Unbiased stereological estimates of neuron number in subcortical auditory nuclei of the rat
Introduction
The structural complexity of the mammalian auditory system has been appreciated since the early anatomists described the numerous nuclei that comprise the ascending pathways, and also the great diversity of their constituent neurons’ morphologies. Modern auditory neurobiology relies heavily on the quantitative analyses of structure and function. However, limited knowledge of the structural composition of auditory nuclei renders the data produced in many studies difficult to interpret. To date, only a few investigations have attempted to determine the number of neurons in nuclei of the central auditory system (see Section 4), and most of these have relied on biased and therefore potentially unreliable counting methods. Therefore, a systematic investigation of neuron numbers in the central auditory system of a mammalian species is warranted.
The goal of this study was to provide unbiased, accurate estimates of the total number of neurons in subcortical auditory nuclei of the rat. This species is commonly used for physiological and anatomical investigations, and substantial background information pertaining to its central auditory system is available (reviewed in Webster, 1995). Knowledge of the number of neurons located in auditory nuclei, coupled with an understanding of their physiological response properties, connectivities and neurochemical phenotypes will enable the formation of accurate quantitative models of auditory circuits. This information may also prove useful in a number of other ways. Knowing the number of cells located in an auditory nucleus enables an assessment of the percentage of cells that are visualized by selective labeling methods such as immunocytochemistry, in situ hybridization, tract-tracing or experimental degeneration approaches. For instance, immunocytochemical probes commonly reveal a single neuron type in a particular brain region, and occasionally even a subpopulation of a cell type that expresses the marker under investigation. Well-documented examples include the cartwheel cells of the dorsal cochlear nucleus (DCN), which represent the only neurons in the guinea pig cochlear nuclear complex that are immunoreactive for PEP-19 (Berrebi and Mugnaini, 1991), or the cartwheel cells of the rat DCN, only a percentage of which are glycinergic (Gates et al., 1996). Similarly, in tract-tracing experiments the percentage of retrogradely labeled cells in a given nucleus is influenced by the number of its neurons that actually participate in the pathway under study, and by various technical artifacts resulting from the effective size of the injection site and the efficiency of the retrograde transport of the chosen tracer. Thus, only with knowledge of the entire population of cells contained within the structure under study can one make quantitative assessments of cells expressing a particular marker or projecting to a particular brain region. For example, by combining immunohistochemical and retrograde tracing data with unbiased stereological estimates of neuron number, it has recently been demonstrated that greater than 90% of neurons in the superior paraolivary nucleus (SPON) express glutamic acid decarboxylase and project to the ipsilateral inferior colliculus (Saldaña and Berrebi, 2000, Kulesza and Berrebi, 2000).
Design-based stereology is a technique that provides reliable estimates of the number and properties of neurons (Howard and Reed, 1998). The method has a rigorous mathematical foundation and provides an indication of the precision of the estimates obtained. Stereologically-based estimates are considered ‘unbiased’ if the chosen test probe is employed using appropriate rules for systematic random sampling of the structure of interest. Once the so-called sampling and systematic sources of bias are eliminated, no assumptions need to be made about the size, shape or distribution of the elements under study. Implementing unbiased stereology is largely automated, thus maximizing efficiency and eliminating human errors. Therefore, we have applied design-based stereology to determine the number of neurons present in the cochlear nuclei, superior olivary complex (SOC), lateral lemniscal nuclei, inferior colliculus and medial geniculate body of the rat. We also produced a three-dimensional structural model of the SOC in this species.
Section snippets
Animals and tissue processing
Adult female Wistar rats (190–210 g body weight) were anesthetized with an intraperitoneal overdose of sodium pentobarbital (120 mg/kg body weight) and perfused through the ascending aorta with a vascular rinse composed of calcium-free Ringer’s solution (pH 6.9). Fixation was accomplished first with 4% freshly depolymerized paraformaldehyde and 0.1% glutaraldehyde in 0.1 M phosphate buffer, followed by 4% freshly depolymerized paraformaldehyde in the same buffer. Brains were then dissected and
Cochlear nuclei
The cochlear nuclei (CN) were subdivided into the anteroventral cochlear nucleus (AVCN), the posteroventral cochlear nucleus (PVCN) and the DCN as depicted in Fig. 1. In the case of this large complex, delineating the boundaries of the three nuclei was easily accomplished by using the entry of the eighth nerve root, the taenia choroidea and the granule cell lamina as guides (Osen, 1969, Osen, 1988, Brawer et al., 1974, Mugnaini et al., 1980, Lorente de Nò, 1981). The counts from each
Choice of the optical fractionator tool
The field of stereology encompasses more than a dozen so-called ‘tools’ for the study of biological entities or other particulate structures. When properly applied, each of these tools renders quantitative estimates that are efficiently obtained, accurate, and unbiased (Howard and Reed, 1998). We selected the optical fractionator method in our study because this particular tool is relatively simple, is unaffected by tissue shrinkage and can be applied to tissues processed by most, if not all,
Acknowledgements
The authors gratefully acknowledge the pre-submission critiques of the manuscript provided by Drs. George Spirou and Aric Agmon. This work was supported by research grant DC02266 to A.S.B. from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health, and by grants BFI2000-1358 and CyL SA 079/01 to E.S. R.J.K. was supported by a graduate teaching assistantship from the West Virginia University Department of Neurobiology and Anatomy. A.V. was supported
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