Science topic

Neuron - Science topic

Explore the latest questions and answers in Neuron, and find Neuron experts.
Questions related to Neuron
  • asked a question related to Neuron
Question
3 answers
We are trying to differentiate various neuronal populations via immunofluorescence in FFPE human cortical tissue. There are various good markers for inhibitory neurons in general (GAD65/67) and certain subpopulations (Parvalbumin, Somatostatin ...), but we are struggling to find markers that reliably stain the somata of (ideally close to all) excitatory neurons.
vGLUT1 and 2 stain synapses, not cell bodies, glutamine synthetase and glutaminase are not specific to neurons. NMDAR1 seems to regionally co-label e.g. Parvalbumin and Calbindin positive interneurons.
Our current plan is to just label NeuN for (nearly) all neurons, subtract inhibitory neurons and assume that all others are excitatory, but that is imprecise and rests on very shaky assumptions, of course.
Does anyone, by chance, know of a good marker that might just fit the bill?
All the best and thanks in advance!
Relevant answer
Answer
Hi Lucas Durieux , CamKIIalpha antibodies are a challenge to work with when doing IF, but you could try the one from Thermo-Fisher, (MA1-048). I've used it at 1:200 with acceptable results in the cortex and semi-acceptable in the hipocampal formation. The problem with CamKIIalpha in the hippocampus is that it gives a strong signal from dendrites and this can severely cloud somatic staining. In the cortex, where cell density and topology are markedly different from the hippocampal region, this is not as problematic.
  • asked a question related to Neuron
Question
1 answer
I am working to differentiate human iPSCs into mature neurons (ideally a mix of glutamatergic and GABAergic neurons), but am having some issues with the final steps. I am able to successfully differentiate the iPSCs into NPCs (Nestin+ and OCT4-) using Stemcell Tech's Neural Induction with SMADi kit. However, when I then seed the NPCs into a 24 well plate (with coverslips coated with PLO/Laminin) they initially grow well for a few days, then begin to slowly become unhealthy and die off by about a week after plating. I am culturing them in brainphys base medium supplemented with SM1, N2, 20 ng/mL GDNF and BDNF, 200 nM Ascorbic acid, and 1 mM cAMP (this recipe is based on Stemcell tech recommendation). I am using 500 uL medium at first, then adding another 500 uL 24 hours after seeding, then performing 1/2 medium changes every other day. I initially seed 40,000 cells/well. I was able to get some B-tub III and NeuN positive neurons the last time I tried (cells made it almost 2 weeks), but they were <1% of the total cell population, and the cells looked very unhealthy overall, preventing me from performing any type of actual experiments or analysis. I am using the PGP1 cell line, but would like to use others as well going forward. Any advice would be greatly appreciated!
Relevant answer
Answer
Hi Matthew. Not sure, if you got sorted. I am generating neurons from NPCs as well. The differentiation medium seems quite similar, however I'm using a different coating (matrigel, cultrex or geltrex).
  • asked a question related to Neuron
Question
6 answers
Being inspired and encouraged by the quantum science revolution in the early 1990s up until our present time, lately I've been following the research by Roger Penrose and Stuart Hameroff on consciousness and mind-matter duality where the extent of knowledge gained thus far concerns how neurons and neural networks in the brain give rise to the phenomenon of human consciousness. My research seeks to go further by proposing that it is non-material information external to the body that interacts with neurons in the brain that gives rise to the mind-body duality. So my specific question is this: What is the mechanism or process through which information interacts with neurons and neural networks in the brain so that a quantum moment becomes a classical action or behavior?
Relevant answer
Answer
“…Being inspired and encouraged by the quantum science revolution in the early 1990s up until our present time, lately I've been following the research by Roger Penrose and Stuart Hameroff on consciousness and mind-matter duality where the extent of knowledge gained thus far concerns how neurons and neural networks in the brain give rise to the phenomenon of human consciousness….”
- really there is no necessity to pay some attention to existent in mainstream science solutions of the problem “consciousness and mind-matter duality”. The “consciousness on Earth” phenomenon, which resides on every living being on Earth, including humans, and governs the beings, is fundamentally non-material phenomenon, and any structure of practically material neurons and neural networks in the brain fundamentally cannot give rise to the phenomenon “consciousness”, including the human’s this consciousness version.
Including so that
“….My research seeks to go further by proposing that it is non-material information external to the body that interacts with neurons in the brain that gives rise to the mind-body duality. So my specific question is this: What is the mechanism or process through which information interacts with neurons and neural networks in the brain so that a quantum moment becomes a classical action or behavior?…..”
- essentially isn’t correct. When a living being’s, let here a human’s, consciousness, obtains information about external, that is/are in everyday practice and mainstream sciences practically always completely material interactions of completely material sources with practically completely material body’s sensors [say, when light that is radiated by a material object interacts with retina molecules in eyes].
Further the practically material electric pulses pass through practically material neurons, and hit in some “well lesser material” structures, in this case in brain and in some structures where some “well lesser material” reflexes are written.
Reflexes respond the pulses “automatically”, the corresponding “well lesser material” structures in brain process the pulses so that the obtained information is processed first of all in non-material modes of the just consciousness operation, up to the highest, and completely non-material – “mind mode of operation”, which in everyday practice and mainstream philosophy and sciences is called “mind”.
So really there is no any “mind-matter duality” – in the mainstream that as a rule is “mind-body” problems” and “solutions”; that exists in the mainstream because of in the mainstream all really fundamental phenomena/notions, first of all in this case “Matter, “Consciousness”, “Space”, “Time”, “Energy”, are fundamentally completely transcendent/uncertain/irrational and so in every case, when mainstream addresses to some really fundamental problem, the result completely obligatorily logically is nothing else than some transcendent illusory mental constructions.
The fundamental phenomena/notions above can be, and are, scientifically defined only in framework of the Shevchenko-Tokarevsky’s “The Information as Absolute” conception
https://www.researchgate.net/publication/260930711_the_Information_as_Absolute, and more concretely in models of Matter and Consciousness; relating to the thread question see first 11 pages, including section “What is Life” in
- more about what is consciousness see first approximation consciousness functional model https://www.researchgate.net/publication/329539892_The_Information_as_Absolute_conception_the_consciousness
Cheers
  • asked a question related to Neuron
Question
2 answers
I am working towards to design a study to asses the efficacy of different LRRK2 inhibitor compounds in neuron. I have been meaning to ask if there is any gold standard to check LRRK2 enzymatic activity.
Thank you
Relevant answer
Answer
Thank you for your answer
  • asked a question related to Neuron
Question
1 answer
I have a project with human neural stem cells but the ones that I was planning to use is from ATCC and currently they are out of stock. Does someone has another company or commercial source to recommend? I am planning to differentiate them in neurons, astrocytes and oligodendrocytes.
Relevant answer
Answer
  • asked a question related to Neuron
Question
1 answer
Hi,
I am currently working on optimizing the protocol in my lab for 2P imaging using a water dipping objective. I'm using mice with implanted GRIN lenses that are 0.6x7.3mm, imaging amygdala neurons (injected at approx -4.7mm depth from bregma). I'm having trouble maintaining the meniscus between the objective and the lens. Are there other compounds that people use or any methods to maintain the meniscus that I may be unaware of? Thanks for any advice!
Relevant answer
Answer
Hi,
I am not sure if you tried that, but what we did was 1. create a "well" using dental cement around our window 2. on the edges of the dental cement well we applied a thin layer of vaseline to keep the water within the well. Just a tip: you should clean the objective between mice because vaseline might stay on the objective.
Good luck!
  • asked a question related to Neuron
Question
4 answers
In recent years there has been a tremendous surge in neuroimaging research, and in my experience the most exciting aspects lie in:
  • exploring how neural systems are able to process and integrate multiple inputs,
  • elucidating how complex neuronal circuits can be understood by computational modelling of simplified models (both static as well as dynamical),
  • elucidating what is the mechanism for synaptic plasticity and the mechanism underlying how brain regions communicate (the neurodevelopmental and plastic brain models of cognitive and computational processing and brain connectivity).
  • Understanding what is the underlying computational basis for the generation of complex neural activity in a given brain region. For example, can neurons with similar inputs and identical synaptic parameters but different weights in a given layer of the brain show a qualitatively distinct neural firing rate pattern?
To understand these kinds of neuronal computations at an integrative level, a systems view is a powerful framework to provide both mechanistic insight, while taking advantage, as a complementary method, also allowing the possibility of modelling the system in a rigorous and detailed way and providing insight into its behaviour. What's your (qualified) opinion?
Relevant answer
Answer
i think the most important future development in neuroscience will be an understanding of how the brain controls focus of attention. This is crucial to an understanding of the operation of the brain and consciousness.
Richard
  • asked a question related to Neuron
Question
1 answer
Hi, does anybody know if there is an endogenous nucleus marker specific for glutamatergic neurons which can be used to purifie nuclei from mouse brain by sorting?
Thank you!
Relevant answer
Answer
In my master, I used AAV viruses expressing a nuclear targeted fluorophore under CamKII promoter. But you need stereotaxic surgeries for this and then your area must be defined. I could ask my former supervisor if you need the constructs.
Good luck,
  • asked a question related to Neuron
Question
1 answer
I was wondering whether NAD+ (not nicotinamide riboside or nicotinamide mononucleotide) can enter cells when supplementing the medium of a neuronculture with NAD+
Relevant answer
Answer
Hello Fa Ro
You may please refer to the research article attached below. It will be helpful.
Best.
  • asked a question related to Neuron
Question
2 answers
Good day,
I have some questions about the MEA experiment. We recorded electrical signals from hiPSC-derived neurons using 48 wells plate with Axion MaestroPro.
Here's what we faced,
1. The data is not consistent. Does anyone have the same issue?
You could see completely different activities if you are recording MEA after changing the culture media.
Even if you are not changing your media completely like removing half and adding half, you would see the differences anyway.
It didn't matter how I cared and how I did slow when changing the media. Data was changed.
We are trying to buy the impedance module from Axion company to check the resistance of neurons which would lead to finding how well the neurons are attached to the electrodes.
If you guys have any comments or advice, please share the info.
2. The size of each well of a 48-well MEA plate is really small which means there is not enough space to culture the neurons without interrupting the reference electrode that is located by the side of the well.
We plate the NPCs on the MEA plate and differentiate them into neurons for around 8 weeks.
In the end, the neurons are going to cover the entire surface of each well.
It seems almost impossible to culture the neurons only in the middle of the well where the recording electrodes are located.
I assume that interfering with the reference electrode could affect the recording of the signal..
I would be highly appreciated it if you comment on anything for me.
Relevant answer
Answer
@Sercan Deniz
Thanks for your answering the questions.
I do understand that the network has heterogenous manner.
We haven't tried to culture with less density of cells, because in order to make happy NPCs to successfully differentiate to neurons, we have to plate a certain amount of cells into the wells.
We ordered the bigger MEA plate which like 12 wells plate and we'll try to plate the cells only in the middle of the well.
FYI, when after changing culture media, the signals are disappeared. We do suspect that it could happened because of detachment of cells from the recording electrodes while changing media or could happened by diluted neurotransmitters.
And also the MEA plates seem like unstable itself. If you try to measure MEA with empty well includes PBS, you could see unexpected signals which is not noise.
Contamination of the bottom of the MEA plate? = NO
Contamination of the bottom of the each well? = NO
We will find very good neurons as a positive control to check whether this machine and plates are working properly as they advertised or not.
  • asked a question related to Neuron
Question
5 answers
I want to isolate neurons from mice, but the neurons should be from adult mice instead of the pups. Has anyone isolated neurons from adult mice before or is there any neuronal cell line which is not from tumor can be used as a replacement? Thank you!
Relevant answer
Answer
Dear Dr. Yiguo
Thank you for raising the discussion on the culture of primary neurons. I have the same query. If you have been able to find a suitable protocol for culturing neurons for more than a week, I request you to guide me regarding the same.
Yours sincerely
Sampurna Dutta
  • asked a question related to Neuron
Question
14 answers
Dear colleagues, I’ve started working with cultured primary neurons and came across a problem.
I need to depolarize neurons for different time intervals (up to 1 hour) and then use them for an assay 2 days after. The problem is that most of them are dying after depolarization.
I culture neurons in complete neurobasal medium (Neurobasal + 2% B27 supplement + 1%Glutamax +1% penstrep) with 1/3 media change every 3 days. I depolarize them on DIV11-14 by swapping media on Tyrode’s solution (45mM KCl) for up to an hour. Then I wash the cells with Tyrode’s solution (5mM KCl) twice and swap the collected media back.
45mM K+ Tyrode’s: 100 mM* NaCl; 45 mM KCl; 1 mM MgCl2; 1.8 mM CaCl2; 1.04 mM Na2HPO4; 26.2 mM NaHCO3; 10.9 mM HEPES; 10 mM D-glucose
* NaCl is used to adjust osmolarity of the solution, so concentration varies.
Since in the current setup I depolarize cells in 5%CO2 incubator I used buffering formula of Neurobasal media. I adjust the pH to Neurobasal’s pH=7.7 and checked that in CO2 incubator it equilibrates to pH=7.4. And I adjust solution’s osmolarity to match the current neuron’s medium too.
Also, I depolarized neurons in live cell imaging using GCaMP6s to monitor calcium elevation and after minutes I can see that some neurites are destroyed (Fig.1, attached) and after 0.5-1 hour cells don’t look good and most of them die afterwards (Fig.2).
At the same time, I keep seeing papers with no explicit details on solution and osmolarity where cultured primary neurons are stimulated with KCl for hours. For example, here 6 hours of 55mM KCl (https://www.nature.com/articles/nature09033).
I guess there are a lot of people routinely working with primary neuronal culture. Could you please help me, what am I missing?
Relevant answer
Answer
Hi there,
maybe you already solved your problem. In case you did not, here is my thought. I did some depolarization experiments myself and encountered the same problem (rat hippocampal neurons). Later, I found this publication addressing the high cysteine concentration of Neurobasal formulation leading to excitatory death of neurons:
So what I did in my experiments is to split the supernatant of the cells and use 1 half spiked up with my desired final concentration of KCL and the second half to replace the depolarization solution after the intended period of "stimulation". In my case this did the trick! Also, for any refeeding or change of medium I only used the first 10 DiV to do so with Neurobasal medium. I hope this might be helpful
  • asked a question related to Neuron
Question
3 answers
My lab ordered GCaMP 8f a while back, but during imaging there was not a lot of activity (1:10 dilution in PBS) in the ~10 mice we tested. We cannot use 8s because we are trying to image PV neurons, which have very fast spiking activity so we want as much temporal resolution as possible. Has anyone used GCaMP8f successfully? We were just wondering if we got a bad batch somehow or there was a different dilution we could try.
Relevant answer
Answer
Hi Christian,
Have you had any luck with your virus at a different dilution?
We are having a similar problem with the cre-dependent GCaMP8s we ordered from addgene at the end of 2021. We are imaging pyramidal neurons using 2P through a GRIN lens. We have not diluted and I believe the titer is around 2.9 x10E13vg/ml so we initially were thinking that we have too high a concentration. I am about to try a lower concentration. I have a colleague who has said that FLEX GCaMP8f worked well for him at 1:10 dilution, although has not tested it extensively. I am also worried about a problem with the virus batch so it would be great to hear if you have had any success!
  • asked a question related to Neuron
Question
2 answers
Hello everyone, I'm looking for a media composition to differentiate primary human progenitor stem cells into neuron. I found a protocole to differentiate immortalized one. Can I use the same differenciation media on primary cells and obtain the same effect ?
Best regards,
Aurélie Cotten
Relevant answer
Answer
Thank you Chandra ! Does it work on human cells ?
  • asked a question related to Neuron
Question
2 answers
Hi all,
I want to know how long the fluorescent signals of Fos- or Arc-TRAPed neurons lasts.
TRAP (Targeted Recombination in Active Populations) reference papers shows that the animals were sacrificed one week after injecting tamoxifen or 4-hydroxytamoxifen and behavioral induction. However, I think it is permanent and the fluorescent signals of Fos- or Arc-TRAPed neurons wll be visible after a week. Is it fine to detect FosTRAPed neurons 3 or 4 weeks after behavioral induction?
I am looking forward to hearing from you.
Thanks,
Tae-Yong
Relevant answer
Hi Tae-Yong Choi,
With TRAP and TRAP2 mice the expression of the TdTomato will be indefinite after Cre recombination since it is driven by the CAG promoter if I remember correctly. You could image months after tamoxifen exposure and you will still have florescence. It is advisable to include controls without tamoxifen so that you can see the percentage recombination as that could potentially be higher the longer you wait.
Hope this helps,
Ezequiel
  • asked a question related to Neuron
Question
1 answer
I'm wondering what the half life of green fluorescent protein is, and how quickly it is degraded and removed in a neuron. IF we express it via a promoter only active in progenitor cells, could we still detect it in daughter cells and for how long?
Relevant answer
Answer
In my experience once the progenitor cells differentiate they tend to lose GFP expression. I do not know what the half life of GFP is, but it should be considered that GFP is regarded by mammalian cells as a foreign molecule and the cell
wants to get rid of the expression of GFP as soon as possible.
  • asked a question related to Neuron
Question
1 answer
I ask this out of curiosity.
Relevant answer
Answer
Are you talking about primary neurons preparation? If yes.....In my hands, primary Hippocampal/Cortical neurons from P0 pups in a low-density culture, co-cultured with an astrocyte feeder layer could easily survive for 3 weeks. Longer periods are usually not required in experimental settings.
  • asked a question related to Neuron
Question
4 answers
I have created a deep neural network for the classification of breast lesions. There are two possible outcomes. The lesion is either benign or malignant. My output layer is defined as below:
model.add(Dense(2, activation='sigmoid'))
My query is that should the number of layers in the output layer be 2 or 1? I have read a few articles where in some cases, the number of neurons in the output layer is the same as the number of class labels. Whereas in some blogs, the number of neurons should be 1 for a 2 class label(binary) problem. Any suggestions would be appreciated.
Relevant answer
Answer
Dear Warid,
as you already mentioned both of the cases can be implemented. However, please note that for the such problem which is a binary classification problem if you use 2 neurons in the output layer, you should use 'Softmax' as the activation function of the output layer, and if you use 1 neuron, 'Sigmoid' would be a good choice.
Generally, for a classification problem, it is common to have neurons in the number of classes, in the output layer which is thus followed by a 'Softmax' layer.
Hope it helps you!
  • asked a question related to Neuron
Question
3 answers
Recently, there are various neural simulation software (eg: NEURON, NEST, etc.).
Is it possible to predict the effects of drugs by using them?
For example, for a compound that is known to activate/inactivate a certain channel in a screening assay, can I predict how the compound affect neurons using the above simulation software?
Thank you.
Relevant answer
Answer
sorry for my delay in response. ell you have many applications such as BRIAN which is based on Python (a specialized library of Python), there is NEST also a good and very commonly used simulation app, GENESIS is also a simulation app yet not very famous like the others. You can also build your own functions using MATLAB, For me, I used BRAIN and it is good and easy to learn. Good luck and if you have other questions please do not hesitate to ask.
  • asked a question related to Neuron
Question
2 answers
If there were the quantum mechanical equivalents of individual neurons and of larger networks of neurons, and if quantum mechanisms of error correction worked on those level, you could get something like consciousness. This is because information could (in principle) flow between neurons - that means you have a mechanism of some sort of distributed computing inside the brain. What's your view?
An alternate (rather elaborate) discussion about the two can be found below. However this particular idea just emerged once I started rethinking about information in general.
Relevant answer
Answer
Navjot Singh I think you are absolutely right to conclude that the key to understanding the operation of the brain is a better understanding of fundamental physics.
However I don’t think quantum theory will help. The Spacetime Wave theory indicates that there are two ways in which neurons can affect each other. One way is direct neuron network connection and the other way is the collective effect of electromagnetic wave action.
Richard
  • asked a question related to Neuron
Question
1 answer
Hi there!
I'm trying to perform an experiment to prove one specific protein interacts and activates NMDAr. For this purpose i'm trying to use Fura-2AM dye to see changes in Ca2+ levels inside the neurons and two photon microscopy. As a positive control, I used different concentration of NMDA (from 0.1 uM to 100 uM) which I applied to the medium with neurons. Surprisingly (?), i see little activity from the neurons.
I noticed that usually people use magnesium free medium while performing this Ca2+ measurements for NMDAr activity. I suppose the reason being Mg ions block the channel and Ca2+ cant go thru. I'm just curious if 1) Mg presence in the medium could completely explain almost no activity from my neurons in regards to Fura 2AM intensities and
2) what is the relevance of such an experiment when my other experiments were done in normal neuronal medium (with Mg ions)? Can I claim effects of the protein X is due to NMDAr binding and activating when I can't get any activation signal unless I use magnesium free medium?
Relevant answer
Depend on your experimental preparation, mine was the in vitro chick retina and magnesium needed to stabilize excitability. In this preparation, phosphate buffer was deadly, by contrast, TRIS was OK. Conclusion, know your preparation, read about its developpment.
  • asked a question related to Neuron
Question
5 answers
Hi folks,
I need to find a stain/tracer to label the neurons within the recording sites. I was thinking to use DiI, but I am not sure that it will be helpful cause it requires a longer time in fixed tissue. As I am not interested in only detecting the electrode location, coating the shanks directly will not help me either. Any help is appreciated! TIA!
Relevant answer
Answer
Since you mentioned you tried DiI, I imagine you're trying to deliver the fluorophore with a needle/cannula to a specific region. In that case, Martijn Sierksma idea for viruses is a really nice bet. Another strategy I can think of is to use AM ester dyes. The AM probes I used so far were for calcium imaging (oregon green or Indo), but it looks like there are some that have potential to help you achieve what you're looking for:
Keep in mind they're gonna label every cell membrane though. So IDK how good they will be when identifying neurons and glial cells in your prep.
That's where the viruses would come in very handy, since they're specific to cell types.
Good luck!
André
  • asked a question related to Neuron
Question
3 answers
Hello Scientific Community,
I've been facing a problem to merge the different images (non-fluorescent) of the Golgi-stained single neuron into a single image to see the full branching pattern of the dendrites and axons. I'm aware of merging fluorescence images in ImageJ but not able to correctly merge these simple Golgi stained images into one. Is there any other possible way to do so?
I'm looking forward to hearing from you!
Kind Regards
Aleem
Relevant answer
use arithmetic, add the pictures, if they were still and match pixel by pixel, if not you have to make they match
  • asked a question related to Neuron
Question
6 answers
I am working on primary hippocampus neurons from mice. I am trying to look at the effect of amyloid beta on the neurons. I am looking at parameters like cell death, number of neurons in a field of view, neuronal length. I also want to try to analyze the neuronal coverage or area occupied by neurites to understand how amyloid beta affects the neurons.
Does anyone know how to measure the neuronal coverage or area occupied by neurites?
Thank You.
Relevant answer
Answer
The neuronal culture can be either live or fixed but would prefer to determine the coverage of neurites in live conditions.
Thanks in advance.
  • asked a question related to Neuron
Question
3 answers
GtACR1 can be activated by light around 500nm to 600nm roughly and peak sensitivity is ~515nm. Activation of GtACR1 in neurons can inhibit neuronal activity.
I aim to activate GtACR1 for several hours to inhibit neurons expressing them.
Is it OK to constantly apply light to GtACR1 or I have to use pule stimulation?
I found several papers apply constant light to acitvate GtACR1 for up to 10 mins.
For over hours activation, I found on paper
(Mechanosensory input during circuit formation shapes Drosophila motor behavior through patterned spontaneous network activity)
It uses pulse stimulation 600ms duration with 1s interval. However, the author did not mention why they choose it (as least I did not see the clear explanation).
I know constantly activated Arch channels can become inhibitory and NpHR can become desensitized. So far I did no see any report talked about that GtACR1 can be strongly desensitized with constant light stimulation.
Any one has any idea should plus stimulation be used to activate GtACR1 for hours and what frequency should be used?
Best
Geng
Relevant answer
Answer
A new opsin has been developed that should perform exactly as you want. "Lamplight" is similar to the SSFO's that have a very long open time, which means that a single light pulse produces prolonged activation. Lamplight is, to my knowledge, the only inhibitory opsin that works in this way.
  • asked a question related to Neuron
Question
1 answer
I'm looking for the best strategy to obtain in vitro cells that once transplanted can be optogenetically activated and opto- or chemogenetically inhibited. However it is necessary that the same cell that is activated could be aslo inhibited, so I need a virus that contains both Chr2 and the inhibitory protein sequences. I only found the AAV eNPAC 2.0. It incorporates Chr2 and NpHR genes. Anyone of you has some experience with it? Are there any better?
For my porpouse the best inhibitory strategy would be chemogenetic, but I didn't find any virus that contains both chr2 and hM4Di sequences.
Any suggestions are really appreciated.
Relevant answer
Answer
Could you not just transfect your cells with two viruses? Also, I'm not a fan of the hM4Di, it just never seems to really do much, no matter how good the transfection (and it's possible this is caused by accumulation in the cytoplasm https://blog.addgene.org/tagging-optogenetics-and-chemogenetics-receptors-fluorescent-proteins-and-other-options).
There is an alternative opsin that can produce activation or inhibition of a neurone depending on the wavelength of light used. I've never used it, but it looks good: https://www.addgene.org/154951/
  • asked a question related to Neuron
Question
1 answer
Does anyone know a source where I can purchase it premade or if you have a spare one laying around and willing to donate?It is a microdot stamp to create microisland cultures of astrocytes and autaptic neurons.
Please help
Sam
#astrocytes
#neurons
#synapse
Relevant answer
Answer
Hi Sam did you find one? We would like to purchase one too! Thank you!
  • asked a question related to Neuron
Question
2 answers
What are some of the particular website you will search? And hopefully there are more details such as the connection type and neurotransmitter used?
Relevant answer
Answer
You should go to wormbase (https://wormbase.org) or wormatlas (https://www.wormatlas.org/).
  • asked a question related to Neuron
Question
2 answers
I want to use laser microdissection (LCM) to capture neurons from tissue slide samples for RNA extraction. In order to do LCM, dehydration using ethanol must be performed before, and I am wondering if this step will lead to loss of RNA from the sample because of RNA precipitation?
Relevant answer
Answer
From what I understand, the dehydration avoid RNAse activity. And you afterwards resuspend your sample in the buffers of interest.
  • asked a question related to Neuron
Question
4 answers
Hi everyone, I am a postdoc at Mount Sinai and I have recently applied the RNAScope protocol to human fetal brain FFPE tissue. I want to quantify the nuclear RNAs for two genes involved in neurodevelopment across the tissue starting from the top of the cortical plate, which contains mature neurons that have completed migration, and then go deeper into the tissue, where there are progenitor cells that are migrating and differentiating into neurons. I imaged multiple tiles in an automated fashion with the Zeiss Axioimager Z2 epifluorescence microscope using a 63x 1.4 NA oil objective. Every tile was imaged in 40 200 nm z-stacks across 3 channels (Cy5, Cy3, DAPI). There are a total of 230 tiles. I would like to open these tiles individually so I can analyze the data. When I try to open on FiJi, I see the entire dataset with all the tiles, and the options to go through the channels and z-stacks, but it's not clear how I can extract the individual tiles. The dataset is very big, about 213 GB. Any advice will be appreciated. Thanks!
Relevant answer
Answer
Actually you can just easily export the tiles from the Zeiss Zen software.
  • asked a question related to Neuron
Question
4 answers
Dear all,
I'm doing primary hippocampal neuron cultures from E17.5 mice embryoes. I use L-15 medium for dissecting buffer, and 1mg/mL papain supplemented with DNase I and L-cysteine for digestion (~20min, 37℃ waterbath). I use a Neurobasal medium supplemented with B27 and Glutamax to culture neurons. I plate 100,000/well neurons on coverslips pre-coated with PDL and laminin.
Things all worked well before, but recently I am not able to grow nice neurons. They looked fine after seeding, but they remained round and didn't grow after 24h, and died a lot. I was wondering if this was because of the over digestion (But papain is a very mild enzyme and the digestion time is not so long I think...). Could anyone give me some suggestions or recommend some protocols and troubleshooting tips? Any suggestions would be appreciated!
Please find the pictures in the attachment. Thanks a lot!
Relevant answer
Answer
Francesco Roselli Thanks for the advice! Actually I used recycled PDL and laminin this time, but I also had another group of neurons digested by 0.125% trypsin side by side and they looked fine. So I would consider there were some problems with the digestion process of papain group. I'll use freshly made PDL and laminin next time to improve the quality of coating as well. Thanks!
  • asked a question related to Neuron
Question
2 answers
Hi all,
There are obstacles that I have faced during electrophysiology recording for the neurons derived from stem cells. Neurons were fell out once I started patching cells. I cannot record any actional potential and/ or sodium or potassium current. Although I have good neurons in my culture. I would appreciate it if you can give me advice to overcome this issue. Thank you.
Relevant answer
Answer
Hi Randah. Cultured neurons can be a little finicky to work with. From what I understood, you can't get anything from them. So let me ask: how is the seal (leak current)? And the resting membrane potential in current clamp? Did this preparation work smoothly before? What's the Na concentration in your culture media?
Andre
  • asked a question related to Neuron
Question
3 answers
Why does my neural network perform better with more input neurons than features/variables ? Now if I use the exact input neurons to features/variables the neural network performs much worse. For example, I have 6 dimensions of data that are 200 in length (or 200 samples). Within that data there are groups of 6 data sets - is this why my NN with 36 neuron inputs performs better than 6 neuron inputs? The dimensions should suggest I just need a NN with 6 input neurons. The hidden layers for the 6 inputs are 12 and 6 and for the 36 inputs, are 72 and 36 respectively.
It's been a long time since I last used NNs and so many thanks for answers or pointers on this?
Relevant answer
Answer
Thanks for the question and the clarification answer.
Best regards
  • asked a question related to Neuron
Question
2 answers
Hey every one,
I am staining Dorsal root ganglion neurons with beta 3 tubulin antibody but I have autofluroscence problem with the background and I should do tracing for neurites of neurons and the program traced also these noises in the background and the results are not accurate in this case . Also ,I used a confocal microscope to take photos. So please, if someone has any suggestion to overcome this problem, I will be so thankful.
Relevant answer
Answer
Dear.Daniel Kiselev,
Thanks for your question.I incubated neurons with Primary antibodies at room temperature for around 20 hours.
  • asked a question related to Neuron
Question
2 answers
We have a strange result: We transduce neurons with an AAV vector with synapsin promoter then DIO-ChrimsonR-mRuby - meaning there are double loxP-lox2722 sites flanking the inverted ChrimsonR-mRuby fusion construct. We then introduce a plasmid by single-cell electroporation in a lentiviral backbone that has cre-recombinase and everything was great - huge photocurrents from the opsin in the red fluorescent neurons. Now we package the second plasmid into an Ecolenti (i.e. a lentiviral vector with a serotype that only enters mouse cells) and we have red fluorescent neurons with no photocurrents. My suspicion is that the ChrimsonR must be mutated or something - the promoter and a start codon must be active or there would be no red fluorescence but it is very strange that the first part of the protein is now non-functional. I want to PCR and sequence the DNA but I am very challenged when it comes to designing primers that will be specific for the DNA in cells with active cre as I don't want to also amplify from the cells in which the non-recombined plasmid is. Anyone know a forward primer that would be specific for the inverted and excised DNA? Many thanks! Chris
Relevant answer
Answer
Thanks Shiqiang!
  • asked a question related to Neuron
Question
2 answers
My laboratory had purchased a set of IMU sensors (perception neuron 3) to capture the motion while performing certain motions. The software can output csv and bvh files. Is it possible to transform the 3 dimensional data to relative joint angle?
For example, to calculate the angle between hip and upper thigh, and thigh and lower leg while performing sitting down motion?
thank you in advance!
Relevant answer
Answer
measure :)
  • asked a question related to Neuron
Question
2 answers
Hello guys!
I am working now with an eGFP female mouse and using her embryos, specifically the embryo's brain.
I want to know if there is any marker I can use in immunohistochemistry to label only maternal immune cells in contrast to neuroblast/neurons and glial cells?
Thanks for the attention!
Relevant answer
Answer
John Hardy Lockhart thank you for your answer! I am already using this methodology to obtain the contrast between maternal cells in the embryo's brain. But, sometimes there are pregnancies where the majority of embryos is eGFP. In that case, I was looking for a way to use the eGFP embryos through immunohistochemistry instead of wasting all this rich biological material. I appreciate your attention!
  • asked a question related to Neuron
Question
1 answer
Hi all,
I have a question about my lip3000 transfect neurons. I transfected my primary neurons with a control(mRFP) plasmid. Then I did immunostaining with primary proteinX antibody and Alexa 488 secondary antibody. Interestingly, for my control group, the neurons that were transfected with mRFP plasmid showed a very low signal of green fluorescence; for the neurons that were not transfected with mRFP, the GFP signal is normal. I am curious why it will happen? Ideally, it shouldn't see the difference between transfected cells and nontransfected cells. I just feel like the cells that are transfected are not likely to be immunostained. Did anyone have the same issue as mine? Will the lip3000 affect the permeability? I used 0.2% TriX-100 for 5min.
Thank you so much!
Relevant answer
Answer
Dear Chen,
Well-observed. Besides obviously dead cells or severely affected cells (just due to transfection. You can do a transfection/water control for this instead of transfection/control plasmid), which you obviously need to recognize and avoid, this problem actually occurs more frequently than one would think and usually nobody really reports this. We also sometime see this when we immunostain for some endogenous proteins in cells transfected with RFP or GFP. I guesse it is an effect of the strong promotor driving expression of these fluorescent proteins but we have not studied that systematically. Dealing with a strong viral promotor (you may be using pCMV, I guess!?), the cells then may just not have the energy and the supplies anymore to keep up the expression of many other (endogenous) proteins (visuable first for the ones that have high turn-over rates!). You will see the same for even viral-promotor-driven expression of two proteins; they will be lower than expression of one of them alone.
There is not much, you can do about it. We usually use a cut off and exclude cells that are below threshold expression of an unrelated, endogenous marker.
The problem is much less reduced when other vectors are used (e.g. H1 promotor instead of pCMV). For some reasons, it also seems less when mCherry is used (lower expression?!).
Hope that was helpful. Keep up spirit with your neuronal transfections - it is in general not the easiest cellular assay one can to in cell biology...
Best,
Michael
  • asked a question related to Neuron
Question
4 answers
Hello friends,
We are trying to depolarize neuronal cell by kcl, research used this way didn't elaborate much about preparing kcl. I read that we can prepare kcl medium, but I don't have a clear idea about that.
Please any one can give me a hand?
thanks in advanced
Relevant answer
Answer
Marcelo A Catalán Thank you very much. This was pretty helpful.
  • asked a question related to Neuron
Question
2 answers
Hello, I would like to ask from everyone's perspective what is the biological relevance and impact if the neurons that are being affected by an exogenous stimulus is (1) peptidergic or non-peptidergic neuron, (2) and their respective class of nerve fibers?
Currently, I am still consolidating and distinguishing these concepts because I think these are important research questions in molecular and cellular neuroscience projects.
Relevant answer
Answer
If these are people, then a clinical response to the administration of naloxone is likely. If the experiment... is a microelectrode neuronal response also using blockade of opiate receptors.
  • asked a question related to Neuron
Question
3 answers
I heard that a group did a 3D EM reconstruction showing that some neurons could form both excitatory and inhibitory synapses, but I have been unable to find it.
Relevant answer
Answer
Yes, there are a number of papers that have come out showing that the same neuron can release both GABA (inhibitory synapse) and glutamate (excitatory synapse). Marisela Morales's group has shown this for VTA to Lateral Habenula projections (Root, et al. Cell Reports 2018 v23 p3465) and it's also been shown for Hippocampus (?) and one or two other areas as well I believe.
  • asked a question related to Neuron
Question
3 answers
Hello
I am new to the field and I would like to ask on what is the criteria to say whether a photoswitchable compound or optogenetic molecule has fast kinetics and high spatiotemporal resolution at the cell-free model, cellular/in vitro model, in vivo and ex vivo model? Is there a consensus criterion to quantitatively qualify if a compound has a fast kinetic and high spatiotemporal resolution in these models?
For instance, if a compound becomes fully activated when turned on by light in less than 30 min, does it have fast kinetics?
On the other hand, if a compound can precisely activate certain neuronal regions in the brain but it has off-target activations in the surrounding regions around 20 uM from the region of activation, does it have high spatiotemporal resolution?
I may have mixed-up some terms here, I will be glad if this will be clarified in the discussion.
Thanks.
Relevant answer
Answer
That's the least I could do.
  • asked a question related to Neuron
Question
1 answer
If trans-epithelial Na+ transport were to increase at the synapse in a neuromuscular junction, how would that manifest itself?
  • Would this translate to higher firing rate or result in more frequency of miniature end-plate potentials?
  • Would this also affect the rise time of action potential? Or its threshold?
Any paper or link would be appreciated.
Relevant answer
Answer
??? The only cells at a NMJ are a motor neuron and a myofiber.
  • asked a question related to Neuron
Question
3 answers
What is the simplest way to compute a single cell spike from multi unit activity recorded as local field potentials?
Relevant answer
Answer
Checkout our paper and software. ROSS ( https://github.com/ramintoosi/ROSS ) is fully automatic spike sorter tool with various visualizations and automatic (5 methods) and manual sorting modules. It is based on our recently published paper (https://www.nature.com/articles/s41598-021-93088-w).
  • asked a question related to Neuron
Question
9 answers
I am trying to differentiate hiPSC-derived neurons. I noticed that at the very early stage, normally the first 3 days of differentiation, cells can be hardly observed. Day 5 onwards more neuron-like cells are developing, but they always clump together. Besides that, other than the neuron-like cells, there are another different morphology of cells appearing. Are potential neurons supposed to clump together while differentiating? Are the big cells without a very defined and obvious axonal processes some other cell types? I am using 96-well plate to differentiate neurons, could it be the 96-well plate has a round bottom which cells tend to accumulate in the centre?
Relevant answer
Answer
It is pretty normal to get clusters of progenitors that give rise to neurons in a cluster like this. The small colonies and really tentative projection formation could be due to poor coating of the matrix you are using for the plates.
Try extending the coating period overnight before you use the plates. Also check the viability of your coating matrix. If it is temp sensitive make sure it has not gelled up before usage.
  • asked a question related to Neuron
Question
3 answers
Dear All,
I am currently performing a stemcell differentiation into neuronal progenitors. However, I`ve realized that the incubator stopped heating yesterday so that my cells were kept under 23 degree for 15h. CO2(5%) and humidity was okay.
There is no cell death, and the cell morphology is still as it is suppose to be. I am in day 6 of differentiation which I need 7 days more.
Only problem was medium was used less than it should be used( color was not orange-yellow enough, indicating cell metabolism was slower).
Do you think that I should trash everything and start differentiation from 0? That will cost me so much time and money, since I am differentiating 9 different colony of stemcell together. Or should I just continue to procedure?
I also had stemcells in the incubator, their morfology was also completely normal, no unspesific differentiation, completely round edges. But less medium usage as well
Best Regards
Yagiz
Relevant answer
Answer
It should be Okay to Continue without Trashing them!
Your differentiation sounds like your cells are at neural stem cell stage / neural epithelial induction stage; the meso-endoderm cells at this stage tend survive well as you are nearing the maturation stage for neural differentiation. However, If you are so keen to publish the data from this batch (from IS/WB) I would recommend to just re-check with the specific lines that works now.
Good luck brother!
Regards
Sri
  • asked a question related to Neuron
Question
2 answers
Hi everyone,
I'm looking for markers to identify excitatory and inhibitory neurons in the nucleus accumbens via immunofluorescence in rats.
I've come across mainly GAD65/GAD67 for inhibitory and vGlut1/vGlut2 for excitatory neurons.
I was wondering whether one of them is representative enough, or is there no escape from using all of them/others?
And also - has anyone had experience with a GAD65+67 antibody, such as this: https://www.abcam.com/gad65--gad67-antibody-epr19366-ab183999.html ?
Thanks in advance!
Relevant answer
Answer
Thanks Antonio!
  • asked a question related to Neuron
Question
2 answers
Hello everyone,
I am looking for a nuclear marker, like a transcription factor, expressed in human enteric neurons.
I would like to characterize my human iPSC-derived culture with immunofluorescence stainings. The problem is that my neural progenitors give rise to enteric neurons and enteric glia (GFAP+), so I cannot use Sox10 throughout the whole maturation. So far I haven't found anything this specific.
Does any of you have any suggestions about a good antibody, or a marker that would be useful in this case?
Thanks a lot in advance.
Relevant answer
Answer
Dear Alexandr,
Thank you for the answer. Unfortunately, I already know these works really well!
I can't use neural crest markers in IF as most of them are either cytoplasmic or downregulated before the enteric neurons become terminally differentiated.
Sox10 works quite well up to a certain point, but it's absent in mature neurons: its expression is maintained in enteric glia only.
  • asked a question related to Neuron
Question
1 answer
I am currently using the Warner RC-49MFSH (https://www.warneronline.com/perfusion-chamber-with-field-stimulation-rc-49mfsh) which has two platinum wires placed parallel approximately 10mm away from each other. I have a Grass SD9 Stimulator connected across these electrodes, which allows me to generate pulses of varying parameters (0.2-200Hz, 0.02-20ms, 0.1-100V). ~1 mm below my electrodes I have a glass coverslip, upon which I place an isolated murine dorsal root ganglion (DRG) (total length of say 1 mm), with a nylon mesh placed on top to hold it in place. I then immerse the tissue and the two electrodes in artificial cerebrospinal fluid (aCSF). I then do calcium imaging on the neurons in the DRG.
My problem is that whereas previously the lab members were able to see a high amount of responding neurons, after we switched a strain of mouse (from Advillin promoter to Thy1), I have had very spotty results with responsiveness in the neurons, despite testing a wide range of parameters and going up to the maximum voltage allowed. I might see one or two neurons respond, as opposed to the ~100 that was found previously.
What are some factors that might affect the magnitude of electrical stimulation on tissue in this setup? I saw some other threads that suggested moving the electrodes closer together, which I plan to try very soon. I also checked the resistance across the electrodes and the aCSF bath and found it to be in the range of 100 kOhms to 1 MOhm. The other thread suggested sanding the electrodes in this case. Should I also minimize the volume level of the bath? Does the length of wire immersed in the bath affect the total current flowing through? What happens if I put the electrodes so close together that I can physically touch the DRG to the electrodes? Should I place the DRG closer to the positive terminal since the positive terminal should attract negative ions in the solution, thereby depolarizing the neurons?
I know this is a lot of questions and such a system can get complex quickly. I am still working on understanding the electrode/electrolyte interface. Thank you in advance.
Relevant answer
Answer
Hi Frank,
By sure placing electrodes closer will help.
Also try insulating till the region which is closer to the tissue, this will concentrated the current flow.
Also consider other conductive media that may be into the bath which may form a facilitated path for the current thus impeding current floes through tissue. Leess bath volume will help.
Consider excitability issues. You may depo- or hiperpolaryze the cells varying the K extra concentration.
Probably using random noise added to the square waave pulse may contribute to activate the cells.
Best Regards,
Enrique Soto
  • asked a question related to Neuron
Question
2 answers
Hi! I am interested in a marker for secretory vesicles inside the cell, to mark the vesicles that move out of the golgi to cell membrane. I need to order an Ab but most of the markers that I found they are for neuronal cells. However, I won't be using neuronal cells and it is harder to find a marker that could work.
It would be very helpful if someone has any suggestions.
Thank you in advance!
Relevant answer
Answer
No... I still could not find it.
  • asked a question related to Neuron
Question
2 answers
Could you explain to me what are the most common challenges using Multi-Electrode Array (MEA) during work with neurons and cardiomyocytes?
Relevant answer
Answer
Thanks a lot Vanessa! :)
  • asked a question related to Neuron
Question
5 answers
I´m looking for a kisspeptin antibody to stain kp neurons in mice tissue. Do you have any suggestions? I´ve tried the Anti-Kisspeptin Antibody from Merck (AB9754) and it works perfectly, but they are having problems producing it. Have you try any of the other available kisspeptin antibodies: from abcam, novusbio or bioss? or any other you may know/tried. I appreciate your help. Thanks.
Relevant answer
Answer
Kiss1 antibody from SCBT works. It can ve used for detection of the protein by both IF and IHC, is a mouse monoclonal.
  • asked a question related to Neuron
Question
4 answers
I am beginner of electrophysiology.
I got to know that nucleus accumbens neurons have inward rectification as its characteristic property, and I investigate the property by whole-cell patch clamp.
However, I don't find what kind of function the character can contribute in neurons .
In other words, what will happen to a neuron when the inward rectification is inhibitied or activated?
Are there reviews about inward rectification of nucleus accumbes and its function?
Thank you for your reading.
Relevant answer
Answer
Dear Ryota,
You could think it this way: Kir (potassium inward rectifying channels) are important for maintaining the resting membrane potential of excitable cells.
Indeed if by chance, the membrane potential (Em) hyperpolarizes at potentials more negative than the K+ reversal (EK), Kir channels would conduct an inward current that would bring back the Em closer to EK. At the same time, small depolarizations, are "blunted" by Kir current since in this case, they would conduct a smaller but important outward current. So overall (simplifying and generalizing), they tend to stabilize the membrane potential of excitable cells.
This one is more general on the physiological role of inward rectification and Kir
This other probably better addresses your question.
  • asked a question related to Neuron
Question
4 answers
Dear All,
In most protocols who maturate neurons from IPSC-derivered NPCs (neuronal progenitor cells); they use NEAA in stemcell stage and NPCs induction phase, some of them use NEAA in NPC medium as well. However only a limited number of them using NEAA during neuronal maturation from NPCs.
Is there a spesific reason that we dont use NEAA during neuronal differentiation or is it just because initial paper was like that and people followed it?
Because it seems like NEAA are harmless and support cellular growth in all cell types.
thank you for answers
Yagiz
Relevant answer
Answer
For neural cell culture, it is not optimal to use certain amino acids.
For example, you really should not use neuroactive compounds such as glutamic acid, which can cause excitotoxicity, despite their clear advantage as an energy source. Aromatic amino acids may also exhibit some signaling effects.
  • asked a question related to Neuron
Question
6 answers
Does anyone know what's the main reasons for a neural network to classify certain classes and the others not. For example: let say we have a MNIST dataset, my network will classify images from 0 to 4 and the rest classes will be misclassed. On other execution will choise other classes and the rest will be misclassed
Infos: I have balanced datasets. I tried to use regularizations, different learning rates and changed the number of neurons in the hidden layer
My network is Elman network for time-serie classification problems with single hidden layer
Relevant answer
Answer
Dear Dr Mohammed Elmahdi Khennour . Neural networks help us cluster and classify. You can think of them as a clustering and classification layer on top of the data you store and manage. They help to group unlabeled data according to similarities among the example inputs, and they classify data when they have a labeled dataset to train on. See the link: https://wiki.pathmind.com/neural-network
  • asked a question related to Neuron
Question
1 answer
Dear all
Have you ever use glutamate to induce primary cultured cerebellar granule neurons injuryI have recently made a lot of this assay but all failed. In a word, every concentration of glutamate can’t injury CGNs. There are some questions that I have meted.
  1. Does the different kind of glutamate influence it’s effects in injury CGNs? I have tried L-Glutamic acid , L-Glutamic acid hydrochloride and L-Glutamic acid monosodium salt monohydrate(sigma), but nether are effective. And L-Glutamic acid is hardly to be dissolved.
  2. In some protocols, they use kinds of solution during dissection, such as HHGN dissection solution(see attached pdf), does it matter? In my operation, I just put the cerebellum in HBSS with 5 mM D-Glucose. Plates cells in the density of 10^5 per well in 96-wells plates with medium( 10% AUS FBS + KCl + GlutaMax + Pan-Strep + BME).
  3. I haven’t change the medium during culture, and add Ara-C in 1 DIV, and add 5 mM D-Glucose every three days. Glutamate was added in 8 DIV(also tried in 9,10,11 DIV). But none of them show injury.
  4. Could you please give me a stable method in this model
Thanks for your any advice, and forgive my grammar.
Relevant answer
firts read this: Chapter 12: Neurotransmitter and Cell Death
  • asked a question related to Neuron
Question
7 answers
Hello ! I am a beginner . I want to write an article on the topic "Effects of heavy metals on neurons" and i don't have idea how to gather enough material . So please i you will guide me it will be a great pleasure for me .Thank you
Relevant answer
Answer
Search for a recent review on this topic. After reading it, select the relevant citations and read these. Repeat this procedure over and over again, till you do not find any new articles anymore.
  • asked a question related to Neuron
Question
18 answers
I am trying to obtain Neuronal Progenitor Cells (NPC) from human induced pluripotent stem cells and I would appreciate the opportunity to briefly discuss the protocol with anyone who has had hands-on experience with this type of cells. I have tried various protocols so far and at the moment I am using the above-mentioned media and I am having some trouble with obtaining a robust homogeneous cell population. Any help would be appreciated! Thank you very much!
Relevant answer
Answer
Note that for most PSC cell lines, EB protocol is a little more robust than the monolayer protocol. If you really need to use monolayer for a particular cell line, there are several options to improve the performance. Plating density, multiple passages (3X passages over weeks) in STEMdiff SMADi differentiation media can help. For detailed methods, check out STEMCELL Methods Library here:
I think you will find it updated with everything that you are looking for to optimize neural differentiation in the monolayer protocol.
Thanks
Jason
  • asked a question related to Neuron
Question
3 answers
Kindly share the protocol for primary Neuronal culture from Adult Rat brain ?
After surfing the net for 2-3 hours, all i could get the protocols with embryos of rat only/rat pups.
I could not find the protocol for Neuronal culture from adult rat brain.
Relevant answer
Answer
Hi,
Please go through the following article. I hope this helps.
Thanks,
Alpana
  • asked a question related to Neuron
Question
2 answers
There is a commercial cell available for POMC secreting cell line - mHypo-POMC/GFP. Similarly I would like to use a cell line for Oxytocin.
Thanks in advance
Ramanan Devaraj
Relevant answer
Answer
I am also interested in such OT neuronal cell lines rather than primary culture. Did you find one ?
Thank you,
  • asked a question related to Neuron
Question
4 answers
It is said that due to the cytoskeletal structure of mature neurons, it is very difficult to obtain pure RNA with a high concentration. I am trying to optimize TRIzol protocol to overcome this issue. I observed waiting with TRIzol on ice before homogenization and isopropanol incubation at -20 helps. Do you have any more suggestions?
Relevant answer
Answer
Thank you very much for your valuable time.
Dear John Hildyard , I am trying to isolate RNA from hiPSC derived neurons. RNA isolation efficiency decreases after 20 days of culture. I attached some results from my trials and a bright field photo of my neurons.
Dear
Sebastian Schmitt
and Soner Öner-Sieben , I pipette 1,2 ml TRIzol onto a 35 mm plastic dish and scrape with a cell lifter. In my last attempt, I let it sit on ice for 5 minutes, homogenized by pipetting with first a P1000 pipette, then P200 pipette, and then vortex for 30 seconds. Afterward, I passed the lysate from a 1ml syringe once and added carrier RNA. It was from a 42 days culture. Now, I will isolate RNA from a 65 day old culture which I am a bit nervous about. I am keeping TRIzol on ice.
Best regards,
Özlem
  • asked a question related to Neuron
Question
3 answers
Recently Jamali et al., at Ziv Williams' lab in Harvard published an intriguing paper in Nature:
regarding the cellular basis of theory of mind which I believe is one of the first hand evidence to prove mentalization at cell and circuit level. The methodology was based on single cell electrophysiology which seems interesting yet tricky as it may spark this philosophical dilemma of systems neuroscience that complex behaviors such as theory of mind may be originated from synchronized population activity in downstream path that may not be represented or evoked in individual neuronal activity in dmPFC. However, Jamali et al. could rationally and intriguingly conclude and investigate such a complex behavior at a single cell level. Interestingly, they indicated that dmPFC neurons can predict whether contents of one's beliefs in the big picture would be true or false.
  • While study of theory of mind at cellular level seemed almost impossible before this, and studies shifted also to cellular and circuit level with this landmark publication, what would you think should be the future research on theory of mind? What is the big question and hypothesis if we want to use multi-modal neuroimaging approaches?
  • What methodology and approaches would better decipher impaired theory of mind in psychiatric diseases such as schizophrenia and autism spectrum disorder?
These are just a couple of questions that may emerge but feel free to discuss and contribute to this topic from any aspects that you would think would give us a better view of the underlying mechanisms of theory of mind.
Relevant answer
Answer
Bahman Sadeghi thank you for sharing this article, the methodology is so thoroughly described that it made it possible for me (with my limited knowledge of brain research methods) to get to a fair appreciation of the extent to which the study is supporting theory of mind. It also made me think of the value of the theory more broadly, so much so that I decided to draw from it for my own research !
Gabriele Scheler with the risk of sounding ignorant in assuming I can make any contribution to the topic given that I have a very different kind of scenario (from state-of-the-art lab research) in mind when trying to make sense of the theory, I would very much be interested in being part of the conversation :)
R. R. Poznansky I've found your answer very reassuring, the scope of accounting for unconscious processing in explaining the "mind" is precisely the reason I have decided to use theory of mind in my research, because I'm looking at a phenomenon which is poorly understood in terms of causality as a result of researchers trying to avoid the intentionality dilemma when investigating human behaviour.
  • asked a question related to Neuron
Question
7 answers
I use 0.1% Sudan Black B in my brain immunofluorescence staining, even though it reduces autofluorescence of background but also gives low non-specific staining. It looks like neuronal marker staining.
