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I am interested in Neurodegenerative diseases, particularly Dementia, and I would like to study the disease basic neurobiology. This will be a helping hand for me to study Dementia well.
Recommendations could be Books, Articles, Lectures, Animated videos..etc
Thanks in advance!
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Hi Mohab,
You could have a look at the NEURONET Knowledge Base
It provides a summary overview of the Innovative Medicines Initiative neurodegeneration research portfolio through an interactive dashboard, the NEURONET Knowledge Base includes links to over 500 publications and more than 380 publishable deliverable reports, acting as a one-stop shop to explore the diverse projects and outputs of the portfolio.
On the project website you can also find an overview of the ongoing (also completed) public-private research projects in the area of neurodegeneration (including dementia & Alzheimer's disease): https://www.imi-neuronet.org/ongoing-projects/
Hope this helps a bit.
Best wishes,
Chris
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Hi everyone,
When I search for the dilution ratios of a number of primary antibodies for IHC (immunohistochemistry) studies, I usually see that the ratios are somewhere between 1/50 and 1/500. Apparently, this range (1/50 - 1/500) generally fits for IHC studies. In case of those antibodies, when it comes to applications for other methods such as western blot, immunofluorescence, the more diluted stock solutions still seem to work (somewhere between 1/500 - 1/1000). Is this just a coincidence, or is there really a linear relationship between application method and dilution ratios ?
If so, for a IHC study, I would like to know if it's a good idea to prepare several dilutions (more diluted) different than the manufacturer's suggested dilution. If the solutions diluted more than the recommended ratio still work just fine, then it would be better to use this way, because by doing so, much less compound would be wasted.
Kind regards,
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Yes, besides the factors mentioned in the previous answers, it is always recommended to test a new antibody (or tissue from a new experimental setting) using series of dilutions. The best dilution is sometimes quite far from those recommended by the manufacturer. (In our studies we are usually using the dilutions of the primary ab ranging from 1:100 til 1:20000, depending on used Ab or type of the tissue).
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I'm looking to collaborate.
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I am Assistant Professor at Department of Zoology, Goa University, Goa, India. Along with my teaching assignment, I do my research in the area of developmental neurogenesis using chick embryos and making an attempt to move ahead with combination of signal transduction involving neurotransmitters and neuromodulators during developmental neurogenesis. Had also worked with rat pups in past. Do not have full fledged neuroscience lab but having animal cell culture facility and enough requirement of lab to sustain work in developmental neurogenesis.
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I am curious about two specific things:
- Why do pseudounipolar neurons have one axon (as opposed to a dendrite + axon like multipolar neurons)? How does this structure reflect sensory function?
- How do potentials propagate through the axon? Since there is no axon hillock for summation, does that mean no summation occurs? Is there still a threshold potential that needs to be met? Or does every graded potential get transmitted through the axon?
Can someone familiar with any of these questions help out or provide a resource I can refer to?
Thank you!
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Hi Sehej
Pseudounipolar pattern of sensory neurons acts as low-pass filtering, potentially regulating sensory information reaching the spinal cord. Thus, by impedance mismatch between membrane point in the vicinity of T-junction, this has been recognized as a site where spike propagation may fail.
As for action potential propagation through the axon, this is a more broad question. Actually, in a really simplistic summary, sensory neurons have "transductors" (specialized proteins that converts physical energy - thermal, mechanic, chemical - into electrical signal) in their peripheral end; these transductors creates a "generator potential", according to specifical thresholds. If, and only if, these generator potentials reach some specific area in the membrane with a larger density in voltage gated channels (as Na channels) within these second step threshold, an action potential (spike) is generated and conveyed until the "T-junction" filter described above.
Some references for better comprehension
1 -Al-Basha, Dhekra, and Steven A. Prescott. "Intermittent Failure of Spike Propagation in Primary Afferent Neurons during Tactile Stimulation." Journal of Neuroscience 39.50 (2019): 9927-9939.
2 - Sundt, Danielle, Nikita Gamper, and David B. Jaffe. "Spike propagation through the dorsal root ganglia in an unmyelinated sensory neuron: a modeling study." Journal of neurophysiology 114.6 (2015): 3140-3153.
Regards,
Tiago Avelar
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Does anyone have experience with the Olympus cellVivo weather chamber for monitoring neurite growth over hours/days?
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sorry not
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I'm trying to get subcellular fractions (specifically, I only need the nuclear fraction) from neuronal tissue.  However, all my current samples were flash frozen and stored in -80C subsequently (for whole cell lysate, which was my original intent for the samples). 
Does subcellular fractionation for nuclear protein require fresh tissues and cells?  Will flash freezing might cause ice crystals to form and pierce the nuclear membrane, so I wouldn't get good separation?
Thank you in advance.
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Late to the party but I have performed sub-cellular fractionation on frozen heart tissue with good results - yet to be published. This paper describes liver tissue frozen and subjected to fractionation also with good results.
Just give it a try, most of the important nuclear proteins complex with a structural binding partner (DNA, Lamins) anyways, so will be probably remain in the nuclear fraction even if the membrane has been a bit pierced with ice crystals. also, careful flash freezing i.e. in isopentane, should limit the number of crystals that form.
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It is a very interesting project. Are you also working in this project in new forms of neuro-scanning (e.g. using deep neural networks to analyze images and signals of new MRIs, or TMS)? We do "non-invasive neuroscience" and computational molecular neurobiology and I wonder if it is related to your project...Thanks.
Juan, IGN
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Thank you Juan, and my pleasure. Apologies for my slow responses lately (conferences + meetings + travel). I do update this project from time-to-time, so I will look forward to your insights. I work closely with Alice Richardson on these projects. Best wishes, Brett Lidbury.
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Does anyone know how/if its possible to measure erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) in living humans?
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آسف السؤال ليس من اختصاصي
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Does anyone prepare big master mix batches (that means primers, buffer, magnesium chloride, dNTPs, water; so everything except for the Taq) to final concentration for genotyping purposes?
What I mean is, what happens if I prepare 1000 reactions, aliquot it and then unfreeze the reactions I need to run the genotyping PCR, add taq, and then use? Do the dNTPs or the primers get ruined if they are frozen at low concentrations?
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If anyone wanted to know, I am doing this regularly and it works just fine. No more pipetting a hundred different things. Just the master mix, primers and taq and that's it. As long as all your PCRs have the same amount of Mg in them. If not, I just add some Mg.
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Hi,
I extracted RNA today via the trizol method. My 260/280 ratio is 1.95 while 260/230 is 1.44. I washed the pellet using isopropanol and ethanol. The cells grown and RNA extracted from were human cells.
I have somewhat shaky hands especially when anyone watches me extract RNA. However, I did not notice any debris enter my pipette tip during aspiration of the supernatant.
Would it be possible to continue and synthesize cDNA from this RNA for qPCR? Would the qPCR be accurate?
Attached: My Nano Drop
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Sounds like som contamination with phenol but since you should do DNase digestion, precipitation, washing, reconstitution and cDNA-Synthesis, your problem is likely to be gone afterwards. Just continue...
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Other than knocking down genes, is there any other way we could demyelinate neurons of particularly postnatal mice.
