Reduced programmed cell death in brains of serotonin transporter knockout mice Antonio M. Persico, CA Alfonso Baldi, 2 Maria Luisa Dell’Acqua, Rainald Moessner, 3 Dennis L. Murphy, 4 Klaus-Peter Lesch 3 and Flavio Keller 1 LaboratoryofMolecularPsychiatryandNeurogenetics; 1 LaboratoryofDevelopmentalNeuroscienceandNeuroplasticity,InterdisciplinaryBiomedical ResearchCenter,UniversityCampusBio-Medico,ViaLongoni83,I-00155Rome; 2 DepartmentofBiochemistry,SectionofPathology, IIUniversityofNaples,Italy; 3 DepartmentofPsychiatryandPsychotherapy,UniversityofWuerzburg,Germany; 4 LaboratoryofClinicalScience, NIMH/NIH,Bethesda,Maryland,USA CA CorrespondingAuthor:a.persico@unicampus.it Received11October2002; accepted3January2003 DOI:10.1097/01.wnr.0000058244.21747.83 Serotonin(5-HT)isknowntoreduceapoptosis in vitro andinro- dent models of brain ischemia. Modulation of programmed cell deathduringneuraldevelopmentwas assessedinearlypostnatal brains of serotonin transporter (5-HTT) knockoutmice, charac- terizedbyelevatedextracellular5-HTlevels.Thenumberofapop- totic cells visualized at postnatal day-1 (P1) by ISELþ orTUNEL staining was signi¢cantly reduced in the striatum, thalamus/hy- pothalamus, cerebral cortex and hippocampus of 5-HTT knock- outmice,comparedtowildtypeandheterozygotemice,withdif- ferencesdisplayinganincreasingfronto-caudalgradientandregio- nal speci¢city. These ¢ndings underscore 5-HT roles in the regulation of programmed cell death during brain development, andspurinterestintopharmacologicalinterventionsaimedatre- lieving pathological apoptosis by potentiating serotoninergic neurotransmission. NeuroReport 14:341^344  c 2003 Lippincott Williams&Wilkins. Key words: Apoptosis; Development; Homologousrecombination; Knock-out; Monoamine; Naturallyoccurring cell death; Programmedcell death; Serotonin;Serotonintransporter INTRODUCTION Developmental roles of serotonin (5-HT) are known to include modulation of cell proliferation, migration and maturation [1,2]. During development, 5-HT affects the proliferation of neural progenitor cells and the maturation of postmitotic neurons, either by directly binding to 5-HT receptors located on neuronal cells, or by stimulating the release of astroglial neurotrophic factors [1,2]. In addition, concentration-dependent 5-HT effects on cell migration have been consistently recorded in several non-neural tissues, including craniofacial mesenchyme, heart, endothelium and vascular smooth muscle [3–5]. Programmed cell death by apoptosis represents another critical step in neural devel- opment, actively contributing to shape brain circuitry by linking cell survival to neuronal activity and to availability of target- or afferent-derived neurotrophic factors [6,7]. Increasing evidence of apoptotic cell death modulation by 5- HT has come largely from in vitro studies and from animal models of ischemia [1,8]. The current study was undertaken to explore potential modulatory effects of 5-HT on pro- grammed cell death during neural development, initially focusing on early postnatal life. To this end, we assessed the number and distribution of apoptotic cells in brains of newborn mice characterized by an excess of extracellular 5-HT due to 5-HT transporter (5-HTT) gene inactivation [9–11]. MATERIALS AND METHODS 5-HTT heterozygote mice from an F6 backcross into C57BL/ 6J were mated to obtain two litters of 9 and 11 neonatal mice. Twelve pups matched by litter and genotype (4 wt/4 hz/4 ko) were killed by decapitation at postnatal day 1 (P1), with P0 corresponding here to the first 24 h following delivery. Brains were quickly dissected, frozen in dry ice and stored at 801C; tails were also removed, frozen and stored for DNA extraction and 5-HTT genotyping, per- formed by PCR as described [12]. Brains were cut into 10 mm coronal sections, and apoptotic cells were stained using the ISELþ method [13] on 2–4 sections from each one of three coronal planes, corresponding to figures 23–27, 42–44, and 52–55 of the Franklin and Paxinos Atlas of the Mouse Brain [14] (see Results). Briefly, sections were postfixed for 5min in 4% paraformaldehyde, extracted with 0.6% Triton X-100 in 2  SSPE, acetylated, dehydrated in graded ethanol, air dried for 1 h, and either stored at 801C in dessicant, or labeled using 150 U/ml terminal-deoxynucleotidyltransfer- ase (Gibco BRL) in 100 mM potassium cacodylate, 2 mM 0959-4965  c LippincottWilliams&Wilkins Vol14 No 3 3 March 2003 341 DEVELOPMENTALNEUROSCIENCE NEUROREPORT Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.