Name: A Comparative Approach for Quantitative Cell Counting Studies in Widely Different Mammalian Brains (Video) | JoVE Journal
Uploaded: 2026-01-16T13:15:00.000+00:00
Duration: 7 min 14 s
Description: Explore comparative methods for quantitative cell counting across mammalian brains. This approach addresses technical challenges in large-brained species and reveals evolutionary patterns in neuronal development.

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We thank Matteo Piumatti and Chiara La Rosa for having contributed to defining the protocol reported here for cortical immature neurons. The work was supported by Fondazione CRT - Cassa di Risparmio di Torino (grant RF=2022.0618) to LB; PRIN2022 (grant 2022LB4X3N) to LB; National Science Foundation (grants EF-2021785, DRL-2219759) and National Institutes of Health (grants NS092988, AG067419) to CCS.

<ol><li>Brenowitz, E. A., Zakon, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Emerging+from+the+bottleneck%3A+benefits+of+the+comparative+approach+to+modern+neuroscience%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Emerging from the bottleneck: benefits of the comparative approach to modern neuroscience.</a> Trends Neurosci. 38 (5), 273-278 (2015).</li>
<li>Bolker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Animal+models+in+translational+research%3A+rosetta+stone+or+stumbling+block%5BTitle%5D)+AND+%22Bioessays%22%5BJournal%5D)">Animal models in translational research: rosetta stone or stumbling block.</a> Bioessays. 39 (12), 1-8 (2017).</li>
<li>Moreno-Jiménez, E. P., Terreros-Roncal, J., Flor-García, M., Rábano, A., Llorens-Martín, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evidences+for+adult+hippocampal+neurogenesis+in+humans%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Evidences for adult hippocampal neurogenesis in humans.</a> J Neurosci. 41 (12), 2541-2553 (2021).</li>
<li>Sorrells, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Positive+controls+in+adults+and+children+support+that+very+few%2C+if+any%2C+new+neurons+are+born+in+the+adult+human+hippocampus%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Positive controls in adults and children support that very few, if any, new neurons are born in the adult human hippocampus.</a> J Neurosci. 41 (12), 2554-2565 (2021).</li>
<li>Simard, S., Matosin, N., Mechawar, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+hippocampal+neurogenesis+in+the+human+brain%3A+updates%2C+challenges%2C+and+perspectives%5BTitle%5D)+AND+%22Neuroscientist%22%5BJournal%5D)">Adult hippocampal neurogenesis in the human brain: updates, challenges, and perspectives.</a> Neuroscientist. 31 (4), 141-158 (2024).</li>
<li>Bao, H., Song, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treating+brain+disorders+by+targeting+adult+neural+stem+cells%5BTitle%5D)+AND+%22Trends+Mol+Med%22%5BJournal%5D)">Treating brain disorders by targeting adult neural stem cells.</a> Trends Mol Med. 24 (12), 991-1006 (2018).</li>
<li>Ghibaudi, M., Zanone, A., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+structural+plasticity+in+large-brained+mammals%3A+not+only+narrowing+roads%5BTitle%5D)+AND+%22Neural+Regen+Res%22%5BJournal%5D)">Brain structural plasticity in large-brained mammals: not only narrowing roads.</a> Neural Regen Res. 21 (5), 1669-1680 (2026).</li>
<li>Bonfanti, L., La Rosa, C., Ghibaudi, M., Sherwood, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Adult+neurogenesis+and+%26%2334%3Bimmature%26%2334%3B+neurons+in+mammals%3A+an+evolutionary+tradeoff+in+plasticity%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">Adult neurogenesis and &#34;immature&#34; neurons in mammals: an evolutionary tradeoff in plasticity.</a> Brain Struct Funct. 229 (8), 1775-1793 (2024).</li>
<li>Paredes, M. F., Sorrells, S. F., Garcia-Verdugo, J. M., Alvarez-Buylla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+size+and+limits+to+adult+neurogenesis%5BTitle%5D)+AND+%22J+Comp+Neurol%22%5BJournal%5D)">Brain size and limits to adult neurogenesis.</a> J Comp Neurol. 524 (3), 646-664 (2016).</li>
<li>Parolisi, R., Cozzi, B., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Humans+and+dolphins%3A+decline+and+fall+of+adult+neurogenesis%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Humans and dolphins: decline and fall of adult neurogenesis.</a> Front Neurosci. 12, 497(2018).</li>
