Localization of Choline Acetyltransferase in the Developing and Adult Turtle Retinas LYNETTE T. NGUYEN, 1 JOAQUIN DE JUAN, 2 MARIA MEJIA, 1 AND NORBERTO M. GRZYWACZ 1 * 1 The Smith-Kettlewell Eye Research Institute, San Francisco, California 94115 2 Departamento de Biotecnologı ´a, Facultad de Ciencias, Universidad de Alicante, 03080 Alicante, Spain ABSTRACT Acetylcholine has important epigenetic roles in the developing retina. In this study, cells that expressed choline acetyltransferase (ChAT), the enzyme that synthesizes acetylcholine, were investigated in embryonic, postnatal, and adult turtle retinas by using immunofluores- cence histochemistry. ChAT was present at stage 15 (S15) in cells near the vitreal surface. With the formation of the inner plexiform layer (IPL) at S18, ChAT-immunoreactive (-IR) cells were located in the inner nuclear layer (INL) and the ganglion cell layer (GCL). In the INL, presumed starburst amacrine cells were homogenous in appearance and formed a single row next to the IPL: This pattern was conserved until adulthood. In the GCL, however, there were multiple rows of ChAT-IR cells early in development, and this high density of labeled cells continued during the embryonic stages, until around birth. The high density of ChAT-IR cells in the GCL was due in part to a population of cells that expressed ChAT transiently. In postnatal stages and adult retinas, the presumed starburst amacrine ChAT-IR cells formed two mirror-like rows of homogenous cells on both borders of the IPL. Two cholinergic dendritic strata that were continuous with these cells were observed as early as S18, and their depths in the IPL were relatively stable throughout development. A third population of ChAT-IR cells was observed toward the middle of the INL around S25 and persisted into adulthood. Finally, cells in the outer nuclear layer (ONL) were ChAT-IR during the embry- onic stages, were less immunoreactive during the postnatal stages, and were not immuno- reactive in the adult retinas. J. Comp. Neurol. 420:512–526, 2000. © 2000 Wiley-Liss, Inc. Indexing terms: amacrine cells; choline acetyltransferase; cell density; development; fluorescence microscopy; immunohistochemistry, photoreceptor cells; retinal ganglion cells; starburst amacrine cells; transient population; turtles; vertebrates A small population of amacrine cells in the adult verte- brate retina synthesizes acetylcholine (Masland and Liv- ingstone, 1976; Masland and Mills, 1979) and releases acetylcholine (Masland and Livingstone, 1976; Masland and Mills, 1979; Massey and Redburn, 1982). To date, cholinergic amacrine cells have been identified in neonatal and adult vertebrates, including human (Hutchins and Hollyfield, 1987), monkey (Mariani and Hersh, 1988), cat (Schmidt et al., 1985; Pourcho and Osman, 1986; Dann, 1989), rat (Voigt, 1986), pig (Eckenstein and Thoenen, 1982), rabbit (Masland and Mills, 1979; Masland et al., 1984), tree shrew (Conley et al., 1986; Sandman et al., 1997), chicken (Millar et al., 1985, 1987), dogfish (Bran- don, 1991), goldfish (Famiglietti and Tumosa, 1987), and turtle (Criswell and Brandon, 1992; Guiloff and Kolb, 1992). These cells make direct connections to ganglion cells (Famiglietti, 1983b) and, thus, directly shape how visual stimuli are processed. In the adult retina, these cholinergic cells are synonymous with the well-described starburst amacrine cells (Famiglietti, 1983a; Vaney, 1984; Tauchi and Masland, 1984), which have a role in direc- tional selectivity (Ariel and Daw, 1982; Vaney, 1990; Grant sponsor: National Eye Institute: Grant numbers: EY08921 and EY11170; Grant sponsor: DGICYT; Grant number: PB-96-0414. *Correspondence to: Dr. Norberto M. Grzywacz, The Smith-Kettlewell Eye Research Institute, 2318 Fillmore Street, San Francisco, CA 94115. E-mail: nmg@ski.org Received 30 March 1999; Revised 13 January 2000; Accepted 13 January 2000 THE JOURNAL OF COMPARATIVE NEUROLOGY 420:512–526 (2000) © 2000 WILEY-LISS, INC.