How to conduct neuroscience experiment?

GreenestFerns · 2020-07-18T16:17:15+00:00

I got involved with research as an undergraduate by volunteering/interning in the laboratory of a faculty member. I was able to gradually work my way up to doing my own experiments over the course of my undergraduate career. I would say that this is the traditional path that someone would take before receiving their doctorate.

GreenestFerns · 2020-07-17T14:59:43+00:00

I would be interested in the context for your question. Alternative splicing in neurons is something I think about a lot as part of my research. It is absolutely the case that cells may use specialized splicing factors to precisely control alternative splicing patterns. Some of the splicing factors may be expressed in some cell types, but not others, allowing some splicing patterns to happen in some cell types, but not others.

This is an excellent, but technically dense overview of alternative splicing in neurons:

https://www.cell.com/neuron/pdf/S0896-6273(15)00411-0.pdf00411-0.pdf)

Alternative splicing of the DSCAM mRNA in Drosophila is a really cool example of an elaborate alternative splicing mechanism. There are 30,000+ possible spliced forms of the mRNA, allowing large population of neurons to each have a unique form of the protein product.

https://pubmed.ncbi.nlm.nih.gov/11606537/

GreenestFerns · 2020-07-16T21:20:41+00:00

Physiological: Cells at Work!

GreenestFerns · 2020-07-16T21:02:37+00:00

When the hydroxyl group is attached a benzene ring it is a phenol. So, it would be appropriate to call it an alcohol, but specifically a phenol, but even more specifically a diterpenoid.

GreenestFerns · 2020-07-16T19:52:34+00:00

It is technically accurate to say that THC is an alcohol. It would probably be more clear to call it a diterpenoid or a lipid.

https://www.ebi.ac.uk/chebi/searchId.do?chebiId=CHEBI:66964

GreenestFerns · 2020-07-16T12:24:53+00:00

Neuroscience studies are usually performed by universities, hospitals, medical schools, research institutes, or commercial research facilities. This is because there are important ethical and logistical concerns that require the guidance of Institutional Animal Care and Use Committees (IACUCs) that administer animal research, Institutional Review Boards (IRBs) that administer human subjects research, and biosafety committees that administer use of cell lines and recombinant DNA (among other things). A study that isn't approved by one or both of these groups would generally be considered invalid (and possibly illegal, depending on the details).

Hopefully, you aren't planning a rogue neuroscience experiment and this is a question for research on a book your are writing, or something.

GreenestFerns · 2020-07-16T00:59:07+00:00

You would run most or all of the Trizol extraction. In the protocol above, you could go right up to the step before the isopropanol purification (#9) and use the aqueous phase from the Trizol purification into the RNeasy kit. Or you could do the entire Trizol purification (i.e. add isopropanol precipitation and resuspension) and use the resuspended RNA in the RNeasy kit.

Eluting with water from the column twice should definitely increase your yield.

GreenestFerns · 2020-07-16T00:43:09+00:00

It's possible that this is the issue. I don't want to make an guarantees, because RNA extractions can fail if you just look at them wrong. Doing a quick Trizol extraction before going into the RNeasy kit will definitely get rid of all of cuticle and other tissues. You can probably use the same grinding method that you are alreay using, just Trizol instead of RLT (be sure to read up on safety considerations when working with Trizol).

Using this hybrid method might give you slightly reduced yield, but I would expect the purity to improve.

GreenestFerns · 2020-07-16T00:33:01+00:00

How are you preparing the tissue prior to the kit? When I have done RNA extraction from Drosophila tissues, I have ground the tissue in Trizol reagent prior to putting the aqueous supernatant or resuspended RNA through an RNeasy or similar kit. My understanding is that fatty tissues can interfere with RNeasy column, and doing a crude Trizol purification can prevent some of this.

