Science method

Neuroanatomical Tract-Tracing Techniques - Science method

Explore the latest questions and answers in Neuroanatomical Tract-Tracing Techniques, and find Neuroanatomical Tract-Tracing Techniques experts.
Questions related to Neuroanatomical Tract-Tracing Techniques
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
2 answers
Dear all,
I would try for the first time the DiI crystals for tracing neuronal pathways. I'm wondering if the Dil labelling, applyed post-mortem and post-fixation period, should pass the chemical synapses or not, as biocitine does. Just because I'm interested in labeling only the first neuron of the neural pathway, and not the second or the third etc.
Thank you in advance
Riccardo
Relevant answer
Answer
In most cases, if you biolistic delivery it does not. Unless you have severely shrunk samples.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
4 answers
I'm stuck on some basic mouse anatomy for a project I'm doing looking at descending motor pathways that are involved in forelimb function. I'm trying to figure out if the medullary reticular formation in the mouse brain contains the lateral reticular nucleus. I'm also trying to tease apart which areas contain the gigantocellular, parvoceullar, and magnocellular areas. 
Relevant answer
Answer
Terminations of reticulospinal fibers originating from the gigantocellular reticular formation in the mouse spinal cord.
I think, this is a good reference for your question.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
6 answers
When I add a drug, I have an increase in amplitude and frequency of sEPSCs (in presence of Gaba blocker) and sIPSCs( in presence of glutamate blocker). What does it exactly mean? What can I speculate from this result?
Relevant answer
Answer
Strictly speaking, if a drug changes the rise and decay time of a miniature synaptic event, you could conclude it is having a post-synaptic action. HOWEVER, if you see slowing of both the rise and the decay, I would think it was about 10 times more likely that your effect is due to loss of voltage clamp. I say that because it is very uncommon for a drug to alters the rise kinetics of synaptic events. On the other hand, changes in series resistance happen very frequently. Changes in series resistance will make your your events slower, (both rise and decay) and should also make them smaller. Though it should have a more pronounced effect on the rise kinetics of the event, rather than the decay (because they're faster). Anyway, if you find that your drug has no effect on the amplitude of the events, but makes them rise and decay more slowly, that would be a pretty good rebuttal to anyone claiming what you are observing is a loss of voltage clamp.
Theoretically, there are other explanations as well, for instance a change in the cable property of the dendrites could effect your ability to voltage clamp distal compartments, which could also change kinetics (though again, you'd also expect a change in the amplitude in this case).
Have a look at this page, if you don't understand why I say your results could be explained by a loss of voltage clamp.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
8 answers
We are working on Acute hippocampal slices for electrophysiological recordings using MEAs (Multichannel Systems) in rats.
We could not find any evoked or spontaneous firing of slices (350-400 microns) made with McIlwain tissue chopper either in young or adult rats.
Without spontaneous firing, can we proceed for further data streaming to analyse EPSP and population spikes???
We have a doubt, whether NMDG can be added directly to the conventional aCSF solution to get better response and prolonged viable slices.
Please advise and share your thoughts in this regard.
Thanking you,
Best Regards,
Grandhi V Ramalingayya
Relevant answer
Answer
From the point of my view  important is very quick excision of brain from skull and short cooling of prepared hippocampus in ice-cold aCSF solution before cutting (cca 1 min).  McIllwain tissue chopper is quite sufficient for cuting slices for electrophysiology, we use it also. Try to set it to such speed that you can gently take each slice separately with wet brush from the blade. I also argee with the most previous suggestions and I would added that temperature of aCSF solution is suitable up to 33-34 oC during the rest of slices and  recordings in the measuring chamber.  Good luck.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
7 answers
I am interested in identifying the brain region displaying relatively high immuno-reactivity for a protein of interest (red) in the attached images of a mouse brain (co-labeled for NeuN-green). I can identify that the signal is adjacent to the caudate, mostly posterior-medial to it. But as there many small structures in this region I am unfamiliar with, I'm not 100% confident picking one out from a mouse brain atlas schematic. My best guess based on the atlas is the globus pallidus - but if anyone can help confirm or refute, that would be great. I have not looked at many other potential regional markers, but the red-IR also colocalizes with GAD67 in this region.
Relevant answer
Answer
Hi Andrew.
I´m more familiar with the rat brain. But looking to the first image I can make a guess that you are staining the amygdala. Also, it makes sense to me that your red-IR colocalizes with GAD67, because all the amygdala (Central amygdala, Basolateral amygdala and Medial Amygdala) is very rich in GABA neurons. But according to the images, I would say that you are staining the central amygdala and the basolateral amygdala.
Also, I think that you are staining the globus pallidus too.
Best wishes.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
6 answers
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!
Relevant answer
Answer
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.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
6 answers
I used DiI and I traced very well some fibers, but a disadvantage is that when it's exposed to UV light in a microscope, loses fluorescence very quickly. I need a tracer in fixed tissue because it's easier to administrate the tracer in the zone that I'm studying than in stereotaxic. Thanks.
