NSCI 300 Lab Exam PDF

Resting Membrane Potential Neuron: receives, conducts, and transmits signals Neuronal membrane: can generate and transmit electrical signals - Ions are unequally distributed across the membrane - Presence and movement of ions determines neuronal firing - OUTSIDE the cell: more sodium...

Resting Membrane Potential Neuron: receives, conducts, and transmits signals Neuronal membrane: can generate and transmit electrical signals - Ions are unequally distributed across the membrane - Presence and movement of ions determines neuronal firing - OUTSIDE the cell: more sodium ions Na+ - INSIDE the cell: more potassium ions K+ Membrane Transport: - Passive Transport: down the concentration gradient (channel or transporter) - Active Transport: against the concentration gradient (transporter/pump & ATP) Membrane potential: the difference in charge across the membrane - No movement of ions across membrane → Membrane potential = 0 - Permeable only to K+ - Concentration gradient rushing K+ OUT - Electrical gradient rushing K+ IN - Positive charge accumulated outside the cell membrane - Nernst Potential: membrane voltage generated when an ion is in equilibrium - Concentration and electrical gradients are equal but opposite - K+ typical Nernst value = -94mV - Na+ typical Nernst value = +61mV - Diffusion across the membrane: - Transporters are SLOW: must bind solute, undergo conformational changes, then transfer solute - Ion Channels are FAST: ions diffuse through the channel, no binding or conformational change needed 1 Action Potentials Receptor Potentials: generated when sensory neurons are activated by external stimuli Synaptic Potentials: arise during neuron-to-neuron communication at synaptic junctions Action Potentials: generated by membrane depolarization and travel along neuronal axons, crucial for signal transmission Discovery of AP Mechanism: - Hodgkin and Huxley recorded AP from a giant squid axon using a voltage clamp - Received 1963 Nobel Prize for Physiology/Medicine Voltage Clamp Technique - Uses electrodes to inject current into an axon or neurons to keep the membrane potential at a set value 1. One electrode inside, one electrode outside monitoring transmembrane voltage and changes in Vm 2. Voltage clamp amplifier compares Vm to the desired (command) potential (Vcomm) a. When they are different, the amplifier injects current into the axon through a third electrode 3. The current flowing back into the axons and across its membrane can be measured - During action potential firing: - Voltage-gated Na+ channels open FIRST for depolarization - K+ channels follow, opening for repolarization Voltage-gated Channels - Sensitive to voltage changes and open only once reached a certain threshold that alters the channel protein’s conformation Voltage-gated Na+ channels: - Resting (-70mV): activation gate closed, inactivation gate open - Activated (-70 to -35 mV): both gates open, selectivity filter allowing Na+ to enter - Inactivated (+35 to -70 mV, delayed): inactivation gates closed Voltage-gated K+ channels: - Resting (-70mV) - Slow activation (+35 to -70mV) - Depolarized = Pore open and K+ can leave - Hyperpolarized = Pore closed and K+ cannot leave At resting membrane potential, these channels are closed, but as it depolarizes the likelihood of these channels opening increases. Generation of Action Potentials: happens by depolarizing the resting membrane potential to a specific threshold, triggering an action potential Passive and Active Current Flow in an Axon: - Passive conduction decays over distance 2  - Active conduction is constant over distance (is renewed) Direction of AP Propagation - Unidirectional propagation of APs is ensured by the inactivation of voltage-gated Na+ channels during the refractory period → so no new AP can be fired during that time - Others, APs can typically propagate in both directions in most biol systems Phases of an Action Potential 1. Resting phase: neuron at its resting membrane potential 2. Depolarization phase: membrane potential becomes less negative and moves to the threshold potential a. Vm: -70mV → +10mV (2 milliseconds) b. Fast acting voltage-gated Na+ channels open (activation) millisecond c. Those Na+ channels automatically close (inactivation) 3. Repolarization phase: membrane potential starts to go back to negative a. Voltage-gated K+ channels open (activation) slower than Na+ channels b. When all K+ channels are finally fully open, all Na+ channels have closed 4. Hyperpolarization phase: membrane potential temporarily becomes more negative than its usual resting potential a. K+ channels remain open throughout the duration of the positive membrane potential (depolarization+repolarization\) and do not close until the membrane potential is negative again (hyperpolarization) - Absolute Refractory period: time during AP when a neuron cannot fire a second action potential, regardless of the strength of the incoming signal due to inactivation of Na+ channels - Relative Refractory period: at time after ARP when a neuron can fire a second AP, but only if the incoming signal is significantly stronger than usual. Conductance over Time of an Action Potential: 1. Sharp spike in Na+ conductance 2. Delayed, smaller, more gradual increase in K+ conductance 3 Action Potential Conduction Velocity: - Larger diameter & more myelination = faster conduction velocity Electrophysiology 1. Electric Charge (Q): physical property of matter that causes it to experience a force when placed in an electromagnetic field. The unit is coulomb (C). 