Research seminars on techniques

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  • Created by: Sarah
  • Created on: 30-05-19 17:56
why do we need multiple techniques to study living cells?
we need to establish WHEN and WHERE an event occurred and we need multiple characteristics to do that
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what are the 5 characteristics needed?
1) high temporal resolution 2) high spatial solution 3) scalability 4) high sensitivity 5) minimally invasive
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why do we want high temporal resolution?
want to resolve events on a short timescale eg the AP is msecs long
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why do we want high spatial resolution?
we want to look at events inside a cell so we need out technique to resolve very small sizes
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why do we want scalability?
technique needs to be scalable so we can look at one single cells or a group of cells intact tissue, entire tissue or entire organ
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what is high sensitivity?
give a good signal compared to the noise of the measurement (signal to noise ratio)
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what is fluorescence?
the emission of light by a substance that has absorbed energy in the form of light or other EM radiation
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what is a simplified definition of fluorescence?
fluorescence is a substance that absorbs and emits light
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what is light made up of (physics concept needed for fluroscence)?
light is made up of photons and each photon has a specific wavelength and the wavelength is linked to the colour of light
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what is at the short wavelength end of the visible light spectrum?
ultraviolet
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long wavelength part of spectrum?
infrared
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what does each photon carry?
a certain amount of energy and this energy has a relationship to the wavelength
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what is the energy inversely proportional to?
the wavelength
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the longer the wavelength the what?
the lower the energy of the photon
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what do we use to see the energy of a photon and see different energy levels a molecule can have?
Jablonski diagram
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is the energy of a molecule continous or discrete?
it is quantitised which means they have discrete values you cannot be between energy
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what happens once the fluroscent molecule thats at ground state absobs energy?
energy is absorbed in the form of a photon, absorb energy from photon and transitions from ground state to excited state
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from moving to an excited state what is lost in this process?
some energy is lost as heat
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why does the molecule go back down to ground state?
the excited state is usually unsatble so your molecule spontaneously goes back to the ground state and loses energy
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when a fluroscent molecule loses energy and goes back to the ground state what can be seen?
fluorescence is lost as an emitted photon
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why is it important that the energy jump (the energy amount) is discrete?
the energy provided by the photon needs to be the exact amount so this jump in energy can happen, because energy of a photon is connected to wavelength for the jump you need to provide light of the correct wavelength
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some energy is lost in the process but what is the energy of the emitted photon always less than?
the incoming photon and for this relationship the wavelength is usually longer so you excite with blue light (low wavelength) and you obtain green light (longer wavelength- less energy of emitted photon)
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the light thats emitted always has what?
a longer wavelength (therefore the energy of the emitted photon is always less)
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example of a fluroscent substance?
fluorescein
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what does fluorescein do?
substance that absorbs light in the blue spectrum and emits light in the green spectrum
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why do you get no fluorescence if you excite fluroscein with red light?
red is not enough light to excite fluorescence
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what happens when you excite fluroscein with blue light?
correct wavelength to excite fluorescence so it emits yellow light
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what is this basically how what works?
Simple example but basically how a fluorescence microscope works
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what does a fluorescent microscope have to provide?
light of the correct wavelength to excite your fluorophore under the objective
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how does the fluorescent microscope light of the correct wavelength to excite your fluorophore under the objective?
you have a light source which can be a light bulb then we have a laser and filter that selects the correct wavelength you use to excite fluorescent dye
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what does the dichromatic mirror in the fluorescent microscope do?
separates out light that you use to excite the flurophore from the light you obtain from your sample
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how does the dicrhomatic mirror work?
it reflects shorter wavelengths and lets the longer wavelength go through and the longer wavelength is always the light emitted as there's always less energy from the emitted photon
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how is the endpoint of the fluorescent microscope?
a detector which can be a video camera
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technique we use for fluorescence microscopy?
confocal microscopy
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what is confocal microscopy?
a special type of fluorescence microscopy technique that employs different techniques to restrict light collection to the focal plane
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what is the problem with normal fluorescence microscopy?