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Nijin,
There is also a non-autoimmune approach to demyelinating neurons in animal models. Cuprizone is a toxin that can be injected as a way of killing oligodendrocytes and simulating the neuropathology of demyelination. Though this approach does not induce an auto-immune effect like EAE, it does generate an inflammatory response that is associated with MS pathology. After treatment is ended myelin will be restored after 3-6 weeks, as OPC's that survive the treatment repopulate the region of insult. There is controversy in the field over what model best suits MS research (see second link). It really depends on what effect your intervention is hypothesized to demonstrate. It is important to carefully sort out the intentions of your study before selecting your model. See the link below for a helpful review of the Cuprizone model, and the other for a debate over MS models in general:
and...
Good Luck,
Ben
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In a situation where a myelinated neuron (at any location in the nervous system) gets demyelinated, what are the changes that occur in the neuron. May be there are three phasic changes. Pre-Demyelination changes, Intra-Demyelination changes and Post-Demyelination changes. What are these changes? Can we ennumerate them.
Important: Does any change in the size/length of the neuron occur at any given instant during the process of demyelination?
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Look, it's a good question ... I try to give a ontological answer but also (above all) a functional one and, in the end, it is the applied chemical stoichiometry that will tell us if budgets exist that do not return and in those mysterious areas we must go to investigate. Chemistry will not support the needs of dialectics even if expressed by authoritative scientists. Truth wins in science, and it is evident that chemical stoichiometry makes this research more fruitful.
The cell body of the neuron needs a lot of energy and the mitochondria can not provide everything that is needed. However, the mitochondria contained in the cell body are very few and this is just a conundrum: in fact it is paradoxical that a cell that consumes a lot of energy with a request for high oxygen supply for biological combustion has few mitochondria!
Different facts lead us to think that, thanks to the probable fusion of the mitochondria with the Nissl substance (Nissl: the endoplasmic reticulum of the neuron-soma) this gives the soma the “machine” that synthesizes all the ATP it needs but… in the very long axon that happens? The endoplasmic reticulum does not penetrates the axon and it’s true-like that myelin support this energy demand, especially for firing (see " Monitoring ATP dynamics in electrically active white matter tracts” Elife. 2017 - PMID: 28414271) let us not forget, is energetically dispersive. So the white matter, but also the gray matter, energetically supports the axon with myelin. Among other things, there is evidence that neural activity stimulates myelin growth. Keep in mind that the brain has a serious deficit, compared for example to the muscle: it does not possess myoglobin which is slow reserve of oxygen so much that it is extremely affected by ischemia, while for the muscle this criticality does not exist, thanks to myoglobin.
So myelin is home of an active energy metabolism with aerobic degradation of glucose, which, through the Krebs cycle - which we have shown to be very active in myelin (see our article “Tricarboxylic acid cycle-sustained oxidative phosphorylation in isolated myelin vesicles” PMID: 23851157), supports "outside" the neuron's energy needs, above all to support firing.
But there's more!
In fact, for go on the combustion the myelin need a lot of oxygen and that's why the myelin has the highest content of carbonic anhydrase (CA) of the whole organism. Let us remember that CA is the enzyme that combines the carbon dioxide produced in gaseous form with water to make it soluble in the form of bicarbonate. Remember us that if the CO2 remained gaseous (let's remember that in this state it is produced by the Krebs cycle) in the brain we would have terrible seizures! Furthermore, again to be in the subject of oxygen, myelin dissolves oxygen quite well and therefore constitutes a slow reserve of oxygen to support neuronal combustion, even if it is not as efficient as myoglobin. In fact, white matter resists better the hypoxia of gray matter.
So many data would then remain mysterious if we do not take into consideration the chemical reactions we have now mentioned are operative in myelin.
At last to answer with the ontological profile, I hope that I have answered your question, and we can realize that all this chemical support to the axon from myelin could not be supported by the neuronal cell alone. I believe that it is possible to look at the issue from a teleological point of view, but if we talk about it we will talk about it another time…
Yours sincerely,
Sandro
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I have flash frozen brains in the past using isopentane with dry ice in it but I found that some of them cracked. What is the optimal time to leave the brain in the isopentane such that it is frozen solid but the cells are still viable?
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Er....what?
If all you're using these brains for is western blotting, cell viability is irrelevant: all you care about is protein.
You'll be crushing the tissue under liquid nitrogen and then dissolving some tissue powder in some sort of lysis buffer (RIPA or SDS or similar).
You don't even need to use isopentane: you could just drop the brains straight into liquid N2, or place them on dry ice. The time taken to freeze solid is far shorter than the half-life of virtually every protein you might be interested in.
The only time isopentane is really necessary is for rapid freezing: to preserve cellular architecture for histology (liquid n2 alone freezes too slowly, due to leidenfrost effect).
If you want cell viability (!?) then you shouldn't be freezing them like this at all, you should be isolating and amplifying the cells of interest in culture, then freezing them as for normal cultured cells.
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Dear sirs, I have been studying much recent literature on alpha-synucleinopathies, and I still cannot find a clear answer to the question below.
I am not so sure that a clear answers exists, but I would like to share such question with you. I am sure your expertise will help me clarify my doubts. I thank you very much in advance for your help!
Premise
Much has been said about the physiological role of alpha-synuclein. For instance, it has been demonstrated that it promotes SNARE-complex assembly, that it regulates the membrane localization of MATs, etc.
We also know that the main toxic effects in alpha-synucleinopathies are due to a gain-of-function, taking place when alpha-synuclein aggregates into soluble oligomers.
Some forms of familial PD are determined by a quantitative increase of alpha-synuclein expression (gene duplication/triplication). As a result, the physiological functions of alpha-synuclein should be exacerbated. Is it possible that some of the pathological effects of such "quantitative" forms of PD are determined NOT by a gain-of-function due to increased aggregation, BUT RATHER by an exacerbation of the physiological role of alpha-synuclein (eg.: excessive induction of SNARE-complex assembly)?
Are there any evidences in support or against such hypothesis?
Are there any evidences of reduction or exacerbation of such physiological roles, in the forms of PD caused by a qualitative mutation as well (eg.: A53T, A30P, …)?
Do such qualitative mutations alter the concentration of active alpha-synuclein species? By active I mean the alpha-synuclein species that are able to carry out the above physiological functions.
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Thank you very much for your answer. Really interesting!
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Hello everyone,
I just completed RNA extraction from brain tissue (PFC) using the RNeasy Lipid Mini kit from QIAGEN. One particular sample gave a very low concentration of 66 ng/ul RNA (in a total elution volume of 30 ul) after measured with NanoDrop.
I want to synthesize cDNA by using the QuantiTect® Reverse Transcription kit. The reaction is for 1ug RNA in a well plate of 20 ul maximum volume each well. After my calculations, the water I need to add to dilute this sample has a negative value and the volume of RNA solution to be added to the well is 15,19 ul. I am a bit puzzled as I don't seem to find any examples anywhere to help me with what I should do.
I haven't encountered this before as this particular sample was not very well isolated and the final tissue piece was very small.
Any ideas or solutions would be highly appreciated!
Thank you in advance,
Lydia
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one way to go would be to precipitate the RNA with ethanol (5 ul Na Acetate pH 5 plus 125 ul ethnaol) pellet the RNA and resuspend it in a smaller volume.
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As we know that, there is about 5-6 Noble prize on Drosophila melanogaster 's Brain. I am really surprise, it is really helpful to understand and solve the futuristic problem of human brain.
If is it so, then how? Could anybody explain with the good example?