2. Voltage (V): potential difference in electrical charge between two points. Unit: Volt (V) 3. Current (I): the flow rate of electric charge. Unit: Ampere (A) = 1C/sec 4. Resistance (R): force that counteracts the flow of electrical charge/current. Unit: Ohm (Ω) 5. Conductance (g): the inverse of resistance. Unit: siemens (S) - Membrane conductance is a property of membranes that determines how much charge can go through the membrane 6. Capacitance (C): the ability of a substance to hold an electric charge. Unit: Farad (F) - Depends on surface area, distance between surfaces, and permeability of the insulator Ohm’s Law: V = IR - Membrane voltage (V) = membrane current (I) x membrane resistance (R) Equipment for Cellular Electrical Signal Measurement: 1. Microelectrodes - target cells are small, measure electrical signal in cell 2. Amplifiers - amplify and clear the electrical signal 3. Microelectrode Puller - used for preparation of microelectrodes 4. Micromanipulator + head stage - adjusting the microelectrode to the desired micrometre-scale location 5. Stereo Microscope - used to dissect specimen and to guide and observe the placement of the microelectrodes 6. PowerLab and LabChart - acquire and analyze data Microelectrode Puller and Microelectrode Preparation Manages various factors to make the electrodes - Heat: generates and sustains a precise T to melt glass tube - Velocity: employs modifiable speeds to evenly stretch the glass electrode under consistent load. - Time: regulates the timing for when the heat is deactivated, the duration of cooling, and the activation of the pulling mechanism - The ‘borosilicate glass capillary tubes’ are made predominantly of silica (silicon dioxide) and boron trioxide - Boron trioxide imparts a very low coefficient of thermal expansion to the glass, so it can withstand extreme temperature shifts without cracking - The tip of the electrode is a few micrometers(µm) in size 4 Microelectrode Holders and Head Stages - Turn screw cap counterclockwise to unscrew and insert/remove electrode - Turn screw cap clockwise to secure seal - Do not touch the head stage itself when moving the micromanipulator Handling Micromanipulators and Headstages - Start with all control knobs at midpoint position (allows for equal movements along axis) - To move the micromanipulator, secure the bottom with one hand and un-magnetise it to move to new location, then magnetise again to secure its position - Use the fine adjustment knobs for the rest Microscopes Light Microscopes: 1. Stereo Microscope a. Uses low magnification b. Used for viewing opaque specimens c. Uses a high working distance d. Ideal for viewing 3D objects: enable depth perception 2. Compound Microscope a. Uses high magnification b. Used for viewing transparent specimens c. Small working distance d. Ideal for ultra-thin samples light can pass through Adjusting the Microscope 1. Set both diopter rings to approximate middle of their range 2. Start with just one eyepiece (left or right) 3. Adjust focus with Focus control knob 4. Look through the other eyepiece 5. Adjust the focus using the diopter adjustment ring 6. Adjust distance between the eyepieces to create a same field of vision To change magnification, use the Focus control knob (not diopter) 5 PowerLab and LabChart - A hardware system and its software - Data acquisition unit (purpose to acquire, store, & analyze data) - Current Injection = Output + Isolated Stimulator - Signal Collection = Input + Bio Amp Ways to Acquire Data: - Transducer = mechanical signal - Electrode = electrical signal PowerLab Data Acquisition System 1. Raw input signal → analog signal to PowerLab hardware 2. PowerLab modifies signal through signal conditioning (amplification, filtering, sampling at regular intervals) 3. Signal converted to digital before transmission to computer and LabChart 4. LabChart usually displays data directly a. Plots sampled & digitized data points b. Reconstructs original waveform by drawing lines between points LabChart Software - Data analysis - Data trace in Chart Window may show different peaks from Input Amplifier dialog - Sampling: - PowerLab (digital recording system) records the value of the signal at regular time intervals (not continuously) - Sampling too slowly = faster parts of the waveform (pulse peak) may be missed LabChart Interface Icons 1. Sampling rate popup menu: 2. Start/Stop button: 3. View buttons (time scale/compression): Compress or expand the time axis to see more or less of a waveform 4. Marker tool: Use w/ waveform cursor to make relative measurements 6 Finger Pulse Transducer: - Piezo-electric element to convert force applied to the active surface of the transducer into an electrical analog signal - Measures the speed at which the pulse moves between 2 points - Aka. aortic pulse wave velocity Neurophysiology - Intracellular Recording The study of the nervous system’s functions and how it processes information. Focus of Neurophysiology 1. Neural Circuits: neuron networks for information processing and CNS function 2. Sensory and Motor Systems: studies processing sensory inputs and their influence on movement and coordination. 