Relevant answer
Answer
Greetings,
From what I understand of your question, DiI is working for you, but you are having issues with photobleaching. Unfortunately, DiI is actually one of the more photo-stable dyes out there. You may want to try optimizing your imaging technique. I am not entirely sure why you are seeing bleaching form from UV since DiI is excited primarily by blue/green light. To minimize bleaching you can try some of the following:
1: Reduce imaging time (try performing your analysis on pre-imaged sections or virtual slices)
2: Minimize excitation energy (photo-bleaching of DiI follows 1st order kinetics and is dependent on excitation energy (see link). Also, at some point more excitation does not equal more fluorescence instead you just get more background and bleaching, and is very easy to do with laser excitation.)
3: Using a mounting agent with an anti-fading agent. These work by preventing oxidation of dyes. They do reduce the amount of fluorescence, but they also increase their longevity.   
4: Double check your filters and light source. Mercury HBOs tend to have a lot of energy in the UV emission spectra. If your UV excitation filter lets the 405nm peak through this may have a strong impact on bleaching. 
Best of luck.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
8 answers
I am looking for a trans-synaptic anterograde tracer - does anyone know if wheat germ agglutinin conjugated to an alexa fluor is able to cross synapses?
I would like to do an in vivo injection into the ventral Hippocampus and expect to see labeling in the mPFC... I know that this is a distance, but I only truly need WGA to get to the second order neuron, so I feel like a viral system (such as HSV129) is overkill. 
Has anyone tried WGA-fluor for a similar application? Thanks in advance! 
Relevant answer
Answer
Just to add to the information mentioned above about WGA:
- It apparently tends to induce inflammatory responses in the injection site moreso than other tracer chemicals.
- It is allegedly taken up by some glial cells between first and second order neurons.
- Not only does circuit length (distance the tracer must travel) make things difficult in estimating survival time, but WGA transcytoses on a rather fast but variable time scale, ranging from one to four days. This effect seems to be concentration-dependent. You'd have to test out time courses and concentrations in several mice to determine the optimal transsynaptic spread. 
With its bidirectional spread, I'm not sure that WGA would be appropriate. You wouldn't be able to isolate transsynaptic anterograde labeling from transsynaptic retrograde labeling. You may want to look into using recombinant Tetanus Toxin C Fragment (TTC), which is preferrentially retrograde and transsynaptic, and tracing from PFC back to vHipp. The main issue there is that recombinant TTC is prohibitively expensive to purchase.
One last general thing - using IHC staining will provide superior visibility of labeled cells compared to visualizing the fluorescently-conjugated tracer as-is (in most cases). This may be important if transcytosis substantially dilutes your tracer over synapses.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
9 answers
Hi. I am planning on doing unbiased counting of TH positive neurons in the substantia nigra region in our animal model. I would like to know if there are any good software out there to do unbiased counting. 
Relevant answer
Answer
There are some plugins for ImageJ that allow automatic cell counting and, best of all, they are free so might be worth trying out in your system. The ones I know of are:
I'm not sure how well either package works. I used automated detection once in the Fiji distribution of ImageJ (
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
4 answers
We would like to keep a substantial sample of aldehyde fixed rat brain samples (perfusion, PFA & GA mix) for later EM studies. What medium should we use to maintain ultrastructure and antigen properties as well? How long can we keep these brains at -80 degrees for TEM and IHC? Do you have any experience with the effects of long term, low temp storage on synaptic ultrastructure? Any advice or protocols are appreciated.
Relevant answer
Answer
Thanks, I am going to try this, sounds very reasonable. 
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
20 answers
Hello,
We are injecting tracer into the mouse brain using a picospritzer.  Our target region is the bed nucleus of the stria terminalis which is in close proximity to the lateral ventricles.  My concern then is that perhaps during injection or when the injection pipette is raised, we are getting tracer into the ventricular system which is then creating false positive projection sites by infiltrating neurons along the ventricular wall.  Has anyone determined a method of ruling out or otherwise assessing this off-target effect?
Relevant answer
Answer
Hi Justin, you can avoid the spread in different ways.
1) if you have a pico-injector that allows you to apply a slightly negative pressure, once you lower the pipette you won't lose tracer from the tip. 
2) if you load a tiny drop of some neutral unsoluble solution after your tracer (mineral oil) this, even if really low in quantity, will efficiently block the spread.
3) if your injection is low in volume (an injection every minute, for a volume of 20nL ca) for a total of 1-3 injections and if you wait at least 10-15 minutes after the last before extracting the pipette from the tissue, you should be able to avoid the spread that usually occurs at the end of the injection protocol.
to improve specificity and precision some people even start injecting 5 minutes after lowering the pipette, which in this case is a step that takes 1 minute each 200-300 micrometer (usually I am not that slow but this could allow the tissue to come back in shape, specially if you reach a deep region)
relatively to your problem, depending on which BNST nucleus you are targeting you almost can't avoid the ventricles. best would be to enter from the cortex above the CPu with an angled injection (you can easily re-adjust coordinates with pitagora) which would totally avoid the issue, roughly could be 30 degrees. that's what I do. depending on which dye/tracer/virus you inject the spread in the ventricles could also be negligible because it would get diluted. of course most important is to know whether the tracer is anterograde or retrograde so that you know where you have to look for cell bodies or fibers you won't be 100% precise anyway, but testing on different animals will give you the right control, by comparison. 
