Discussion Flashcards
Why do you think the correlative assessment for TF photometry did not work?
What approaches are you trying to rectify this?
- The cone of light from the TF is not homogenous. At higher regions it can spread downwards in a cone across deeper regions of the TF. Therefore, quantifying the way we are by measuring a cylinder, may miss plaques that are captured with photometry.
- The estimation/assumption of the light protocols have not taken into account the difference of light profile within brain tissue due to light scattering. This means some light power may be over/under-estimated.
We are working with collaborators to try understand more about the light properties of the TF.
By getting 2p values in the slice experiments to get a vlue of the fluorescence that is acquired at each galvo in tissue.
Also, by using the exact voltages of laser applied along the galvo and normalising against this as this can account for these variations.
What did the solicon prove recordings in Soula et al.
- measure
- how did they measure
- what did they find?
Craniotomies were made for the hippocampus CA1, V1 or EC. Silicon probes were mounted on custom-made three-dimensionally (3D)-printed drives and were inserted into the target brain region.
3D headcaps and copper mesh was connected to a screw in the skull and served as a Faraday cage.
A 10-min baseline was recorded first, followed by 40-Hz stimulation for 30 min and concluding with a second 10 min of no-stimulation control epoch.
They then spike sorted and did LFP analysis.
failed to modify native local field potential (LFP) gamma-band power in V1, EC or CA1. Instead, it induced a steady-state response in a narrow band at 40 Hz in V1 (14 sessions in 5 mice) but not in EC (7 sessions in 3 mice) or CA1
As a population, V1 neurons showed an overall significant increase in firing rates during the 10-s-on 40-Hz epochs, compared with the interleaving stimulus-off epochs (P = 0.0066; paired t-test), whereas changes in EC and CA1 populations were not significant
a substantial fraction (132 of 458; 28.8%) of V1 neurons were significantly phase-locked to 40-Hz stimulation (Fig. 4i–l). In contrast, only a small fraction of EC (7 of 211; 3.3%) and hippocampal CA1 (126 of 1,874; 6.7%) neurons showed significant phase-locking of their spikes (Fig. 4k,l)
What other therapies do you believe this approach would be transferrible for?
The new anitbody therapies.
Other new drug targets.
How are you getting the value 36 um resolution?
Because the length of the TF is 1.8 mm and we were able to record a max of 50 measures from -5 to 5V galvo.
By dividing the total length of the TF by 50 this provided 36 um.
But this will vary depending on the galvo voltages used. So for me with a galvo of -1 to 4.5V and 50 measures, by resolution was roughly ~39/40um as this is 0-4.5 which is ~1600 um for 41 measures.
How can you increase the number of measures on ther TF?
By increasing the sampling frequency of the galvo mirror to make it work faster and protrude light from increasing regions along the fibre.
However, this can be damaging to the galvo and can be computationally challenging before even thinking of the difficulties extracting a pure light profile.
You mention that TFs are the best, but what other ways could you measure?
There has a group in DEEPER consortium which have developed a fibre that is cleaved at an angle at the end of the fibre and use this to imagine inside the brain. They are able to image from the brain surface to the deepest region and are working on establishing this for chronic procedures.
Also groups working on 3D scans and new probes for making the plaque but these can genreally have challenges with resolution and acquititon times, and freely behaving conditions.
Another group have been working on improving endoscopes into a microendoscioe format which would allow imaging.
Why are the TFs limited to 1.8mm in depth?
This is the limit my collaborators can achieve for the tapered region to be able to have a reliable light collection.
We found that the light-sensitive region along the taper, defined as the collection length L, grows with increasing fiber NA and decreasing ψ.
But the signal propagation can increase as long as you do not mind about signal collection.
What is a microLED array? What does Scharf et al develop?
A microLED array is a probe that can be implanted into the brain and has several LEDs mounted on the shank. By electronically controlling these, we can deliver light into the brain in a depth-resolved manner.
They were the first to develop a high-density silicon-based μLED array, that had up to ninety-six μLEDs emitting at a wavelength of 450 nm. They confirmed their use by inserting them along side a multishank silicon probe and montioring optogenetic changes in neuronal activity. (Chr2-PV)
How is the NA and TA a limitation of the TF?
NA and TA can limit the depth resolution of the TF.
increased NA and decreased TA best length - ~ 2 mm
However NA at 66 is extremely brittle and difficult to handle for implants.
How could you increase the solubility of M04?
Mixing with DMSO alone and then creating a smaller stock solution.
When you made new batches of M04, how did you ensure it was the same concentration?
We did test calibrations using the new dilutions of M04 and compared the fluorescent profile.
It was mainly consistent across batches.
How do you know that M04 is susceptible to photobleaching?
We done a pilot experiment in vitro where we would have the optic fibre in a concentration of m04 and applied continuous light to the solution.
We found the fluorescence declined rapidly, within several seconds.
Why would targeting specific stages of AB be important?
Because they can represent dfferent toxicity.
For example, previous GENUS papers found differences in soluble AB like monomers and oligomers, but not insoluble after acute stimulation.
Why does the fact its a blue shifted dye mean its susceptible to AF?
Its been found at these shorter wavelengths are absorbed more by tissue.
What other neurodegenerative molecular hallmarks could this be adapted for?
In terms of AD, this could be AB or tau.
In terms of other neurodegenerative disorders this could be alpha-synuclein, TDP-43
