Neuro Final PDF

Neuro Final EXPERIMENTAL DESIGN: extraneous variable: any variable other than independent variable that could influence dependent variable if not controlled confounding variable: type of extraneous variable that is correlated with both...

Neuro Final EXPERIMENTAL DESIGN: extraneous variable: any variable other than independent variable that could influence dependent variable if not controlled confounding variable: type of extraneous variable that is correlated with both independent + dependent variables directly impacting cause-and-effect relationship between them and confounding interpretation of experiment between-subjects study: each subject is assigned to only one condition within-subjects study: repeated measures, subjects experience all levels of independent variable types of data: nominal: categorical data without any intrinsic order; no quantitative distinctions; no meaningful order ex. color, brand, type of stimulus (visual, auditory) ordinal: ordered categories; no equal intervals between categories; arbitrary or absent zero point ex. rankings, clothing sizes, education level interval: ordered categories, equal intervals between categories; arbitrary or absent zero point ex. temperature (Celsius, Fahrenheit), time of day, IQ ratio: ordered categories; equal intervals between categories; true zero point ex. temperature (Kelvin), height, number of correct answers unpaired t-test v. paired t-test v. ANOVA BUFFERS: chemical solution: homogenous mixture of 2+ substances Neuro Final 1  solute dissolved into solvent pH = potential of hydrogen = measure of acidity of solution accuracy: how close a measured value is to true/known value precision: how close 2+ measurements are to each other monobasic = weak acid dibasic = weak base buffer = monobasic + dibasic buffer: solution that resits changes in pH when small amounts of acid or base are added important b/c they help maintain the pH balance of a system by acting as “shock absorbers” help antibodies + enzymes keep their shape by maintaining pH of solution blood is natural buffering system buffer solutions can be made by either mixing a weak acid with its conjugate base or mixing a weak base with its conjugate acid → acid: forms H+ ions when dissolved in water → proton donor → base: forms OH- ions when dissolved in water → proton acceptor phosphate buffer (PB): monobasic sodium phosphate (weak acid) + dibasic sodium phosphate (conjugate base) monobasic phosphate neutralizes OH- ions from base, buffering against drastic elevation in pH that could occur with addition of a base dibasic phosphate neutralizes H+ ions from acid, buffering against drastic drop in pH that could occur with addition of an acid buffer capacity: ability of buffer to resist changes in pH measure of amount of acid or base that the solution can absorb without a significant change in pH influenced by concentration of acid + base as well as starting pH of solution Neuro Final 2  when buffer capacity is exhausted, the buffer's pH will change dramatically HISTOLOGY: [the study of microscopic anatomy of cells, tissues, and organs in plants + animals] Neuro Final 3  P1000 = 924 P200 = 157.6 P20 = 12.55 histology is used to study: cell + tissue morphology presence, location, + abundance of organelles, RNA, protein neural circuitry experimental location stains are used to label particular cell structures ex. Nissl stain: labels cell bodies + allows visualization of cell layers binds to neg. charged nucleic acids (DNA, RNA) labels nucleolus, free polyribosomes, + ribosomes on rough ER ex. cresyl violet ex. DAPI stain: labels cell nuclei binds to DNA regions rich in adenine + thymine used to differentiate cell layers ex. Golgi stain: stains entire neuron leaves silver chromate deposit in cells randomly stains only small percentage of neurons good for studying neuronal morphology ex. Injectable dyes: used to mark brain locations in vivo (via surgery) ex. fluorescent dyes labeled probes target mRNA transcripts/proteins → used to locate + quantify gene expression in situ hybridization (ISH): labeled nucleic probes bind complimentary RNA Neuro Final 4  immunohistochemistry (IHC): chromogenic or fluorescently labeled antibodies bind target proteins tracers: molecules that are injected into nervous system that allow researchers to determine where axons project to/from in the brain retrograde = away from axon terminal anterograde = toward axon terminal transgenic: organism with genetic material from another species histology process: 1. fixation: preserves + stabilizes tissue a. strengthens molecular interactions between cell structures b. disrupts enzymes c. kills microorganisms 2. cryoprotection + embedding: prevents formation of ice crystals (which cause holes to form in the tissue) a. tissue is put in solution of sucrose 3. sectioning a. cryostat: tissue is embedded in paraffin and then frozen b. microtome: tissue might be embedded in paraffin or gelatin and then frozen i. what we used in lab c. vibratome: tissue does not have to be embedded or frozen Neuro Final 5  4. staining: uses immunohistochemistry to bind and label target proteins using antibodies antibody: protein produced by immune system that binds to specific substance that the body considers foreign antigen: foreign substance to which particular antibody binds epitope: the precise region on antigen where antibody binds variable region binds to a specific antigen constant region is similar among all antibodies from particular organism where secondary antibody binds to make an antibody, you inject a target protein (antigen) into an animal → immune system produced antibodies against antigen → antibodies are unified from whole blood samples antibodies can be modified with a visible marker or enzymatic tag that can be used to visualize the location + abundance of a target protein in a tissue sample types of