3. Synaptic Transmission: neuron communication at synapses (intracellular record. focus) Methods used in Neurophysiology 1. Imaging and optogenetics: Uses MRI, PET scans, EEG, and light-responsive neurons 2. Neural Mapping/Behavioural Studies: correlates brain region function with behavior 3. Electrophysiology: involves recording electrical activity from neurons - intra and extra Intracellular Recording: - Used to measure the electrical activities (potential changes) inside a single cell - It helps in understanding the basic mechanisms of neuron function, such as how neurons generate and propagate electrical signals Challenges and Limitations: - Technical Complexity: requires precise techniques and skilled handling - Stability Issues: stable recordings over time is hard, minor movements can disrupt the connection between the electrode and the neuron - Risk of Damage: risk of damaging the cell during electrode insertion, which can alter functioning and affect the accuracy of data - Limited Duration: invasive technique → recordings are limited in duration, as prolonged electrode presence can compromise cell health - Muscle cells allow for 3 hours of recording Which Ions Contribute Most in resting potential? [K+]: maintaining a proper balance of [K+] inside and outside our cells is very important to the functioning of neurons and skeletal and heart muscles. Hyperkalemia - High [K+] outside the cell Diet rich in K+: cantaloupe, honeydew melon, banana. Certain Medications: beta-blockers, calcium channels blockers, AG-II receptor blockers Certain Disease: diabetes, heart disease, kidney disease Excessive loss of water: sweating, vomiting, diarrhea 7 *Kevorkian’s suicide device uses an injection of high [K+] saline to stop the heart. If the RP follows the Nernst Potential for a certain ion, then we can say that that ion is the primary contributor to maintaining the RP. To experimentally determine which ion contributes most to the resting potential: - Monitor RP after altering the ion concentration in the extracellular fluid - OR monitor RP after altering the ion concentration in the intracellular fluid [K+] contributes most to the resting membrane potential - At resting state, K ions are mostly permeable. Therefore, we expect changes in [K+], will cause the RP to shift towards a new Ek. Crayfish as Model Animal for Intra/Extra Cellular Recording Why this animal model? 1. Relatively easy dissection for learning dissection techniques for live preparation 2. The muscle is stable for several hours (3h) in minimal normal saline 3. The muscle preparation (fast-acting) is fairly robust when external [K+] is altered for short time periods 4. Due to large muscle size, it is relatively easy to obtain stable intracellular recordings - Extensor muscles: straighten the tail - Flexor muscles: bend the tail - Subdivided into phasic and tonic muscles - Phasic muscles: used for rapid tail flips for escape - Tonic muscle: control fine movement of tail to maintain posture 8 Intracellular Recording of Crayfish Muscle Cell (superficial flexors SF) 1. Dissection a. Remove head with big scissors b. Remove swimmerets with small scissors and a coarse forceps c. Remove the kin with fine forceps, scalpel, and Vannas Scissors d. *Segments for Dissection (best to worst): 2 &3, 1&4, 5 e. Make a midline incision with scalpel f. Grasp skin with fine forceps and separate skin from lower segment to avoid damaging the muscles g. Remove skin with scalpel or vannas scissors without damaging the SF muscle attachment point i. Might encounter second skin layer (hypodermis) 2. Microelectrode Preparation a. Place microelectrode in a small beaker with 3M KCl for back-filling microelectrode via capillary action b. Use fine syringe to fill the electrode and holder with 3M KCl 3. Charge Conduction Through Electrode and Holder a. No charge conduction possible without filament due to air bubble b. c. Charge conduction possible cause no air bubble d. e. Silver (Ag) has the highest electrical conductivity of all metals, and it does not spark easily - Ag defines conductivity, so it ranks as 100. f. Ag/AgCl electrodes are very stable because of chloridation and are essential non-polarizable. They can pass µA currents yet still remain stable for extended periods of time 9  4. Equipment Set-up a. Apply the settings; Low Pass: Off, DC Offset knob: Counterclockwise, DC Offset (+/Off/-): Off, Current Injection (CONT/OFF/MOMEN): OFF, Current Injection polarity (+/-): Either, Capacity Compensation: Counterclockwise, Meter: Probe. 