Ch 5 Flashcards
Why might the post-mortem assessment affect the analysis of GENUS?
As we only have a single read-out post-mortem. As plaque pathology is different across mice, we would need to see a pre- and post-measure to illustrate an actual change in pathology.
Why would the singular approach be different?
The original papers that complete the multisensory stimulation show that this modulated neuronal activity across wider brain regions with pronounced effects. Therefore, it may be more effective at reducing plaque pathology.
What is the hypothesis and aims?
Hypothesis: novel approach allows monitoring of plaques caused by GENUS
Aim: to expose AD models to GENUS and monitor changes in AD pathology
Why were the same animals used as before?
for 3Rs. They were used adter PK to ensure they are being used maximally.
Why were the electrophysiological recordings not completed on the FP mice?
There has been papers, including my group, that have done dual photometry and ephys experiments by mounting an electrode along the fibre shank.
However, currently, TFs are just too fragile for this approach for this to be feasible. However, there are now being TFs made with electrode channels along this.
Another consideration is the 40-Hz noise that would occur due to the LEDs, which required an extra internal faraday cage which would not be feasible for long-term recordings as it is not freely behaving.
Why were walls lined with black tape?
So that it limits reflection off the walls and ensures the light being delivered to the mouse is the 40hz light.
This is the same as the landmark study.
Why white LED light?
Broad Spectrum: White LEDs emit light across a broad spectrum, covering a wide range of wavelengths. This broad spectrum ensures that the flicker stimulation affects a wide range of photoreceptors in the eye, including both rods and cones. This comprehensive stimulation can be beneficial for certain research or applications where a broad response is desired.
Why did you choose 20-cm and 70-dB for the speaker?
20-cm was the same as the original study
70-dB is high enough for the c57 background to hear according to shuzo previous study that monitors the ABR in c57 mice.
Why did you use a 50% duty cycle?
So, during a 40 Hz stimulation with a 50% duty cycle, the LED is on for 0.0125 seconds (or 12.5 milliseconds) during each cycle.
This is like previous papers.
What does 100kHz mean for stimulation?
Full range of mouse hearing so we are activating the whole tonotopic auditory pathway
Why can the NIDAQ not provide the sufficient current?
Because the NIDAQ has current safety limits which are below what is required for dlivery of the 5V LED with ~3A current flow.
What is a
- MOSFET
- gate resister
- pull down resister?
Mosfet is an electrical component that is used as a switch to control the current flow through a circuit. This was used to control the current flow to the LEDS. It has a gate, source and drain. The date is the gate resistor, the source is the DAQ connection and the drain is the pull down resistor.
Gate resistor is used to control the overcurrent of the system. So the impedence is chosen so that it limits the current flow to protect tge system from damage.
Pull down resistor works as the drain of the mosfet as its connected to the pin it will have some voltage so this works to a low voltage value.
What is the function of the gate resister? Why 1kOhm?
prevents overcurrent from teh NIDAW as this can damage the components
This minimises the current range to only a few milliAmps.
What is the function of the pull-down resistor? Why 10kOm?
switch to the MOSFET, where if the DAQ is on, it will allow the current ro flow
as its higher than the gate resistor, allowing a good amount of voltage at the gate - it needs to be high enough to allow sufficient voltage flow.
Can you explain how this electrical breadboard works?
Each pin on the board allows a connection to be formed.
The two outer channels represent power rails which are positive and negative to allow power flow and this power will flow horizontly.
Then to transfer the power to the terminals, a connection must be made and this allows power accross horizontal channels.
Then, the DAQ connection will contact the pulldown resister. THE DAQ ground on MOSFET and the LED on the MOSFET.
This voltage flow will then traveel along the row to turn on LED drip.
What are broadband noise and puretones?
Broadband noise contains all frequecies of sound.
Pure tones is a sound that has just one frequency.
Why does the sound need to be amplified before reaching the speaker?
It is required to amplify the analog signals into sound.
Why does the microphone require an amplifier?
The microphone picks up small vibrations that are converted to a voltage signal that is amplified and recorded.
Why do you need to use the root mean squared voltage to play a broadband noise?
When playing a broadband noise from a speaker, you need to control the voltage that drives the speaker to ensure that the sound produced has the desired characteristics, such as amplitude and intensity.
Audio signals are dynamic and can vary in amplitude over time. The RMS voltage takes into account these variations by averaging the square of the instantaneous voltages over time and then taking the square root. This provides a measure of the effective voltage level that drives the speaker, which is more representative of the overall power delivered than simply using the peak voltage. Therefore, it represents the average voltage signal.
What is a bipolar electrode and why did you use it?
This is two conductive electrodes that are mounted together. They have a small offset and allow recording of electrical signals at the same time.
We did this as it provides a hippocamal measurement at two regions, allowing improvment of targeting success.
Why did you do EEG over the PFC?
PFC provides a hub of brain functionality and activity.
Also, previous papers discuss the spread of gamma oscillations to the PFC.
Why did you have EMG?
These mice were used for other recordings for a seperate project.
This was a sleep project where they required the measurement of muscle tone.
Why did you have two ground connections?
Incase one is faulty, the other one can be used. This prevents the loss of the animal without recording.
What is the difference in EEG/ground and EMG wires?
EMG are coated in silver to allow better preservation as they are implanted into the muscle.
Why do you use stainless steel wire instead of the copper wire for electrodes?
Copper will oxidise within the brain and can be toxic. Whereas stainless steel is conductive, non corrosive and non toxic.
Why was an offset of 0.1 cm used?
The target was the CA1 and so to ensure the targeting was appropriate.
Why was the electrode wire attached to the pin using epoxy instead of soldering?
Because the stainless steel doesnt conduct with solder, instead the epoxy allows good connection to the pin.
Why do you cover the connections with dental cement?
This works as extra insulation of the pins to reduce electrical noise.
Why were the electrodes superglued together?
To maintain the shape of the electrodes and to make insertion into the brain easier with the electrodes being sturdier.
Why do the electrodes need to be within a certain impedance?
The resistence controls the current floe through the electrode.
If the resistence is too high this will prevent the current flow and reduce signal strength.
If it is too low, it may be noisy.
Why were ephys animals housed alone?
For the seperate project, they were recorded from their home cages.
Why were the ground screws above the cerebellum?
Because this has the least neuronal activity so will not influence the recorded signals.
Why was that age range used for ephys recordings?
Because these mice on the c57 background begin to become deaf from around 5-months.
Why do you use a preamp and amp for plexon?
Because the signals that we are recording are on a miniscule range and are therefore greatly susceptible to noise.
Therefore, they need amplified as qucikly as possible. Thus, they are amplitified on the skull by preamp.
Then, they are amp 100 times by amp.
Why were the signals amplified 1000 times?
As signals are so low so they need maximised so they are on an identifiable range.
Why was ephys recorded at 1-kHz?
Gamma oscillations are fast rhythmic fluctuations in neural activity, with cycles occurring within milliseconds. A higher sampling frequency provides better temporal resolution, allowing you to capture the rapid dynamics of gamma oscillations with greater precision.
And to preserve the signal by oversampling
Why was the internal faraday cage required?
Because the treatment chamber has the LEDs which will provide 40-Hz noise. So the mice are within the internal chamber so ground from them.
How can you ensure the signals were not noise from 40-Hz?
First we tested with the connections in salineinside the internal faraday cage and did not find this.
Then, we found that the signal intesntiy varied acrtoss the duration of treatment on which suggests this is not due to noise as we would expect that to be more stable.
Why did you perform the treatment for ephys in a randomised order and how was it randomised?
To be sure that the effect being seen was not due to the same order being presented.
Using a built in labview generator.
Why did you only supply the treatment fo 60s? As this is different to the treatment recordings will this not be different?
Because the change in brain state can occur rapidly.
We do not expec it to be
What is int16?
Its a data type for storing infomration.
This approach allows lowest memory to be used and as these files are large we use this.
Why was the sync pulse 0.5 v used?
Becuase the speaker was driven at 0.77V and the led at 5V which meant that both sync pulses will show a voltage greater than 0.5v in their on periods.
Allows detection of the stimulation in the ephys channels.
Why do you have three different frequency ranges for ephys analysis?
The first was to show if we see an increase in 40-Hz over the duration of the treatment and therefore focussed surrounding the 40-Hz band which is why we choose 35-45.
Second was to see changes in the frequyency bands across treatments which is why we choose a wider band - 10-60.
Lastly, we wanted to quantify a specific target of 40-Hz to see the exact change in 40-Hz power which is why we choose 39.9-40.1
Was the mean not calculated for the heatmap analysis for ephys?
Yes it was, it shows the mean PSD analysis across mice.
Why did you use shapiro-wilk tests and what does this do?
We wanted to check the normality ofg the data in order to present and analyse it properly. It tests to see the goodness of fit of the data to a normal distribution
We choose this normality test because this is more suitable for smaller groups of data.
Why did you do a one-way anova on ephys data?
Because I wanted to compare the 40-Hz power across each treatment, which is only one indepdentn vairable.
As the data was normally distributed this was the best choice.
Why did you do bonferroni corrections?
If the one-way anova showed that the test was significant, we wanted to determine what treatment groups showed a statistically significant difference to one another.
bonferroni is a good way to do so as it is conservative and will adjust the p-values appropriately to avoid false positives.
How was the treatment order randomised if the same two treatments had to be completed as the last recordings?
A recording schedule was planned before the recordings began, always placing the AC or NS as the last rreatment. We then used a random gonline generator.
Do you worry that your older mice cannot hear the sound?
We did consider this when designing the experiment as our group has previously assessed the hearing abilities of c57 mice.
They found that around 6-months their hearing threshold should be around 35dB so should hear it well. Also, the orifinal paper has mice this age and also see an effect.
Why do you only end on AV or NS?
Because as our approach has not been fully confirmed for real-time assessment using a treatment, we wanted to confirm the effects seen with histology.
For this, due to our low sample size, we choose to have a non treatment control group and a treatemnt group.
As we hypothesise that the multisensory approach should be most effective, we choose this.
Will the fact the mice have underwent all treatments not effect the histology analysis?
The original paper that show the effects on soluble AB levels with ELISA show that the effects recover betweeen 12-24 hours. As all treatments were seperated by at least 48-hours, this should not effect this?
This will require alot of redosing of M04… what will this affect?
The clearance of m04 is unclear.
However, we maintained fluorescence and so it is liekly it will continue to bind to new oodiogmers in the brian.
Why did you use different powers?
At first, we had a spell where we were concerned that the higher powers were causing bleaching of m04 and therefore we reduced the powers.
However, this minimised our signal intesntiy and we increased this again.
Why are the number of reps different?
Because we have issues with laser stability, this means our signals can contain noise and therefore, variability across our recordings.
To increase the iterations to allow a better median signal and to try reduce this noise, we increased the number of reps.
