Molecular Neurobiology - Science topic

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

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).

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.

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

sorry not

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.

آسف السؤال ليس من اختصاصي

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.

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...

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

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

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.

Thank you very much for your answer. Really interesting!

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.

@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?

We do have this line and currently characterizing it.  This is the line from Allen brain institute.

Have you checked this paper? http://www.jbc.org/content/289/23/16148.long

It's a discontinuous process but can be very fast (0.2-1 um/s)

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.

please, try to read

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.

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:

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.

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

Try to make your antibody titration in PBST (5% milk). I think that should help.

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. 

I want to prepare the single cell suspension from brain especially hippocampus to measure ADP/ATP Ratio.

thanks a lot!

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

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

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.

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.

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

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

 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.

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

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

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

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

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

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.

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/

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

I did post-fix and I like what I'm seeing, thanks :) Looks like the epitope is more visible. 

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.

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

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

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.

Thanks Rafik for the advice

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.

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.

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).

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

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.

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.

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.

AAV DJ serotype is also good. 

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.

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

Your high background is caused by the DTT in the buffer, which reacts with the DTNB. Try the reaction without the DTT.

I send you one article with title "Genetics of autism spectrum disorder & BDNF gene" by

Usha P. Dave

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

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.

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? 

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!

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.

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.

Hi Laura. Can you provide the details/citations for the conflicting studies?

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.

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.

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.

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.

Ideally, you can infuse over hippocampus, because as mentioned by Giovanna you can induce gliosis in hippocampus.

Inactivation of endogenous peroxidase with H2O2 does not improve non-specific labelling when you are doing immunofluorescence. Endogenous peroxidase reacts with hydrogen peroxide to reduce the 3,3'-diaminobenzidine (DAB) substrate or other peroxidase substrates, resulting in non-specific staining of the tissue.

The spinal cord is in fact very soft tissue and it requires some experience to divide it longitudinally and isolate a desired section. I manage to do it successfully in rat spinal cord. The mouse spinal cord is much smaller, however, I think that it is still doable. My suggestion would be: 1. use a stereomicroscope; 2. place your lumbar section of spinal cord in 100 mm Petri dish; 3. put the dish on a black plastic sheet to provide a contrasting background; 4 keep that all on ice – dividing may take a while. You can consider to freeze your sample and divide it when it's still frozen. That should eliminate the problem with tissue softness.

Hope that helps,

Best wishes

What method are you planning to use for spinal cord isolaton?

Very strange.  You shouldn't get two phases with such a buffer, but if I had to guess:

1) The "thick" layer is your genomic material. SDS lyses the nucleus and the chromosomes form a thick, very viscous and sticky liquid.  I've never seen it layer like you say, but simply sonicate your sample, or pass your lysate through a small gauge syringe quickly (2-3 times), or just freeze and thaw your sample and see if that fixes it.  

2) Too much material for the amount of SDS buffer.  SDS binds at a specific ratio of 1.4g SDS per 1g protein.  Most people forget this and try to lyse too much with too little detergent.   In this case you might get a floating layer of unsolubilized protein.  Just add more buffer.

Unless you are interested in seeing what mycoplasma infected microglia do, don't use such cells for two reasons. First, mycoplasma is very likely to affect the responses of microglia and other cells in the culture.  Second, it will infect other cells In the culture and other cultures in your incubator and facilities.  You should not only discard the cells but discard all cells that have been in the same incubator.  When we had a mycoplasma infection in our facility, we had to throw out all cultures from the incubator.

IEGs are surely not simple activity markers, but reflect "above threshold" significant stimulation e. g. a sensory stimulus like a taste or smell do not elicit IEG expression when familiar but do so when experienced for the first time. So the novelty adds the significance. It does not make sense to alter gene expression for every "normal" signal transmission, but only if something exceeds basal activities like coincidence of stimulation.

Hi Brian,

I'm doing this kind of experiment from months and the best solution to obtain a nice single cell suspension is using the neural tissue dissociation kit by MACS Miltenyi.

I prefer using the kit with Papain because is more gentle and maintain the surface markers of the cells. After that I separate the cells from the myelin by a Percol gradient and they are ready to be stained and sorted.

I used the sorted cells to perform the gene expression microarray and the results are really nice.

If you need some tip, just let me know

I agree to the previous answers. The most important fact is, that BV2 cells have a different gene expression pattern than primary microglia. In general, these cells seem to be pre-activated and might behave different upon stimuli. Thus, results obtained from BV2 cells may not be the same in primary microglia cultures and should be handled carefully.

A High Pressure Xenon Arc Lamp will do the work. You can also use a laser source but that would be probably a bit more expensive. the rest of the setup is exactly as a classic patch clamp rig with Ca2+ imaging capability. You can make it as fancy (more expensive) or as simple (less costly) as your budget can permit. The usual companies are known: Zeiss; Olumpus, Leika...etc. Of course, the Axon 200B could be used but any other amplifier could do as well.

Hi

A Nature Protocol paper shows very detail protocol step by step for microfluidic culture of neuroscience research. Also, they provide good troubleshooting information, enclosed please find it. And, JoVE also provide good VIDIO for how to generate a microfluidic culture platform for the compartmentalization of neuronsoma and axons (http://www.jove.com/video/261/fabrication-microfluidic-device-for-compartmentalization-neuron-soma).

With best,

Actually I'm working on blue native but the problem I face is that the presynaptic fraction contains triton x-100. The presynaptic fraction is the supernant part which I get after separation. Therefore when I compare the presynaptic and the PSD fraction (is the pellet)  I have to maintain the equal amount of protein (Does it make sense to compare both the presynaptic membrane fraction and the PSD fraction?). But always I find that after staining the gel the presynaptic fraction is  less protein concentration than the PSD fraction despite that I get more more concentration OD value for the presynaptic fraction. How far dialysis would help me that I don't want to remove completely triton x-100 from the presynaptic fraction as I need to run blue native and also I need to compare it with the PSD fraction in which case we need to extract (extraction buffer will contain the detergent) the proteins from the pellet. But the Amicon filtration doesn't help me.