<li>Snyder, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recalibrating+the+relevance+of+adult+neurogenesis%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Recalibrating the relevance of adult neurogenesis.</a> Trends Neurosci. 42 (3), 164-178 (2019).</li>
<li>Bonfanti, L., Charvet, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+plasticity+in+humans+and+model+systems%3A+advances%2C+challenges%2C+and+future+directions%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Brain plasticity in humans and model systems: advances, challenges, and future directions.</a> Int J Mol Sci. 22 (17), 9358(2021).</li>
<li>Duque, A., Arellano, J. I., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+assessment+of+the+existence+of+adult+neurogenesis+in+humans+and+value+of+its+rodent+models+for+neuropsychiatric+diseases%5BTitle%5D)+AND+%22Mol+Psychiatry%22%5BJournal%5D)">An assessment of the existence of adult neurogenesis in humans and value of its rodent models for neuropsychiatric diseases.</a> Mol Psychiatry. 27, 377-382 (2022).</li>
<li>Gómez-Climent, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+population+of+prenatally+generated+cells+in+the+rat+paleocortex+maintains+an+immature+neuronal+phenotype+into+adulthood%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">A population of prenatally generated cells in the rat paleocortex maintains an immature neuronal phenotype into adulthood.</a> Cereb Cortex. 18 (10), 2229-2240 (2008).</li>
<li>Rotheneichner, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cellular+plasticity+in+the+adult+murine+piriform+cortex%3A+continuous+maturation+of+dormant+precursors+into+excitatory+neurons%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">Cellular plasticity in the adult murine piriform cortex: continuous maturation of dormant precursors into excitatory neurons.</a> Cereb Cortex. 28 (7), 2610-2621 (2018).</li>
<li>Sorrells, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immature+excitatory+neurons+develop+during+adolescence+in+the+human+amygdala%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Immature excitatory neurons develop during adolescence in the human amygdala.</a> Nat Commun. 10 (1), 2748(2019).</li>
<li>La Rosa, C., Parolisi, R., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((structural+plasticity%3A+from+adult+neurogenesis+to+immature+neurons%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">structural plasticity: from adult neurogenesis to immature neurons.</a> Front Neurosci. 14, 75(2020).</li>
<li>Bonfanti, L., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+PSA-NCAM-positive+%26%2334%3Bimmature%26%2334%3B+neurons%3A+an+old+discovery+providing+new+vistas+on+brain+structural+plasticity%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">The PSA-NCAM-positive &#34;immature&#34; neurons: an old discovery providing new vistas on brain structural plasticity.</a> Cells. 10 (10), 2542(2021).</li>
<li>Benedetti, B., Couillard-Després, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+would+the+brain+need+dormant+neuronal+precursors%5BTitle%5D)+AND+%22Front+Neurosci%22%5BJournal%5D)">Why would the brain need dormant neuronal precursors.</a> Front Neurosci. 16, 877167(2022).</li>
<li>Luzzati, F., Bonfanti, L., Fasolo, A., Peretto, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DCX+and+PSA-NCAM+expression+identifies+a+population+of+neurons+preferentially+distributed+in+associative+areas+of+different+pallial+derivatives+and+vertebrate+species%5BTitle%5D)+AND+%22Cereb+Cortex%22%5BJournal%5D)">DCX and PSA-NCAM expression identifies a population of neurons preferentially distributed in associative areas of different pallial derivatives and vertebrate species.</a> Cereb Cortex. 19 (5), 1028-1041 (2009).</li>
<li>Palazzo, O., La Rosa, C., Piumatti, M., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do+large+brains+of+long-living+mammals+prefer+non-newly+generated%2C+immature+neurons%5BTitle%5D)+AND+%22Neural+Regen+Res%22%5BJournal%5D)">Do large brains of long-living mammals prefer non-newly generated, immature neurons.</a> Neural Regen Res. 13 (4), 633-634 (2018).</li>
<li>La Rosa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phylogenetic+variation+in+cortical+layer+II+immature+neuron+reservoir+of+mammals%5BTitle%5D)+AND+%22Elife%22%5BJournal%5D)">Phylogenetic variation in cortical layer II immature neuron reservoir of mammals.</a> Elife. 9, e55456(2020).</li>
