Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

TiN AW > Electrophysiology > Flashcards

Electrophysiology Flashcards

1
Q

Extracellular and intracellular recordings

A

Both used in animals:
Intracellular = single channel recordings in anaesthetised animals to keep them still
Extracellular = implanted in awake/live animals

Multi-unit extracellular recordings can be done in vivo - in whole organism

Electrode placed in anaesthetised rat brain

Recording area = LGN = lateral geniculate nucleus

With electrode in place, a flash of light is used to activate neurons

Largest action potential (electrical signal) from one neuron very close to electrode and smaller action potential from a cell further away from electrode = simultaneously recording from x2 different cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is electrophysiology?

A

Recording of electrical activity of whole brain tissue, a single neuron or a single ion channel in the brain

Immunotechniques/immunostaining/immunodetection/immunolabelling = to visualise specific molecular targets - mostly proteins in tissues

Targets are antigens visualised by use of antibodies raised/made against antigens

High temporal resolution approach

Can be used in vivo (in animals)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Patch-clamp technique:
On-cell patch
Cell-attached patch

A

Fill electrode with solution containing pore-forming antibiotic

Electrodes go to the cell’s surface and interact with the membrane and forms a gigaseal = on-cell patch

Make gigaseal - just wait for antibiotic to diffuse to membrane and form pores in it by itself (so do not need electrode to break membrane)

Pull electrode away after making gigaseal - this pulls patch of membrane off the cell to give intracellular access

Can apply drugs and record single channel activity

Perforated patch

Single channel recording - cell attached patch - x2 single channels in the patch

Cell-attached patch = break through membrane with electrode attached to whole cell (whole cell clamp) to access inside cell to record from all ion channels in the membrane of that cell (whole cell recording)

Patch-clamp in vivo - anaesthetised animal - put electrode into brain - force air into back of electrode

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Which electrophysiology techniques used in humans?

A

Only Extracellular recordings in vivo humans as part of existing treatment

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Halle Berry neuron

A

Single unit recordings in human brain: recording of neuron responses to x3 photos of Halle Berry BUT no response to photo of Michelle Pfeiffer = association neurons

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

MEA’s: multi-electrode arrays

A

Electrodes bedded in dishes next to neurons grown in dishes in incubator = non-invasive to monitor electrical activity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

IR: Intracellular recordings

A

Use sharp electrodes to penetrate plasma membrane to stimulate small, brief pulse to cause action potential to occur

To record activity in single cells using sharp electrodes or patch suction electrodes

Single channel recording - SCR - recording activity of single ion channels and uses patch-clamp electrodes

Field potentials/whole nerve recordings/multicellular recordings/single unit recordings/MEA’s = multi-electrode arrays

Electrodes are outside and very close to the neuron and pick up field potentials

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Luigi Galvani (1790) frogs

A

Discovered nervous system uses electrical activity to function

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

Large electrical voltages in animals

A

Animals such as electric eels/rays generate large voltages to stun prey

Up to 700 volts

Electric fish = electrophorus/torpedo - produce electricity

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Negative resting membrane potential

A

= 70 millivolts

Comparing outside of cells to inside

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

Post-synaptic potentials:

A

Up to 40 millivolts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Action potentials:

A

100 millivolts
Large/fast/all or none fashion

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Electroplaques

A

Membrane sodium-potassium exchanges

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is immunohistochemistry?

A

Study tissue anatomy and cytoarchitecture

Study distribution of proteins within tissues

Study pathological changes of disease in tissues and cells

Essential for clinical diagnostic neuropathology and basic/translational neuroscience research

Tissue sources: animal models/post-mortem/pathology samples/surplus surgery material

Antibodies raised against a protein (antigen) in a different animal to bind to it and generate a complex - visualised by cutting out enzymatic reaction on a section sample.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Why use animal models in immunohistochemistry?