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
3 answers
If so, you might be interested in our development of such future devices.
We are a university group and think about starting a company in future. Don´t worry, this is not an advertising or so, I do not want to sell you anything.
We would just like to cordially invite you to take part in a very short poll to get very first impressions of the academics´ and other professionals´ needs.
Please answer six short questions regarding the academic and the pharmaceutical version we have in mind. This takes just five minutes of your time per poll.
For you convenience, you can use the following two survey links:
For the ACADEMIC system (LOW throughput, SEMI-AUTOMATED):
For the PHARMACEUTICAL system (HIGHER throughput, FULLY AUTOMATED):
Let´s shape the future of patch clamp analysis together! You are free to leave your personal data, if you would like to test our prototypes in future (2-3 years)! You can visit our university project site on the following website link:
Relevant answer
Answer
Hi Gabriele,
the mentioned nanion products do not allow for the analysis of cellular networks. This product family and ALL other APC systems availbale were developed and optimized for cells in suspension, cells freshly detached from culture flasks or cells from thawn vials. These APCs cannot analyze cultured cellular networks and were not built for that issue. Let me know if that´s not right.
Don´t get me wrong, nanion and the other APC producers did a great job with their APC development. Chapeau! The devices are great if you need higher troughput. I guess the new nanion 384-channel machine will have a good response in the market. Congrats to nanion!
But if you need network data or a higher organisation grade like in a cell network (some ion channels are only expressed when the cells are attached to a substrate or to other cells...) or in a brain slice, you are limited to manual patch clamp. And we all now that using a manual rig does not allow for higher throughput (5-20 data points per day if you are very experienced). Hence, we believe (and know from our potential customers already) that our future systems are complementary to conventional APCs. Further, our system will drastically increase the parallel number of patched cells, up to 16 at once with one chip! If we get that done: world record! I am sure that the information of the cell-cell-communication analyzed with our systems will be of very high value in future.
By the way, Prof. Markram´s 1 billion € project uses a 12-channel setup (http://www.2045.com/news/31235.html). The rig can be seen in the last figure in this article. The parallel patch clamp of neurons is an important tool for his research.
Well, with our short survey we would like to know the point of view of scientists regarding our technology to define our product development when we start our company in future.
For further information, please contact me under philipp.koester@uni-rostock.de
Best regards, Philipp
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
2 answers
The Economist magazine (Aug. 3, 2013) published the following statement in the article entitled ‘The Machine of a New Soul’:
“An important property of a real brain is that it is what is referred to as a small-world network. Each neuron within it has tens of thousands of synaptic connections with other neurons. This means that, even though a human brain contains about 86 billion neurons, each is within two or three connections of all the others via myriad potential routes.”
Does anyone have a reference for the statement ‘each [neuron] is within two or three connections of all the others via myriad potential routes’?
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
2 answers
I am a beginner user of Neurolucida and Neuroexploler
I am placing different markers on a dendritic tree either on the dendritic shaft or onto the bifurcation points of that previously traced dendrite. Later after saving and trying to open the file by Neurolucida explorer to get the branched dendogram I found some problems:
1- some markers are shift away from its proposed position on the dendogram, for example away from the bifurcation point or even the shaft itself on which I surely attached that marker in neurolucida.
2-sometimes, some or all my markers can not be displayed on the dendogram even I checked marker counts /object markers on segment in the display menu of the explorer.
3- frequent difficulty to attach markers onto certain points of the dendritic shaft, for instance attaching markers every 50 microns using neurolucida but the dendogram displayed later with the markers has not accurate distances.
Relevant answer
Answer
Thanks you very much Dr.Oliver,
I had a problem of matching the placed markers into their points on the dendritic tree, the dendogram obtained is not accurate. later I found that the Z position of that markers was not matching with these points but now I fixed it.
  • asked a question related to Neuroanatomical Tract-Tracing Techniques
Question
8 answers
Lesionectomy can influence positively disturbed neurological functions by influencing neural neworks.
Relevant answer
Answer
Let me point you to an article recently published by the neurosurgery department of Geneva University Hospitals, Switzerland, that focused on neuropsychological outcomes following extra-temporal lobe resections. An interesting finding is that neuropsychological improvement did not depend strictly on seizure freedom.
El Hassani Y, Fournet M, Momjian S, Pollo C, Seeck M, Pegna A, Schaller K.
Neuropsychological outcome after extra-temporal epilepsy surgery.
Acta Neurochir (Wien). 2012 Aug;154(8):1337-42.
Of note, neuropsychological improvement following surgery in children with severe epilepsy of early onset is not systematic and depends on multiple factors. Cf. Roulet-Perez E, Davidoff V, Mayor-Dubois C, Maeder-Ingvar M, Seeck M, Ruffieux C, Villemure JG, Deonna T. Impact of severe epilepsy on development: recovery potential after successful early epilepsy surgery. Epilepsia. 2010 Jul;51(7):1266-76. http://dx.doi.org/10.1111/j.1528-1167.2009.02487.x.