immunohistochemistry: direct IHC: primary antibody is tagged with a fluorophore or enzyme faster process than indirect IHC indirect IHC: a tagged secondary antibody binds to primary antibody Neuro Final 6  signal amplification chromogenic IHC: primary or secondary antibody is tagged with enzyme that produced a color product used with light microscopy fluorescent IHC: primary or secondary antibody is tagged with fluorescent molecule/protein used with fluorescent or confocal microscopy what we used in lab IHC process: 1. antigen retrieval: uses heat, chemical detergents, or proteases to unveil the reign on the antigen containing the epitome 2. permeabilization: incubating tissue in a solution that contains a detergent/soap a. removes lipids + disrupts the cell membrane, allowing large molecules (antibodies) to penetrate cells + gain access to intracellular antigens 3. blocking: incubating tissue in blocking buffer to prevent nonspecific binding of primary and secondary antibodies to off-target proteins a. blood serum- contains general antibodies that will bind to off-target proteins b. albumin- protein that binds to a wide range of substances and will “block” off-target proteins c. PBS is the solvent used b/c ions mimic those found in natural tissue 4. primary antibody incubation: tissue is incubated in solution of primary antibodies in blocking buffer 5. secondary antibody incubation: tissue is incubated in solution of secondary antibodies a. ex. inject primary rabbit antibody (antigen) into a goat + extract goat antibodies → “goat anti- rabbit” antibodies → process: Neuro Final 7  1. apply unlabeled primary antibody 2. unbound primary antibody washed away 3. apply secondary antibody conjugated to fluorophore 4. unbound secondary antibody washed away 5. fluorescent signal detected wherever antigen is located → fluorescence: ability of certain chemicals to emit light after absorbing visible light or other electromagnetic radiation → fluorophore: fluorescent chemical compounds that absorbs light of short wavelength + emits light of long wavelength in return 6. counterstain: provides contrast to principle stain + allows structure of entire tissue section to be visualized 7. mounting a. mounting media helps preserved tissue + contains chemicals that prevent bleaching of fluorescent labels 8. image analysis → wash with PBS after each step to clean Neuro Final 8  SOCIAL ISOLATION EXPERIMENT: cFOS protein is a marker of neural activity neurons that are activated due to stimulus will express cFOS 45-90 min later the brain regions that have differences in neural activity between co-housed and isolated groups include: dopaminergic reward systems → basal ganglia (substantia nigra, ventral + dorsal striatum) regions dealing with emotion → amygdala, BNST dopaminergic neurons in DRN are more active during social interactions after a mouse has been isolated DRN is located in midbrain + pons DRN functions: pain, sociability, reward, anxiety/depression activation of dopaminergic neurons in DRN causes increased sociability DRN neurons send axons to BNST + central amygdala (as well as mPFC, hypothalamus, etc.) axons release glutamate + dopamine, causing excitation therefore, we expected to see increased cFOS in BNST + central amygdala of socially isolated mice EQUATIONS: W7-EntryTicket-PreparingSolutions.docx molarity (M): number of moles of solute per liter of solution M = mol/L g = MM x M x V 1 g = 1 mL Neuro Final 9  molar mass (MM) = molecular weight (MW) = formula weight (FW) normality (N): number of mole equivalents per liter of solution determine how many moles of H+/OH- dissociate volume percent (% v/v): volume of solute per 100mL of solution mass(weight) per volume (%w/v): mass of solute per 100 mL of solution c1v1 = c2v2 for solutes with multiple solvents ELECTROPHYSIOLOGY: [the study of electrical properties of cells] allows researchers to study how neurons communicate with each other + form complex networks used to measure action potentials in living neurons either intracellularly or extracellularly electricity describes the flow + interactions of charged particles Ohm's Law: V = I x R voltage: electric potential energy per unit of charge (difference in amount of charge at two locations) potential energy: capacity for doing work which arises from position/configuration if you have more negative particles in location A compared to location B, the particles at location A will push against each other and try to move to B current: rate of flow of electric charges resistance: inability of a charged particle to move through a material how electrical signal is generated in neurons: 1. membrane potential = voltage a. ions can only travel across cell membrane through channels → different ion concentrations inside + outside cell → voltage Neuro Final 10  b. measured across membrane (one recording electrode inside + one outside cell) 2. ion flow across membrane = current a. through diffusion + electrostatic force 3. action potential = spike a. signal that conveys info over distances b. occurs in axon of cell c. brief + rapid depolarization of membrane (inside becomes pos. charged) d. all-or-none 4. electrochemical synapses a. action potentials travel down axon → Ca channels open → neurotransmitter releases from presynaptic terminal → neurotransmitter binds to postsynaptic neuron membrane b. EPSP vs. IPSP how is electrical activity in neurons measured? 