A

To examine tissue throughout course of disease from early to end stages

Animal models are generated based on specific genetic mutations found in humans

To analyse if potential treatment strategies have any effects

Animal models do not fully replicate human disease so need to interpret results carefully

Welfare of animals must be considered

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Human post-mortem tissue:

A

Reduces need to use animals

Better material to study human diseases especially if no animal model exists

End stage study of disease only

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Autolysis/necrosis:

A

Once tissue is removed from living beings/post-mortem donor, irreversible autolysis/necrosis occurs = cell damage

Post-mortem tissue = cellular damage more likely

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Tissue preservation: chemical fixatives:

A

Chemical fixation:

Formaldehyde = best balance - morphology and staining quality

Chemical fixatives stabilise proteins and macromolecules enmeshed amongst proteins, eg carbohydrates

Cross-linking fixatives, eg Formaldehyde/glutaraldehyde create covalent bonds between proteins in tissue

Fixation masks/alters epitopes (foreign proteins) to stop them binding to primary antibody

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Cryopreservation :

A

Preservation of tissue structure/components by rapid freezing without fixation but can fix tissue and freeze it.

Stops tissue degradation by rapid cooling sample with dry ice/liquid nitrogen

Does not permanently fix tissue - over time degradation will occur rapidly if not stored at minus 80 degrees in freezer.

Snap-freezing = does not change protein structure

BUT: morphology poorly preserved as ice crystals form that damage cell structure.

However, if rapidly cooled to below minus 70 degrees using liquid nitrogen, liquid water converted to vitreous (glass-like) water without crystalline phase so minimises cell damage.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

Covalent bonds:

A

Between proteins in tissues lead to good preservation of cell morphology = anchors proteins close to each other inside cells and between cells

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

Precipitating fixatives:
Perfusion:

A

Ethanol/Methanol

Disrupt hydrophobic bonds between proteins causing irreversible precipitation = create a solid from a solution

Fix tissue by immersing in chemical fixative as a liquid = diffuse through tissue over time

Fixation is slow

Larger samples need stirring (agitation)

Can inject fixative as fluid into circulating system = perfusion - requires in-tact circulation system

22
Q

Perfusion:

A

Injection of fixative quickly via blood vessels = preferred method

Changes in pH affect reactivity of fixative

Rate of fixative diffusion determines length of incubation in the fixative

Size of specimens = dissected to no larger than 5mm cubes for optimal fixation

Temperature: very important to fixation, eg 4 degrees C = retards degenerative changes but reduces penetration rate of fixative

OR room temperature fixation accelerates fixation penetration but also degenerative changes

23
Q

Embedding:

A

In solid medium to give extra support to enable tissue sample to be cut into thin sections

Many embedding available: in study of neural architecture, use harder plastic resins so you can cut much thinner

Paraffin wax = most common embedding media and prevents tissue distortion = allows for cutting of different thicknesses

Softer embedding = agarose/gelatine = cut thicker slices

24
Q

Tissue processors:

A

Dip and Dunk machine = with 12 stations - specimens in basket and stay at stations for set times with agitation (stirring) and automatically transfer from graded alcohols - xylene/Histo-clear - melted paraffin wax before embedding in fresh molten paraffin wax.

Enclosed tissue processor = a chamber in which ingredients pumped in/out under vacuum.

Paraffin wax embedding station = tissues placed in metal moulds filled with molten wax and then placed on cold plate to set the wax.

25
Q

Microtomes and vibratomes:

A

Plastic cassettes = to enclose tissue - lids removed in embedding - then metal moulds

Sectioning done on microtome - cutting/slicing of samples

Bench-top rotary microtome = used to cut sections from paraffin embedded tissues - thickness ranges from 3-10 microns

Sledge microtome

Paraffin wax sections can be cut as ribbons and floated onto 40 degrees C water to soften wax around samples so samples lay flat so can mount onto slides

Vibratome = used for samples embedded in softer media - NOT paraffin wax but agarose/gelatine instead

Vibratome = 50-100 micron sections can be cut
Sections cut from vibrating blade at high speed but advancing slowly into samples

Stained onto slides/free-floating sections

Cryostat = used for sectioning snap-frozen tissues - cut using refrigerated cabinet at minus 20 degrees C and contains rotating microtome - 8-50 microns

OCT = optimal cutting compound freezes at same density as soft tissues

Sliding microtome = used to cut frozen samples - fitted to bench top, tissue blasted with CO2 gas to keep frozen - 15-200 micron sections

26
Q

Nissl staining:

A

Dye staining achieved by ionic interaction between dye and tissue

Developed by Frank Nissl - end of 19th Century to identify neurons

27
Q

Luxol Fast Blue:

A

Acidic dye used to visualise central nervous system myelin axonal sheaths in paraffin wax sections - myelin is stained deep blue

28
Q

Counter stain:

A

Produces contrasting background colour to main stain

29
Q

Golgi stain:

A

Metal precipitation (solid formed out of liquid)

Developed by Camillo Golgi (1783)

Modified by Santiago Ramon Y Cajal to draw neurons/circuits - founding study of neuroscience

Small piece of formalin-fixed tissue immersed in potassium chromate then SILVER nitrate

Small random subsets of neurons in exquisite detail - neurons are stained in dark black/brown showing dendritic spines. Colour produced by silver salt deposits in the neuron.