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@Matthias R. Schaefer, thank you...the people most commonly facing the complex neurological disorder, like Dementia: At what time and which state it converts into other of its sub-type?
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Hi! 
Please let me know if you or your lab in Europe have  Ndnf-IRES2-dgCre-D transgenic mice. I will be extremely grateful!
Many thanks!
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We do have this line and currently characterizing it.  This is the line from Allen brain institute.
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I'm trying to estimate the amount of transport time needed to map out specific circuits in the spinal cord using a rabies-glycoprotein pseudotyped lentiviral vector. Does anyone know the kinetics of retrograde or anterograde transport for any lentiviral vectors? I've been trying to find information on this, ideally I'm looking for how many micrometers or millimeters/day these vectors can be transported through axons, etc. 
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It's a discontinuous process but can be very fast (0.2-1 um/s)
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I want to contruct a primer for assay of scavanger receptor marker MARCO , or CD163 for rat animal model. So my question is that how to consttruct a primer for this ?
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so are we talking PCR based detection? If that is the case check out taqman probe availability for this receptor. I am pretty sure they provide kits where you can design whatcha need.
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Any good paper/publication regarding this would be helpful.
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Hello! Has somebody used the ED1 clone of the CD68 marker of microglia in rat brain homogenates? I'm planing to use it as a marker of "activated" microglia in WB
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We use it frequently for IHC on mouse and rat frozen brain sections. It works very well to mark activated microglia/macrophages. I have not tried it for western.
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I’m looking at the active zone for docking/priming/fusion between the nociceptor’s membrane and vesicles.
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Hi Laura!
Definitely, SNARE is involve in the nociceptive pathway. In our lab, we publish several papers speaking about its implication and even develop a peptide called DD04107 that interfere with the induced exocitosis of TRPV1.
Here you have some of this references:
An inhibitor of neuronal exocytosis (DD04107) displays long-lasting in vivo activity against chronic inflammatory and neuropathic pain
Bradykinin Induces TRPV1 Exocytotic Recruitment in Peptidergic Nociceptors
αCGRP is essential for algesic exocytotic mobilization of TRPV1 channels in peptidergic nociceptors
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I would like to measure dopamine release in anaesthetised rats with continuous amperometry, what anaesthetic is most suitable for this experiment considering that I cannot use urethane? Thank you!
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There is a publication from Mark Wightman group comparing DA overflow (release and reuptake) in the caudate after stimulation of the DA neurons during anesthesia and in the same awake animals. They used FCV. As far as I remember release was not much affected, but uptake was a bit slower after chloral hydrate. To make amperometry less noisy you may use much more sensitive carbon fiber electrodes as for example electrodes manufactured from 30-32 micron fiber. Indeed, this will not improve selectivity.
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I want to measure brain damage in EAE mice but i dont hve any experience about that
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I can send you LFB protocols on paraffin or frosen sections, as you prefere. It worked in rat and cat brain. But I am really curious how you will quantify the brain damage. And why don't you try Myelin Basic Protein Staining? LFB stains not only myelin. MBP is more specific
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In my experiment, I am using isolated mice brain mitochondria from control and Ppif DKO mice and running a BN-PAGE. After transfer, I probe for anti-Ppif antibody, but the antibodies I have tried are showing non-specific binding to Ppif DKO (negative control).
Has anyone seen this before and can help me troubleshoot this?
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Try to make your antibody titration in PBST (5% milk). I think that should help.
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Many kits are declared just for plasma or cell culture samples. Thank you!
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Hi Katerina, I see that others are already recommending the Biosensis ELISA kit. A recent paper from Enrico Tongiorgi's group published in Nature Scientific Reports compared most kits and found the Biosensis kit to be superior in several respects. Details are on the Biosensis data sheets and protocols. However, there are no kits that have been fully validated for CSF. Biosensis scientists have begun to examine CSF so you might want to contact them directly. Please note that I have been involved in the development of this Biosensis kit as an advisor, and that I am also declaring that I a shareholder in the company. 
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How to prepare a single cell suspension from without disrupting the membrane integrity for ADP/ATP ratio measurement?
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I want to prepare the single cell suspension from brain especially hippocampus to measure ADP/ATP Ratio.
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Did we use denatured protein or undenatured protein?
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thanks a lot!
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Dear all, I have run my tPA zymography protocol for more than 1 year without problems. Last week it stopped working. I made all new buffers and still is not working. Anyone had the same experience?
Here is the protocol: 12.6% SDS gel with casein and plasminogen (both substrates are stored in aliquotes at -20 or -80C). Running at 110V in the cold room. 2x30 min rinses with 2.5% triton-x solution, 3x10 min rinses with water. Incubation in glycine/edta buffer pH 8.3, coomassie blue R250 staining overnight, destaining, rinses in water.
Thank you in advance!
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Dear
Please check the following publication to find another interesting informations: Use of Gel Zymography to Examine Matrix Metalloproteinase (Gelatinase) Expression in Brain Tissue or in Primary Glial Cultures. Harald Frankowski et al. 2012. Methods Mol Biol. 2012; 814: 221–233. Directlink: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3670093/
Hoping to be helpful
Marius
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I am Working on Primary Neural Cell culture from SVZ of the Postnatal Mice Can anybody Help me to Provide Some guidance how to Precisely cut this part. and How to recognize SVZ in Brain of Postnatal mice. If you have some Videos Illustrative Diagram I could be better for me?
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Hi Masood,
I wonder if you have seen this JOVE article?
This is a protocol for whole-mount immunostaining but you can follow it's steps of dissection. It is mostly applicable to all postnatal brains. For new born (p0-5) the brain shapes slightly differently, but the general gist is there.
Let me know if you need more info!
Xiwei
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We have been performing a thioflavin-T dose-response curve on alpha-synuclein fibrils with very consistent results for some time now.
Recently, we received a new batch of fibrils produced by the same method as before, but by a different person as the original person has moved elsewhere. Since then we have not been able to get the same dose-response curve.
If you look at the attached graph, curve E7 was obtained using the original fibres and curves E16 1 and E16 2 using the new batch. Does anyone have any idea what might be happening?
I have had a lot of help with this experiment on here, so I hope someone can help with this question too.
Previous discussions on this experiment are below.
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I forgot to mention in the question that the concentration of the fibrils is kept constant and varying concentration of thioflavin-T is used to obtain the curve.
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I am trying to decide how to place some samples that will be sequenced in a Hiseq2500. I want to be sure these are randomized and that I have enough replicates to interpret my results. I know publishing this data is tricky and reviewers always have concerns about replicates. So I want your opinion before I spend a lot of money on something that wont be published later.
I am trying to validate the use of blood transcriptomes as a proxy for brain transcriptomes. My genes of interest are found in the brain but I do not have access to many of these samples, hence I will use blood. I have collected samples like so:
Brain individual A, Blood Individual A (collection site 1)
Brain individual B, Blood Individual B (collection site  1)
Brain individual C, Blood Individual C (collection site 2)
Brain individual D, Blood Individual D (collection site  2)
Blood from 12 Individuals from collection site 1
Blood from 12 individuals from collection site 2
I can afford 3 lanes in the HiSeq2500. And because I want deep coverage I will not pool more than 12 samples per lane.
Thank you!
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Hello Angelica.
I was in a course of RNA-seq. In this course they said that the ideal was three biological replicates. If you can not at least do two biological replicates of each condition.
Good look.
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Anaerobic exercise: lab animal models
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Dear Nirmala,
The recent publication entitled "Swimming Physical Training in Rats: Cardiovascular Adaptation to Exercise Training Protocols at Different Intensities " contained in the following link contains a protocol for anaerobic exercise models using lab animals:
Hoping this will be helpful,
Rafik
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Can anyone suggest how can be used microglial cells derived from newborn mouse brain for parkinsons desease research? to study a role of alpha synuclein and exosomes?
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Hello David Aphkhazava
please check the pdf related to your question
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hi guys,
my differentiated PC12 cells do not stick nicely to poly-d-lysine coated well plates. They are easy to wash out after a few washes by pbs or hbss solution. Any idea why and/or what to use instead? Could the adherence change while treating undifferentiated pc12 and incubating with NGF 100ng/ml?
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Hi Marcela and Sean,
Nice to hear your practical solutions. I am getting very good response (Neurite outgrowth) with NGF 5O ng/mL. Serum starved (for 4 hrs) Cells were seeded after an hour coating with NGF and followed by single wash with sterile PBS. 
But, the issue is cells are detaching from the surface after staining with cell membrane stain supplied by Molecular Probes, which I am not able to image under the microscope
Do you guys have any experience with this kind of work 
Thank you
Raju
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Hello,
I need to objectively do counts of neurons labeled with green and red fluorophores.  I want to know what portion of those cells labeled with the green fluorophore are even dimly red above a certain intensity.  The amount of red cells that are also green is not important.  The fluorophores do not need to be colocalized, they only need to be in the same neuron.  Is there a way to do this in ImageJ or FIJI?
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 As far as I know, there is no direct way or plugin can do this in Fiji. But of course you can achieve this using Fiji, with the assist of macros and scrips. Briefly, you have to use 'threshold' methods to binary green/red/neuron channels, then 'analysis particle' can be used to extract the signal's position information on each channel. Finally you have to write scripts (Javescript or Jython) to implement a loop function to check the overlay of green/red signals with neuron.
I also have some matlab scripts developed several years ago which can do similar stuff. Pls let me know if you are interested.
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Hello everyone !  I got some strange result following ischemic experiment in mice. After 3 days of MCAO/reperfusion microglia are believed to be activated and morphologically transformed to the amoeboid shape. In some MR brain section I got the expected results, however in other brain sections there was complete blank in ischemic core and no single microglia was detected. For the further clearance I have attached the iba1 stained photographs of ischemic ipsilateral hemisphere as below. Have you ever experienced such strange results?
What might be the reason that I could not detect any microglia in the ischemic core though I got the result in some other mice brain challenged with same MCAO procedure?
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Thanks Bhakta,
It is interesting. Do you know if the DAPI stained cells in the item core primarily neurons or glia/astrocytes? If you have primarily neurons than you may question the effectiveness of MCAO. If it is glia/astrocytes then something interesting or unusual may be happening. Although it is frustrating, but you may be into something interesting with your research.
Best wishes, Refik
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Can anyone explain to me why I got a large amount of myelin staining when I did IHC for Bax (Ser184) using the ABC method and DAB? I know Bax is associated with apoptotic neurons, which is what I was looking for (and did find a few nicely labeled cells), but why do I also have so much labeling of myelin?
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Dear Vincent,
I agree with Imam in that it looks like blood vessels.  You might be able to get rid of the endogenous reaction of DAB with red blood cells by incubation new sections in a higher concentration of H2O2 (0.5%) before immunolabeling, but I haven’t had much luck with it.  I suggest that if you need to use any remaining sections from these animals, you try swithching to an amplification system that doesn’t rely on HRP.  Are you able to use fluorescence?  If so, you can replace the ABC with a fluorescently-tagged streptavidin.
Good Luck!
Jill
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I'm interested in investigating the nuclear role of ChAT, however all the published information I've been able to find is for human ChAT in the nucleus.  Anyone know if mouse ChAT also traffics into the nucleus?  It does have a predicted NLS, but I haven't found any studies specifically identifying ChAT in mouse nuclei.
I'm new to neuroscience work, so this may be a well-known thing that I'm just oblivious to
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Dear Maureen,
How did the previous studies identified/localised ChAT in the nucleus? What methods did they use specifically. If it is just immunolabelling, I would be very sceptical about it. In unhealthy cells, the nuclear membranes are disrupted and permeabilised to let antibodies and probes in the the nucleus. See my previous discussions on that subject below.
best wishes, Refik
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Using immunohistochemisty, I want to determine if a postsynaptic protein is juxtaposed to C-fibers in lamina II of the spinal cord superficial dorsal horn.  There are plenty of marker proteins for differentiating C-fiber terminals but I was also wondering if there are any postsynaptic proteins that mark synapses formed with C-fibers- the idea being to co-localize my protein of interest with this hypothetical marker, to show it is located at a C-fiber synapse.
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Hello Steve, it has been a while since I have worked in this area.. But this review paper (Neural Plasticity
Volume 2013 (2013), Article ID 654257, 19 pages
Review Article
Ionotropic Glutamate Receptors and Voltage-Gated Ca2+ Channels in Long-Term Potentiation of Spinal Dorsal Horn Synapses and Pain Hypersensitivity
Dong-ho Youn,Gábor Gerber, and William A. Sather)
indicates that when C-fibers terminate in Lamina I or II they either use glutamate or substance P.  For glutamate, the post synaptic receptors are ionotropic glutamate receptors.
So if you know which system you want to look at (glutamate or substance P), you could colocalize your protein of interest with an antibody for a specific receptor class on the interneurons. As below, ionotropic glutamate receptors have multiple subunits. So, you can make your staining a little more specific if you stain for a subunit of the receptor subtype that you are sure is there.
There are many types of GluRs. Further, they underwent a terminology change after their discovery. So the earlier papers use older terminology. For this area, you just need to consider AMPA receptors (GluA1-GluA4).
There is a good review paper on glutamate receptors that lays out their structure and terminology, just look at the AMPA receptor sections: Traynalis et al.,  Pharmacol Rev. 2010 Sep; 62(3): 405–496.
doi:  10.1124/pr.109.002451
PMCID: PMC2964903
Glutamate Receptor Ion Channels: Structure, Regulation, and Function
They say: "The glutamate receptors assemble as tetrameric complexes of subunits (Laube et al., 1998; Mano and Teichberg, 1998; Rosenmund et al., 1998; Greene, 2001; Matsuda et al., 2005; Nakagawa et al., 2005; Sobolevsky et al., 2009), and functional receptors are formed exclusively by assembly of subunits within the same functional receptor class (Partin et al., 1993; Kuusinen et al., 1999; Leuschner and Hoch, 1999; Ayalon and Stern-Bach, 2001; Ayalon et al., 2005). Glutamate receptors are grouped into four distinct classes based on pharmacology and structural homology, including the AMPA receptors (GluA1–GluA4), the kainate receptors (GluK1–GluK5), the NMDA receptors (GluN1, GluN2A–GluN2D, GluN3A, and GluN3B), and the δ receptors (GluD1 and GluD2). The AMPA receptor subunits GluA1 to GluA4 can form both homo- and heteromers. "
In terms of the distribution of the receptors on the Rexed Lamina I or II interneurons, I am sure there are  newer papers than the ones below (ones that use the newer terminology for the glutamate receptors) that can tell you what specific type of glutamate receptor is found on the interneurons. Then you can use an antibody for a specific subunit in that receptor on those cells.(the receptor itself is a tetramer, and you can stain for just one part of it)
R. C. Spike, D. J. Maxwell, and A. J. Todd, “GluR1 and GluR2/3 subunits of the AMPA-type glutamate receptor are associated with particular types of neurone in laminae I–III of the spinal dorsal horn of the rat,” European Journal of Neuroscience, vol. 10, no. 1, pp. 324–333, 1998. View at Publisher · View at Google Scholar · View at Scopus
H. S. Engelman, T. B. Allen, and A. B. MacDermott, “The distribution of neurons expressing calcium-permeable AMPA receptors in the superficial laminae of the spinal cord dorsal horn,” Journal of Neuroscience, vol. 19, no. 6, pp. 2081–2089, 1999.
I hope this helps. feel free to call me if you have questions. 703-371-2733
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Can someone tell me if it Is possible to identify a synaptic dysfunction by open field test?
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Hi Simon
Behavioral tests, such as open field exploration test, assess the behavioral output of experimental mice. Generally speaking, this behavior is a result of specific brain regions and neural circuits activation. For example learning and memory, as assess by Morris water maze, is mainly related to the hippocampus, while anxiety-like behavior can be assessed by open-field exploration test, and this can be related in part to the activity of the amygdala, for example. 
Now, in case you see abnormal behavior (in your case, abnormal exploration pattern in the open-field), it can be the result of many things, such as abnormalities in connectivity between brain regions associated with the behavioral task, abnormal genes expression that affect the functionality of the brain region, impaired development of the brain region, synaptic dysfunction (as you asked), and many more.
The direct (!) link between behavioral outcomes and synaptic dysfunction can not be directly identified in a behavioral test, so my short answer to you is that, no, it is impossible.
You can use behavioral tests to focus your research on specific brain regions that are known to govern the behavior measured, and then characterize these regions and manipulate them, to assess their synaptic transmission properties. But to learn directly on synaptic dysfunction from an open field exploration test it is impossible.
Best
Boaz
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I will likely be using the hM4Di variant of DREADD and hope to avoid stressing the rat with an IP injection so close to a behavioral study that requires attention. IP will further be made more difficult by a headstage containing implanted electrodes that I would hope to avoid knocking around, so I am hoping to get suggestions for trying to administer CNO orally by gavage.
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Hi!
Actually, oral gavage can more stress animals than ip. It raises blood pressure, heart rate, and plasma corticosterone levels (Brown AP, Dinger N, Levine BS (2000). Stress produced by gavage administration in the rat. Contemp Top Lab Anim Sci 39: 17-21).
What if you habituate rats prior experiment with saline injection? In 7-10 days of habituation you reliably reduce stress levels.
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Sphingosine-1-phosphate1 (S1P1) regulates various molecular and cellular events in different body parts. Its expression pattern and functions are also varied dependent upon the cells types where it is expressed. Does anyone have any information regarding the effect of S1P1 in brain vasculature? Specially in ischemic condition (many paper suggests activation of S1P1 is neuroprotective in ischemic condition, however they lack the discussion on effect of S1P1 in brain vasculature). So if you have any information regarding the S1P1 signaling in brain vasculature, please do share !!!
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S1PR1 is one of the targets of the multiple sclerosis drug FTY720 (Fingolimod). Here is a review article about S1P and S1PRs: http://www.hindawi.com/journals/mi/2016/8606878/
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Hello, I am trying to figure out how to induce and silence the same set of neurons in brain using dreadds. I am running into the problem that CNO activates both Gq and Gi dreadds. If I infused the Gi and Gq in the same animals, then both get activated at the same time after CNO injection. I am trying to find out the population of behavior relevant (like in open field test) inhibitory and excitatory neurons involved in regulating the behavior in the same animals by using in-vivo electrophysiology. Any help will be highly appreciated.
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I realize this is a very old thread, but the solution to your problem is now available in KORD http://www.cell.com/neuron/abstract/S0896-6273%2815%2900289-5 which can be used to silence while hM3Dq can be used for stim. Good luck.
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It is quite well established that DHPG perfused in acute hippocampal slices causes a transient decrease in synaptic transmission in hippocampal CA1 region (DHPG 100 micoM is perfused for 10 minutes in Palmer et al. 1997) but i haven't found the molecular mechanism that is proposed to underlay this short depression (i did find a lot of mechanisms by which mGluR-LTD could occur, including retrograde transmitters hypothesis)
It seems to be related to a reduction in calcium influx in pre-synaptic terminals but i don't know how this can happen upon group 1 mGluR activation induced by DHPG....
Thanks!
Celia
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Dear Celia, may I suggest you check the paper:
Switch from facilitation to inhibition of excitatory synaptic transmission by group I mGluR desensitization.
Rodríguez-Moreno A, Sistiaga A, Lerma J, Sánchez-Prieto J.
Neuron. 1998 Dec;21(6):1477-86
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I am setting up a protocol for IF on endomembranes in hippocampal neurons. Thus far, it has been difficult to obtain crisp clear image even when using saponin permeabilization, as it seems that some structures get destroyed, especially the smaller ones. I found that in the literature MES buffer was used instead of PBS. Do you have any experience in that? What could you recommend?
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I did post-fix and I like what I'm seeing, thanks :) Looks like the epitope is more visible. 
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Dear all,
I would like to know if there is a proportion in the concentration of any compound to compare iv / ip / icv administration in a mouse model.
Thanks
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I think iv and ip sometimes can be the same. It depends on the solubility of the compound. The icv dose we used was more often 1/100 of the iv one. Again it depend how it passes over the liquor-brain barrier.
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I am wondering if anyone has used and characterized the expression of Cre in mature astrocytes vs neuronal cells using the B6.Cg-Tg(Gfap-cre)77.6Mvs/2J line of GFAP-Cre driver? http://jaxmice.jax.org/strain/024098.html
It seems that inducible lines have issues getting complete astrocyte coverage (and determining the right timing and dosage of tamoxifen) and non-inducible lines hit progenitors and neuroblasts early in development resulting in too much expression in neurons. The aforementioned line is relatively new, so I was wondering if anyone had characterized it as yet?
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Hi Natalie
B6.Cg-Tg(Gfap-cre)77.6Mvs/2J seems to be the best so far. 73.12Mvs/J is not good, as it expresses Cre in adult neural stem cells.
In B6.Cg-Tg(Gfap-cre)77.6Mvs/2J, 80-90% of the cortical and hippocampal astrocytes express Cre. Cre expression in astrocytes in other brain regions is relatively sparse. Important to note, there is also very sparse neuronal expression of Cre in the cortex of P21 mice.
Good luck
Boaz
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Hello Everyone,
First I do apologize for posting my question here. I just signed up and am quite new. I just started recording fEPSP from hippocampal CA1 region and the problem is that the signal amplitude I get is very small (between 0.2-0.5 mV). I do sagittal sectioning (300 µm) in cold ASCF and incubate for 1-1:30 h at 30 degrees. I work with 2-4 month-old mice. When I put the slices under the microscope, all the layers can be easily distinguished. The fiber valley is very very small which I think can be good sign.
