As an alternative to purely counting neurons, one may choose to study neuronal terminals or varicosities. Stereology was used to count the vesicular glutamate transporters and tyrosine hydroxylase-immunostained terminals in a study investigating the effect of chronic or repeated deprivation of alcohol on the dopamine (DA) and glutamate (GLU) terminals in the extended amygdala (4). Immuno-staining for tyrosine hydroxylase (TH) labeled DA terminals, and vesicular glutamate transporter 1 (vGLUT1)/vesicular glutamate transporter 2 (vGLUT2) labeled GLU terminals, which delineated regional boundaries of subdivisions in the extended amygdala due to their innate expression. The heterogeneous distribution of TH, vGLUT1, and vGLUT2 within a brain region rendered a unique challenge for stereological sampling, but it emphasizes that stereological analyses remain valid even though regional variation exists in terms of intensity or distribution of the signals. Figure 2 reveals the heterogeneous distribution of TH+ terminals in the shell of the nucleus accumbens, septum, and anterior commissure. Heterogeneous distributions require more sampling for analysis than a homogeneous distribution, for any given level of precision in the final estimate.

Neuroscience methods may be combined to use some of the available options packages contained in many stereology software packages. Stereo Investigator features tight integration with the Olympus DSU spinning disk confocal microscope (in addition to many other microscope models, including Zeiss microscopes with the ApoTome) and allows for easy acquisition of brightfield, widefield fluorescent, or multichannel fluorescent image stacks. In a study on retinal ganglion cell ocular hypertension, confocal images stacks of N-methyl-d-aspartate receptor 1 (NR1) and glutamate receptor 2/3 (GluR2/3) fluorescent immunostaining were obtained from confocal laser scanning microscope, quantified using stereology, revealing alterations in glutamate receptor expression (5). Additional discussion on 3D volume analysis is available in Section 7.

The following two arrangements may be taken into consideration before starting a stereology analysis. First, remove the user bias by having the individual performing the analysis blinded to the samples and treatments represented on the slides. If the difference between the two groups appears to be large, different sampling parameters may be used for each group to increase efficiency. Furthermore, an independent second person should run the stereological analysis using identical procedures in order to reduce personal bias and increase validity. If inter-rater variability turns out to be high, further sampling is needed if the users are both properly trained. If inter-rater variability is low, this definitely lends confidence to the validity of the results.

Performing many common stereology protocols, such as the Optical Fractionator, using immunostained or counterstained tissues, it is suggested that the user confirm the depth of penetration and measure the final mounted section thickness. While the final mounted and original section thickness are not required in Stereo Investigator, if the original cut thickness is supplied, the software can estimate the volume of the ROI as a bonus. A pilot test of 5–6 sections may be measured as described below. This can be easily accomplished within the Stereo Investigator software by following the Focus Position Meter. First, focus at the top of the section, where the cells first start to come into focus. Move down through the section following the Focus Position Meter to the point where the staining starts to fade or is less clear; if focusing down further does not bring more cells into focus, that point is the bottom of the section. The depth traversed provides information about the shrinkage of your tissue, a common occurrence for immunostained and counterstained tissue that is dehydrated through graded alcohol-xylene-based washes. Confirmation of depth of penetration and total section thickness is helpful prior to starting stereological counting. It may be advisable for new stereology users to learn how to measure the final mounted section thickness using differential interference contrast (DIC) microscopy or other means using brightfield microscopy. For stereological counting using brightfield, the user must use an objective lens with a sufficiently high numerical aperture (commonly 1.4 N.A.), achieve Kohler illumination, and then open the condenser aperture all the way, in order to get the thinnest possible depth of field. Without this step, the counting results will be far less robust than expected. Guard zones are established at the top and bottom of the section to allow for sampling in tissue where there are no “lost caps” or “craters” and also avoids sampling from an area where the tissue may have been badly deformed by the cutting process in a number of different ways. The guard zone is determined empirically for each study.

Equipment: The essential equipment required for a stereology system includes a microscope (e.g., a modern Leica, Zeiss, Nikon, or Olympus) with a motorized x-y-z stage interfaced with a dedicated cooled monochrome for fluorescent work and a color camera for brightfield work, and a microcator (Z focus position linear encoder) with the stage controller connected to a PC (Intel® Quad Core Xeon Processor W3540 2.93 GHz, 12 GB DDR3 SDRAM, 2  ×  500 GB hard drives, 1 GB nVidia 9800 GT video card, DVD drive, USB and serial ports, Windows 7, 64-bit operating system). This configuration is based on the current hardware needed for Stereo Investigator. Other systems may have different hardware criteria. Whichever stereology software is used, the ability to generate a grid of sampling frames to overlay the microscopic images on the monitor for individual signal counting is required.

Sequential procedures for counting using Stereo Investigator software (MBF Bioscience) and the Optical Fractionator probe are detailed below. Open the Stereo Investigator software and from the Probes menu choose the Optical Fractionator Workflow. In the next window, select Start a New Subject or Load subject from existing data file. At the bottom of the Set up the Subject panel (Fig. 4a), enter the fresh tissue cutting thickness. When starting the first section for a new subject, select a reference point. This may be any point in the section or within the region of your interest. The reference point should be easy to identify, allowing the software to perform parfocal and parcentric corrections when switching to high power. At low magnification (usually ×1 to ×10), trace the contour of the area you desire to sample. Anatomical or regional landmarks ensure the same area is traced and measured in each section. The actual cytoarchitectural ROI will be retraced on each subsequent section. On a two-button mouse, right clicking brings up a menu and you can select Close contour to end the tracing and close any gap between the beginning of the trace and the end (Fig. 4b). Switch to a high power (×40 or ×100, 1.4 N.A. oil objective) on the microscope. For brightfield work, it is critical to have the thinnest possible depth of field, requiring Kohler illumination and a fully open condenser aperture as well.

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