Only some neurons react in Golgi method

Golgi used to study neuronal morphology

Can be used on very thick brain sections up to 500 micrometers or on whole brains.

Used to quantify number of dendritic spines a neuron has - eg loss in schizophrenia

30
Q

Immunofluorescence:

A

Visualised using fluorescent dyes

31
Q

Immunocytochemistry:

A

Enzymatic reaction used to visualise antibody antigen complexes on cells grown in laboratory in tissue culture dish

32
Q

Monoclonal and polyclonal antibodies:

A

Monoclonal = single antibody that recognises specific single epitope (a foreign protein) on antigen molecule

Produced by immunising/injecting animal with antigen (the protein to generate antibody against)

Polyclonal = produced by injecting an antigen of interest into rabbit - rabbit’s B cells will produce antibodies against the antigen - found in serum of rabbit.

A protein has many foreign epitopes and different B cells will make antibodies against different epitopes = polyclonal

33
Q

Antibodies:
Direct/indirect

A

Y-shaped and have x4 polypeptide chains - belong to the immunoglobulin protein family

x2 heavy chain copies and x2 light chain copies

Y arms contain antigen-binding site

IgG/IgM/IgA/IgD/IgE antibodies

Most antibody reagents are IgG or IgM (added to cause chemical reaction)

To visualise proteins in sections (in membrane of cells) = direct/indirect

Direct = primary antibody applied to tissue sample to bind directly to antigen - only for highly expressed proteins as reporter molecule signal is weak if very few epitopes seen.

Indirect = significantly amplify reporter molecule signal - primary antibody binds to antigen BUT antibody has no reporter linked to it - section incubated with second antibody that binds to primary antibody - second antibody has reporter linked to it.

Secondary antibodies are polyclonal so will react with epitopes all over the primary antibody.

Signal amplified as all secondary antibodies carry reporter molecules.

Reporter molecules are: enzyme/fluorochrome

Fluorescence = antibody (primary/secondary) linked to fluorochrome which is reporter molecule that emits coloured light at wavelength when illuminated by UV light.

Fixation masks/alters epitopes (foreign proteins) to stop them binding to primary antibody

34
Q

Fluorescence microscopes:

A

View fluorochromes (reporter molecule that emits coloured light at wavelength when illuminated by UV light)

To view multiple proteins using multiple antibodies

Enzymatic detection = antibody linked to enzyme, eg horseradish peroxidase (HRP)

Substrate added to tissue - enzyme and substrate interact to generate insoluble coloured product at site of antibody-antigen complex visualised under light microscope

Substrate = chromogen

35
Q

Positive and negative controls:

A

Always use positive controls to assess fidelity of technique and primary antibody

Negative control = is making a primary antibody

Immunostaining needs 1) positive/negative controls, 2) antigen retrieval, 3) block non-specific binding

36
Q

HIER: heat-induced epitope retrieval:

A

Carried out with sections immersed in buffer solution = resists change to pH (acidity/alkalinity)

37
Q

PIER: protein-induced epitope retrieval:

A

Sections incubated with enzymes, eg Proteinase-K, Trypsin, Pepsin.

False-positive signal = antibodies bind to non-specific components in cells/tissues - low strength

38
Q

Tissue and cell culture:

A

Cultivation of eukaryotic (cells contain nucleus in organism) tissues or dissociated cells outside of organism in growth media

Cell culture = culturing of dissociated cells

Tissue culture studies cell biology to diagnose chromosomal disorders from blood or amniotic fluid samples, to generate monoclonal antibodies to produce vaccines (Kohler & Milstein, 1975) and IVF (Steptoe & Edward’s, 1977).

Roux, 1885, neural tissue from chick embryos maintained in saline solution for short time.