5. Measuring Electrode resistance: use “ELEC TEST” to inject 1 nA current and record voltage change. By Ohm’s law, 1 mV of output from 1 nA of current indicates an electrode resistance of 1 MΩ. a. Resistance Issues: - Resistance under 5MΩ = broken electrode - Resistance over 30MΩ = blocked electrode b. Make sure that the second, Ag/AgCl wire is touching the saline solution (Data Interpretation and Experiment Details in Notebook 4) Neurophysiology - Extracellular Recording - Focus on sensory and motor signals - Motor neurons: control muscle contraction Signal transmission in sensory and motor neurons: - Sensory: afferent (to CNS) - Motor: efferent (from CNS) - Unipolar: 1 neurite - Bipolar: 2 neurites - Multipolar: >2 neurites - The diameter of an axon is correlated with its conduction velocity Electrical Circuits and Effects of Diameter - Neurites function like electrical wires, with semi-conductive fluid (cytosol) inside an insulator (membrane) and their current flow can be modeled as an electrical circuit. - Re/R0 (External Resistance): extracellular fluid offers minimal resistance to electronic current flow - Rm(membrane resistance): reflects plasma membrane resistance and leakage through open ion channels - Ri (internal resistance): cytoplasm resists the flow of electronic current 10  - - When you increase the diameter of a neurite - Increased cytoplasm volume: leads to reduced internal resistance Ri, due to more space for ion movement - Expanded membrane surface: this provides more points for ions to leak through per unit length, thereby reducing the membrane resistance - AP propagation involves both active and passive/electrotonic current, which is continually influenced by both Rm and Ri. We also know that the effect of diameter is greater on Ri than Rm. Motor System in Crustaceans - Every tail segment contains 1 ganglion - The superficial flexors SF is innervated by a small set of motor neurons whose axons travel in the superficial branch nerve 3 of each tail ganglion - Superficial Branch Nerve 3: - Contains only 6 axons and shows spontaneous activity with easily identifiable AP classes. - All are from motor neurons; all APs are motor commands for tail contraction - Has a wide range of axon diameters, so most axons produce APs of different amplitudes - USED TO DETERMINE THE NUMBER OF AXONS IN NERVE 3 BASED ON EXTRACELLULAR RECORDING DATA. - Equipment Set-up (extracellular recording): 1. Uses an AC/DC differential amplifier 2. Apply the settings; High Pass: 300 Hz, Notch: Off, Low Pass (low resistance): 10 kHz, Capacity Compensation: Fully counterclockwise, DC Offset Coarse and Fine knob: Fully counterclockwise, DC Offset (+OFF-): OFF, Gain: 1000, Input Probe: Headstage Attached, Input Select Switch: Diff, Ω Test: Off, Mode Select Switch: Record (REC) 11  3. Crayfish/Lobster Dissection 1. Remove Head 2. Remove swimmerets 3. Make a T cut along the midline of a segment to expose Nerve 3 Suction Electrode - To make, with coarse forceps break the tip (60-70%) of the electrode - Apply heat with a torch lighter to achieve a desired tip size - Insert electrode to ensure the wire remains free from twists - PIPETTE POLISHING IS VERY IMPORTANT! - Crucial to achieve an optimal seal between the nerve and the glass pipette → a more uniform seal - Without it, the current flowing through the axon’s extracellular space may leak –? UNDETECTABLE SMALL SIGNALS BY THE RECORDING ELECTRODE Proper Alignment for 3-Way Luer Connector - Do not push/pull syringe in alignment 1 or 2 - Alignment 3 is the setting for suction to nerve 3 - After completing suction of nerve, adjust to alignment 4 to secure retain the nerve within the electrode Extracellular Recording - Electrode not inserted – electrode is placed in the extracellular fluid, NEAR the cell of interest - 12  - 1. Lower suction electrode into saline, suck up saline to the level of the wire inside the tube 2. Saline provides electrical contact between nerve and amplifier 3. External wire must rest in the saline to complete the circuit 4. Suck in a small loop of the nerve into tip (watch signal to determine optimal seal) Audio Set-up - Sources: 1 Extracellular; Play while sampling ON, Automatic Gain on High Essential Considerations for Extracellular Recordings 1. If no recording or extremely high noise: a. Check that the saline reacher electrode internal wire b. Check that the external wire is in the saline 2. If excessive force is required to suck the nerve into the electrode: a. The electrode pipette tip may be too small or clogged, replace it b. Tubit between electrode and syringe may be bent, unbend it. 3. If suction won’t hold: a. The electrode tip may be too big or is not firmly placed in the electrode holder 4. Weak recording with small or no AP despite intact circuit: a. The electrode tip may not be forming tight seal with the nerve Data Analyses - Calculate the AP amplitude and rate/frequency during baseline and reflex activity - Identify number of axons for each recording - Measuring Amplitude: - Marker placed on peak of the spike, waveform cursor on the lowest point (valley) Firing Rate rate(SPM) = ((n-1)/diff T)*60s 13 This equation calculates the firing rate of action potentials, which is the number of spikes per minute (SPM). It is used to analyze how frequently a neuron fires action potentials over time. rate (SPM): The firing rate of action potentials in spikes per minute (SPM) n: The total number of action potentials (or spikes) observed diff T: The total time interval over which the action potentials were recorded, in seconds 60 seconds: A conversion factor to convert the firing rate from spikes per second (SPS) to spikes per minute (SPM) Firing Frequency f = SPM/60s This equation calculates firing frequency, which, similar to firing rate, describes how frequently action potentials occur. f: The firing frequency in Hertz (Hz) SPM: The number of spikes per minute, calculated from the firing rate equation, in SPM 60 seconds: The conversion factor from minutes to seconds Electrical Signalling in Sensory Reception Peripheral Nervous System (PNS) - Sensory neurons carry information from peripheral sensors to the nervous system for perception and motor coordination - Has two components - Somatic nervous system: connects to the skeletal muscles, skin, and joints, enabling VOLUNTARY movements - Autonomic nervous system: connects to internal organs, blood vessels, and glands, regulating involuntary functions Sensory Nerve: - Carry info from the body’s peripheral sensors into the nervous system for the purpose of both perception & motor coordination - To help coordinate movements & maintain proper muscle tone, sensory system uses proprioceptors to monitor the positions of body parts Sensory Nerve in Skin: - Skin has dynamic sensory properties to sense different sensations - Pain, itch, pleasure, heat, touch, vibration Mechanoreceptors: - Subset of somatosensory receptors that transmit external stimuli to intracellular signal transduction by means of mechanically gated ion channels - short receptors (1 cell per 1 neuron): receptor potential spreads passively from the sensory region to the synaptic pole (does not require APs) - long receptors: receptor potential gives rise to trains of APs whose duration and frequency code information code information about the duration and intensity of the original stimulus Membrane Voltage Affect AP Firing Rates 14  - Stronger depolarizations trigger faster firing rates, up to a maximum firing rate. - Increasing the STRENGTH of the depolarizing stimulus barely changes the SHAPE or SIZE of the APs, but does INCREASE the FIRING FREQUENCY up to a maximum firing rate where the frequency no longer increases with stronger current. - The maximum firing rate exists due to the AP refractory periods Proprioceptors: - Some are stretch-sensitive receptors embedded in or parallel to muscle fibers - Humans = Muscle spindle organs - Crustaceans = Muscle receptor organs - Sensory organ reports position & helps guide body movements (kinesthetic sense) Types of Somatic Receptors: - Proprioceptors - Exteroreceptors = tactile, thermal, and pain sensation - Enteroceptors = regulate breathing, thirst, hunger, blood pressure Adequate Stimulus = form of physical energy w/ the lowest threshold to activate a receptor Membrane Potential & Chronic Pain: - Changes in resting potential or a lowered action potential threshold can make neurons hyperexcitable - This means they react more intensely or to stimuli that normally wouldn't cause pain, leading to allodynia. Crustacean Paired Receptor Organ - Generates neural signals when the body wall is stretched - Has a set of two sensory structures that work together to detect changes and respond to envi stimuli - Rapidly Adapting: a stretch produces receptor potential that can trigger a series of APs - Slowly Adapting: the receptor potential is not maintained, and during a large stretch the AP frequency declines *to record JUST the sensory signals of a mixed nerve, just cut the nerve and record from the lower segment (synapsing with the muscle/tissue) Muscle Receptor Organ In Crustaceans - Nerve 2 – extensor muscles (both sensory and motor) - Sensory Receptors – MROs: - MRO1 – slowly adapting receptor - MRO2 – fast adapting receptor Crayfish Dissection 1. Remove head 2. Remove ventral part of shell a. - carefully cut the abdominal shell to remove sternite, preserving as much tergite as possible 15  3. Remove deep-flexor muscles (avoid damaging the MROs) a. Ensure that the connection between the extensor muscles and tergites remains intact 4. Attach a thread to tail (to invoke stretch) Equipment Set-Up - Very similar to extracellular nerve recording Electrical Noise: - Biological signals will be in μV - Most noise is 60 Hz (frequency of alternating current in power lines) High Signal-to-Noise Ratio: - High signal = getting the best possible recording - Low noise = decreasing noise reaching the electrode - Filtering = electronically removing noise that reaches the amplifier Data Analyses - Measure action potential amplitude and firing rate - Compare these findings during a brief vs prolonged stretch Neuroplasticity - Habituation Neuroplasticity: the ability of neurons and neural networks to change and adapt due to experience - Learning a new ability - Information acquisition - Neuronal damage/dysfunction - Environmental influences 1. Functional plasticity – the brain’s capacity to transfer function from an impaired region to unaffected areas 2. Structural plasticity – the brain’s capability to alter physical structure in response to the process of learning Hebbian Theory: - “Cells that fire together, wire together” - When 2 neurons are