Ž . Developmental Brain Research 103 1997 171–183 Research report Zinc-positive presynaptic boutons of the rabbit hippocampus during early postnatal development J.V. Sanchez-Andres a , J.J. Palop b , C. Ramirez b , J. Nacher b , A. Molowny b , C. Lopez-Garcia b, ) a Fisiologia, Facultad de Medicina, UniÕersidad ‘Miguel Hernandez’, Elche, Elche, Spain b Neurobiologia, Biologia Celular, Facultad de Ciencias Biologicas, UniÕersidad de Valencia, 46100.-Burjassot, Valencia, Spain Accepted 1 July 1997 Abstract The evolution of vesicular zinc-containing boutons in the developing rabbit hippocampus has been studied during early postnatal life using the selenite–Danscher histochemical method. By P3, large immature mossy fiber boutons with labeled synaptic vesicles were seen in the hilus of the dentate gyrus and in the stratum lucidum of the CA3–CA4 hippocampal areas. After P5, smaller boutons with labeled vesicles were identified in the stratum oriens and stratum radiatum of all hippocampal areas, and even transiently in the stratum lacunosum of P6 animals. Vesicular zinc-containing boutons increased in number and underwent ultrastructural maturation; light microscope densitometric–volumetric measurements were used to quantify their presence in every hippocampal lamina. Electron microscope stereologic analysis permitted accurate estimation of the actual numbers along early postnatal development. Three main phases of zinc-positive bouton growth were detected during the first postnatal month. The first phase, starting at P5, is characterised by an abrupt rise in vesicular zinc content which at P8–P9 begins to decrease. The second phase is characterised by a consistent rise in vesicular zinc content from P10 to P12 to a level which is maintained until P18; this steady period is the result of partial and sequential elimination Ž of zinc-positive boutons in some areas i.e. oriens of CA3 by P11, radiatum of CA3 by P13, and radiatum of CA1 and lucidum of CA3 by . P15 while they continue to increase in other areas. The final phase is a continuous increase to almost adult levels. q 1997 Elsevier Science B.V. Keywords: Zinc-positive presynaptic bouton; Ultrastructural maturation; Stereological study; Absolute numbers estimation; Densitometry 1. Introduction Zinc is one of the most abundant cations in nervous Ž . w x system tissue 0.15–0.2 mM in the mammalian brain 14 . Zinc is found in proteins such as nuclear replication and transcriptional enzymes, transcriptional regulators, and a great variety of cytoplasmatic enzymes and growth factors w x 8,48 . In the intracellular environment, free ionic zinc interacts with many regulatory enzymes and, similarly to calcium, could act as an intracytoplasmatic messenger the w x homeostasis of which seems relevant 9,38 . Perhaps this is because many neurons have developed vesicular compart- ments with specific sequestering and storage mechanisms ) Ž. Corresponding author. Fax: q34 6 386-4781; E-mail: lopezc@uv.es. w x w x 32,36 , i.e. zinc-enriched neurons 15,19 which accumu- late zinc inside their synaptic vesicles. Zinc-enriched neurons are abundant in the mammalian telencephalon and particularly conspicuous in the hip- w x pocampus 16 . From an ontogenetical point of view, zinc-enriched neurons are usually late generated neurons which emit short-medium length axons giving rise to w x association synaptic fields. The Timm 47 and Danscher w x 11 histochemical methods clearly define these zinc-en- riched synaptic fields thus permitting clear evaluation of w x their size and development 50,54 . The role of vesicular zinc during synaptic transmission is not clear. Zinc-enriched synaptic boutons seem to be w x glutamatergic 31 and a zinc–glutamate interaction has w x been hypothesised 45 . Zinc appears to be accumulated w x inside synaptic vesicles 24,28,37 and is released by exo- w x cytosis 2,26 thereby reaching the synaptic cleft. In the 0165-3806r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.