I have been struggling with this problem for a couple of weeks and tried many things to get bigger signal but nothing seems to work. The last thing that recently came to my mind was that maybe there is something wrong with the stimulation protocol. This is my first recording experience and I have no idea about how to make a protocol. The one that I am currently using is the one used by one of the students in other lab for recording EPSP from cortex and is apparently working perfectly for him. 
Can anyone explain what each of the following is:
First level, delta level, first duration, delta duration, digital bit pattern (#3-0) & (#7-4), train rate...
Maybe I will be able to make a protocol for hippocampal fEPSP if I have a better idea about each of the above term and the best possible range they can be within.
Also, does anyone have any recommendations for the positioning of the stimulating and recording electrodes? Is there any particular area to get the best possible response?
I do appreciate anyone's help in this regard.
Thank you all in advance
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digital bit pattern (#3-0) & (#7-4): this is trickier. it's asking you which output to send the stimulation through. look at your digital outputs as a grid: 3210 top row, 7654 bottom row. the bit pattern is a 0 for outputs you do not want to use, 1 for those you do. 
so for channel 2 it would be 0100 and 0000
for channels 1 and 7 it would be 0001 1000
for channel 5 0000 0010
or try * to enable a bit if you use train and it can keep up with it
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I plated a 96 wells plate with SK-N-AS cells, which I then treated to induce a lysosomal storage disease. Then, I treated the cells (in threefold) with different drugs that would hypothetically lower cholesterol levels. After, I measured cholesterol levels and, because the cell growth seemed to be influenced by certain drugs, protein levels of the cells.
How do I use the protein assay results to cancel out any differences in cell confluencies?
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I already measured the protein in micrograms/ml. I divided [cholesterol] by [protein]. Now all cholesterol seems about the same (also, none of the results are normally distributed). I guess I should treat the cells with a higher concentrated solution.
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Dear Researchers,
I have to use the nuclear, cytoplasmic and mitochondrial extract for my studies involving m-RNA and protein in brain isolated parts (striatum, substantia nigra, hippocampus, prefrontal cortex).
I have no much budget to purchase kits for the same like of Thermo scientific (http://www.thermofisher.com/order/catalog/product/78835?ICID=search-78835)
I wish to prepare chemical solutions for extracting them.
Kindly tell me the exact good and short reliable protocol to perform that involving ease and reproducible results...
Thanking you
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Thanks Rafik for the advice
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Any help would be highly appreciated :)
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Thanks Yu, much appreciated ! The problem with NMDAR is that they become toxic for the cells so it's a bit tricky in that manner as compared to expressing a protein that won't harm the cell.
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I want to know what neuronal molecules are sulfonated in brain samples. Does anyone know how to identify and quantify all neurotransmitters and their sulfonate conjugates in a sample? or how to enrich sulfonated neurotransmitters/neurohormones? Thank you!
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We would also like to know. Some neuropeptide was sulfated, such as SKs. And phosphorylation and acetylation was intensively studied in different organisms. However, little is known about sulfonation.
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Can someone please give me some names of voltage sensitive proteins that are present in the voltage gated ion channels? Which have been widely studied so far?
What I have found so far was mucin, cadherin and ferritin and XIAPs.
Do anyone know anything more than this?
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Dear Maedeh,
I think you are looking for voltage sensing domains of voltage-gated ion channels. If you do a pubmed searh under this phrase you will get 437 hits, and if you also include review  in your search phrase then you get only 47 articles. Perhaps initially reading reviews may help guide you.
For example, check this review out:
Molecular pathophysiology and pharmacology of the voltage-sensing module of neuronal ion channels. By Miceli F, Soldovieri MV, Ambrosino P, De Maria M, Manocchio L, Medoro A, Taglialatela M. Front Cell Neurosci. 2015 Jul 15;9:259.
best wishes,
Refik
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I'm running experiments and I need to silence the mouse DRG in culture. I'm thinking of either Lidocaine or Toxin cocktail (TTX with conotoxins). Does any one have experience in this and willing to share some information regrading protocol conc. duration etc.
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Sorry I just realized that you are doing it on cultured neurons. Why not in vivo? I would just use TTX in that case around 1-10 micromolar should block action potentials. See my 2005 JPET electrophysiology paper on cultured DRG neurons. Another drug you may consider is riluzole, an FDA approved drug, though it does not block all action potentials (see my 2013 NeuroMethods paper).
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I'd like to fluorescently stain for activation of GABAergic neurons in the mPFC- but have a few questions.
1) When looking at the mPFC, would it be best to use antibodies for FOS and GABA, or FOS and GAD67? What are the advantages or disadvantages to each? 
2) Are there any particular antibodies you would recommend for this?
3) Any tips and tricks as far as staining goes? TBS or PBS? Dilutions?
Thank you in advance!
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Hi Sara,
I would use FOS and GAD67 antibodies. GAD67 will label all the cells that are GABAergic wheres, GABA will also label the vesicles and axon terminals that contain GABA not just the cells. For antibodies I would check Journal of Comparative Neurology Antibody Database at:
The rest should be just standard double-labelling immunohistochemistry.
Best wishes, Refik
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Brain
Frozen at -80 degrees since 2011 (no reason to believe samples were tampered with)
activated PPARy ELISA 
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Hello..Its very difficult to get result of such a long time stored sample. But I suggest you may try, again centrifuge the sample aliquot and then perform the ELISA. May be it will work.
Gud luck..
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Has anyone observed ThT-positive or -negative aggregation for Ab1-40 fragments depending on the supplier? We have and cannot understand why. The supplier of the ThT-negative fragment denies the use of any additive and the salt (TFA) is the same than that of the ThT-positive fragment purchased from another brand. Thanks in advance for your help.
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Hi Ana, this is a known phenomenon documented in literature. Solid-phase synthesis of hydrophobic peptides yields impurities that may be responsible for differences between different batches from the very same supplier. I would suggest to use recombinant Abeta if possible.
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There is less information available for human cortex about optimal golgi staining than for mouse or rat. But the tissue I am working with is human and has been in formalin for many years.
I am specifically looking for dendritic spines for classification purposes.
Any protocol ideas or publications for the optimal human cortex golgi staining protocols?
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We have been using the Golgi-Kopsch modification with potassium di chromate in paraformaldehyde and silver nitrate for the impregnation for archived human brain tissues.Hope this helps.
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At the moment I am trying to isolate Purkinje cells from mouse cerebellum. I have a protocol which contains steps like 3% PFA perfusion, homogenization of the cerebellum with douncer, filtering through 100um pored nylon filters and stainings for FACS sorting. Main buffers are HBSS and PBS.
After the sorting (I sorted over Parvalbumin, although I would have GFP expressing Purkinje cells) I extracted RNA from my cells of interest by usin the RNA Kit of Macherey-Nagel. I got in total 198ng RNA with a RIN of 3.1 and bad purity. The extraction of the RNA happens 2 days after the perfusion. Until then I stored the homogenate at 4°C.
Now I am trying to shorten and improve the protocol. Does anybody has any experience with isolation of specific cell type from brain followed by extraction of RNA? I will need the RNA for RNA-Seq to compare wildtype with a knockout.
Thanks for any suggestions.
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Have you tried to sort them directly into triazol buffer in 1.5 ml size microtube; then, right away do the standard triazol-chloroform RNA extraction?. It did work for mouse neuro progenitor cells (around 30-50 thousands cells) for RNA-seq.
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which serotype of AAV-shRNA is best for rat mid-brain neurons? Thanks.