activated simultaneously, the connection between them is strengthened Learning: the acquisition of new knowledge Associative learning Changes in behavioral response - Classical conditioning by the formation of associations between events or two stimuli - Operant Conditioning Non-associative learning Changes in animals’ behaviour as - habituation (negative mem) a function of experience with 16  particular stimuli - Sensitization (positive mem) Model Organism - Aplysia californica - Are annual animals (live only a year) - Breathe through their gills - Gills are covered by an over hanging mantle shelf - Water is drawn from the front and leaves through the siphon - The respiratory apparatus is further protected by the parapodium What makes Aplysia the perfect model system for neuroscience? - Nervous system is simpler (than humans at least) - Have relatively large nerve cell bodies (largest reaching up to 1mm) - Identified neurons = consistent location of individual neurons from one animal to another - Their capability for various behaviors and simple forms of learning Siphon-induced gill withdrawal reflex A defensive reflex in Aplysia where it retracts its gill when its siphon is touched Habituation: the process that causes an animal to become less responsive to repeated occurrences of a stimulus - NOT due to muscle fatigue - Spontaneous contractions are the same strength as the original stimulated contractions - NOT due to involvement of the motor synapse - Before & after habituation the gill response is the same size (when stimulating the motor neuron intracellularly) - NOT due to skin sensitivity - if it was a loss of skin sensitivity unblocking the nerve should have still showed a decreased response = shows the changes are at the synapse between sensory and motor neurons 17 The mechanism of short-term habituation in Aplysia: - Results from an activity-dependent presynaptic depression of synaptic transmission - Quantal analysis revealed that the amount of glutamate released from presynaptic terminals of sensory neurons decreases Habituation and Aplysia Experiment Procedure 1. Preparing the Apysia a. Container filled with seawater b. Aplysia placed in the container 2. Habituation set-up a. Allow aplysia to acclimate for 10+ minutes b. Minimize water disturbance and shadow changes before and during testing, as these can trigger behavioral responses c. The animals should not be disturbed during testing 3. Habituation Training a. Stimulate Aplysia’s siphon with a brisk flick from a paintbrush (target the inner siphon) for a short duration (~1sec) i. Should be strong enough to elicit a response lasting over 12 seconds b. Start the timer at the moment the aplysia is flicked c. Repeat the stimulus at regular intervals (as soon as its ready for the next trial), targeting the SAME spot on the siphon – and record the responses. d. Try to maintain a similar level of insertion and flick speed 4. Data collection a. Trial number - code - response quality - response time - % response time reduction b. Code: i. GT = Good Trial ii. >30 = trial time over 30 seconds iii. NCR = no clear relaxation iv. BT = Bad trial 1. Natural movements or reflexes 2. Responses to water movement or to shadows 3. Touched different part of siphon or body 4. Double pump (siphon/gill) 5. variation in the intensity or speed of siphon stimulation 6. Other factors c. Take notes if: i. Aplysia inks ii. Aplysia is non-responsive iii. Aplysia laid eggs iv. Trials missed or other issue arose d. Don’t record bad data: i. Make sure to code BTs ii. Better miss data than record bad data 18  5. Data Analysis a. Calculate percentage reduction in response over time: 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑇𝑖𝑚𝑒− 𝐹𝑖𝑛𝑎𝑙 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑇𝑖𝑚𝑒 i. 𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑇𝑖𝑚𝑒 × 100% ii. Create a graph of the habituation process by plotting the stimulus number on the X-axis and response time on the Y-axis Neuroplasticity - Sensitization A type of non-associative learning where an animal becomes more responsive to a stimulus after exposure to a noxious or arousing stimulus - Type of positive memory The extent of sensitization depends on - Age of the animal - Intensity of the stimulus - Frequency of exposure to the stimulus Neuronal signal in short-term sensitization: - A noxious stimulus can produce sensitization (positive memory) of the GWR - Tail shock activates tail sensory neurons - Tail sensory neurons excite modulatory interneurons that synapse with siphon sensory neurons and cause facilitation of the synaptic pathway Molecular mechanism for short-term sensitization: 1. Stimulus shock activates tail sensory neuron 2. Sensory neuron releases glutamate onto serotonergic modulatory interneuron (aka facilitatory interneurons 3. Interneuron releases serotonin onto presynaptic terminal of siphon sensory neuron (at interneuron and motor neuron synapse) 4. Serotonin binds G-protein coupled receptor & initiates signal cascade a. Serotonin upregulated cAMP & activates PKA in the sensory nerve terminal b. which phosphorylates various proteins, including ion channels, leading to increased neurotransmitter