<li>Sherwood, C. C., Gómez-Robles, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Brain+plasticity+and+human+evolution%5BTitle%5D)+AND+%22Annu+Rev+Anthropol%22%5BJournal%5D)">Brain plasticity and human evolution.</a> Annu Rev Anthropol. 46, 399-419 (2017).</li>
<li>Piumatti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-newly+generated%2C+%26%2334%3Bimmature%26%2334%3B+neurons+in+the+sheep+brain+are+not+restricted+to+cerebral+cortex%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Non-newly generated, &#34;immature&#34; neurons in the sheep brain are not restricted to cerebral cortex.</a> J Neurosci. 38 (4), 826-842 (2018).</li>
<li>Pattaro, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phylogenetic+variation+of+layer+II+cortical+immature+neurons+in+dog+and+horse+confirms+covariance+with+brain+size+and+neocortical+surface%5BTitle%5D)+AND+%22Brain+Struct+Funct%22%5BJournal%5D)">Phylogenetic variation of layer II cortical immature neurons in dog and horse confirms covariance with brain size and neocortical surface.</a> Brain Struct Funct. 230 (6), 115(2025).</li>
<li>Ghibaudi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Consistency+and+variation+in+doublecortin+and+Ki67+antigen+detection+in+the+brain+tissue+of+different+mammals%2C+including+humans%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Consistency and variation in doublecortin and Ki67 antigen detection in the brain tissue of different mammals, including humans.</a> Int J Mol Sci. 24 (3), 2514(2023).</li>
<li>Ben Abdallah, N. M., Slomianka, L., Vyssotski, A. L., Lipp, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Early+age-related+changes+in+adult+hippocampal+neurogenesis+in+C57+mice%5BTitle%5D)+AND+%22Neurobiol+Aging%22%5BJournal%5D)">Early age-related changes in adult hippocampal neurogenesis in C57 mice.</a> Neurobiol Aging. 31, 151-161 (2010).</li>
<li>Ghibaudi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+changes+in+layer+II+immature+neurons+of+the+murine+piriform+cortex%5BTitle%5D)+AND+%22Front+Cell+Neurosci%22%5BJournal%5D)">Age-related changes in layer II immature neurons of the murine piriform cortex.</a> Front Cell Neurosci. 17, 1205173(2023).</li>
<li>Workman, A. D., Charvet, C. J., Clancy, B., Darlington, R. B., Finlay, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modeling+transformations+of+neurodevelopmental+sequences+across+mammalian+species%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Modeling transformations of neurodevelopmental sequences across mammalian species.</a> J Neurosci. 33 (17), 7368-7383 (2013).</li>
<li>Charvet, C. J., de Sousa, A. A., Vassilopoulos, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Translating+time%3A+challenges%2C+progress%2C+and+future+directions%5BTitle%5D)+AND+%22Brain+Res+Bull%22%5BJournal%5D)">Translating time: challenges, progress, and future directions.</a> Brain Res Bull. 221, 111212(2025).</li>
<li>Bartges, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AAHA+canine+life+stage+guidelines%5BTitle%5D)+AND+%22J+Am+Anim+Hosp+Assoc%22%5BJournal%5D)">AAHA canine life stage guidelines.</a> J Am Anim Hosp Assoc. 48, 1-11 (2012).</li>
<li>Ghibaudi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multispecies+characterization+of+immature+neurons+in+the+mammalian+amygdala+reveals+their+expansion+in+primates%5BTitle%5D)+AND+%22PLoS+Biol%22%5BJournal%5D)">Multispecies characterization of immature neurons in the mammalian amygdala reveals their expansion in primates.</a> PLoS Biol. 23 (8), e3003322(2025).</li>
<li>Ghibaudi, M., Boda, E., Bonfanti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+mice+to+humans%3A+a+need+for+comparable+results+in+mammalian+neuroplasticity%5BTitle%5D)+AND+%22Neural+Regen+Res%22%5BJournal%5D)">From mice to humans: a need for comparable results in mammalian neuroplasticity.</a> Neural Regen Res. 20 (2), 464-466 (2025).</li>
<li>Smulders, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+relevance+of+brain+evolution+for+the+biomedical+sciences%5BTitle%5D)+AND+%22Biol+Lett%22%5BJournal%5D)">The relevance of brain evolution for the biomedical sciences.</a> Biol Lett. 5, 138-140 (2009).</li>
<li>Keck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bridging+theory+and+experiment+in+%28statistical%29+learning%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Bridging theory and experiment in (statistical) learning.</a> Curr Opin Neurobiol. 94, 103106(2025).</li>
</ol>

