We use doublecortin (DCX) as a marker to quantify the density of young neurons in adult zebra finches.
Below, I list some technical difficulties we encounter. I very much value suggestions or references addressing one, or more of the following:
We do not get clear, consistent labeling.
In the same bird, we can find clearly labeled, dark staining, and then faint labeling, or dark pigmentation on the neuron's soma contour, but faint colored soma.
How can we determine whether these differences are due to the immunohistochemical treatment?
We have a set protocol for DCX IHC. What are potential factors that contribute to such variability in labeling?
Is it also possible that some of these cells with faint labeling may be due to downregulation DCX expression, and upregulation of other markers of maturity like NeuN?
In songbirds, DCX is expressed in immature new neurons of a largely migratory population. The age of these neurons vary from 3 days old to 3 weeks old. Is it possible that these neurons with faint label are older? Should they be quantified/analyzed separately?
One solution for future studies is to use double or triple labeling. But for these tissue sections that are solely labeled with DCX--what are some ways we can proceed to quantify and measure soma size/shape?
I appreciate any insight, comments and whatever assistance into these matters.
I think a pool of young neurons in either organism consists of cells in a number of differentiated states, some being immature and others (partly) maturated. As neurons pass through these different states, it is known (in embryonic development) that expression of DCX changes. DCX is also linked to neuronal migration, and the position of neurons can also explain the observed differences. Because DCX is associated with microtubules, also cell shape can have an infuence on the signal pattern of each cell.
You can always add a negative control to check whether the heterogen signal is due to background staining. Also, there are very good commercial antibodies for NeuN available, and I would suggest to try this. Adding a positive control can help to determine if the staining pattern is biased because off the immunohistochemical method used, cause you know where to expect the positive signal. By combining the DCX staining with for example a NeuN staining, one can determine in which differentiated state a cell is, there should be some kind of balance between the DCX and NeuN signal: Young neurons --> DCX+, NeuN- and more mature neurons --> DCX-, NeuN+. Other neuronal markers can be used as well.
A possible way to quantify is to use the program ImageJ which is freely available online, and has some tools to measure cell dimensions.
Hope this helps!
I think a pool of young neurons in either organism consists of cells in a number of differentiated states, some being immature and others (partly) maturated. As neurons pass through these different states, it is known (in embryonic development) that expression of DCX changes. DCX is also linked to neuronal migration, and the position of neurons can also explain the observed differences. Because DCX is associated with microtubules, also cell shape can have an infuence on the signal pattern of each cell.
You can always add a negative control to check whether the heterogen signal is due to background staining. Also, there are very good commercial antibodies for NeuN available, and I would suggest to try this. Adding a positive control can help to determine if the staining pattern is biased because off the immunohistochemical method used, cause you know where to expect the positive signal. By combining the DCX staining with for example a NeuN staining, one can determine in which differentiated state a cell is, there should be some kind of balance between the DCX and NeuN signal: Young neurons --> DCX+, NeuN- and more mature neurons --> DCX-, NeuN+. Other neuronal markers can be used as well.
A possible way to quantify is to use the program ImageJ which is freely available online, and has some tools to measure cell dimensions.
Hope this helps!
Pieter, thank you for your response.
These two facts you've stated are the most interesting and relevant to the problem we are trying to solve: 1. The position of a neuron can account for observed differences, and 2. The cell shape influences the signal pattern of the given cell.
Do you recommend any publications or reviews which expand on this?
Do you know the studies from Balthazart?
Brain Behav Evol. 2014;84(1):1-4. doi: 10.1159/000362917. Epub 2014 Jul 17.
Doublecortin is a highly valuable endogenous marker of adult neurogenesis in canaries. Commentary on Vellema M et al. (2014): Evaluating the predictive value of doublecortin as a marker for adult neurogenesis in canaries (Serinus canaria) . J Comparative Neurol 522:1299-1315.
Balthazart J1, Ball GF.
(http://www.ncbi.nlm.nih.gov/pubmed/25034511)
J Comp Neurol. 2014 Dec 15;522(18):4100-20. doi: 10.1002/cne.23661. Epub 2014 Aug 20.
Endogenous versus exogenous markers of adult neurogenesis in canaries and other birds: advantages and disadvantages.
Balthazart J1, Ball GF.
(http://www.ncbi.nlm.nih.gov/pubmed/25131458)
By the way, if you are using exogenous proliferation markers, keep in mind that some of them may be toxic (see suppl mat of :
Proc Natl Acad Sci U S A. 2013 Mar 12;110(11):E1045-54. doi: 10.1073/pnas.1219563110. Epub 2013 Feb 21.
Cell cycle and lineage progression of neural progenitors in the ventricular-subventricular zones of adult mice.
Ponti G1, Obernier K, Guinto C, Jose L, Bonfanti L, Alvarez-Buylla A.
(http://www.ncbi.nlm.nih.gov/pubmed/23431204)
Good luck!
Giovanna
I don't have a review where it is clearly stated that these are the reasons for altered cellular signal, however it is reasonable to think that the difference in cell shape and position in immature versus mature cells is responsible for a heterogeneous location of antigenes, and thus immunohistochemical signal. For example, if you would follow Purkinje cell development, the position of these cells in the developing cerebellar cortex changes, as does cell shape, and these changes are reflected in a change in immunohistochemical signal. To prove that signal can vary, maybe these publications can help:
http://onlinelibrary.wiley.com/doi/10.1111/j.1460-9568.2004.03813.x/pdf
http://onlinelibrary.wiley.com/doi/10.1002/cne.10874/full
I hope you can sort it out!
Pieter
Dr. Ponti--yes, we are familiar with Balthazart's work, as well as the controversy posited by Manfred Gahr, where he shows some of the disadvantages of using DCX for the study of adult neurogenesis. Thank you for references--much appreciated.
Pieter, thanks for the references. By position--do you mean in terms of location in a brain region or nucleus? Or do you mean position in terms of stage of development?
Thank you both for your very helpful and useful comments.
Both can be true. Immature cells often reside in another brain regions before migrating to their final destination. Once fully matured, brain regions or nuclei can contain specific cell types with a specific expression profile. So, the word 'position' can be in terms of development (time) and location (space). I hope this is an answer to your question?
Regards,
Pieter
Dear Alicia,
In my experience (only mammals and a couple of lizards) DCX immunoreactivity is rather clear and strong in newly generated cells. The same is true for a population of non-newly generated DCX+/Tbr-1+ cells that we and others have described in ldorsal lateral/ ventral pallium derivatives of mammals. Faint cells in which the staining is restricted to the cell body can be occasionally observed, but in my experience they are not newly generated. You have to check with BrdU, there is no other way to understand if they are newly generated.
To analyze areas and shapes check fiji! , You may simly manually trace a diameter or, after segmentation make more detailed analyzes of areas and shapes.
Take a look also at the biovoxel plugins
http://fiji.sc/Fiji
http://fiji.sc/BioVoxxel_Toolbox
Dr. Luzzati,
Thank you kindly for answering my question and sharing your insight based on your experience.
Best,
Alicia
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