Neuroanatomical Tract-Tracing Techniques - Science method

In most cases, if you biolistic delivery it does not. Unless you have severely shrunk samples.

Thanks for the advice!

I have tried a washout using aCSF with blockers,  for about 10-15 min but there wasnt much recovery from the test drug i used. However, in washout using aCSF up to an hour in aCSF, it shows some recovery. 

I understand that 10-15 min of washout using aCSF with blockers may not be long enough though.  

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.

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.

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.

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.

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.

Hi! You may use ImageJ software, which is free and essentially performing as well as expensive commercial solutions. Following links on the website you will find multiple tool packages for different applications including automated cell counting. Certainly, documentation and support is better in commercial software. The same often applies to user-friendlyness. You will have to decide, whether this is worth several thousand dollars. I would suggest to download some free trial versions and compare them to the free imageJ as a basis for your decision.

Best, Just

Thanks, I am going to try this, sounds very reasonable. 

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. 

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

This phenomenon can be observed in cell culture:

Other:

Regards,

Joachim

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.

http://dx.doi.org/10.1007/s00701-012-1379-0.

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.