Abstract
Stereology is a group of mathematical and statistical methods that allows the extrapolation of three-dimensional structural information from two-dimensional sections (or slices). This allows researchers to derive important quantitative structural information, such as the volume, surface area or numbers of particular particles (e.g. cells) within defined regional boundaries. The need for such quantitative information in biology is of particular importance when evaluating the influence of various experimental treatments on specific organs, tissues and cells in the body. Knowledge of such changes has given important insights into the neural substrates that may be responsible for the functional and behavioral consequences of a disparate range of experimental treatments. Here, we describe some of these methods as applied to quantifying the total numbers of cells in defined regions of the hippocampal formation. The methods used for this evaluation were, first, the Cavalieri principle, which was used to determine the volumes of the various subdivisions of the rat hippocampus, and, second, the ‘physical disector’ method, which was used to estimate the numerical density of neurons within each subdivision. Once these values were derived, it was but a simple task to multiply them together to obtain estimates for the total numbers of cells in the given hippocampal region. We found that 16-and 30-day-old normal male rats had 176 800 and 152 700 pyramidal cells in the CA1 region, respectively. This decrease in the neuronal number was statistically significant. However, in the CA2+CA3 region, there were approximately 169 300 and 149 600 pyramidal cells in 16- and 30-day-old normal male rats, respectively, which was not significantly different. In the dentate gyrus, there were approximately 36 700 neurons in the hilus region and 483 000 granule cells in the granule cell layer, irrespective of the age of the rats. There were no significant differences between these estimates of hilus neurons and granule cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Amaral D, Witter M (1995) Hippocampal formation. In: The Rat Nervous System. (Paxinos G, ed.). Academic Press, San Diego, 443–93.
Andrade JP, Madeira MD, Paula-Barbosa MM (1995) Effects of long-term malnutrition and rehabilitation on the hippocampal formation of the adult rat. A morphometric study. J Anat 187, 379–93.
Angevine Jr JB (1965) Time of neuron origin in the hippocampal region. An autoradiographic study in the mouse. Exp Neurol 11 (Suppl. 1), 1–70.
Baddeley A (2001) Is stereology ‘unbiased’? Trends Neurosci 24, 375–6.
Bartheld AS (2001) Comparison of 2-D and 3-D counting: The need for calibration and common sense. Tr ends Neurosci 24, 504–6.
Bayer SA (1980a) Development of the hippocampal region in the rat. I. Neurogenesis examined with 3H-thymidine autora-diography. J Comp Neurol 190, 87–114.
Bayer SA (1980b) Development of the hippocampal region in the rat. II. Morphogenesis during embryonic and early postnatal life. J Comp Neurol 190, 115–34.
Bedi KS (1984) Effects of undernutrition on brain morphology: A critical review of methods and results. In: Current Topics in Research on Synapses, Vol. 2. (Jones DG, ed.). Alan R Liss, New York, 93–163.
Bedi KS (2003) Nutritional effects on neuron numbers. Nutr Neurosci 6, 141–52.
Benes M, Lange N (2001) Two-dimensional versus three- dimensional cell counting: A practical perspective. Trends Neurosci 24, 11–17.
Calverley RKS, Bedi KS, Jones DG (1988) Estimation of the numerical density of synapses in rat neocortex. Comparison of the ‘disector’ with an ‘unfolding’ method. J Neurosci Methods 23, 195–205.
Coggeshall RE (1999) Assaying structural changes after nerve damage, an essay on quantitative morphology. Pain 82 (Suppl. 1), S21–5.
Cruz-Orive LM, Weibel ER (1990) Recent stereological methods for cell biology: A brief survey. Am J Physiol 258, L148–56.
Elias H, Hyde D, Scheaffer R (1983) Section thickness and the ‘Holmes Effect’. In: A Guide to Practical Stereology. (Elias H, Hyde D, eds). Karger, New York, 121–6.
Evans SM, Howard V (1989) A simplification of the ‘step’ method for estimating mean section thickness. J Microsc 154, 289–93.
Gundersen HJG (1977) Notes on the estimation of the numerical density of arbitrary particles: The edge effect. J Microsc 111, 219–23.
Gundersen HJG (1986) Stereology of arbitrary particles. A review of unbiased number and size estimators and the presentation of some new ones, in memory of William R Thompson. J Microsc 143, 3–45.
Gundersen HJ, Jensen EB (1987) The efficiency of systematic sampling in stereology and its prediction. J Microsc 147, 229–63.
Gundersen HJ, Bendtsen TF, Korbo L et al. (1988a) Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96, 379–94.
Gundersen HJ, Bagger P, Bendtsen TF et al. (1988b) The new stereological tools: Disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96, 857–81.
Hof PR, Schmitz C (2000) Current trends in neurostereology: Introduction to the special issue ‘Recent Advances in Neurostereology’. J Chem Neuroanat 20, 3–5.
Kaplan M, Bell D (1983) Neuronal proliferation in the 9-month- old rodent: Radioautographic study of granule cells in the hippocampus. Exp Brain Res 52, 1–5.