The authors have nothing to disclose.

The method described here provides comparable quantitative data when diverse species widely differing in brain sizes and neuroanatomy are considered. It can be used to assess the consistency of cell populations in the whole brain of diverse mammals, as well as to follow their development in a single species across subsequent stages, from postnatal to aging. It starts with the assumption that a relatively high number of brains (whole hemispheres) and species will be involved, being processed by the same lab following qual...

Neuroscience research largely uses inbred mice and rats to translate results to humans. Despite many molecular, cellular, and even anatomical similarities, important interspecies differences also exist, which can be misleading for the interpretation of preclinical data1,2. Though comparative studies are needed, they are hindered by technical and practical difficulties when large-sized brains are involved (e.g., from an average of 0.5 g in mice to 1300 g in humans; Figure 1A). Moreover, heterogeneous experimental approaches by different laboratories can limit the comparison of results, especially quantitative analyses, sometimes generating controversies. This is the case of brain structural plasticity, with special reference to neurogenic processes3,4,5, a topic that is of interest in understanding developmental neuroplasticity and brain repair6,7.
Recent discoveries in the field of brain plasticity strongly suggest that substantial differences emerged during evolution in the occurrence, rate, and anatomical location of different types of neurogenic processes8. An example is given by the evident decrease of stem cell-driven neurogenesis occurring in adult large-brained mammals compared with laboratory rodents5,9,10, likely linked to different expansion of brain regions and functions as an adaptation to ecological pressures7,8, as well as to different neurodevelopmental time courses11,12. The rate of neurogenesis appears to differ remarkably between mice and humans, thus raising the issue of translation13.
On the other hand, a novel kind of "neurogenesis without division" represented by neurons blocked in arrested maturation (so-called immature or dormant neurons14,15,16,17,18,19) has been hypothesized to increase from small-brained to large-brained species20,21. Interestingly, the stem cell-independent population of immature neurons is hosted in the cerebral cortex (a site of high-order cognition in gyrencephalic species22,23) and far from the canonical neurogenic sites, which are mostly linked to olfaction and spatial navigation, which retain great importance in lissencephalic species7,8. These differences across brain structures and mammal species go beyond sheer comparative interest since they can be relevant in translation by shifting the potential for plasticity in mice and humans. For these reasons, it is crucial to assess in a "comparable way" to what extent the differences represent a phylogenetic variation across widely different mammals, with special reference to laboratory rodents and primates.
An approach recently established to study the interspecies variation of a population of layer II cortical immature neurons (cINs) in mammals widely differing in brain size, gyrencephaly, and socioecological niche22,24,25is described. This method is based on three principles: i) to consider several (diverse) mammalian species to be processed in parallel using the same procedures; ii) to minimize variables that cannot be fully standardized in widely different brains (fixation procedure, postmortem interval, age) and to address the species-specificity of antibodies for immunocytochemistry by using widely shared features of the cell population under study as internal positive controls; and iii) to establish corresponding anatomical structures as reference points for performing the cell counting on correspondent brain levels of different species (Figure 1B and Figure 2). Data obtained (e.g., cell densities) by counting cells identified with the well-established marker doublecortin (DCX15,17,26) can be mapped onto phylogenetic trees to reveal evolutionary patterns and analyzed for covariance with different parameters, such as brain size, gyrencephaly, and cortical surface area.
<img alt="Brain size evolution chart and cerebral cortex diagrams; mammalian species comparison; lissencephalic vs. gyrencephalic brains." class="xfigimg" src="/files/ftp_upload/69446/69446fig1.jpg" data-altupdatedat="1784001604000" data-alt="Figure 1"> 
Figure 1: Processing of widely different mammalian brains for assessing the distribution and amount of cINs in the cortical mantle. (A) Mammals belonging to different species and orders are characterized by widely different brain sizes and degrees of gyrencephaly and cortical expansion. Brain icons reproduced with BioRender license. (B) Representative Toluidine blue-stained coronal sections cut from brains of widely different sizes and cortical mantle extension. Histologically stained serial sections (cryostat cut, coronal, 40 µm thick) are used to identify the main neuroanatomical structures in each animal species (see Figure 3); note the remarkable differences in brain size and cortical perimeter. The animal icons in panel A have been reproduced with permission from La Rosa et al.22. <a href="https://www.jove.com/files/ftp_upload/69446/69446fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Finally, since all plastic processes generally undergo progressive reduction with increasing age27,28, one further issue in comparative neuroplasticity is to assess the different time courses of such a reduction, which may be related to different time courses of neurodevelopmental processes across mammals12,29,30. As a matter of fact, the existence of fast-maturing (mouse) versus slow-maturing species (primates) is another reason why the most common model system, the laboratory mouse, is limited in resolving challenges in biomedical sciences. The method described here can also be useful to consider cell populations at different ages across the lifespan of each species and then compare the resulting trends among species. Phylogenetic variation in the cIN density, as well as differences in their persistence through ages, were found between small-brained and large-brained mammals22,28.

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a comparative approach for quantitative cell counting studies in widely different mammalian brains

A Comparative Approach for Quantitative Cell Counting Studies in Widely Different Mammalian Brains

Most neurobiological studies are conducted on laboratory rodents. Despite many similarities across mammalian brains, important differences also exist, which can be misleading in translation. Marked interspecies differences have been found in brain plasticity, particularly neurogenesis. Different neurogenic processes can be prevalent because of evolutionary tradeoffs, displaying variation across brain structures and mammals and shifting the potential for plasticity in divergent species, such as mice and humans. Comparing widely different species raises multiple issues: comparative studies encounter technical difficulties when large-sized brains are involved; the use of heterogeneous experimental approaches by different laboratories can limit the comparison of results; heterogeneity may be related to different time courses of neurodevelopmental processes across mammals, thus adding variables to the comparison.
To tackle these limitations, an approach to study the interspecies variation of a population of layer II cortical immature neurons in mammals widely differing for brain size, gyrencephaly, socioecological niche, and age was established. Despite some variables that cannot be fully standardized, a method that combines reduced heterogeneity in collecting brains and the establishment of common anatomical structures as reference points for performing the cell counting on corresponding brain levels is proposed. Data obtained (e.g., cell densities) can be mapped onto phylogenetic trees to reveal evolutionary patterns and analyzed for covariance with neuroanatomical features (e.g., brain size, cortical surface area). This approach has demonstrated remarkable variation in the number of cortical immature neurons between phylogenetic groups and uncovered covariation with brain size. The method can also be used to quantify differences through different developmental stages in the same species and can be extended to other diverse mammals and biological processes to map comparable results that allow for more accurate quantification of different cell populations in adult brains to support plasticity.

