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

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.

Figure 1

Outline of Substantia Nigra pars compacta used to determine cell number. Forty micron free-floating cryostat sections were stained for tyrosine hydroxylase expression. The sections shown are spaced every 240 microns and thus represent every 6^th section from a single brain. White outlines indicate areas used to define the SNpc for the purposes of stereology. The rostral portion of the SNpc starts with the first TH positive cells near the end of the subthalamic nucleus (STh) and the caudal SNpc is where the retrorubral field (RRF). The outlines used include the substantia nigra lateralis. The mediodorsal boundary of the SNpc is defined by TH expression. The dorsal portion of the SN pars reticulata (SNR) defines the ventrolateral boundary. The anterior medial boundary is defined by the ventral tegmental area (VTA) and by size and orientation of stained cells. DA neurons of the SNpc are larger than ventral tegmental area DA neurons, and SNpc DA neurons orient along the long axis of the SNpc. The posterior medial portion of the SNpc is defined by the medial lemniscus (ML).

Figure 2

A representative overlay with individual dots representing single dopaminergic neurons used for serial reconstuction of the SNpc. (A) The SNpc in a 10 micron paraffin section stained for tyrosine hydroxylase was outlined. Inset: Higher power of box outlined in yellow. (B) Individual dopaminergic neurons within an outline of the SNpc on each section were labeled with a green dot. Inset: Higher power of box outlined in yellow.(C) Once all cells were marked in a single section, the adjacent section was overlaid on the previous section and aligned (D). Once aligned, the opacity of the overlying section was reduced so that the cells marked from the underlying section were visible. Inset: Higher power of box outlined in yellow. (E) Dopaminergic neurons in the adjacent section that contained cells not marked as present in the previous section were labeled with red dots. Inset: Higher power of box outlined in yellow. (F) A representative overlay with the dots the represent total dopaminergic neurons in 2 sections. Scale bar = 60 microns, Scale bar, inset= 30 microns

Figure 3

Bilateral distribution of dopaminergic neurons (number ± SEM) in the SNpc based on serial reconstruction of four individual brains. The majority of the dopaminergic neurons are located 400–700 microns (rostral to caudal) within the SNpc.

Figure 4

Effect of section interval and number of sampling sites on neuron number estimation in SNpc. (A) Using an oversampling technique described by Slomianka and West (Slomianka and West, 2005) we determined the estimated deviation of dopaminergic neurons within the SNpc using different sampling intervals in a single C57BL/6J mouse. Counting every section through every 6^th section gives numerical estimates within 10% (B) Using the same oversampling technique we estimated that an investigator could sample up to every third site and generate estimates within 10%.