Kaplan MS, Bell DH (1984) Mitotic neuroblasts in the 9-day-old and 11-month-old rodent hippocampus. J Neurosci 4, 1429–41.
Ling E, Paterson J, Privat A, Mori S, Leblond C (1973) Investigation of glial cells in semithin sections. I. Identification of glial cells in the brain of young rats. J Comp Neurol 149, 43–71.
Maier SE, West JR (2001) Regional differences in cell loss associated with binge-like alcohol exposure during the first two trimesters equivalent in the rat. Alcohol 23, 49–57.
Mayhew TM (1992) A review of recent advances on stereology for quantifying neural structure. J Neurocytol 21, 313–28.
Michel RP, Cruz-Orive LM (1988) Application of the Cavalieri principle and vertical sections method to lung: Estimation of volume and pleural surface area. J Microsc 150, 117–36.
Miki T, Fukui Y, Uemura N, Takeuchi Y (1994) Regional difference in the neurotoxicity of ochratoxin A on the developing cerebral cortex in mice. Dev Brain Res 82, 259–64.
Miki T, Fukui Y, Takeuchi Y, Itoh M (1995) A quantitative study of the effects of prenatal X-irradiation on the development of cerebral cortex in rats. Neurosci Res 23, 241–7.
Miki T, Fukui Y, Itoh M, Hisano S, Xie Q, Takeuchi Y (1997) Estimation of the numerical densities of neurons and synapses in cerebral cortex. Brain Res Protoc 1, 9–16.
Miki T, Harris SJ, Wilce P, Takeuchi Y, Bedi KS (2000) Neurons in the hilus region of the rat hippocampus are depleted in number by exposure to alcohol during early postnatal life. Hippocampus 10, 284–95.
Miki T, Harris SJ, Wilce P, Takeuchi Y, Bedi KS (2003) The effects of alcohol exposure during early life on neuron numbers in the rat hippocampus. I. Hilus neurons and granule cells. Hippocampus 13, 346–56.
Miki T, Harris SJ, Wilce PA, Takeuchi Y, Bedi KS (2004) Effects of age and alcohol exposure during early life on pyramidal cell numbers in the CA1-CA3 region of the rat hippocampus. Hippocampus 14, 124–34.
Mouton PR (2002) Principles and Practices of Unbiased Stereology: An Introduction for Bioscientists. The Johns Hopkins University Press, Baltimore.
Pakkenberg H, Gundersen HJG (1988) Total number of neurons and glial cells in human brain nuclei estimated by the disector and the fractionator. J Microsc 150, 1–20.
Schlessinger AR, Cowan WM, Gottlieb DI (1975) An autoradio- graphic study of the time of origin and the pattern of granule cell migration in the dentate gyrus of the rat. J Comp Neurol 159, 149–75.
Schlessinger AR, Cowan WM, Swanson LW (1978) The time of origin of neurons in Ammon’s horn and the associated retrohippocampal fields. Anat Embryol 154, 153–73.
Seifert (1983) Neurobiology of the Hippocampus. Academic Press, London.
Sousa N, Madeira MD, Paula-Barbosa MM (1997) Structural alterations of the hippocampal formation of adrenalectomized rats: An unbiased stereological study. J Neurocytol 26, 423–38.
Sousa N, Madeira MD, Paula-Barbosa MM (1998) Effects of corticosterone treatment and rehabilitation on the hippocampal formation of neonatal and adult rats. An unbiased stereological study. Brain Res 794, 199–210.
Sousa N, Paula-Barbosa MM, Almeida OFX (1999) Ligand and subfield specificity of corticoid-induced neuronal loss in the rat hippocampal formation. Neuroscience 89, 1079–87.
Sterio DC (1984) The unbiased estimation of number and sizes of arbitrary particles using the disector. J Microsc 134, 127–36.
Swaab DF, HBM. Uylings (1987) Density measures: Parameters to avoid. Neurobiol Aging 8, 574–6.
Warren MA, Bedi KS (1984) A quantitative assessment of the development of synapses and neurons in the visual cortex of control and undernourished rats. J Comp Neurol 227, 104–8.
Weibel ER (1980) Stereological Method, Vol. 1. Practical Methods for Biological Morphometry. Academic Press, New York.
West MJ (1993) New stereological methods for counting neurons. Neurobiol Aging 22, 275–85.
West MJ (1999) Stereological method for estimating the total number of neurons and synapses: Issue of precision and bias. Tr ends Neurosci 22, 51–61.
West MJ (2002) Design-based stereological method for counting neurons. In: Progress in Brain Research, Vol. 135. (Sutula T, Pitknen A, eds). Elsevier Science, Amsterdam, 43–51.
West MJ, Slomianka L, Gundersen HJ (1991) Unbiased stereo-logical estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231, 482–97.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Miki, T., Satriotomo, I., Li, HP. et al. Application of the physical disector to the central nervous system: Estimation of the total number of neurons in subdivisions of the rat hippocampus. Anato Sci Int 80, 153–162 (2005). https://doi.org/10.1111/j.1447-073x.2005.00121.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1447-073x.2005.00121.x