39
Q

Harrison (1907):

A

Removed sections of frog embryos, embedded them into blood clots to allow microscope evaluation - he observed growth of nerve cells over many weeks.

40
Q

Burrows & Carrel:

A

Continued growing cell lines - embryonic chicken hearts.

41
Q

Cell lines:

A

Can grow indefinitely in culture BUT via genetic mutations and chromosomal abnormalities.

42
Q

Lewis & Lewis (1911):

A

Grew embryonic chick tissue in liquid media other than blood.

43
Q

Rous & Jones (1916):

A

Trypsin - dissociate tissues into cells in culture.

Trypsin can cause cell death so now use Accutase or EDTA.

44
Q

Vessels for tissue culture:

A

Carrel & Baker (1923) = new vessel for tissue culture called ‘Carrel flask’ - angled neck to stop airborne particles.

Modern tissue culture vessels = plastic, sterile, single use plates/flasks/dishes

Dishes = up to 15cm diameter
Plates = 384 wells
Flasks = 25 cm squared - 175 cm squared
Flasks have vented lids to stop airborne particles entering but allow free exchange of gases

45
Q

Biological safety cabinets and tissue culture incubators:

A

Reduce risk of microbial contamination of cultures

(1909): simple ventilated hood to prepare Tuberculin for mycobacterium tuberculosis = class 1 cabinet

Class 1 = give protection to user/environment from sample BUT do not protect sample from airborne particles.

Class II = most commonly used since 1960’s - rely on continuous uniform flow of clean, filtered air over the sample, air flow splits and flows through grills front and back. Unfiltered air enters and out via filtered exhaust without touching sample.

Class III = hoods - completely enclose sample and have integrated gloves in cabinet

Tissue culture incubators = constant temperature/humidity and levels of gases, eg CO2/oxygen

46
Q

Hayflick & Moorhead (1961):

A

First cell strains of human fibroblasts W1-38 - they saw primary cells/cell lines/cell strains:

Primary cells = normal tissue and grown without passaging

Cell strains = derived from primary cells

Cell lines = grow indefinitely with abnormal carrier types.

47
Q

Gey (1951):
HeLa cells:

A

Henrietta Lacks had cervical cancer - Gey generated cell line from a single cell called HeLa cells.

48
Q

Martin Evans (1982):

A

Isolated embryonic stem cells from mice blastocysts.

49
Q

Jamie Thompson:

A

Isolated human embryonic stem cells to generate neurons in large numbers.

50
Q

Western Blotting:

A

To detect specific proteins from mixture of proteins

Transfer proteins in electric field from polyacrylamide gel onto a membrane which blots the proteins from the gel.

Put gel and membrane together in a sandwich between two electrodes.

Sandwich filled out with filter papers/sponges

Membrane made from PVDF/nitrocellulose

Electronic current ran through sandwich and proteins move out of gel and onto membrane

Nitrocellulose and PVDF are very sticky to proteins

Proteins immobilised on membrane and membrane is blocked using milk and add primary antibodies to bind to proteins.

Following adding primary antibody, incubate membrane with secondary antibodies to bind to primary antibody

These secondary antibodies either have:
1) fluorescent tag - gives off light in wavelength
2) have enzyme, HRP (horseradish peroxidase) attached

Add substrate for HRP, enzyme catalyses a chemical reaction that produces light.

Can detect light using photographic film or an imager to pick up signal.

Picture of gel and Western Blot - in blot is only the protein we are interested in due to using specific antibodies.

51
Q

Advantages of Western Blotting:

A

Takes 1 and a half days - just need glass plates, running and blotting tanks and power supply

Cheap and easy to make - gels and buffers

Works for wide range of proteins and needs a good antibody so all you will see in the gel is your protein you are studying

Can use ANY cell type or tissue - can use to look at changes in protein levels too

However, antibodies often do not bind specifically and pick up proteins not wanted

Sometimes proteins get changed by cell

More difficult if larger proteins or smaller:

Larger = move slowly and do not transfer well

Smaller = run fast and need high % gels

52
Q

Disadvantages of Western Blotting:

A

Need at least 10% change in protein levels to see on Western Blot

Other techniques, eg QRT-PCR/ELISA more sensitive to measure exactly how much protein

Approximate changes only

Artificial system = cell is making more protein than usually does and the fluorescent tag affects the way the proteins behave at times.

TiN AW (4 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions