Elsevier

Neuroscience

Volume 161, Issue 4, 21 July 2009, Pages 1082-1090
Neuroscience

Neuroanatomy
Research Paper
A comparison of model-based (2D) and design-based (3D) stereological methods for estimating cell number in the substantia nigra pars compacta (SNpc) of the C57BL/6J mouse

https://doi.org/10.1016/j.neuroscience.2009.04.031Get rights and content

Abstract

The substantia nigra pars compacta (SNpc) is a compact brain structure that contains a variable distribution of cells in both medial to lateral and rostral to caudal dimensions. The SNpc is the primary brain structure affected in Parkinson's disease, where loss of dopaminergic neurons is one of the major hallmarks of the disorder. Neurotoxic and genetic models of Parkinson's disease, as well as mechanisms to treat this disorder, are modeled in the mouse. To accurately assess the validity of a model, one needs to be assured that the method(s) of analysis is accurate. Here, we determined the total number of dopaminergic neurons in the SNpc of the C57BL/6J mouse by serial reconstruction then compared that value to estimates derived using model-based stereology and design-based stereology. Serial reconstruction of the SNpc revealed the total number of SNpc dopaminergic neurons to be 8305±540 (±SEM). We compared this empirically derived neuron number to model based and design-based stereological estimates. We found that model based estimates gave a value of 8002±91 (±SEM) while design-based estimates were 8716±338 (±SEM). Statistical analysis showed no significant difference between estimates generated using model- or design-based stereological methods compared to empirically-derived counts using serial reconstruction.

Section snippets

Animals

Adult 3–4 month old male and female C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were used in this study. All studies were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80–23) revised 1996 and were approved by the SJCRH Animal Care and Use Committee. The authors further attest that all efforts were made to minimize the number of animals used and their suffering.

Serial reconstruction of the SNpc

Eight 3–4 month old C57BL/6J

Serial reconstruction of the DA neurons in the SNpc of the C57BL/6J mouse

The total bilateral number of DA neurons (±SEM) in the SNpc from the four serially reconstructed brains was 8305±539 (n=4, range 7464–9763). On average, the rostral to caudal length of the paraffin embedded SNpc was 1150 μm. The distribution of the cells demonstrates that the majority of cells are present 400–700 μm into the SNpc (rostral to caudal) (Fig. 3). This is similar to the distribution of C57BL/6J SNpc DA neurons previously reported (Nelson et al 1996, Hamre et al 1999).

Model-based (2D) stereological estimates of DA neurons in the C57BL/6J SNpc

Model-based

Discussion

Establishment of a gold standard for the number of neurons within the SNpc of mice is necessary in order for investigators to both assess the accuracy of the estimated values of the given experiments and allow comparisons between experiments. This is especially important with regard to determining the efficacy of particular treatments that purport to cause or prevent neuronal loss. Here we used serial reconstruction of the entire adult SNpc of the C57BL/6J mouse to reveal that it contains

Conclusion

In conclusion, we believe that the empirically-determined number of DA neurons of the SNpc reported here, along with the multiple methods for estimating this value will be beneficial to many using this system to assess disease models and potential treatments. Surprisingly, we found that the accuracy and precision of the design-based stereological methods were equal to that of the method-based analysis. Thus, since both methods can be used in obtaining accurate neuronal estimates, one needs to

Acknowledgments

The authors acknowledge support from the National Parkinson's Foundation, NIH (NS39006 to R.J.S.) and P-30 CA 21765 and the American Lebanese Syrian Associated Charities (ALSAC). The authors thank Haeman Jang for assistance with photomicroscopy and Yun Jiao for assistance with histology. We thank the staff at MicroBrightField Inc., especially Dan Peruzzi, for comments on this paper.

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