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AAV DJ serotype is also good. 
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Thank you,
Matt
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Axon 200b is a patch-clamp amplifier. If you want to do whole-cell recordings in voltage-clamp mode, it's perfect.
If you want to do current-clamp recording, it's possible but not ideal.
If you want ot do extracellular (field) recording, it's not the best solution.
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I have had this ICC working for several years and have used it for multiple experiments, but recently it has stopped working. I am now having nearly every cell in the brain labeled in every trial I do. I initially tried ordering a new lot of the primary antibody, but that made no difference. I have modified the concentration of the secondary and the time in the DAB and neither of those helped. I tried doing a no primary control to rule out nonspecific binding of the secondary and a no primary or secondary control to rule out ABC binding to endogenous biotin in the tissue. Both of those trials came out clean, with no staining, suggesting that the problem is not with the secondary or the ABC. We have tried replacing the paraformaldehyde (our brains are not perfused, so fixation is the first step of our IHC), the methanol, the H2O2, the normal goat serum, and the DAB tablets and none of these things have solved the problem. We also thought that contamination in the water may have been the problem, but I got the same result when using all solutions made with HPLC grade bottled water. Finally we thought there may have been a problem with the tissue since it had been in the -80 freezer for 2+ years and may have experienced temperature variation, but I recently included sections from a brain that was collected and sliced within the previous week and that did not solve the problem.  I have run out of ideas for what could be causing the excess staining.  Does anyone have suggestions on what I could try?
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The steps you are following to identify the culprit are all right. Write down a flow chart of the problems. Some suggestions for it: Are you getting the same high background when using other atbs? If NO, follow recommendation from Srinivasan, above. If YES, go to a colleague who is now running (successful) IHC and repeat your experiment in his/her setting. If GOOD results there, start replacing reagents, one by one, etc.Good luck
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I am doing a Citrate synthase activity assay in synaptosomes enriched fragments from mouse brain. The synaptosomes are isolated using Syn-PER™ Synaptic Protein Extraction Reagent (87793 from thermo fisher scientific) giving functional synaptosomes. The assay works, and I find a significant difference in citrate acid activity between my two groups, however my background measuring before adding oxaloacetate to the reaction is very high - higher than the reaction after adding oxaloacetate. There is not significant difference in the activity of the enzyme in the background measurements (before the oxaloacetate is added). Does anybody know how I can solve this problem? Can I trust the measurements of my enzyme if the background is so high? Is the background important for the assay if it is “the same” (not significantly different) in all samples?
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Your high background is caused by the DTT in the buffer, which reacts with the DTNB. Try the reaction without the DTT.
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How BDNF regulates the Wnt2 and shank protein in autism
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I send you one article with title "Genetics of autism spectrum disorder & BDNF gene" by
Usha P. Dave
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Everyone is on and about neuron image analysis in 3D (stacks of 2D images). And the reasons are legitimate (reconstruction in 3D is general, it is a standard). I wanted to ask is how much in practice scientific labs work with 3D data. It seems as most of the neuron morphology analysis in practice is actually carried out with in-vitro samples that are 2D. Do you have any reference where it would be possible to see the statistics - how many studies involve 2D and how many 3D neuron reconstructions to make conclusions in neurobiology. Would anyone have anything to add regarding their own opinion/experience? 
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Dear  Miroslav:
 I will give an indirect answer to your question. In Remote Sensing, we always use the stacked images in the form of multi-spectral and hyper-spectral images and there is a plethora of literatures that can be adapted to your field of study. Neuron or Neural networks are computational models to capture the underlying dependency in any data set in the form of supervised or unsupervised modes of learning. Hopefully this answer may shed some light to your question.
Regards,
Gamal
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We are trying to visualize fibers from a BDA anterograde tracer in the cortex. However, despite trying multiple different ways of staining using DAB, we are unable to get fiber staining. Any suggestions for modifications to the protocol?
The BDA tracer we are using is biotinylated, so we have tried using an ABC kit followed by DAB (which gives us no stained fibers), and we have also tried using streptavidin, and we still don't have visualization of fibers (though, this method does give us some background staining, though not fibers). 
Are we missing a step somewhere? Since the BDA is biotinylated, we are under the impression that we don't need to use a primary or secondary antibody...rather we should be able to just use the HRP (with an HRP against the biotinylated species) step so that we can get a reaction when using the DAB. 
Any insight/suggestions would be very helpful and appreciated! Thanks!
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To eliminate the possibility that there is a complete detection failure with DAB procedures, consider localising the injection site with a fluorescently conjugated avidin or streptavidin. You may also consider including a fluorescent marker in with the BDA in the pipette next time. However, I agree that the most likely issue is that the BDA did not leave the electrode.
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2. which drug can also induce seizure in adult wistar rat?
it is an immunohistochemical studies on the brain
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This is not my field of study, but I know that pilocarpine is a popular model to induce epilepsy in rats. Some of my colleagues administer this drug i.p. dissolved in normal saline at 280-290 mg/kg. 
The orexin system regulates sleep in human. I did a quick literature search "rat model of insomnia". Some labs inject orexinB conjugated to saponin toxin and report depleting 90% or more of orexigenic neurons. 
Is your goal to find a drug that induces both insomnia and epilepsy, or do you want two separate drugs for each model? 
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I need to extract proteins from mouse brain to run western blot and check the level of expression of different post-synaptic proteins.
Do I necessarily have to run synaptosomal preparation?
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Hi Giulia,
To answer shortly, it is not "mandatory" to use synaptosomal preparation but very often you have to in order to enrich your sample with synaptic proteins and get reasonable signal.
Here is a recent method paper you might be interested looking at http://www.sciencedirect.com/science/article/pii/S2215016114200276
Hope this helps. Good luck!
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There are different receipts of internal solutions for acute slices (cortex, hippocampus etc ). There is no standardized receipt in the field. Some people include phosphocreatine and spermine in the internal solution, but some do not. What are their functions?
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Dear Man and Jei
it is important which age of neurons has been used: in young (9-15 days old) versus "adult" (after 22 days) in terms of neuron and astrocyte maturation. Particularly in rodents, guinea pig and rabbits after 21 days, the potassium inwardly rectifying channels (Kir4.1) are developed (while others declined (Schopf et al., 2004)) and Kir4.1 requires spermine to be rectified (Oliver et al., 1998; Skatchkov et al., 2000).
In neurons rectification of Kir2.x channels are basically provided by magnesium because the synthesis of spermine is stopped in neurons at adult neurons (Krauss et al., 2006; 2006). The only remnant spermidine synthesis was found in some synaptic terminals (Krauss et al., 2006; 2007) where it is probably important to keep AMPAR  rectified.
Also, spermine is necessary to keep glial Cx43-gap junctions open (Skatchkov et al., 2015) that is very important for astrocyte-to-astrocyte communication (Bendikt et al., 2012) otherwise function of glial syncitium will be failed.
So, if working on adult neurons you may ignore spermine or add about 0.1 mM, but not when patching glial cells that you need to use 1 mM (Kucheryavykh et al., 2008, Benedikt et al., 2012).
Cordially, Serguei.
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Hi All,
Recently I want to study neurogenesis, and try to use Edu as a cell marker for proliferation. However the edu not working very well so I decided change it to other markers, such as Ki67 or PCNA. Some friends told me ki67 only label S phase, as neuron no longer divided, I won't get Ki67/Tuj1/NeuN labeled cells in my CNS neurons culture. But wiki said Ki67 will label all cell phase except G0, which confused me a lot. Any one get Ki67 labeled neurons maker (i.e Tuj1/NeuN) yet?
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Ki67 should not co-label with NeuN, as they are by definition post-mitotic. Also bear in mind that as Ki67 is expressed thorough the cell cycle, it will label more cells than just EdU. What protocol are you using for the EdU? It's normally pretty reliable.
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I would like some clarification on focal adhesions (and stress fibers).  Do proliferating cells express more or less vinculin-immunopositive focal adhesions (and phallodin-positive stress fibers)?  
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Hi Laura. Can you provide the details/citations for the conflicting studies?
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I'm new to this type of analysis and am unsure of the identification of everything in this image (see attached). This is a 100x image.
Is there a clear picture of a phagocytosing macrophage? Schwann cells that have detached from their myelin sheaths?
I'm wondering if I can only quantify degenerating fibers and perhaps use EM for cell analysis...
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This is actually a pretty good image, although it would be easier to interpret in color since the lipid in the myelin looks black while proteins in the cytoplasm are more of a dark blue.  There are lots of degenerating axons in this photograph.  To me, they have a very obvious crumpled appearance.  I can see a close grouping of 4 large prominent degenerating myelinated axons in the upper right quadrant of the photograph. (The ones that look like squashed spirals.) Just below this group of degenerating axons is part of a phagocytic cell (it could be a macrophage or a Schwann cell) engulfing several bundles of myelin debris. The nucleus of the phagocytic cell is not visible in this section.  There are also 2 1/2 nice looking intact myelinated axons int the upper right quadrant (thick black bands surrounding pale centers containing a few small dark mitochondria) and lots of unmyelinated  axons (small pale circles).  Also there is one presumptive Schwann cell cell not attached to an axon in the lower center of the image that looks almost like a sperm in this section and another one with tree processes in the upper left quadrant. (These are just the  cells where the nucleus is visible in the sections.  A lot of the other process are probably Schwann cells as well but could be parts of macrophages or fibroblasts.)   You cannot however distinguish between Schwann cells that have become detached from their axons and those which never were myelinating unless you catch them right in the act.  EM makes it much easier to count unmyelinated axons and to look at the state of any remyelinating fibers as well as to more convincingly identifying some other cell types by their organelle and cytoskeletal compositions.  However, in both light and EM sections, you can only see a part of the structure so cell type identification can be somewhat ambiguous.  For speed of counting, light microscopy is usually easier.  By the way, I have generally found it easier to count the numbers of spared intact myelinated and unmyelinated fibers than to count degenerating fibers, since by definition, degenerating axons tend to be lost.  Good luck.
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If I want to transfect some optigenetic constructor in cultured hip neurons, and then light-induce action potential in axons, what is the best option?
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That answer depends a little on what you want to achieve from your experiment. You can use an AAV vector to transfect the cultured neurons with your opsin of choice (ChR2 or one of its variants). I've only ever transfected neurons in vivo so you might want to check out this reference for specific tips on doing this in cultured neurons: http://www.ncbi.nlm.nih.gov/pubmed/22367821
The choice of promoter that you use will be dictated by your experimental aims: if you want to transfect all neurons, use an AAV-hSyn-ChR2(H134R)-XFP construct, but be aware that that you will be eliciting both excitatory and inhibitory postsynaptic responses, although use of appropriate antagonists will get around this problem. If you want to transfect specific subtypes of neuron, use a floxed ChR2 construct and a mouse expressing Cre under a specific promoter (Emx1 for pyramidal cells, PV for parvalbumin interneurons, etc etc).
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I want to have channelrhodopsin expression only in the subthalamic nucleus (STN) to infect specifically the neurons in the STN.
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Hi Pietro,
Thank you so much for your reply.  I am performing the viral injection(Ch2-syn) protocol to target the subthalamic nucleus (STN). But i get a lot of expression in and around STN. B'se of this it is difficult to convince that the projections i am looking at is specific to the STN.
I should make it more clearer. I am searching for a cre-dependent tg-mouse model to target only the neurons in the STN by using a specific inducible viruses. In this way i can target only the STN projection neurons that are glutamatergic.
Thanks for the papers.
Ramki.
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In fact, I am have a good struggling with coinjection of fluorogold &BDA in the same brain region. My protocol for this combination is quite similar to which L. Swanson reported in PNAS, 2010 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2930585/), but with smaller injection current (+1-2 µA) and longer injection time (15-20 min). I can, however, get barely BDA labelled neurons or dendrites, but always very nice retrograde FG labelled cell bodies.
I almost always oberseve FG gets precipitated, especially up to first 5mm of glass pipette from its tip. I consider this might be a reason for a lack of BDA labeling in my case.
Any suggestions or comments? Thanks in advance!
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In my experience, the best choice for dissolving fluoro-gold is to use 0.1 M cacodylate buffer. This results in a crystalline solution without any trace of precipitates. By going this way you can inject FG either by iontophoresis or by pressure. This buffer also works for BDA. Just search PubMed for my earlier papers.
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Hexokinase I is predominant form of hexokinase family in mammalian brain. But under hypoxic conditions, there is no obvious change in its expression. In contrast, hexokinase II, which is mainly expressed in insulin-sensitive tissues and malignant tumors, experiences a robust upregulation. I think it is a norm that only when a protein's function has been compromised,  then  its subsitute may undergo a change to adjust to this situation.So my question is why hypoxia exposure can induce a significant uptegulation of hexokinase II whereas it has no effect on hexokinase I in brain? Is it possible that it may play some other roles in hypoxic situations? Thanks ~~
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Dear Yuan Lee,
Hexokinase II binds to the mitochondrial membrane through interaction with the outer membrane voltage-dependent anion channel (VDAC), preferentially at contact sites between the outer and inner mitochondrial membrane. This location is important for the integration of glycolysis with mitochondrial energy metabolism, since VDAC interaction with Bcl-2 antiapoptotic proteins controls the rate of release of mitochondrial intermembrane space proteins that activate the execution phase of apoptosis.
Therefore, Hexokinase II binding to VDAC suppresses the mitochondrial permeability transition and inhibits apoptosis, thereby contributing to the neuronal survival of hypoxia. That is why hypoxic conditions upregulate expression of this enzyme.
On the other side, Hexokinase I has no effect on Bcl-2 and apoptosis regulation, which makes it not useful in survival of hypoxic cellular stress.
I hope this helps you.
For more info see:
Chiara, Federica, et al. "Hexokinase II detachment from mitochondria triggers apoptosis through the permeability transition pore independent of voltage-dependent anion channels." PloS one 3.3 (2008): e1852.
Pastorino, John G., and Jan B. Hoek. "Hexokinase II: the integration of energy metabolism and control of apoptosis." Current medicinal chemistry 10.16 (2003): 1535-1551.
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I am planning to perform a pharmacological study to assess hippocampal neurogenesis via IGF-1 receptor inhibitor. But most studies were performed  in vitro or in vivo (systemic). So whether this inhibitor of IGF-1R (such as AG-1024 or picropodophyllin) can be  intracranially injected by stereotaxic localization and what is the optimal dose? Thank you!
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Ideally, you can infuse over hippocampus, because as mentioned by Giovanna you can induce gliosis in hippocampus.
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