we want light to come just from the focal plane of where our cells are but when you excite with light you get light from above and below your focal plane
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what is the result of getting light above and below your focal plane?
the organ of corti labelled with a fluorescent tag wanted to see where a protein was located- if you don't use confocal microscopy get a huge background and difficulty seeing protein localisation
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what do you get less of with confocal microscopy?
less background
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what is one particular confocal microscopy technique used in the lab
2 photon microscopy technique
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what does the 2 photon microscopy technique allow us to do?
use longer wavelengths to excite the fluorescence from under the focal plane
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why is fluorescent microscopy so important in neuroscience?
developed a huge amount of fluorescent sensors that are sensitive to diff cellular processes
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what are the 4 different catergories of fluorescent sensors?
1) ca sensors 2) voltage sensors 3) fluroscent molecules sensitive to changes in the rec channels on the PM 4) fluorescent molecules sensitive synaptic + vesicle release
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what are voltage sensors sensitive to?
voltage = the MP of the cell
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what was the first fluorescent sensor developed?
ca sensor
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why are ca sensors so important?
ca is a ubiquitous 2nd messenger it is involved in a variety of biological processes including exocytosis, contraction, metabolism and transcription
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when changes in cellular processes happen what do you usually have a change in? why?
IC ca concentration increases- as ca usually low IC nm levels
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what are monitoring when we are using ca imaging?
monitoring all these processes by monitoring changes in ca conc inside the cell#
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how can ca enter the IC cytoplasm?
VG ca channels, released from IC stores following a stimulus
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what do we use for ca sensors?
calcium dyes
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example of a calcium dye?
Fluo-3
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how is Fluo-3 composed?
1 part chelates calcium which is based on a molecule called BAPTA which is a calcium cheator 2nd part is the fluorescent part is GFP
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what is a chelator?
bonding of ions to metals
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describe the 4 steps of a fluorescence microscope?
1) select light of the correct wavelength to excite the dye 2) focus the light on the preparation 3) collect fluorescent light emitted 4) separate emitted light from excitation light 5) record emitted light
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what are ca indicators?
fluorescent molecules that can respond to the binding of ca ions by changing their fluorescent properties
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what is a calcium dye?
a calcium dye is a fluorescent molecule whose spectral properties change when it binds to ca
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what happens when Fluo-3 binds to ca?
it causes a conformational cahnge which causes the fluorescence emission to increase (so more fluorescence)
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what we record and quantify the amount of fluorescence emitted with a ca sensor we generally assume what?
the fractional change in fluorescence we record is proportional to the change in ca concentration
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by calibrating our dye we are able to do what?
precisely determine how the ca concentration has increased inside the cell
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3 techniques used to deliver ca dyes (and other dyes) into the cells?
1) patch pipette miroinjection 2) AM ester loading 3) genetically encoded indicators
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how does patch pipette microinjection to deliver ca dyes work?
fill micropipette with a solution containing the dye you want to inject inside the cell and then use the patch clamp technique to inject the dye inside the cell
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example of patch pipette microinjection?
vestibular cells- researchers were interested in localising the site of M where ca enters the cell so they used patch clamp to depol cell and fluorescence imaging to look where ca was entering
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what is AM ester loading stand for?
acetyoxymethyl esters of calcium dye
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why can acetyoxymethyl esters calcium dyes pass through the membrane?
they are non polar
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what happens once they've diffused across the membrane into the cell?
ester groups get cleaved by IC esterases which traps the ca indicator inside the cell
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how do we load acetyoxymethyl esters and the ca dye in?
we prepare a solution with the normal EC solution inside in cell and then put the dye at several concentrations then incubate cell with solution and dye, squash out loading solution
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what is the final method of delivery of ca dyes?
genetically encoded indicators
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how do you get a genetically encoded ca dye?
generate transgenic animals that express the fluorescent indicator of interest in the cell population you're intested in studying
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why is this very useful to use genetic tools?
you can target these fluorescent indicators at a specific cell population you want to study for example just want to look at cochlear IHCs but not supporting cells other methods would load entire tissues but this method allows it in specific cells
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what did they express in the mouse cochlear hair cells using a genetically encoded dye?