release 5. Increase in the amount of glutamate released when the siphon is touched 19  - Experimental Procedure 1. Equipment Set-up a. Materials: Aplysia, seawater, paintbrush, timer, constant DC current stimulator, container/dish, hand towel, electrode. b. Only change Power (On/Off), plug in the negative and positive terminals 2. Preparing the Aplysia a. Place two aplysia into separate containers with seawater b. Expose their respiratory systems c. If the aplysia ink, gently rinse with seawater two to three times d. Start habituation training (similar to earlier lab) 3. Sensitizing Stimulus and testing a. Once aplysia are determined to be habituated: b. Administer an electrical stimulus to Aplysia 1 by making contact between the tail pin and the electrode (delivering 60-100 mA DC current) i. Touch the tail pin with the electrode (make sure both ends are in water) ii. Turn the power switch on, check the current in the monitor, then turn off the switch iii. Ensure the electrode only touches the tail pin of aplysia c. Aplysia 2 acts as a control for this trial 4. In Vivo Serotonin Exposure in Aplysia a. Aplysia 2 will have its water replaced with a serotonin solution (5HT) 5. Data collection a. Measure gill withdrawal response times for both Aplysia before and after the sensitizing stimulus or serotonin exposure b. Essentially, repeat the stimulation trials after the treatment c. Calculate the percentage change in response time for each Aplysia: [𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒 − 𝑓𝑖𝑛𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒] i. % 𝑟𝑒𝑑𝑢𝑐𝑡𝑖𝑜𝑛 = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒 · 100 [𝑓𝑖𝑛𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒 − 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒] ii. % 𝑖𝑛𝑐𝑟𝑒𝑎𝑠𝑒 = 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑒𝑠𝑝𝑜𝑛𝑠𝑒 𝑡𝑖𝑚𝑒 · 100 20  d. Create graphs plotting trial numbers (X-axis) against gill withdrawal response times (Y-axis) for all conditions (control vs. electrical stimulation, control vs. serotonin incubation) e. Compare the response times and percentage changes between the experimental and control groups to determine if sensitization occurred and to what extent f. Consider the sample size limitations and interpret the data within the context of potential variability Adjusting Expectations: Sample Size and Data Analysis - Standard scientific experiments usually require a minimum of n = 3 samples per group to ensure statistical reliability - Various factors can affect experimental outcomes - Differences in animal physiology - Variations in experimental procedures - Set-up configurations - Understand these constraints and interpret data within the context of these limitations Psychophysiological Methods - EMG Psychophysiological Methods: - Study the relationship between psychological processes & physiological responses Brain and Behavior - John Harlow's case study of Phineas Gage emphasized the connection between distinct brain regions and various psychological behaviors - Psychosurgery = frontal lobe ablation was a routine treatment for mental disorders - Phineas Gage = iron rod through his left frontal lobe changed his personality Neuronal Control of Emotional Response: Emotional stimuli Sensory systems Emotion system Hypothalamus & brain stem Spinal cord & autonomic ganglia 21  Effector cells Emotional responses Spinal Cord / Autonomic Ganglia & Emotional Responses: 1. Somatic motor nerve = skeletal muscle = behaviour (freezing) 2. Autonomic nerve = smooth or cardiac muscle = autonomic activity (rise in BP) 3. Pituitary gland = endocrine gland = hormonal release (stress hormones) Methods Recording/Measuring Implications Electroencephalography Electrical activity in the brain Information on sleep, cognition, EEG and neurological disorders Electromyography Electrical activity produced by Indicates the level of muscle EMG skeletal muscles tension due to emotional or psychological stress Electrocardiography Electrical activity of the heart Information on the effects of ECG psychological states on cardiac function Functional Magnetic Brain activity through changes Understanding brain function Resonance Imaging fMRI of blood flow and the BOLD and structure response Galvanic Skin Response Changes in sweat gland activity Reflective of the intensity of GSR emotional states Eye Tracking Monitors movement and Insight into attention and dilation of the pupils arousal Electromyography - Assessing the health of muscles and the nerve cells (motor neurons) that control them - Revealing nerve system dysfunction, muscle dysfunction, or nerve-to-muscle signal transmission issues Skeletal (striated) muscles = locomotion and support of the skelton - Attach to the bones by = Tendons ( bundles of collagen fibers), aponeuroses, & fascia Skeletal Muscle Structure = Skeletal muscle > fascicle > fibers > myofibrils > myofilaments More muscular person = thicker muscle fibers, more myofibrils, more ATP & glycogen - Not more muscle Motor Unit: - Somatic motor neuron axon from spinal cord + all the skeletal muscle fibers it innervates - Types of motor units: - slow, fast fatiguing, fast fatigue-resistant 22 Timecourse of