BIOC32 Midterm notes (Lecture 1-12)

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Department
Biological Sciences
Course
BIOC32H3
Professor
Joanne Nash
Semester
Fall

Description
⬛ Lecture 1: Physiology ●Physiology:How the body maintains homeostasis -Level of organisation: Gene→Protein→Pathway→cellular mechanism→cells→Tissues→Organ→Organism ●Homeostasis: Maintain balance between compartment -”Homeo” = similar, “stasis” =condition -Have pathologies when homeostasis fails ●Each compartment is comprised of mixture of different electrolytes (salt), sugars, protein ●Internal changes:Dehydration, temperature, hunger ●External changes: Viruses, antibodies, insulin independent diabetes  Understand human physiology ●1) Apply integrated approach 2) Require for hypothesis 3) Experimental design ➣ Integrated approach: Use different method to address a question -Multi-disciplinary approach spans all cell biology/biochemistry etc ➣ Hypothesis = ”to assume”, needs to be logical and informed based on information available -Need to be testable, depends on technologies available ➣ Non testable hypothesis: Consist variable which cannot be quantified ➣ Experimental design: Important to choose a correct system ●Test tube design: Advantages: Cheap, fast, no infrastructure/ethical issues Disadvantages: Circuit not intact, functioned measurement is less direct (not direct answer) ●Animals design: Advantages: Intact system compared to test tube design, reduced inter-species variability and cost -Similar physiological processes to human (e.g. rodent) -Subjects are exposed to same living environment (reduce inter-species variability) Disadvantages: More simple than human, physiological shortcoming ●Human design: Advantages: Most relevant to hypotheses, medically / scientifically most relevant Disadvantages: Consist variability of race, sex, age and lifestyle -High costs: salaries, organization, infrastructure etc. -High # of replicates needed ●Experimental design factors: -e.g. Different level of training in personnel / constant testing environment -# of replicates per group: Are the statistical reliable / cost problems ➣Interpretation ●Variability between subjects: sex, lifestyles, race ●Psychological bias: Either from patient or experimenter, placebo or double-blind study are needed ●Ethical issues: May have potential health risk to subjects or patients Lecture 2: Properties of Excitable Cells ●(Neuro)Physiologist: Explain animal behaviour using anatomical connections & rules of cellular response ●Biophysicist: Explain rules of cellular response/ physical chemistry and electricity -Uses kinetic models, rules of thermodynamics and electrostatics to test out hypotheses ●Julius Berustein: Proposed the Membrane theory 1) Cells are composed of electrolytic interior, surrounded by membrane which is selectivity permeable to ions 2) Have pre-existing electrical difference of potential across membrane when at rest 3) Cell membrane is selectively permeable to potassium K+ ion at rest 4) High intracellular [K+], Low extracellular [K+] ●Concentration of ions in intracellular and extracellular solutions: -Extracellular environment: High in [Na+] and [Cl-], and some [Ca+] -Intracellular environment: High in [K+] and some protein ●Different ion concentrations create concentration/ chemical gradient between IC and EC -Have plasma membrane separates them -More –ve charge inside the membrane compared to outside ●Water and small non-polar molecules able to pass through membrane ●Large molecules (glucose) = Slow diffusion rate || Impermeable to polar molecules ●Chemical gradient is builded by NA/K ATPase pump ➣ NA/K ATPase: Transport 3 Na+ out and 2 K+ into the cell ●Known as electrogenic pump: Produce net movement of +ve charge outside the cell ●Requires ATP since it’s pumping against chemical gradient, will hydrolysed to ADP ●An active transport in cell membrane ●Electrophysiology: Used to measure membrane potential differences (V )mof cell -Voltmeter: measure the voltage difference between recording and grounding electrode -Measure different voltage between 2 points -Ground electrode: Always 0mV -Electrical gradient: or potential difference, difference in charge between 2 compartments (IC and EC) -Potential difference: Driving forces in electrical gradient -Amount of energy (Joules) that drive movement of ions between 2 compartments ➣ Resting membrane potential ●All cells of body have resting membrane potential ●Muscle and nerve are excitable: Potential can be changed ●Have –ve charge inside cell when it’s at rest ●Resting membrane potential difference (RMP): approx. -70mV -Difference in cell of body in charge in between IC and EC -Na+ channel closed, K+ channels open -No Na+ flux and have K+ efflux at resting potential (K+ leak channel) -can be between -9 (red blood cells), -100mV (neurons of spinal cord) ●Generated by: 1) K+ leak channels 2) NA/K ATPase 3) Impermeable cell membrane ➣Ion Channels ●Ion channels are voltaged-gated, and are selective for specific ions -Open and close in response to changes in voltages -Voltage-sensitive: Open at specific voltages ●K+ leak channels at RMP: K+ channel at -70 mV - -ve charged proteins prevent too many K+ leave from the cell -As more K+ leave the cell, ↓electric potential slowing K+ movement out until equilibrium ➣ Equilibrium potential of NA+ at resting ionof +60mV) ●High [Na+] in EC on chemical gradient = NA+ tends to move inward ●Negative charge in IC on electrical gradient= Na+ tends to move inwards ●+60mV must apply inside the cell to prevent NA+ movement ●Membrane is impermeable to Na+ and have Na/K pump ➣Equilibrium potential of K+ at resting ionof -90mV) ●High [K+] in IC on chemical gradient = K+ tends to move out of the cell ●Negative charge in IC on electrical gradient = K+ tends to move into the cell ● -90mV must apply inside the cell to prevent K+ movement -Concentration of K+ is high inside cell, net charge must be more negative -Concentration (Chemical gradient) influences more than charge (electrical gradient) ➣Forces controlling movement of ions between IC and EC compartments: ●Electrochemical gradient: Combined electrical and chemical gradients ●Resting membrane potential (RMP): Reached when ion influx = ion efflux, at equilibrium ●Ion movement is determined by Equilibrium potential (Eion -EionVoltage that has to apply inside the cell to prevent ion flux -Only apply to Galvanic cell: Cell which permeable to only one ion ➣ Nernst equation: E ion61/ z) log ( [ion]out / [ion] in) ●Used to calculate individual equilibrium potential of ions ●Take account of: 1) Ion concentration 2)Valency 3)Temperature ●61 = the absolute temperature at 37 degree ● z = electrical charge (valency) of ion ➣ Goldman-Hodgkin-Katz equation- by Goldman, Hodgkin and Bernard Katz ●Predicts membrane potential using multiple ions and permeability ●Accounts for permeability of K+, Na+, Cl- Lecture 3: Action Potential ➣Nerve and muscle cells are excitable cells ●Open & close of voltage-gated ion channels cause changes in membrane permeability ●Allow ions to move between IC and EC compartments→ Changes in excitability of cell ●Membrane potential difference: Vm in relation to 0 Depolarization: More towards to 0 ●Hypolarization: More negative than RMP Repolarization: Towards RMP when depolarized ➣Depolarization ●Depolarization mainly due to opening of Na+ channels ●Have 2 gates, only one can be closed at any given time (open and close rapidly) ●Activated when activation gate is closed when Vm = -55mV -Cannot be activated when inactivation gate is closed -Na+ ion only flow into cell when both gates are open ●In RMP: Activation gate closed, inactivation gate is open (No Na+ influx) ●Depolarization: Activation gate opens, Na+ ion influx into cell -Then inactivation gates closes after 0.5ms, no more Na+ flux Two types of K+ channels ●i) Leak Channels: Usually open, responds to -70mV resting membrane potential -Direction of K+ flux depends [K+] in IC and EC ●ii) V-Gated K+ channels: Responds and open at -55mV or less negative (e.g. -40) -Open in response to membrane depolarization -Contribute to repolarization and hyperpolarization of membrane -Open and close slowing ●Changes in membrane potential do not cause much changes in homeostasis -100mV changes in membrane potential = 1 in many K ion enter/leave the cell  Neuron ●Presynaptic axon terminal: Release neurotransmitter, and cause depolarization of postsynaptic mem. ●Changes in membrane potential cause open/close of ion channels ●Graded potential: Electrical signaling in neurons -Amplitude of GP = Directly proportional to strength of triggering signal -GPs decrease in amplitude as move away from stimulus -Spontaneous GP and AP are decreased in Parkinson’s disease ●Strong GPs at trigger zone (Axon hillock) will generate AP -GPs will generate AP when it reaches threshold = Less –ve than -55mV -Na+ channels open and stimulate A.P at threshold ●Action potential always approx. the same size Na+ and K+ channels at Action potential Lecture 4: Action Potential ●Luigi Galvani: Discovered electrical signals from lightening, will cause contraction in dead frog’s leg ●Juilus Berstain: Used differential rheotome to study action potential as negative variation -Also studied in frog nerve muscle preparation -Found out A.P almost completed (~1ms) before muscle contract -Found out time course is independent of stimulus ●Hodgkin and Katz: Discovered negative variation (refer as Overshoot) -Proposed the sodium hypothesis: Reverse RMP by Na+ influx -Predicted A.P in giant squid axon with mathematical model -Unmyelinated axon, Leaky= prone to signal loss, Broad axons = conduct better -Beginning of computational neuroscience -Used Voltage-clamp: Deliver current to maintain particular voltage inside cell -Measure how much current is required to keep voltage from changing -Indicates the amount and direction of ion flow -Discover which ions were responsible for currents -A.P is described as Positive variation: Positive inside the cell to negative outside in AP  Voltage-Gated Na+ and K+ channels ●Most Na+ and K+ channels are located in dendrites and cell body ●Sodium channels (Na+): High conc. in trigger zone and between myelin sheaths in node of Ranvier -No Na+ channels located on myelin sheath ●Same type of ion channels used in graded potential and AP ➣Na+ influx creates a Positive feedback loop ●Depolarization by opening of Na+ channels → Open more voltage-gated Na+ and K+ channels ●AP is generated by opening of many Na+ channels ●Positive feedback loop allows conduction of AP down the axon ●Feedback loops stops: Closing inactivation gate of Na+ channel, slower Na+ influx ➣ Refractory periods of AP ●Second AP cannot be generated, no matter the size of the stimulus (~2ms) ●Vm in Refractory membrane: About -70mV to -90mV ●Due to resetting of Na+ gate to default RMP position: activated open, inactivated gate close ➣Relative refractory period ●Need larger graded potentials for initiate another AP ●Some Na+ channels have resetted, not all of them ●Membrane potential is hyperpolarized: Lower than the RMP ●Significance of refractory periods: -AP will not overlap and move backwards, will move trigger zone → Axon terminal  Coding of Stimulus Intensity ●Graded potential is located in cell body/ dendrites -Able to be added and summate, different from AP ●AP cannot be summate together, always the same amplitude no matter the strength of stimulus -Frequency increase with larger stimulus intensity ( ↑ number of AP per sec) ●Current flow: Flow of AP down the axon ➣Conduction in non-myelinated axon (Local current flow) ●Occurs by Local current flow: 1) Graded potential (above threshold) reaches trigger zone 2) V-gated Na+ channel opens, Na+ influx into axon 3) Positive charge flows into adjacent sections by local current flow 4) New membrane depolarized by current flow from active region 5) No backward conduction due to refractory period -Loss of K+ (efflux) from cytoplasm repolarized membrane ●Channels must all the way down the axon for conducting AP ➣Conduction in myelinated neurons ●Flow of action potential is occurred by saltatory conduction -Saltatory conduction: “Leaping” of current between node of Ranvier ●Myelinated membrane acts as an insulator ●Sodium channels are only located between nodes of Ranvier, not on the myelinated axon -Ion channels don’t open in myelinated sections ●Faster AP rate in myelinated axons: Current leak is prevented by myelin sheath ●Multiple Sclerosis: Demyelinated disease, nerves are damaged due to scarred myelin -Nerve cells in brain and spinal are damaged, inability for nervous system to communicate -AP leaks from the axon, resulting abnormal functioning -Plaques are shown as white spots in MRI scan Lecture 5: Chemical Synapses  Three Signaling mechanism in neuronal signaling i) Graded Potential ii) Action Potential iii) Chemical neurotransmission ●Chemical transmission: Conversion of AP into chemical signal -Signal (neurotransmitter) is released from presynaptic axon terminal -Cause graded potential in dendrites/cell body of target neuron at postsynaptic junction ●Number of neurons in brain: ●Neurons have 10,000 to 150,000 synapses on dendrites and cell bodies ➣ Transfer at Synapse 1) Action potential depolarizes axon terminal 2) Depolarization opens V-gated Ca channels, Ca enters cell 3) Ca entry trigger Exocytosis: Synaptic vesicles fuse with the membrane at active zone -Synaptic vesicles release neurotransmitter into synaptic cleft 4) Neurotransmitter diffuses across the synaptic cleft → Binds with receptor on postsynaptic cell 5) Neurotransmitter binding to receptor → Cellular response in postsynaptic cell ●Receptor activation initiates downstream signaling cascade (Signal transduction) -Different types of receptors activate different signaling cascades: 1) Ionotropic Receptor: Chemical/ ligand-gated ion channels -Activated by neurotransmitter, cause movement of ions -Directly coupled to ion channels, allow movement of ion across postsynaptic membrane -Fast postsynaptic response (~ms), fast to inactive 2) G-Protein coupled receptors (GPCR): -Neurotransmitter binds to GPCR, → Activation of receptor → Receptor activates G protein -Slow synaptic response (mins to days), slow to inactive - Signal is amplified by signal transduction: GP → Amplifying enzyme → Second messengers → Cell responses ●One NT binds to one receptor = Activation of many intracellular signaling molecules ➣Postsynaptic response ● 1) Chemically-gated ion channel activation: (by Ionotropic receptor) NT activates ionic channel → Change in membrane potential → (Activates v-gated ion channel) + Ion channels open → More Na influx → EPSP/ Excitatory depolarization - + More Cl influx /K efflux → IPSP / Inhibitory hyperpolarization ●2) GPCR activation: Activates G-protein coupled receptor → Activated G proteins actives amplifying enzymes + Close v-gated ion channel → Less Na influx → IPSP Inhibitory hyperpolarization + Less K efflux → EPSP Excitatory depolarization -Activation of amplifying enzyme → Second messenger pathway →Response -Modification of existing proteins + Protein synthesis + Cell division + protein movement -EPSP= Excitatory post-synaptic response -EPSP + IPSP: Trigger graded potential, trigger structural change in v-gated channels  Signalling molexules downstream of GPCR ➣G-protein-coupled adenylyl cylase: cAMP system Stimulation ●Adenylate cyclase: Catalyzes conversion of ATP to cAMP First amplifier enzymes of cAMP-mediated signal transduction pathway ●cAMP: Function as second messenger in the system 2+ ●Protein kinase A: Phosphorylates Ca channels and pumps NT binds to receptor → Activate Stimulatory G psotein → Activate adenylyl cyclase Adenylyl cyclase activates cAMP → cAMP activates protein kinase A ➣cAMP system Inhibition: NT activates GPCR → GPCR interacts with G pritein → Inhibit adenylyl cyclase Inhibition of cAMP → Reduced activation of protein kinase A ➣G-protein coupled phospholipase: DAG and IP system 3 Activated GCPR stimulates G →Activates PLC → Produces products of IP and DAG q 3 ●Both IP3 and DAG act as second messengers in different branches: (PLC as amplifying enzyme) 2+ -IP3: Binds to Ca release channels in ER membrane 2+ 2+ -Activate channels to open → Allow Ca efflux from ER → Increase [Cytoplasimic Ca ] 2+ 2+ -Ca acts as third messenger and further activates channels with greater Ca efflux -DAG: either remain in membrane or activates protein kinase C 2+ 2+ - PKC: Ca dependent kinases, increased cytopplasmic Ca trigger PKC moves to membrane ➣ Neurotransmitter Cycling in Presynaptic terminal ●NT are transported back to presynaptic terminal via uptake transporter (via ATP pump) ●Some of the NT are recycled by endocytosis at active zone  Neurotransmitter ➣Neurotransmitter: Classified based by chemical composition: 1) Amines: Synthesized in body, derived from amino acid -Hydrophilic molecules and most biogenic amines are packaged into vesicles -Included Catecholamines: Synthesized from Tyrosine -e.g. Dopamine, adrenaline, noradrenaline - Adrenaline: responsible for fight and flight responses in PNS -Noradrenaline: Only formed in CNS - Neurons that secrete noradrenaline are adrenergic neurons: -Serotonin (5HT): Synthesized from tryptophan → 5HT -Taken up by transporter (active reuptake) → Broken down/ repackaged into SV -Large number of 5HT receptor in hippocampus (Involved in memory) 2) Amino acid: GABA and glutamate -Glutamate: Synthesized from glucose and glutamine, found only in brain -GABA: Synthesized from glutamate -Process of making glutamine and GABA: Glutamine → α -ketoglutarate → Glutamate → GABA -Taken up into presynaptic terminal by transporter (active reuptake) → Broken down 3) Acetylcholine: Synthesized in body, an amine which is not from amino acid -Synthesized from choline and acetyl coenzymes A (acetyl CoA) -Primary neurotransmitter at neuromuscular junction of vertebrates -Acetylcholine in synaptic cleft: Rapidly broken down by enzyme acetylcholinesterase -Broken down in synaptic cleft to prevent prolonged contraction -Choline transport back to axon terminal → Used to make more Ach ➣Biosynthesis and Catabolism of Catacholamines 1) Tyrosine transported into axonal terminals 2) Converted to neurotransmitter by series of enzymes: DOPA → Dopamine → Norepinephrine → Adrenaline -Enzyme content of presynaptic terminals determines which types of NT are formed 3) Neurotransmitter in synaptic cleft is taken back by active transport 4) Excess NT is broken down by enzymes in cytoplasm of presynaptic terminal → Into SV ➣Neurotransmitter binding to Receptor ●Have finite number of receptor: -At low concentration of NT: Linear relationship between [NT] and amount of binded NT -At high concentration of NT: Receptors become saturated, no more NT can bind to receptors ●Maximium # of NT bound to receptor = Maximum downstream effect that NT can cause ●Neurotransmitter bind to different subtypes of receptors: -Receptor subtypes is defined by the NT that binds to it 1) Adrenergic Receptor: Binds by noradrenaline or adrenaline -G protein-coupled receptor, with α and β subtypes (α1 and α2, β1, β2 and β3) -Receptors located in heart, blood vessels, movement of intestines - α1: G protein, Activates PLC, increases DAP, IP3, Ca 2+ q -α2: G irotein, Inhibits adenylyl cyclase and ↓cAMP (inhibit cellular response) -β1, β2 and β3: G srotein, activates adenylyl cyclase and ↑cAMP (excitatory cellular) 2) Dopaminergic Receptor: -Gs protein, activates adenylyl cyclase, ↑cAMP (excitatory cellular response) 2) Dopaminergic receptor: binds by dopamine -G-protein-coupled receptor, D1 and D2 subtypes -High levels of receptors found in brain area that control mood and personality 3) Acetylcholine (Ach) receptor: binds by acetylcholine -Contains ionotropic and GPCR subtypes receptor -Nicotinic receptor: Ionotropic Muscarinic receptor: G-protein coupled receptor -Receptors found in muscle (skeletal), movement control 4) Glutamate receptor: Binds by glutamate -Contains ionotropic and GPCR subtypes receptors -NMDA and AMPA receptor: Ionotropic Metabotropic glutamate receptor: GPCR -GABAergic and glutamatergic receptors are found throughout the brain ●Types of neurons = Type of NT released from axon bouton -NT released can go into blood stream to causes effects (e.g. adrenaline) -Different types of receptor are located in different regions of body  Functions of amine neurotransmitters ●Motor control: Responsible by dopamine, affected by Parkinson’s disease which reduces dopamine ●Mood control: Responsible by dopamine and serotonin ●Motivation/reward pathway and Vomitting: Responsible by dopamine ●Increased 5-HT receptor stimulation: Used as anti-depressants, anti-anxiety, Ecstasy ●Dopamine R agonist: Used to treat Parkinson’s disease, as anti-depressants ●Parkinson’s disease: Due to decreased dopamine, increased glutamate and acetylcholine ●Depression: Due to decreased dopamine and 5HT Drug addiction: Increased dopamine ●Schizophrenia: Increased dopamine Anxiety: Reduced 5HT ●Stress and inosomnia: Increased noradrenaline  Functions of Amino acids neurotransmitter ●Motor control: Responsible by glutamate and GABA ●Learning and memory: Responsible by glutamate ●Huntington’s disease: Increased GABA Epilepsy: Reduced GABA ●Alzheimer’s disease: Increased glutamate, decreased acetylcholine ●GABA receptor agonists: Used to treat epilepsy, bipol
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