1. patch-clamp recording: glass pipettes (recording electrode) seals to a portion of the membrane using suction and reference electrode is extracellular in bathing solution a. study membrane properties + individual ion channels i. studies opening/closing of particular channels ii. measure ion current through channels iii. measure voltage changes 2. intracellular recording: recording electrode is in a sharp glass pipette filled with electrolyte solution (placed into membrane of neuron) + reference electrode is in bathing solution (outside the cell) a. measures voltage or current across membrane of single cell i. studies whole-cell ion currents going in/out of cell Neuro Final 11  ii. studies membrane potential (action potential, EPSP, IPSP) b. APs are consistent in amplitude b/c there is little difference in how APs look once they are initiated 3. extracellular recording: recording electrode is a metal electrode or glass pipette in extracellular space near neuron + reference electrode is placed in a brain area farther away a. measures appear inverted compared to intracellular recordings b/c recording electrode is measuring ions entering + leaving extracellular space i. outside is getting increasingly neg. compared to inside instead of inside getting increasingly pos. compared to outside b. measures large changes in voltage due to action potentials i. record from one (single-unit) to a few neurons (multi-unit) at a time c. does not measure EPSP or IPSP d. APs can be of varying amplitude b/c: i. it is measuring electrical activity from multiple cells at one time ii. if multiple axons near an extracellular electrode are sending APs down their axons, resulting data will be larger iii. larger axons can increase amplitude of AP in extracellular setup iv. APs in extracellular electrode will get smaller the farther away the axon is from point of measure 4. multi-electrode arrays: probes that contain many recording electrodes 5. electrocorticogram: recording from the cortical surface of the brain a. can be done in humans b. records local field potentials 6. electroencephalography (EEG): non-invasive measure of changes in electrical potential at the scalp produced by neural activity Neuro Final 12  a. measured voltage is due to activity of many neurons across large areas of the brain cockroach experiment- each segment of cockroach contains region of ventral nerve cord (VNC) VNC: collection of neurons that sends info to muscles of body, while receiving info from sensory organs of periphery information is relayed to + from brain using APs and synapses cockroach leg is covered with large spines along tibia + femur each spine has neuron wrapped around it, which sends APs to VNC (and then brain) pattern + frequency of APs will allow VNC to distinguish strong somatosensory stimulus from a weak one ACTION POTENTIAL: strength of action potential propagation is determined by time constant and length constant length constant: measure of how far voltage will travel before reaching 37% of original peak value rm = electrical resistance of neuron's membrane directly related to length constant ri = resistance of intracellular fluid inside axon inversely related to length constant time constant: measure of how much time it takes for a neuron to fully charge to a stable voltage cm = capacitance of neural membrane (ability to store charge directly related to time constant rm = electrical resistance of neuron's membrane directly related to time constant Neuro Final 13  → smaller rm and cm → smaller time constant → less amount of time needed to change axon's voltage ideal neuron would have high length constant and low time constant myelin: fatty covering that increases rm by a factor of 2 bigger axon diameter: increases conduction velocity by factor of square root axon diameter earthworm: MGF: transmits sensory info about anterior of worm LGF: transmits sensory info about posterior of worm → MGF has a faster conduction velocity than LGF b/c it has a larger diameter, which leads to decreased internal resistance, which increases the length constant sparse coding: nerves fire when you introduce touch and remove touch EEG: EEGs measure post-synaptic potential action potentials are all-or-none APs for particular neuron will have same amplitude post-synaptic potentials result from changes in membrane permeability + ion flow caused by neurotransmitters binding to receptors last longer than action potential multiple PSPs can sum together they are graded responses that can vary in amplitude with stimulus strength electrical dipole: separation of charge over a distance local extracellular charge is opp. of local intracellular charge spatial summation: dipole alignment when neurons are in same orientation Neuro Final 14  temporal summation: when dipoles are generated at the same time/neurons fire in synchrony EPSP: excitatory post-synaptic potential increase likelihood of action potential can result from positive ions flowing into cell IPSP: inhibitory post-synaptic potential decrease membrane potential can result from positive ions flowing out of the neuron, or negative ions flowing into neuron cortical pyramidal neurons form dipoles b/c varying PSPs at their proximal dendrites compared to their distal dendrites creates a difference + separation of charge over a distance current in dipole flows from the positive extracellular side (source) to the negative extracellular side (sink) EEGs cannot detect the dipole of a single neuron factors that impact summation of dipoles across cortical neurons: timing of PSPs in neighboring cortical neurons orientation of cortical neurons same orientation leads to strongest EEG signal alpha block: decrease in amplitude of alpha-band oscillations during the shift from eyes closed to eyes opened states gamma: >35 Hz problem solving, concentration beta rhythm: 14-60 Hz, lower amplitudes mental activity + attention alpha rhythm: 8-13 Hz, high amplitudes awake with eyes closed Neuro Final 15  theta + delta rhythm: 4-7 Hz drowsiness + sleep → power = amplitude squared proportional to energy → EEG rhythms depend on activity in the thalamus → summary: eyes-closed state may be associated with neural inhibition, resulting in dampened processing of external stimuli; eyes-opened state may reflect neural excitability, resulting in heightened processing of external stimuli Neuro Final 16