Relevant answer
Answer
I'm out of ideas what the cause is, but I think you found the best solution already - just do the protocol without the SSB :-)
If you're still trying to troubleshoot: you could try to a) perform the counterstain at different steps in your staining protocol, e.g. after blocking or primary antibody; and b) look at the other wavelenghts.
Sorry I couldn't help more.
  • asked a question related to Neuron
Question
3 answers
Hi,
I'm currently trying to measure action potential according to drug perfusion in DRG neurons.
For the proper pipette solution, I'm searching articles and I found that some researchers use CaCl2-containing internal solutions for AP measuring, while someones don't.
For example,
Maet al. Molecular Pain (2015); used 140 mM KCl, 1 mM MgCl2, 2.5mM CaCl2, 5 mM EGTA, 10 mM HEPES, 2 mM Na-ATP and0.3 mM Na-GTP, then adjusted to pH 7.3 for capsicin-induced AP measure and Chiu et al., Nature (2013); used 140 KCl, 5 NaCl, 2 MgCl2, 0.5 CaCl2, 5 EGTA, 10 HEPES, 3 Na2ATP and 0.1 MgGTP for bacteria-induced AP measure.
What's the role of CaCl2, and what factors/composition about internal solution should be considered between the current clamp and voltage clamp?
I would be so happy if I can get an answer. Thanks!
Best regards, Hayun
Relevant answer
Answer
Hi Hayun,
Even if you will not add calcium in the internal solution, the solution still will have small calcium concentration (may be 1 micromolar) due to contamination by other used by you chemicals that have some calcium residues.
However, calcium is a major secondary messenger that affects ion currents in a very small concentration.
Therefore, if one does not want to suppress the effects of intracellular calcium concentration transients on cellular electric properties, calcium is not added.
Otherwise, people prefer to stabilize internal calcium by adding small amount of calcium and a few times higher EGTA concentration (or any other calcium chelator) to get small but relatively stable free calcium concentration.
You can use maxchelator (https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/) to calculate free calcium concentration, or you can use calibrated calcium-sensitive electrode to measure and adjust free calcium concentration.
My impression is that you are not studying effect of calcium transients, and therefore you should add calcium and appropriate concentration of calcium chelator(s) as in the articles you have mentioned.
Good luck!
  • asked a question related to Neuron
Question
21 answers
I often see statement like "we used cells with series resistance lower than 40 MΩ for experiments." May I ask how this Rs is measured practically? Is it calculated by generating a I-V curve and calculate the inverse of slope value at I=0?
Thank you.
Relevant answer
Answer
A great place to start is Barbour’s ‘Electronics for Biologists’ (https://www.biologie.ens.fr/~barbour/electronics_for_electrophysiologists.pdf). If you want some more background literature, feel free to contact me.
  • asked a question related to Neuron
Question
3 answers
Hello,
I stained mouse spinal cord sections using Nissl stain (cresyl violet). I am wondering how I can count for neurons, and differentiate between neurons and glia. I have attached an image taken using a microscope.
As an additional question: is the attached picture a good Nissl stain?
Thank you,
Suzie
Relevant answer
Answer
Is there a reason why you use the Nissl stain instead of specific markers for neurons and glia (e.g. NeuN and GFAP)? This would probably facilitate automated analysis. In general, a fast way to count cells in large histological images is using a maxima finder, though I'm not sure if this would work well on Nissl or not. For questions on image analysis I can recomment you the image.sc forum. Here is for example a discussion on Nissl: https://forum.image.sc/t/automated-cell-counting/32770/4
  • asked a question related to Neuron
Question
1 answer
Hello,
We are attempting to stain various brain slides with an antibody binding to a protein of interest and compare neuronal counts/density across genotypes. Mouse brains have been sliced coronally.
We would like to stain not every slice, but certain slides at the same position (Bregma ± mm). How can it be determined which slices are at the same position?
The end goal would be Cavalieri estimation, similar to this paper: https://www.frontiersin.org/articles/10.3389/fnagi.2015.00196/full
Note that these authors chose the position of slices prior to Vibratome dissection. Erroneously, our brains have already been Vibratome dissected coronally into thin sections (100+ per brain).
Relevant answer
Answer
How were the slices organised/stored? Usually when I section I put slices into a 12-well plate in a pattern, so that each well contains every 12th slice. If you have done something similar, you might be able to pick a really consistent feature (eg, the first slice that has the corpus callosum fused) and then count slices forwards or backwards from there. If all the slices are in together, your best bet might be to compare slices to a mouse brain atlas and pick ones that look similar, and say that slices were chosen from 'approximately [coordinates]'. If the images from different brains are clearly very similar in position, I think this would be good enough for most purposes.
  • asked a question related to Neuron
Question
3 answers
How can I transfer transfect Culture of hippocampal neurons with GFP?
the more I read I get confused
correct me if I am wrong according to my understanding.
1) the neuron need to extract
2) then culture or transfect using for example Lipofectamine 3000
please if you know any articles that help me to understand share it with me
thank you
Relevant answer
Answer
Thanks, Ryan.
Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons.
Calcium phosphate transfection of primary hippocampal neurons - PubMed (nih.gov)
jove-81-50808.pdf (nih.gov)
  • asked a question related to Neuron
Question
1 answer
I have tried doing a primary embryonic neurons in chamber slide experiment. The neurons will grow when the chamber slide is PDL coated. However, when I try to do immunocytochemistry, the processes wash away/ break. Does anyone do acid etching on chamber slides? If so, what chamber slide brand do you use to do this? What do you do if you do not use acid etching?
Relevant answer
Answer
Hi Britanie,
I use mostly primary hippocampal neurons from embryos and for some exp. I used chambers slide to visualize live neurons. After live acquisition, I tried fixing and staining for ICC. However, I never had this problem.
I used these chambers: Ibidi µ-Slide 4 Well (https://ibidi.com/chambered-coverslips/37--slide-4-well.html) and Ibidi µ-Slide 8 Well high Glass Bottom (https://ibidi.com/chambered-coverslips/252--slide-8-well-high-glass-bottom.html). Once I ordered an already coated chamber (PLL) and they worked pretty fine.
Which protocol do you use for fixation and staining?
  • asked a question related to Neuron
Question
32 answers
Will artificial neural structures become such advanced artificial intelligence that artificial consciousness will arise? Theoretically, such projects can be considered, but to really verify this, artificial neural structures should be created. From research on the human brain, it appears that this is a very complex and yet not fully understood neural structure. The brain has various centers, areas that manage the functioning of specific organs and processes of the human body. In addition, Sylvia is also complex and consists of elements of emotional, abstract, creative, etc. intelligence that also function in separate sectors of the human brain.
In view of the above, does research on the human brain and progress in the construction of ever more complex structures of artificial intelligence lead to synergy in the development of these fields of science? Will the development of these fields of science lead to the integration of research into the analysis of human brain activity and the construction of more and more complex structures of artificial intelligence equipped with elements of emotional, creative intelligence, etc.?
Besides, does the improvement of artificial intelligence lead to the emergence of artificial emotional intelligence and, consequently, to autonomous robots that will be sensitive to specific changes in environmental factors, factors of the surrounding environment? Will specific changes in the surrounding environment trigger programmed reactions of advanced artificial emotional intelligence, ie activation of pre-programmed algorithms of implemented activities and learning processes as part of improving the learning processes of machines.
Therefore, another important question arises in this area:
Is it possible to create an artificial consciousness that will function with the structure of an artificial electronic neural network built in such a way as to reflect the structure of the human brain? In this way, the structure of advanced artificial intelligence will be able to improve on the basis of acquired knowledge eg from external internet databases?
Do you agree with me on the above matter?
In the context of the above issues, I am asking you the following question:
Will it be possible to build artificial emotional intelligence?
Please reply
I invite you to the discussion
Thank you very much
Best wishes