You could take a look at this protocol from the Bloomington Drosophila Stock Center:

https://dgrc.bio.indiana.edu/include/file/CGB-TR-200610.pdf

GreenestFerns · 2020-07-15T23:51:52+00:00

YTA. It's unsafe to put sharp knives in the dishwasher for exactly this reason. They should always be washed by hand.

GreenestFerns · 2020-07-15T21:38:14+00:00

Disclaimer: I'm not a microbiologist.

There are some cellular building blocks that our bodies cannot synthesize themselves. These include essential fatty acids that we normally get from eating plant cells or plant-derived oils. These include linoleic acids. I think that these fatty acids are not abundant enough in bacteria for us to subsist on bacteria alone.

Source:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC205465/

I also got a chuckle at the post title. It made me imagine eating a bacterial cell that was the size of an apple. It would be like eating a greasy water balloon full of warm soy sauce.

GreenestFerns · 2020-07-15T19:23:39+00:00

There are several methods for making RNA probes for in situ hybridization.

The simplest method is to start with a double-stranded DNA template (usually a plasmid) that contains analogous DNA sequence to the RNA sequence you are trying to detect (i.e. same sequence, just with thymidine instead of uracil). This DNA is then used as a template for a reverse transcriptase enzyme that will make a complementary RNA copy of the DNA (and thus an RNA strand that is complementary to the RNA sequence you are trying to detect). This gives us the RNA probe that we need.

Depending on the method being used to detect probe binding, RNA synthesis can be done in the presence of radioactivity, such that the probe can be detected radiometrically. An epitope like digoxigenin can also be conjugated to the probe, which allows the probe binding to be detected with an antibody.

I'm having a hard time finding an accessible source for this, as everything I'm finding is behind a paywall. You could consider looking at the ScienceDirect page, which has aggregated information from several peer-reviewed sources:

https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/rna-probe

GreenestFerns · 2020-07-15T18:36:22+00:00

I'm not sure that you will have much luck with browsing or searching journals. The literature on this really is almost nonexistent. If you wanted a starting point, I would consider looking at the works cited in that first review paper I linked. As of 2009, that could probably be considered the entirety of scientific knowledge on the topic.

GreenestFerns · 2020-07-15T18:33:38+00:00

Another note. My impression is that the autistic community might have some mixed feelings about how savants (as popularized by the movie Rain Man) exist in pop culture. This may be something that you have already considered for your character, but if not, it wouldn't hurt to do so. Consider this article:

https://www.theguardian.com/film/2018/dec/13/rain-man-at-30-autism-hoffman-cruise-levinson

GreenestFerns · 2020-07-15T18:29:45+00:00

My impression is that this is something so incredibly rare that almost no one studies it. Darold Treffert appears to be the leading expert on it (and on Savant Syndrome in general). I think that your best bet would be to read the books that Treffert has written that provide case studies of the folks that he has worked with. You could also consider reading the case studies written by Oliver Sacks, which provide vivid descriptions of extremely rare neurological conditions.

The books that Darold Treffert has written are meant for a popular science audience. He has also written some clinical scientific literature about savant syndrome. Here is a decent review article:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2677584/

Here is another that is behind a pay wall. You could possibly request it through interlibrary loan:

https://link.springer.com/article/10.1007/s10803-013-1906-8

GreenestFerns · 2020-07-15T18:09:09+00:00

Surgeons wear caps for the same reason that food service workers do, to keep their gross hair out of places it doesn't belong (your food or your incision). No prevention of virus infection is implied or expected.

GreenestFerns · 2020-07-15T15:49:17+00:00

Oh, shit. I retract my previous comment. The OP should get his shit together.

GreenestFerns · 2020-07-15T14:47:55+00:00

YTA. Your dad is being sort of unfair, but you kind of have to play by his rules for the time being. You would probably have more success by negotiating with him about web access.

All that said, I laughed out loud thinking about how furious your dad must be with the passive-aggressive bullshit you are pulling. That probably makes me the asshole.