SyPhy a synaptic indicator in hair cells- the indicator was only present in the hair cells- 3 rows of OHCs and 1 row of IHCs but not other cells
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example of genetically encoded ca indicator and where it was used?
GCaMP7- used in the mouse brain in the cortex and the hippocampus only = very useful to target a specific population of cells
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what is another advantage of genetically encoded dyes?
you can get stable expression so you can study the same animal at different points but normal dyes dilute out and are extruded from the cell but GE persists. This technique is also minimally invasive as no loading dyes just expressed
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what can you see by fluorescence imaging recording taken from the cochlear?
there are ca waves in the developing cochlea- each fluorescent dot is an IHC loaded cochlea with ca dye fluo-4
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what is one characterstic of the IHCs?
during development of the cochlea they have spontaneous calcium transient waves of calcium without any stimulation- ca waves travel from cell to cell
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what were they interested in measuring of the ca waves?
the frequency of ca waves, the extension, measuring how they interact with the inner cells and where these ca waves are located in the cochlea
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what did imaging the entire turn of the cochlea allow them to do?
precisely locate which cells shows these ca signals
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what is the beauty of magnification in ca imaging?
by simply changing the magnification (by changing the objective) we can zoom in and look at individual cells
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how can you record the spontaneous ca dependent Aps that OHCs produce during develop?
record the APs using imaging as you can see flashes of ca at the base of the hair cell. During ca AP theres a huge amount of ca entering into cell dye allows you to see flashes which are APs
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where did they see precisely where the ca entry site is?
they are restricted to the synaptic pole of the cell
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what do ZF use to sense water movement around the body?
the lateral line neuromast
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what did they express in transgenic ZF to see the afferent fibres and hair cell?
2 different ca indicators in the hair cell and afferent fibres. Hair cells express RGECO- red ca dye and afferent fibres express GCaMP3 green ca dye- by having 2 diff colours we can look at the same time at ca signal in HC and afferent fibre
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what did they use to displace the hair bundle in the neuromast?
they used a fluid jet
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how did they record the ca response in the neuromast when they stimulated the hair bundle with a fluid jet?
they used fluorescent imaging as they had the 2 ca dyes expressed transgenically so you can simultaneously record ca response in HC + afferent
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what becomes brighter when the afferent is stimulated?
the hair cell
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in this ZF neuromast example what were they interested in?
understanding how the signal is transferred from the HC to the afferent fibre- particularly synaptic depression mechanism
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how did they look at the synaptic depression mechanism?
they present multiple stimuli and look how the response of the fibre decreases with the number of stimuli over time
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why is the ZF a useful model?
it's small and the brain is 1mm so you image the entire brain under the microscope and it's transparent
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what did they express in the ZF neurons to see the whole brain?
GCaMP6
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what were they looking to understand by imaging the whole brain?
how sound is processed in the fish brain- put ZF under microscope play sound and record from which area of the brain responds to sound stimuli
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when do you particularly not have to manipulate the sample during imaging recordings?
when using genetically encoded indicators
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what is the drawback of using imaging?
1) signal to noise ratio 2) sampling rate 3) photobleaching 4) photodamage
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why is signal to noise ratio always a problem in imaging?
you're collecting photons of light from very small structures inside the preparation so num of photons you detect are usually very very small and you have to detect this photon on huge background noise
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why does signal to noise ratio give problems with sampling rate?
if you acquire a few photons you need to expose your camera for a longer time to acquire more photons- taking longer exposure limits the amount of exposure you can take in time which influences sampling rate of your recording
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what is the problem of photobleaching with imaging?
usually fluorescent dyes go through many cycles of excitation and detection they become damaged and don't produce fluorescence anymore and this causes degradation of your signal over time- keep imaging won't see fluorescence anymore
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what is photodamage?
photodamage is when you expose your sample excessively to light you affect some physiological processes and damage things you want to measure
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what is electrophysiology?
electrophysiology is the study of electrical properties of biological cells and tissues
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what are we concerned with when we talk about electrophysiology?
