PNB 2274 Practical II info PDF

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LAB 5 EMG  Power lab hardware is a multi-channel recording instrument for the measurement of electrical signals – it includes a transducer to convert signals  Electrode placement: ensure good electrical contact, away from tendon, avoid hair areas, ground electrode should be placed as far away from recording electrodes as possible  Raw Data: reflects electrical activity of muscle fibers active at that time  *Motor units fire asynchronously thus, EMG is a recording from multiple muscle fibers and their unsynchronized electrical activity  Integrated activity is commonly used in assessment of muscle function because it reflects overall activity of raw EMG and is easier to quantitate.  As you add more weights to your arms (add demand to your muscles) amplitude of EMG trace will increase because more and more muscle fibers are activated with added weight.  Strength of muscular contraction increases –density of action potentials increases – raw signal can represent electrical activity of 1000s individual fibers!  Reciprocal Activation: o Agonist muscle is mainly activated while antagonist is also slightly activated (antagonist is NOT completely silent!)  Co-Contraction: Opposing muscle groups work antagonistically and cooperatively to maintain a joint angle.  What makes a good hypothesis?? o It must be testable experimentally and be capable of being disproven o Should be simple yet specific o Contain two variables, ‘independent’ and ‘dependent’  Q: when the biceps brachii flexes the elbow, which muscles play the role of the synergist? Antagonist? o Synergist: brachialis, brachioradilais o Antagonist: triceps brachii 

LAB 6 ISOMETRIC MUSCLE CONTRACTIONS

o o Measuring Force of Contraction in this lab // muscle tension through a force transducer:  Transduction = conversion of one form of energy to another  Force transducer is needed to convert mechanical force into an electrical signal that can be interpreted by the software  Stimulator = motor neuron  Greater intensity and frequency of stimulation will activate a greater number of fibers. o The strength of muscle contraction can be increased 2 ways!  1: increasing the number of active motor units

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 2: Stimulating active motor units more frequently o Muscle contraction order of events:  AP propagates down axon of motor neuron  Voltage-gated Ca2+ channels are activated  Release of vesicles containing acetylcholine  Acetylcholine binds to receptors on motor endplate  Nonspecific cation channels open  Motor endplate depolarization, muscle fiber action potential  Ca2+ is released from the sarcoplasmic reticulum  Ca2+ ions bind to troponin, troponin removes blocking sites from tropomyosin, active sites exposed  Myosin-actin cross-bridges formmuscle contraction o Effects of Stretch:  In the experimental set up it should be at optimal/physiological length. Therefore as the calf was stretched more, the contractile force should decrease because of limited cross bridges between myosin and actin filaments o Summation as the stimulus interval is decreased; 1st and 2nd muscle contractions fused together and increased contractile force. (second stimulus arrives before muscle has relaxed again causing a second twitch on top of first results in greater peak tension) o o o o o o o o Tetanus  When muscle fibers contract continuously and all contractions merge into a smooth and powerful contraction, stimulus frequency will be very high; muscle does not have time to relax before next stimulation arrives o Fatigue: What contribute to fatigue?  Build up of waste products like lactic acid and CO2,  Build up of potassium ions in T-tubules results in conduction failure,  inhibition of cross – bridge cycling due to build up of ADP and Pi  NOT depletion of ATP Q: What factors influence muscle tension? Overlap of thick/thin filaments & frequency of motor neuron AP Q: In a state of rigor mortis, the muscles are highly contracted and difficult to manipulate. Why does this phenomenon occur? Lack of ATP [Actin & myosin are the central actors in muscle contraction. Muscle contraction begins in the brain with a nerve impulse sent down the spinal cord to a motor neuron. The AP started in the brain is passed on to the muscle fibers through an axon where it is carried to an NMJ. The NMJ, also referred to as the myoneural junction, which releases Ach when the AP reaches the NMJ. When the Ach comes into contact with receptors on the surface of the muscle fiber, a number of transmembrane channels open to allow sodium ions to enter. This influx of sodium ions creates an AP within the fiber, which

triggers a release of Ca2+ ions from the sarcoplasmic reticulum (like the ER, but in muscle cells). Calcium ions filter throughout the sarcomeres, and bind with troponin complexes, causing a shift in the tropomyosin structure (change shape), and exposing the myosin binding sites on the actin.. A “powerstroke” follows, wherein the myosin head drops the ADP and Pi, which hold the heads in a cocked back position, and move laterally thereby moving the actin filament at the same time. Finally, the ATP binds to the myosin heads, thereby detaching from the actin. Upon release from the actin, the ATP breaks down into ADP and Pi, giving energy to return the myosin into initial position, thereby renewing the cycle. Cross bridge theory. The supply of ATP is central in the continuing process of muscle contraction. Death terminates aerobic respiration because the circulation system has ceased. Thereby, the muscles rely on phosphagen and anaerobic metabolism methods to acquire ATP. However, this would supply a significantly less amount of ATP, which disables the myosin heads from DETACHING from the actin.] LAB 7 BRAIN ANATOMY & ACTION POTENTIAL  Brain structures – add pictures from kura cloud here

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o o Cranial nerves

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o Spinal cord anatomy (gray matter = dorsal and ventral horns; & white matter) and neurons have larger nuclei than glial cells

o Nernst equation o Intracellular and extracellular ion concentrations determine reverse potential of that ion: o Nernst equation is used to define voltage produced by difference in concentration of a single ion between extracellular and intracellular

o Parallel conductance equation o This equation allows one to calculate resting potential when more than one permanent ion is involved. o The greater the number of channels that are open, the greater the conductance, G, of membrane for that ion and the greater current produced by the ion o Action potentials

What does each waveform indicate? • Action potential measured adjacent to stimulus on the axon Action potential measured 2.5 cm away • from stimulus • Action potential measured 5 cm away from stimulus • Sodium conductance during an action potential • Postassium conductance during an action potential

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A good experimental design should be: replicable, controlled, randomized, and establish a baseline Effects of TTX

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o o Blocks voltage gated sodium channels  You still have sodium traveling down and depolarizing the axon, but the voltage-gated sodium channels do not open, so there is no massive influx of sodium ions and resulting spike Effects of BTX

o o Prevents inactivation of voltage gated sodium channels  prolonged depolarization/plateau without repolarization

 LAB 8 EARTHWORM ACTION POTENTIAL  earthworm anatomy o 3 Giant Fibers: o cells that make up the fibers are electrically coupled though gap junctions = each fiber behaves as if a single axon!  1 Median  2 Lateral

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How do these differ from the median giant fiber?  higher threshold and slower conduction Intracellular vs. extracellular recording o Intracellular technique is used to record the resting membrane potential, as done last week in metaneuron. Allows for accurate assessment of electrical activity of a single cell, but difficult to do in vertebrate nerve fibers. o Extracellular Recording: Reading is due to the voltage differences between 2 recording electrodes while action potential propagates through the axon  This recording indicates the propagation of an AP  less demanding, and it involves placing one electrode in close proximity to the excitable cell and the reference at another location. This arrangement records potential changes at the membrane surface rather than across the membrane. o Differences:  size of one action potential is smaller in ex. Shape of waveform depends on the exact geometry of its contact with the electrode.  Both intra and extra are biphasic. They both have positive and negative deflections but for different reasons. o The negative phase of the intracellular action potential is attributed to the mechanism of after hyperpolarization. o The negative phase of compound AP is due to the manner in which it is recorded. Two recording electrodes, changes as it picks up on AP.  The situation changes as the CAP travels along the nerve. The shape of the CAP will depend on the relationship between the inter-electrode difference, the length of the axon segments depolarized by action potentials, and the conduction velocities of the axons. potential difference between 2 recording electrodes: (= charge @ pos electrode – charge @ neg electrode) When CAP reaches RI, proximal electrode becomes transiently negative to the distal electrode. The potential difference between the two is detected an the trace is displayed as an upward deflection on the screen. As the CAP progresses between both recording electrodes, the recorded potential returns to baseline ( no voltage difference) As the cap passes R2, a deflection of the same size but opposite sign will be recorded. The sign is negative because of the way the amplifier compares the two inputs.

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Action Potentials fired on a single axon = all or none events

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o When stimulus is below threshold = no response, when AP occurs in response to external stimulus = full sized o No relationship between stimulus strength and response amplitude in single axon Nerve conduction velocity o 1: Conduction velocity = Distance / Time absolute method o 2:Conduction velocity = (D2 - D1) / (LP2 - LP1) difference method Refractory period o From beginning of action potential to restoration of resting membrane potential, the neuron is incapable of producing another action potential  Absolute: impossible to initiate a second action potential (due to conformational changes in the sodium channels)  Relative: stimulus of greater than normal intensity can elicit a response Results of experiments:

LAB 9 COCKROACH AND SENSORY SYSTEMS  Retina cross section o Path of light

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Layer 1: Pigment epithelium is the single layer of cells that helps light absorbing properties of photoreceptors, and have function in phagocytosis of membranous disks of photoreceptors.  Layer 2,3,4,5- Photoreceptors rods and cones have inner and outer segment and fiber.  Layer 6 contain bipolar, amacrine and horizontal cells which helps in synaptic transmission of visual impulse.  Layer7-10: ganglion cells , which axons make optic nerve o Human vs. cow eye  Difference: Cows have tapetum lucidum, a layer of tissue in the eye that lies immediately behind the retina. This tissue reflects visible light back through the retina, increasing the light available to the photoreceptors, though blurring the initial image of the light on focus  This is what makes the cow eye more adapted to night vision Eye anatomy (also vitreous humor not shown)

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o Ear anatomy (also membranous and osseous/bony labyrinth, oval window, vestibule)

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o Record and measure cockroach extracellular action potentials and explore sensory response o Sensory neurons in these organs are responsible for converting external stimuli from the organism's environment into electrical and chemical signals for transmission to the rest of the organism’s nervous system. o Extracellular recordings of action potentials from leg of a roach.  electrophysiology technique that uses an electrode inserted into living tissue to measure electrical activity coming from adjacent cells, usually neurons.