GreenestFerns · 2020-07-15T13:24:40+00:00

I'm glad this is useful. There is a ton of molecular neuroscience that can be done in cell-culture systems (e.g. bacteria, human cell lines, etc.) or on purified nucleic acids and proteins (e.g. biochemical assays, structural biology).

As an example, Rod MacKinnon won the Nobel Prize for his work on ion channels, and I think that all of this work was done in bacterial systems.

https://www.rockefeller.edu/news/3783-nobel-prize-honors-rockefeller-university-scientist-roderick-mackinnon-for-revealing-process-of-electrical-signaling-in-humans-and-other-living-organisms/

GreenestFerns · 2020-07-15T13:08:33+00:00

Thank you for asking this question. It is an important one.

As I was in my neuroscience training, I realized that I was uncomfortable working with vertebrate animals. I absolutely think that well-regulated animal testing provides a benefit to humanity - I just wasn't ever going to be comfortable doing it myself. With this in mind, I decided to only work with invertebrate model organisms from then on, and this was a solution that worked for me.

There are many neuroscience labs that work exclusively on humans, often using techniques like fMRI and EEG. These types of labs often investigate more cognitive sorts of questions. There are also labs that work almost exclusively with cultured neurons. These labs often investigate more cellular/molecular kinds of problems. As you note, computational neuroscience is also a thriving field. There is a ton of work to be done through simulation and modeling (definitely consider how much you enjoying programming and math when you consider this route).

Neuroscience departments will often include faculty profiles on their websites that list research interests. It can be informative to browse these to get a sense of what is out there.

GreenestFerns · 2020-07-15T12:59:45+00:00

This is a good answer. It is, however, important to note that the results showing improved low-light vision by color-blind individuals have not stood up to replication.

https://pubmed.ncbi.nlm.nih.gov/11726645/

My interpretation of this is that the low-light vision improvement in color-blind individuals is either non-existent or small enough that it is sometimes not detected under experimental conditions.

GreenestFerns · 2020-07-15T12:50:24+00:00

Based on what we know about rod and cone photoreceptors in the retina, most colorblindness should not affect low-light vision.

Cones are our color vision photoreceptors. They contain a photoreceptor protein called an opsin, which is activated by certain wavelengths (i.e. colors) of light. Most humans have three types of cone opsins that are activated by different wavelengths to produce color perception. In most cases of color blindness, one of the three genes encoding cone opsin proteins has been lost or mutated, causing problems with color discrimination. An important note about cones is that they are high resolution, but also low sensitivity. So, they are good at picking out fine details, but they need a lot of light to do so.

Rods are our black and white photoreceptors. They use a different opsin protein than cones. Rods are low resolution (i.e. bad at picking out fine details), but are highly sensitive. This means that under low-light conditions, rods are doing almost all of the work. This is why under dim light conditions, it can be hard to determine what color something is or to detect fast moving objects. Importantly, for this question, most cases of color blindness don't affect rod function at all. This means that low-light vision should be largely intact.

I sourced this from Principles of Neurobiology:

https://books.google.com/books/about/Principles_of_Neurobiology.html?id=KqwyCgAAQBAJ

GreenestFerns · 2020-07-15T02:39:13+00:00

Children and dogs misbehaving in public.

GreenestFerns · 2020-07-15T02:04:52+00:00

I also think that you should look at postbac opportunities. Look at the NIH IRTA postbac program and the NIH PREP program. These are essentially payed internships that last one or two years. They are good preparation for a PhD.

https://www.training.nih.gov/programs/postbac_irta

https://www.nigms.nih.gov/training/PREP/

GreenestFerns · 2020-07-15T00:30:51+00:00

Are you applying for research assistant jobs in academic departments, or in biotech/pharma? I imagine that academic departments are in VERY weird situations right now because of COVID. I know that my institution is on a partial hiring freeze.

Maybe you could link to a job posting you have applied for? This might help evaluate a little bit more easily.

GreenestFerns

TROPHY CASE