The DCX+ cells in the cerebral cortex of all species studied consistently share a series of features indicating the existence of a well-preserved population of undifferentiated neurons (Figure 4A). In brief, they: (i) are topographically arranged to form a monolayer along cortical layer II; (ii) show the morphological subtypes described for different maturational stages in immature neurons, (a) with a large prevalence of small, unipolar/bipolar cells and a small fraction of cells with comple...

Modern neurosciences need to explore differences among many species, including laboratory rodents and humans. Yet technical difficulties arise when large-brained mammals are involved. This approach addresses interspecies variation of neuronal populations in widely different mammals, yielding comparable results to be mapped for revealing evolutionary patterns.

Most neurobiological studies are conducted on laboratory rodents. Despite many similarities across mammalian brains, important differences also exist, ...

a-comparative-approach-for-quantitative-cell-counting-studies-widely

Research

JoVE Journal

Neuroscience

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The comparative species approach allows behavioral neuroscientists to explore various neurobiological factors associated with specific behaviors viewed as characteristic of a specific animal model.  Taking advantage of naturally occurring differences in behavior between closely related species, this technique doesn’t require invasive techniques to manipulate the expression of the behavior.

Using a Comparative Species Approach to Investigate the Neurobiology of Paternal Responses

Source: Olesen, M. V., et al. The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy. J. Vis. Exp. (2017). The video demonstrates the technique for quantifying newly generated neurons in brain tissue. Hippocampal tissue slices containing Bromodeoxyuridine (BrdU)-labeled neurons were stained using immunohistochemistry and subsequently quantified across the tissue thickness using the optical fractionator technique.

Quantifying Newly Generated Neurons in Rat Brain Tissue Using the Optical Fractionator Technique

Here, we present a stereological method, the optical fractionator, used to quantify the formation of new neurons, and their survival, in the rat hippocampus following electroconvulsive stimulation. When correctly implemented, the sensitivity and efficiency of stereological methods ensures accurate estimates with a fixed and predetermined precision.

The Optical Fractionator Technique to Estimate Cell Numbers in a Rat Model of Electroconvulsive Therapy

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

This article describes a straightforward method to prepare small animal brains for micro-CT imaging, in which lesions can be quantified and electrodes located with high precision in the context of the whole brain.

A Micro-CT-based Method for Characterizing Lesions and Locating Electrodes in Small Animal Brains

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

Here we present an automated method for semi-quantitative determination of dopaminergic neuron number in the rat substantia nigra pars compacta.

Semi-Quantitative Determination of Dopaminergic Neuron Density in the Substantia Nigra of Rodent Models using Automated Image Analysis

This protocol describes methods for conducting magnetic resonance imaging, clearing, and immunolabeling of intact mouse brains using iDISCO+, followed by a detailed description of imaging using light-sheet microscopy, and downstream analyses using NuMorph.

Whole-Brain Single-Cell Imaging and Analysis of Intact Neonatal Mouse Brains Using MRI, Tissue Clearing, and Light-Sheet Microscopy

Serial two-photon tomography (STPT) imaging is a technique to image a mass of tissue in its three-dimensional shape by combining two-photon imaging with automatic stage control and microtome slicing. Here we describe a protocol for implementing it for marmoset brains to better understand their structural features.

Serial Two-Photon Tomography of the Whole Marmoset Brain for Neuroanatomical Analyses

Explore comparative methods for quantitative cell counting across mammalian brains. This approach addresses technical challenges in large-brained species and reveals evolutionary patterns in neuronal development.

A Comparative Approach for Quantitative Cell Counting Studies in Widely Different Mammalian Brains

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