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Different from intracellular recordings! where we measure electrical activity inside the cell/neuron Why do we see different spike amplitudes?  Many axons are within the spatial detection of pin electrodes in femur = many different action potential amplitudes are visible  Diameter of the axon determines the amplitude size – larger action potentials originate from larger diameter axons Factors that influence amplitude of extracellular action potentials:  Size of axon  Proximity of axon to recording electrodes Action potentials with distinct amplitudes arise from different axons, and it is possible to estimate the number of axons Bin size Action potentials –  negative then positive then negative waveform  triphasic (unlike intracellular APs)  be larger than noise (signal/noise ratio)  duration = 100 microseconds main ideas:  spontaneous activity  stimulating different sensory spines  At the base of each of the leg spines is a single sensory neuron o The cell body of the neuron is under the cuticle of the spine, with the unbranched dendrite of the neuron projecting up to the cuticle.  1) When the spine moves, the dendrite of the neuron is activated, opening mechanically-gated ion channels in the dendrite.  2) This creates a receptor potential, which in turn triggers action potentials. o These sensory neurons function to allow the animal to sense vibrations (sounds, etc.) in their environment  SURVIVAL!  Sensory adaptation  Adaptation = the phenomenon observes in all sensory neurons where the response to a constant stimulus gradually declines

 LAB 10 ENDOCRINE SYSTEM  Anatomy

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o Experiment: o Metabolism chamber  A mouse is placed in a wire cage, and then into the metabolism chamber. As respiration proceeds, O2 is consumed and CO2 is given off by mouse. The CO2 will be absorbed by the CO2 absorbent at the bottom of the chamber, and a sub-atmospheric pressure will be created. The pressure difference results in a fluid movement (bubbles) of the calibrated tube. o Objective: measure metabolic rate as a function of O2 consumption (3 groups of mice – normal chow, thyroid powder, PTU) o Calculating O2 consumption:

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To calculate air consumption rate, divide O2 consumption rate by 0.21 (air contains 21% oxygen) Experimental conclusions: o Thyroid powder  supplement additional T3 and T4, expect an increased metabolic rate (hyperthyroidism) o PTU  decrease synthesis of T3 and T4 by inhibiting iodine and peroxidase from their normal interactions with thyroglobulin to form T4 and T3, expect a decreased metabolic rate (hypothyroidism)

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o Key Steps  Inorganic iodide is accumulated and transported into the follicle cell by NIS on basolateral side.  Inorganic iodide and thyroglobulin are transported across the apical side of the membrane into the colloid.  Organification and oxidation of iodine performed by thyroid peroxidase (TPO)  Coupling of MIT/DIT forms T3 and/or T4  MIT + DIT = T3  DIT + DIT = T4  Thyroglobulin/Thyroid hormone complex is endocytosed back into the follicle through the apical side of the membrane.  Thyroid hormones released from thyroglobulin via hydrolysis and lysosomal degradation  T3 and T4 exported from the follicle through the basolateral side into circulation  T3 and T4 bind to thyroid binding globulin (TBG) and circulate to target tissues (i.e. brain, bones, heart, etc). Thyroid

o Thyroid hormones’ synthesis activator o TSH receptor activation stimulates all steps of thyroid hormone synthesis o Biological effects include gene transcription, specifically production of: Na+/I- symporter, Thyroglobulin, Thyroid peroxidase, Thyroid hormones (T3, T4)

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o TSH receptor activation is absolutely critical to development, growth, and function of thyroid gland Questions: o How many Na+/I- are required for T3? T4?  2. Stoichiometry 2 Na in : 1 I in, so for T3: 3 I, 6 Na. For T4, 4 I, 8 Na. However, net flux ends up being 1:1 considering Na/K ATPase 3 Na out. o What are consequences of locally depressed I-/Na+ levels in circulation?  3. Decreased synthesis of t3 and t4 o Predict what happens when endosome formation efficiency is diminished? Enhanced?  4. Diminished: no thyroid hormones make it to circulation. Enhanced: premature endocytosis may initially lead to increase rate of thyroid hormone release to a point and then thyroglobulin would continue to be endocytosed but faster than the rate of coupling so immature Tg-I complex w no T3/T4. o Theorize effects of increased MIT concentrations in colloid on overall body. DIT?  5. Increase t3 and t4 – more reactant more product, net accumulation of thyroid hormone o What are potential drug targets to treat hyperthyroidism? Hypothyroidism?  6. To treat hyperthyroidism – answers that imply a decreased net release of thyroid hormone would be sufficient. Quiz students on effects of different impairments in various stages of synthesis pathway. Hypothyroidism – same as above but resultant in increased net release of thyroid hormone. Can also supplement T3 and T4 entirely, not directly intervening in the pathway, as PTU does. o Speculate on nature of I- trapping. What happens to thyroid production if I- can diffuse back across basolateral membrane?  7. Negatively impacts the rate of thyroid hormone biosynthesis since iodide is needed. Yes, you may be able to continue synthesis with whatever stays in the follicle, and I that is salvaged from MIT/DIT after step 6, but stoichiometry 2Na/1I influx is critical for correct rate of synthesis....