1) voltage 2) Current
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what voltage is usually measured for cells?
there's an electrical potential difference between the inside and outside of the cell = MP- measured in volts or mV. Usually -ve on inside compared to outside. Use electrophysiology to measure change in MP
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why is current of interest in electrophysiology?
ion channels- let ions in/out of cell. Movement of ions = movement of charge which creates a current
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what do you do with current in electrophysiology?
measure the current that goes across the CM or inject current into that cell to look at the response
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what is current usually measured in?
amps- in cells currents are peako amps to nano amps = very small currents
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what is the technique most used in electrophysiology?
the patch clamp technique
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who founded the patch clamp technique?
Neher and Sakmann- won noble prixe
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before the patch clamp technique how did they measure or change the potnetial in the cell?
needed electrical access to IC side of the cell and people used sharp electrodes where they impaled the cell to measure currents
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why was that technique of impaling the cell not useful?
it didn't allow for stable recordings for a long time
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what did Neher and Sakmann have the idea to use instead?
use a glass micropipette instead of a sharp electrode
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what patch clamp technique is most widely used?
whole cell patch clamp technique
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how does patch clamp work
use a glass micropipette which contains a fluid that mimics the IC fluid present inside the cell and inside the pipette there is an electrode which creates an electrical conduit between the pipette and recording electronic equipment
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what do we use to get the pipette close ot the cell?
microscope- we touch the M and apply suction (negative pressure) to **** a patch of cells inside the pipette forming the gigaohm seal
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why is it called a gigaohm seal?
because there is a very high resitance between the inside of the pipette and your EC solutin which means all your currents you provide will go into your cell and not the EC space
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what can you do from this cell attached configuration setup?
you can record the current going through the channels you have in the patch of membrane inside your pipette so study kinetics of opening/closing of these channels, Po, how much current goes thro, how they respond to stim = cell attached configuration
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how do we get a whole cell configuration from the cell-attached config?
apply more suction and break the patch of membrane that appears inside the pipette and make the inside of the pipette continuous with the cytoplasm
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why can you control the MP in this whole cell configuration?
it creates an electrical connection between the pipette and the cytoplasm- you can inject current and measure the MP and currents
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what is the main disadvantage of the whole cell patch clamp technique?
when you rupture the patch the solution inside the cell is usually washed out very quickly by whatever you put in your pipette so you lose the IC buffer usually present in cells
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what are the 2 pipettes that are important in the patch clamp equipment?
patch clamp pipette (used to enter the cell) and the bath electrode (ground)
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what does the patch clamp pipette do?
used to access the cell
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what is the gorund electrode?
touches the bath solution and creates a continuous electrical circuit between the pipette, equipment and cell
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what is the core of the patch clamp equipment?
the patch clamp amplifier- we measure very small currents to record them we need to amplify them so have this device to do this
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when we use patch clamp technique what 2 recording configurations do we use?
1) current clamp config 2) voltage clamp congif
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What is the current clamp configuration?
use the current clamp we control the amount of current that's injected into the cell and we measure the change in MP as a result of current injection- most natural way to record from a cell
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why is this the most natural way to record from a cell?
this is what the cell naturally does- responds to changes in current by opening of channels current coming inf from ionic channels and changing the MP one example is AP
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what we can also do when we inject the current and see how the cell responds?
record the spontaneous activity- immature IHCs fire spontaneous APs and using current clamp config without injecting any current (So no stim) can record the spontaneous AP freq or how it changes during stim
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what is the other configuration?
