution with syringe --> refill bath with new, higher K+ [C] --> aspirate again -->
refill with same solution?
o To ensure that the new higher K+ solution is not diluted by the previous lower solution
o To allow time for the extracellular space of the muscle to equilibrate with the new solution

• T test: df=2n (observation groups) – 2, 0.05 confidence, if # is > than the # listed, then significant
 Lab 5: Action Potentials of the Earthworm Ventral Nerve Cord

Studying Action Potentials

• This is done for basic science – to find out how nerves operate
• Clinically useful in the diagnosis of neurological
disorders (detecting nerve malfunction)
• Nerve conduction velocity tests  tests to determine
how fast an action potential (AP) is traveling down an
axon. Action potentials are measured at 2 points, A
and B, and the distance between the two points is used
to calculate conduction velocity.
• Nerve conduction velocity tests are useful in the
diagnosis of various neuropathies, especially
demyelinating conditions which result in reduced or non-existent conduction velocities
o Note: EMGs are often performed at the same time to determine whether muscles are functioning
properly in response to the stimuli sent via their connecting nerves.

Why Send Signals with Action Potentials?

• Neuronal APs are not the only way for cells to communicate  other ways include the diffusion of ions
and hormones.
• In single-called animals such as the paramecium, simple diffusion is sufficient for communication.
o The flagella of the paramecium propels its movement by sensing calcium levels
o However, diffusion is proportional to the square of the distance, thus it is too slow for use of
communication in large animals

Mechanism of the Action Potential

• Axons propagate information from one region of the
nervous system to another via brief electrical impulses
(APs)
• APs usually start at the initial segment of the axon and then
propagate down the axon’s length to the presynaptic
terminals
• The AP is a transient depolarizing spike that travels down
the axon.
• At the AP peak, the memorable potential approaches ENa
(the sodium reversal potential)
• The threshold is the minimum stimulation voltage needed
to elicit an AP
o It depends on proximity of the stimulating electrodes as well as the size of the axon
 Larger axons have lower stimulation thresholds
• Steps involved:
1. Voltage-gated sodium ion channels open, depolarizing the cell's membrane potential.
2. Action potentials are All-or-None. The activation threshold is -55 mV.
3. During repolarization, Na+ channels inactivate and V- gated K+ channels open.
 4. When the cell membrane voltage overshoots its resting membrane potential (-60mV), the cell
enters a phase of hyperpolarization.
5. The cell returns to a resting state.
• Activation of voltage-gated potassium channels causes a brief
overshoot in membrane repolarization. This results in a relative
refractory period, during which membrane is farther from
threshold and is thus less excitable.
• The relative refractory period is preceded by a brief period in
which the sodium channels are recovering from inactivation.
During this absolute refractory period, the membrane is
completely unexcitable (sodium channels cannot re-activate).
• Thus these refractory periods are due to:
o The inactivation of sodium channels
o The lag of potassium channel closing (longer effect)
 When both of these occur  absolute refractory period
 When some of the sodium channels return to their active state and can respond to
depolorization again, the action potential hits its relative refractory period. The
membrane remains hyperpolarized because the potassium channels are not as quick to
return to their closed state.
Vertebrate Nerves
• Vertebrate nerves consist of over 100,000 fibers (axons)
• The cell bodies of these axons are located either in the CNS (for motor fibers) or in the peripheral
nervous system (for sensory fibers  In the dorsal root ganglion, for example).
• Fibers may be:
o Large and myelinated (15-25 um)
o Small and unmyelinated (0.2 um)
• All of these nerves conduct non-decremental (all-or-none) action potentials
• However, conduction velocity varies depending on:
o Fiber size
 Increasing fiber size = increasing conduction velocity
o Presence/absence of myelin
 Increasing myelin = increasing conduction velocity

Invertebrate Nerves
• Invertebrate nerves can consist of one single, giant axon
o This is why they are model organisms!
 Ex: squid giant axon is 1 mm in diameter
o These have evolved for rapid conduction of APs
• These axons are important for fast escape responses
• Fibers tend to be larger than those of vertebrates
• Can be myelinated or unmyelinated (more common)
• All of these nerves conduct non-decremental (all-or-none) action potentials
• Earthworms have both large nerve fibers and as well as quiet nerve fibers in the absence of stimuli.
Action Potential Propagation
• Unmyelinated axons: continuous AP propagation
• Myelinated axons: because of myelin, there is
saltatory conduction (node-node conduction)
• The conduction velocities characteristic of
mammalian nerves can go up to 100 m/sec for A-
alpha axons
• The conduction velocities characteristic of frog
nerves can go up to 25 m/sec for A-alpha axons
• Sensory fibers send signals to the brain more rapidly
than C-type (pain) fibers
o Therefore, for example, you feel that you
have stubbed your toe before you register the train

Earthworm Preparation
• The earthworm (Lumbricus terrestis) we will be using
comes from the class of invertebrates known as
annelids.
• The top (dorsal) side of the earthworm is darker than
the bottom (ventral)
• The clitellum is closer to the anterior end of the worm
• The earthworm has 3 fibers in its ventral nerve cord
which all transmit sensory information:
o 2 lateral giant fibers
o 1 medial giant fiber
• The remainder of the nerve cord is largely neutrophil,
where synaptic connections are made
• Each giant axon is formed from many individual
neurons whose axons fuse into a single functional unit
(but the cell bodies remain separate)
• The 2 lateral giant fibers are interconnected by synpases and normally fire together (therefore together
they only contribute to one spike in our recordings)
• All 3 fibers mediate escape responses (“escape withdrawal reflex”), but:
o The lateral giant fibers mediate sensory signals from the posterior (tail  head)
o The medial giant fiber mediates sensory signals from the anterior (head  tail)
 Notice in the diagram on the right that the medial fiber has a larger diameter than the
lateral fibers  thus it has a faster conduction velocity.
• Touching the head (anterior) activates the medial giant fiber to facilitate movements away from the
stimulus
• Touching the tail (posterior) activates the two lateral giant fibers to facilitate movements away from the
stimulus
• How is this done?
 o The worm detects and processes information about a tactile stimulus by converting it into an
electrical signal (an AP)  these APs are then propagated down the length of the worm’s body
 causes succinct muscular contractions
Conduction Velocity
• In the lab, we will conduct electrical stimulation of the earthworm ventral nerve cord using two pairs of
stainless steel pin electrodes that are placed near the nerve, therefore recording the potential difference
between two points on the nerve.
o Stimulate the ventral nerve chord anteriorally  pick up the AP in Channel 3 (Point A)  then
pick up the same AP in channel 4 (Point B).
o If no AP occurs, the recording will pick up a
flat line
o If we only measured the AP at point A, we
would just be able to know that an AP
occurred.
o If we measure at point A and B, we can use the
distance to calculate the conduction velocity
• Conduction velocity is related to the nerve fiber
diameter and myelination.
• In myelinated nerve fibers, the relationship is approximately:
• Velocity (m/s) = Diameter (micrometers) x 2.5

Measuring Extracellular, Biphasic, Compound APs

• Recall the Resting Membrane Potential lab (PHGY 212)  an intracellular recording was performed by
inserting a glass pipette into a cell and measuring the potential with respect to an extracellular reference
electrode
o This measured the potential across the membrane
o This method is best for large, invertebrate cells.
o It is difficult to do in vertebrate nerve fibers and can cause considerable damage to the membrane
around the electrode tip
• In this lab, we will perform an extracellular recording of an AP from the ventral nerve chord of the
earthworm by placing a stainless steel recording electrode near the excitable cell and placing the
reference electrode in the extracellular fluid.
o This records potential changes at the membrane surface rather than across the membrane
o Advantage: easy to do, does not damage the cell membrane
o Drawbacks (as compared to intracellular): AP size is smaller (microvolts vs millivolts), AP
waveform depends on the geometry of the cell contact with the electrode
o Extracellular recordings are best suited to recording 1) whether or not an AP has occurred or 2)
the activity of a population of cells
• In the early days of electrophysiology, intracellular recordings were used to examine the ionic basis of
membrane potential.
• Alan Hodgkin and Andrew Huxley won the Nobel Prize in 1963 for using intracellular methods to
determine the ionic basis of action potentials. However, most experiments that once used intracellular
electrodes are now usually performed using patch clamp techniques due to higher signal-to-noise ratios
and the ability to ask questions about the nature of single ion channels.
 o Indeed, after developing patch clamp techniques and using them to study the function of single
ion channels, Erwin Neher and Bert Sakmann were awarded the Nobel Prize in 1991.
• Both intracellularly and extracellularly recorded APs are biphasic  they both have positive and
negative deflections, but for different reasons.
• The negative phase in the intracellular recording of the AP
is due to the mechanism of after-hyperpolarization
o The large, positive depolarization is due to a
transient increase in Na+ permeability, followed by
a smaller, slower, and negative hyperpolarization
caused by an increase in K+ permeability.
• The negative phase in the extracellular recording of the AP
is due to the way in which it is recorded  two wire
recording electrodes (R1, R2) are placed near the nerve,
each connected to one input of the differential amplifier.
o The extracellular electrode records the AP outside of a single axon by measuring local circuit
currents flowing around the axon as the AP propagates.
o The peak amplitude for these APs ranges from a few microvolts to 1 mV, depending on the size
of the axon (larger diameter axons have larger ionic currents flowing around them) and the
proximity of the recording electrode to the axon.
o We will need to amplify our signals in order to record them on our computers.
• Extracellular recording makes use of differential recording
• The shape of the AP depends on the position of the two pin electrodes with respect to the traveling AP
• At the beginning (before the stimulus is delivered), both pins should be measuring the same voltage 
there will be no deflection (straight line recording) because the amplifier takes the difference between
the two inputs
• The situation changes as the AP travels along the nerve. The shape of the AP that is now picked up will
depending on:
o Inter-electrode depending
o Length of the axon segments depolarized by the APs
o Conduction velocities of the axons
• When the AP reaches the first recording electrode at Ch 3, the proximal Ch 3 electrode becomes
transiently negative to the distal Ch 3 electrode. The voltage between the two is detected and the trace is
displayed as an upward deflection on the screen.
• Then as the AP passes the second electrode (Ch 3, distal), a deflection of equal magnitude but opposite
sign will be recorded. The sign is negative because of the way the amplifier compares the inputs.
• Thereotically, if the electrodes were sufficiently far apart, a short segment of 0 deflection could be
recorded before the AP reaches the second electrode.

Characteristics of the AP
• In the lab, the first biphasic waveform screen is a stimulus artifact. The second biphasic waveform that
appears is the AP.
 o The stimulus artifact results from virtually instantaneous, passive current
spread form stimulating electrodes to recording electrodes.
• Note: because the AP is an all-or-none event, increasing the stimulus will not
increase the size of the AP.
• The following AP characteristics can be measured in the lab:
o Peak amplitude of the AP: voltage at the peak of the AP
o Latency of onset of the AP: the time between the onset of the stimulus
artifact to the onset of the AP
o Latency of the peak of the AP: the time between the onset of the stimulus
artifact to the peak of the AP
o Duration of the AP: time from the beginning of the positive phase to the
end of the negative phase of the AP

Stimulus Strength-Duration Curve

• The threshold for activation of a nerve fiber depends on:
o Stimulus voltage
o Duration of stimulus
• For a short duration stimulus generating a steady trans-membrane current, the charge transferred Q is
proportional to the product of current (I) and time (T):
Q=I x T
• Hence if the amount of charge required to activate the fiber is Qt, and the stimulus duration is D, the
current It required to achieve activation will be:
It= Qt/D
• The ease with which a membrane can be stimulated depends on:
o The strength of the stimulus
o The duration for which the stimulus is applied
 These variables are inversely related: as the strength of the applied current increases, the
time required to stimulate the membrane decreases.
• The rheobase is an asymptote marking the lowest intensity with indefinite pulse duration which
stimulates muscles or nerves
• The chronaxie is the minimum stimulus time required for an electric current the double the strength of
the rheobase
• At 2x rheobase, there is a high stimulus strength with low stimulus duration
• Since the equation is a hyperbola, if the neural membrane was an ideal capacitor, the effective stimulus
intensity should decline asymptomatically towards baseline as stimulus duration increases. In other
words, since stimulus strength required to reach threshold decreases as duration increases, then even a
very small stimulus should reach threshold if its duration is long enough. The hypothetical/theoretical
curve on the left shows this decline to near zero.
o However, this never actually happens because there is always some current leaking across the
cell. The neural membrane is a leaky capacitor, not an ideal one.
o The outward current of K+ leak channels opposes the inward Na+ current induced by
stimulation. As long as the outward leak current is > than the inward stimulating current,
threshold will not be met.
  Hence, below the rheobase, stimulation is ineffective.

Worm Ventral Nerve Cord Experiments (Anterior and Posterior Stimulation) in the Lab
• In the lab, we can activate the medial and lateral fibers with touch or electrical stimulation
• Behavioural responses  touch stimulation
o Will the worm elicit an escape response in response to touch?
• Action potential characteristics  electrical stimulation
• Conduction velocity  electrical stimulation
• Strength-duration curves  electrical stimulation
• Action potential characteristics  touch stimulation
• Conduction velocity  touch stimulation