Study of Tyrosine Hydroxylase Immunoreactive Neurons in Neonate Rats Lactationally Exposed to 2,4-Dichlorophenoxyacetic Acid G. Garcia 1 , P. Tagliaferro 2 , A. Ferri 1 , A.M. Evangelista de Duffard 1,* , R. Duffard 1 , A. Brusco 2 1 Experimental Toxicology Laboratory, School of Biochemical and Pharmaceutical Sciences, National University of Rosario, Suipacha 531, Rosario 2000, Argentina 2 Institute for Cell Biology and Neuroscience, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina Received 24 November 2003; accepted 16 April 2004 Available online 2 July 2004 Abstract Dopaminergic neurons from the midbrain nuclei substantia nigra (SN; A9) and ventral tegmental area (VTA; A10) were investigated by tyrosine hydroxylase (TH) immunostaining in neonate rat brains exposed to 2,4-dichlorophe- noxyacetic acid (2,4-D) through lactation. Dorsal raphe serotonin (5-HT) projections to SN and VTA were also studied by 5-HT transporter (5-HTT) immunostaining and results were quantified by image analysis. Twenty-five-day- old pups exposed to 2,4-D through mothers milk were used. Dams were intraperitoneally administered 70 or 100 mg/ kg/day of 2,4-D from the 9th to the 25th postpartum day. After 100 mg/kg of 2,4-D exposure, a 25% diminution in the SN and a 33% diminution in the VTA neurons’ TH immunostaining along with a significantly 5-HT fiber density diminution were observed. The present work supports previous reports which suggest that exposure to 2,4-D during development has multiple effects on CNS. # 2004 Elsevier Inc. All rights reserved. Keywords: 2,4-Dichlorophenoxyacetic acid; Dopaminergic neurons; Substantia nigra; Ventral tegmental area; Serotonin transporter INTRODUCTION During mammals’ development, environmental and chemical insults could happen producing a reversible or permanent central nervous system (CNS) damage. In rats, the brain growth spurt occurs primarily during the first 2 weeks after birth (Dobbing and Sands, 1979) and it is associated with a large number of biochemical and morphological changes (axonal and dendritic outgrowth and synaptogenesis) that transform the feto-neonatal brain into that of the adult (Eriksson et al., 1992). Different toxicant compounds have been detected in mammals’ milk including humans’milk (Welch and Findlay, 1981; Da Silva et al., 1991). Fetuses, as well as neonates, may be exposed at a greater risk than adults, and newborn development can be modified by direct exposure to environmental chemicals through their mothers’ milk since maternal exposure to environmen- tal chemicals has increased. 2,4-Dichlorophenoxyacetic acid (2,4-D) is a chlor- ophenoxyherbicide widely used in broadleaf and woody plants. We have already demonstrated the pre- sence of 2,4-D residues in stomach content, blood, brain and kidney of 4-day-old neonates rats breast-fed NeuroToxicology 25 (2004) 951–957 * Corresponding author. Tel.: þ54 341 430 2032; fax: þ54 341 480 4598. E-mail address: aevangel@fbioyf.unr.edu.ar (A.M. Evangelista de Duffard). 0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.05.004