Neuroscience Letters 419 (2007) 34–37 Stereologic analysis of cell number and size during postnatal development in the rat substantia nigra Samuel A. Scott a,∗ , Naomi M. Diaz a , Syed O. Ahmad b a School of Physical Therapy, Ohio University, Athens, OH 45701, United States b Occupational Therapy Education, University of Kansas Medical Center, Kansas City, KS 66160, United States Received 4 January 2007; received in revised form 3 March 2007; accepted 20 March 2007 Abstract Parkinson’s disease is characterized by age-related atrophy and loss of dopaminergic neurons within the compact portion of the substantia nigra (SNpc) projecting to neostriatum. Despite numerous studies using rodent models to examine mechanisms underlying this disorder, the fundamental question of whether development- or age-related changes occur in the rodent SNpc remains unanswered. The present study used a three-level, optical fractionator approach to estimate the number and size of SNpc neurons immunoreactive for tyrosine hydroxylase (TH) in eight young (2-month) and eight older (7-month) Sprague–Dawley rats. Following standard protocols for animal care and tissue harvesting, every eighth 60-m section from a gapless coronal series was treated immunohistochemically for TH along with a thionin counterstain. Neither the ventral tegmental area nor the lateral part of the SN was included in the analysis. The total bilateral number of SNpc TH+ neurons (∼8000) was equivalent between groups, whereas mean TH+ neuronal volume decreased significantly in the older group (∼18%). In contrast, volume of the SNpc increased with age by 17%, as did volume of the entire brain (24%). TH+ cells in the SNpc were also significantly larger on the left versus right side of the brain. These data are consistent with the hypothesis that age-related volumetric expansion of the SNpc is accounted for by an increase in the ratio between neuropil and average neuron somal size during intermediate postnatal development. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Stereology; Basal ganglia; Optical fractionator; Tyrosine hydroxylase; Dopaminergic; Cavalieri The compact portion of the substantia nigra (SNpc) is an extrapyramidal motor subnucleus in the tegmental midbrain that regulates voluntary movement tone by way of reciprocal connec- tions with the neostriatum (nigrostriatal and striatonigral tracts). Parkinson’s disease (PD) is known to result from the atrophy and loss of dopaminergic neurons within this structure. Owing to the relationship between PD and aging, as well as the extensive uti- lization of rodent models as a means of studying this disorder, we sought to provide a quantitative, stereology-based descrip- tion of the compact portion of the SN in two age groups of rats. In a recent review by Stark and Pakkenberg [14], focusing on age-related changes in dopaminergic cell populations, one stere- ological study regarding the rat SNpc was cited [10] in which one age group was investigated (28 days). Hence, the latter report was neither a study of aging nor development per se. ∗ Corresponding author at: W-290 Grover Center, School of Physical Therapy, Ohio University, Athens, OH 45701, United States. Tel.: +1 740 593 9567; fax: +1 740 593 0293. E-mail address: scotts3@ohio.edu (S.A. Scott). The present study utilized stereological assessment of neuron number, neuronal volume, and overall nuclear volume in the compact portion of the SN bilaterally in normal rats using two time points (60 and 210 days). Our intent was to provide baseline data in terms of cellular changes that may occur during normal postnatal development of this structure. Sixteen Sprague–Dawley rats (Taconic Laboroatories, Hud- son, NY, USA), 12 males and 4 females, were housed one per cage in a temperature control room under a 12-h light:12-h dark cycle with free access to food and water. Eight of the animals were sacrificed at 2-month whereas the remaining eight were sacrificed at 7-month postnatal. All animals were treated accord- ing to the National Institute of Health Guide for the Care and Use of Laboratory Animals within guidelines set by the Society for Neuroscience, and under the oversight of an Institutional Animal Care and Use Committee. Animals were deeply anesthetized with subcutaneous pentobarbital (120 mg/kg, Sigma–Aldrich, St. Louis, MO) and perfused with a 4% paraformaldehyde, 4% sucrose and phosphate buffer solution using a peristaltic pump at a flow rate of 12 ml/min. Brains were removed and placed in carefully sealed containers with the perfusion fixative for 4 h 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.03.067