Voltage clamp
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what do you do with voltage clamp?
control the MP of the cell and measure the amount of current that goes through the membrane
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why is the voltage clamp config important?
there are many ion channels that open and respond in response to dep and they open and close in a narrow range of MPs values
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what can we determine by using voltage clamp on IHCs?
diff characteristics eg voltage dependence of the current so how the current depends on voltage. eg see currents due to mainly opening of VG K channels in the M and we can determine if they're dep on voltage
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what does measuring the size of the current give us info on?
how many cells there are
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why do we want to study the kinetics of this current?
voltage is stepped up but the current doesn't increase immediately follows certain kinetics, by looking at kinetics we can determine which channels are involved in these currents
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how can voltage clamp be used to separate out the different conductance?
see which different channels are generating the channels- so in the trace see before +ve current there is a -ve part of the current = -ve part is K going out whereas negative part is positive ions like Na and ca going in
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how can you isolate the current eg only record K currents?
do voltage clamp then use blockers for particular channels eg tetrodotoxin to block VG Na channels then we can isolate currents and see which bits where the Na currents and measure properties and characteristics of individual currents
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pros of patch clamp?
1) higher signal to noise ratio compared to imaging (can record v small currents) 2) sampling rate is much better- can track processes in millisecond scale eg APs
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what are the drawbacks of patch clamp?
1) need to insert an electrode in the tissue and manipulate the IC medium which disrupts the cell you're studying 2) recording limited to a few cells bcos you can't patch many cells
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does electrophysiology or imaging have better temporal resolution?
electrophysiology can look at individual APs which are very fast and look at synaptic release very fast EPSP and IPSPs
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does electrophysiology or imaging have better spatial resolution?
imaging- can look at individual part of the cell or the whole organism like ZF
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does electrophysiology or imaging have better scalability?
scale from big to small with imaging- individual synapses in the ZF neuromast and the entire brain
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does electrophysiology or imaging have better sensitivity (signal to noise ratio)?
electrophysiology
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what's more minimally invasive?
imaging- especially if you use genetically encoded indicators
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if you want to establish when an event occurred what do you use?
electrophysiology- better temporal resolution - look at individual channels or AP spiking
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what do you want to use when you want to establish where something happened?
imaging
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what is voltage clamp used for in particular?
to record voltage dependent currents in cells which gives us information about the ion channels present in the basolateral m of the cells
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what can current clamp technique record changes in?
MP such as AP
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why use electrophysiology when temporal dimension is important?
due to the higher temporal resolution of patch clamp electrophysiology can see APs that are very fast msec events
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why use imaging when spatial dimension is important?
high spatial resolution- can look at events inside a cell and due to scalibility of imaging we can look at a single cell or even an organ
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how did researchers combine electrophysiology and imaging to find out where the MET channel is localised?
used fluoresence imaging to see spatial location of MET channel in the stereocilia and at time used electrophysiology to control the MP of the cell to control the precise timing at which ca enters into the cell and to deliver the dye into the cell
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what was the 3 step protocol they used?
1st depolarised cell to ca reversal potential to stop ca entering the cell 2) displaced bundle using mechanical stimulus to open MET channels (Cell depol + no ca entering) 3) hyperpol cell again to a -ve potential driving ca into the cell
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when do you see the ca entering the cell?
when they go back to a negative MP with the patch clamp-hyperpol the cell see where it enters sterocilia
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What did they conclude from this?
that ca enters only in the 2 shortest rows of stereocilia and therefore the MET channel is located at the bottom of the tip link
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what technique did they use to ask if HC spontaneous activity is coordinated by extrinsic factors?
used imaging and recorded the AP activity as the flash of fluorescence in the IHC when they have this spontaneous activity
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IHC before they before they become a fully functional sensory receptor do what?
fire ca dependent APs (to refine auditory pathway) and we can record this AP with electrophysiology and imaging
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at the same time during development what else happens?
non-sensory cells form spontaneous activity which is represented in the form of ca waves that spread from cell to cell
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what could they see in the cell types with imaging?
you can see 2 different kinds of spontaneous activity- the one present in the IHC and the one present in the non-sensory cell here. They don't look independent- see large ca waves in non-sensory cell you see activity in IHC increase
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what do you see an increase in at the same time?
increase in the fluorescence of many IHCs at the same time
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what did this raise the question of?
is there a form of coordination between these different forms of spontaneous activity- is HC spontaneous activity coordinated by extrinsic factors = by ca waves in non sensory cells
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what did they have to measure to answer this question?
how the firing rate of the IHC changes in the presence or absence of ca waves in non sensory waves
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how do you measure the firing rate of the HCs?
count the single APs spikes in a certain time window = get firing rate
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why can't imaging be used for this
they fire really quickly so spikes fuse together can't distinguish indiv spikes, you can't see the individual spikes with imaging as temporal resolution isn't good enough, can only resolve when firing rate is quite low
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what is too low in imaging to see individual spikes?
temporal resolution so use electrophysiology as it has a better temporal resolution compared to imaging
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what did it show when you compare the AP in IHCs with fluorescence imaging compared to electrophysiology?
AP recorded with imaging is much longer compared to the one with electrophysiology as the slow nature of the dye that has to bind+unbind ca during the AP so while the electrical impulse only lasts msecs fluorescence lasts longer bcos of long binding
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what do you lose when recording APs with fluorescence? why?
lose the resolution of individual spikes because it takes long for the dye to bind on and off
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how can we solve this problem?
use fluorescence imaging to record the spatial information from ca waves in non-sensory cells but use patch clamp to record from 1 indiv IHC at the same time with good temporal resolution
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what did this allow them to do?
record the spikes from IHC and see individual spikes and then a ca wave appears in the non-sensory cell (seen with imaging) and the spike firing rate increases and can count the indiv spikes
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what was the firing rate of the IHC correlated with?
with ca signals in the non-sensory cells- when there's a ca signal from non-sensory cells you get an increasing in firing rate of IHCs + can quantify see how much firing rate of IHC is higher during ca waves
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why was it good that they could quantify the firing rate of IHCs in these 2 conditions?
can proceed to investigate the mechanism that lead to this coordinated activity between the 2
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what is optogenetics?
control the activity of cells using light and transgenic animals (in this will be excitable cells- able to fire APs such as neurons or IHCs)
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why do we want to control neural activtiy?
1) activating/silencing neurons informs us of functions or understand the role of a particular neuron/neuronal population in a circuit so activate/silence see what happens to circuit 2) understand role of particular neuron in producing beh
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what was the last reason we want to control neural activity?
understand how the activity of a neuron influences functional changes in the neuron itself- eg activity-dependent changes in synaptic plasticity
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2 ways to control the activity of a neuron?
1) electrophysiology 2) optogenetics 3) chemicals
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how can electrophysiology control the activity of a neuron?
can depol or hyperpol the neuron or cell so decreasing or silencing AP but is limited to usually one cell at a time
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what is the downfall of using EC electrical activity to stimulate multiple cells at the same time using an electrode?
we can insert the electrode in our preparation but this is invasive and gives poor spatial control about which cells you're stimulating as all cells respond to electrical stim so cannot control which cells activating within distance from electrode
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what is another alternative?
chemicals that activate or inhibit the activity of a neuron
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what is the problem with using chemicals?
poor temporal and spatial control as you cannot control the diffusion of these chemicals and when they stop acting on the neurons
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what is the technique that tries to overcome all these limitations?
optogenetics
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what does optogenetics use?
optics- so a characteristic of imaging to control the activity of a cell
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how does optogenetics overcome the limitation of imaging not being able to control the activity of cells?
With imaging we can record the activity of cells but differently from electrophysiology we cannot control the activity of cells so optogenetics tried to overcome this limitation of imaging by using light and optics to control the activity of a cell
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what does optogenetics do?
a technique that integrates optics so light and genetics to directly induce electrical currents into specific cells
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what family of proteins are used in optogenetics?
family of light responsive proteins that belongs to the opsins family
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what are ospins
light responsive channels or pumps that can be genetically encoded into neurons
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when these channels are activated by light what happens?
these channels/pumps depol or hyperpol neurons so by using light to depol/hyperpol cell we control the activity of a cell
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what is more recent optogenetics, ca imaging or electrophysiology?
optogenetics
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what can be used to activate a subset of the cells in your preparation?
optogenetics
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why is electrical stimulation no good to activate a subset of cells?
you have no control in which cells you activate as electrical is spread in tissues and cells respond to electrical stimulation by depolarising and activating
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how does optogenetics overcome the fact that we can't focus an electrical stimulation?
opsins can be expressed only in neurons we want to stimulate and activate
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how do we focus light in optogenetics?
can control spreading of light, to focus light we use lenses so we can focus light to activate only the area of preparation in which the cells we want to activate are present
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where is channelrhodopsin isolated from?
chlamydomonas reinhardtii
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can optigenetics be used to activate or silence neurons?
both, excite (depol)- using channerhodopsin and inhibit (hyperpol) using halorhodopsin
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what is Channelrhodopsin? what does it do?
Channelrhodopsin is a light-gated ion channel that opens in response to blue light
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what light opens Channelrhodopsin?
blue light
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what happens when you shine blue light on channelrhodopsin preparation?
express and open channelrhodopsin
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what goes through Channelrhodopsin?
is a non-selctive cation channel so lets positively charged ions through in particular ca and na therefore depol the cell
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if you express Channelrhodopsin what can you do to AP firing?
can promote AP firing by turning on blue light- these are genetically encoded
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what colour light does Halorhodopsin respond to?
yellow light
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where is Halorhodopsin isolated from
natronomonas pharaonis
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what opsin is expressed to inhibit the activity of the cell by causing hyperpol?
Halorhodopsin
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is Halorhodopsin a channel?
no it's a light-gated pump which activate in response to yellow light (570nm)
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what ion does Halorhodopsin pump?
is a cholirde pump so when its activated it pumps chloride ions from EC to IC side- creates a negative current going into cells so hyperpols cells and reduces AP firing
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can you use Halorhodopsin and channelrhodopsin at the same time?
yes because they're excited by diff wavelengths, Halorhodopsin- yellow and channelrhodopsin blue- so can activate and silence neurons in our preparation
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why use optogenetics?
1) non-invasive 2) activates multiple cells at the same time by shining light 3) tight spatial control- express to certain cell subtypes and focus light 4) fine temporal control 5) can be used in freely moving animals
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why is optogenetics non-invasive?
not damaging cells with electrodes just turning a light on and off
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why do you have fine temporal control with optogenetics?
channelrhodopsin and halorhodopsin cloe and stop pumping chloride ions as soon as we turn off the light so it's readily reversible
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disadvantage of optogenetics?
ineffective in deep brain areas because light can't penetrate deep inside the brain
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why is optogenetics very useful for functional brain mapping?
if you want to map circuit organisation in the brain which neurons are connected in your preparation you can use optogenetic
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how is optogenetics used for functional brain mapping?
want to understand which neurons send inputs to a particular pyramidal neuron in layer 5 of cortex, express channelrhodopsin in neurons of the brain and use patch clamp to record from this particular neuron
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what happens when you focus blue light sequentially in different positions across the preparation and record the response?
light activation induces response in cell see how much the response is when you activate a far away neuron no response or close neuron and get response
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what do you get in the end?
an entire map that shows the receptive field for this particular neuron so you can understand how circuits are organised and which neurons send inputs to neurons in layer V
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what can optogenetics be used to link
neuronal activity to a behaviour or certain output
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why were researchers limited looking at animal behaviour before?
couldn't record from neurons while animals performed a task so not sufficient to know if activity in these neurons are causing this behaviour- not casual link for behaviour
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what do you have to do to establish a casual link?
have to activate these neurons and see whether this influences behaviour
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what did thy show with a mouse expressing channelrhodopsin 2 in neurons of the motor cortex?
opening in skull and blue LED implanted on his head- mouse moves around but when blue light is switched on moves around more and stops when you turn the blue light off
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what is this proof of?
that using optogenetics we influence the behaviour of an animal by activating it's neurons in different parts of the brain
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what response is very well studied in the fish?
the escape response if you touch the fish it tries to escape by firstly bending its tail in order to turn its body and propel away in the opposite direction
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what did researchers wanting to show with channelrhodopsin 2 expressed in somatosensory of ZF?
whether activation of the somatosensory neurons alone without somatosensory input was sufficient to induce the escape response in the fish
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What did they show by expressing channelrhodopsin 2 in somatosensory neurons that are responsible for transducing touch stimuli (when touch fish they try to escape)
embedded the fish in agarose but freed tail so switched on blue light and activated somatosensory neurons and the tail of the fish moves because it initiates the escape response
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what were they able to determine?
only activating the somatosensory neurons were they able to trigger the escape response of the fish
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how did they show that even activating one somatosensory neuron was sufficient to induce the escape response in the fish?
focused light down to a single neuron
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before IHCs become fully functional sensory recs they respond to sound with what
graded receptor potentials
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what do immature IHCs fire?
spontaneous ca dependent APs
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when do you get spontaneous ca dependent APs from IHCs?
well defined time window mouse before the onset of hearing (PN12) and particularly before full maturation of the IHCs
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in the IHCs how can you see the changes happening in dev?
see the change in currents present in IHCs
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during development what happens to the ion channels that are characterstic of immature IHCs?
they are progressively downregulated and some of them completely disappear just at the onset of hearing or right after (SK2?)
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what starts to be expressed at the same time?
ion channels that are characteristic of mature hair cells that are responsible for transducing sound in the HC eg KCNQ4
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what is the q they wanted to address about this AP activity in the IHC in development?
is the spontaneous activity required for IHC dev
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what channel did they overepxress in a mouse model which is responsible for the AP activity in the IHCs?
SK2 channels overexpressed
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what does SK2 overexpression cause?
is a ca activated K channel causes the AP in the cell to become faster and the firing rate to become higher
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what happened because the AP activity in the IHC was altered?
HCs fail to mature properly - eg synaptic properties- synaptic release as a function of ca current by measuirng change in capicitance it remains in the high power relationsip characterstic of immature IHC
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so what happened when IHC spontaneous activity is altered?
IHCs fail to develop properly
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why did they want to control the IHC development in vitro?
1) if we could develop a mature IHC in vitro we may be able to use less animal models 2) if we can create an IHC in vitro may be able to develop therapies for people who lose IHCs due to injury or disease
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in order to create an IHC in vitro what do we need to do?
understand which steps are important for making a mature IHC
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what did they think spontaenous activity was impotnat for?
maturation of the IHCs
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what happens in vitro?
IHCAS lost sponteanous activity 24 hours after you culutre the IHCs
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how do you culture IHCs?
make an organic culture, put cells in a petri dish, leave them in an incubator 24 hrs later they don't fire spontaneous APs anymore
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if you stimulate these IHCs that have lost their spontanous activity because of being in culture can they still fir eAP?
yeah if you stimulate them the cells are able to fire APs if you inject current into the cells
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what happens to the maturations of the IHCs when you culture them?
they don't mature they stay in the same state they were in when you took them out
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what was there hypothesis about why they don't mature properly?
they don't mature properly because spontaenous acitivity is not present in vitro
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how did they test the hypothesis that spontaneous activity is necessary for IHC maturation?
re-introduced spontaneous activity into the culture IHCs using optogeneitcs- expressed channelrhodopsin in IHCs and stim them with light so if this is sufficient to induce maturation of IHC in vitro
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what will channelrhodospin 2 do if expressed in IHCs?
will depolarise and excite the cel
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what do we do when we culture the cochlea and make organotypic cultures of the cochlea?
put it in the incubator where the cochlea is on top of a blue LED, by controlling light from the LED we can control activity of the IHC
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what was the setup?
have array of 6 LEDs- put a mutliwell on top of this array and then in each well we put a cochlea organotypic culture of a cochlea then control in the incubator with an computer progr to control light stim- eg intensity or freq of light pulses
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what are the 5 characteristics needed?

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1) high temporal resolution 2) high spatial solution 3) scalability 4) high sensitivity 5) minimally invasive

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why do we want high temporal resolution?

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Card 4

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why do we want high spatial resolution?

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why do we want scalability?

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