Motor Unit Activity: 1. Motor neuron AP 2. Conduction of AP along nerve fiber 3. NT release at neuromuscular junction 4. Depolarization of muscle membrane w/ resultant contraction of muscle fibers Muscle Contraction = synchronous activity in fibers in the same muscle - Summation = multiple fiber summation or frequency summation - Larger muscle contraction = more frequent action potentials Motor Units = Fire Asynchronously: - Could detect contributions of individual motor units w/ weak contractions - Increasing strength of contraction = density of APs increases Conduction Velocity: - Stimulate the median nerve & record the time it takes for the muscle to contract - Speed of the response is dependent on the conduction velocity (50-60 m/s ish) Abductor Pollicis Brevis Muscle: - One of the thenar muscle group on the palmar surface of the hand - Median nerve = motor nerve that innervates the APB muscle Neurogenic & Myopathic Diseases: - Neurogenic = nervous system dysfunction - Myasthenia gravis - chronic neuromuscular disease that causes weakness in the voluntary muscles - Treatment with an acetylcholinesterase inhibitor - Myopathic = skeletal muscle dysfunction EMG can Locate the Site of Nerve Damage: - Measuring electrical activity in muscles & nerves can help detect the presence, location, & extent of diseases that damage muscle tissue (eg. muscular dystrophy) or nerves (eg/ amyotrophic lateral sclerosis/Lou Gehrig’s disease) Experimental Procedure Techniques/Procedures: ○ Electrode Placement: Surface electrodes are placed on the skin overlying the muscles of interest (e.g., biceps and triceps) ○ Signal Amplification and Filtering: EMG signals are amplified and filtered using a bio-amplifier ○ Stimulation: In evoked EMG studies, a nerve is electrically stimulated to elicit muscle contractions ○ Recording Software: LabChart software is used for data acquisition, display, and analysis Equipment: - PowerLab system, - Bio Amp Cable, - Shielded Lead Wires, 23  - Disposable ECG Electrodes, - Stimulating Bar Electrode, - Dumbbells, Abrasive Gel, - Electrode Paste, - Dry Earth Strap Setup: - Connect electrodes to the Bio Amp, - connect the Bio Amp to the PowerLab, - place electrodes on the biceps and triceps muscles, - secure the dry earth strap around the wrist Measure Voluntary Changes in Contractile Force: - In LabChart, open “Voluntary Change Settings” from the “Welcome Center” - Make strong biceps contraction and adjust signal in BioAmp dialog window - Make strong triceps contraction and adjust signal in BioAmp dialog window - Start recording and follow the steps in the lab module, finish analysis, and record data - Record EMG during various muscle contractions Measuring Alternating Activity and Coactivation - In LabChart, open “Coactivation Settings” file - Practice activating biceps and triceps immediately after one another - Record EMG during alternating and co-contraction tasks 24 Measure evoked EMG activity: - ! people with cardiac pacemakers, or suffer from neurological or cardiac disorders should not do EMG! - Remove the Channel 2 Lead wires from the cable and attach channel 1 lead wires to new disposable electrodes - Place recording and stimulating electrodes on the skin of the wrist Finding the Median Nerve and Stimulator Set-up - In LabChart open “Nerve Effect Settings” - Turn on PowerLab Isolated Stimulator switch and select the On button in the Stimulator Panel. - Look for a stimulating site that invokes the largest twitches - Open “Evoked EMG Settings” and record data - Measure the latency and amplitude of the evoked EMG response - Calculate nerve conduction velocity 𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑠𝑡𝑖𝑚𝑢𝑙𝑎𝑡𝑖𝑜𝑛 𝑠𝑖𝑡𝑒𝑠 (𝑚𝑚) 𝑉𝑒𝑙𝑜𝑐𝑖𝑡𝑦 = 𝐷𝑖𝑓𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑏𝑒𝑡𝑤𝑒𝑒𝑛 𝑙𝑎𝑡𝑒𝑛𝑐𝑖𝑒𝑠 (𝑚/𝑠) - The general range for normal conduction velocities is 50-60 m/s - Interpretation: Relate EMG findings to muscle function, nerve conduction, and potential neuromuscular disorders Imaging Human Nervous System Function - EEG Psychophysiological methods: explore the link between psychological processes and physiological responses - Sleep and cognitive studies - Emotion research - Diagnosis of mental health disorders - Marketing and consumer research - Educational applications 25  - Brain-interface technology Electroencephalography EEG: techniques to measure electrical activity of the cerebral cortex of the brain - Signals can be analyzed to diagnose epilepsy and brain death - Advantages: great simplicity - Limitations: poor spatial resolution because electrodes are separated from brain by scalp, skull, and cerebrospinal fluid - Sources of EEG Signals: EEG signals primarily originate from the synchronous activity of cortical pyramidal neurons - Distinct pyramid-shaped cell body (soma) with an apical dendrite extending towards the cortical surface and basal dendrites branching out from the base - EPSPs and IPSPs summed up to make EEg signal Brain Waves and EEG Signals: EEG signals exhibit rhythmic patterns known as brain waves, which are categorized by their frequency and amplitude - Delta (0.5-4 Hz, 100-200 µV): High amplitude waves associated with deep sleep - Theta (4-8 Hz,