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Neuronal differentiation not only entails the acquisition of various molecular and cellular characteristics but also involves the loss of characteristics that appear only during certain stages of development (1)(2)(3)(4). Calcium ions have been implicated in the regulation ofneuronal development (5-7) and Ca2+/calmodulin-dependent protein kinases have been demonstrated to be an important pathway for Ca2+ signaling (8,9). In this report we describe the developmental pattern ofexpression ofgranule cell-enriched Ca2+/calmodulin-dependent protein kinase (CaM kinase-Gr), which is comprised of Mr 65,000 and 67,000 polypeptides and has been localized to various neuronal populations (10). In the adult cerebellum, this enzyme was found to be concentrated in granule cells but absent from Purkinje cells (10). Because the development and circuitry of these cells have been extensively studied, we attempted to correlate the production of CaM kinase-Gr with different stages of cerebellar development. The conclusions arrived at in these studies were subsequently extended by examining the appearance of CaM kinase-Gr in the hippocampus. The present observations of developmental expression of CaM kinase-Gr typify the acquisition and loss of a Ca2+-signaling pathway during development.MATERIALS AND METHODS Primary Antiserum. Rabbit antibodies were raised against a f-galactosidase fusion product of CaM kinase-Gr expressed in and purified from Escherichia coli. The antiserum was affinity-purified by adsorption to mammalian CaM kinase-Gr isolated from rat cerebellum (10). The resulting monospecific antibody preparation was employed throughout this study.Immunoblots. Cerebella from postnatal day (PND)-3 and adult rats were homogenized with 10 volumes of buffer containing 25 mM Hepes (pH 7.5), 2 mM EDTA, 0.1 mg of phenylmethylsulfonyl fluoride per ml, and 20 ,ug of leupetin per ml. Equivalent amounts of homogenate were electrophoresed in duplicate in SDS/10% polyacrylamide gels (11) and the proteins were electroblotted onto nitrocellulose paper (12). Blots were incubated at 40C for 16 hr in 25 mM Tris HCl, pH 7.5/0.15 M NaCI/1 mg of polyethylene glycol 20,000 per ml/3 mg of bovine serum albumin per ml. The blots were subsequently incubated with the affinity-purified antibody to CaM kinase-Gr in the same buffer for 1 hr at room temperature followed by extensive washing. Control blots were similarly processed but without the primary antibody. Blots were incubated together with alkaline phosphatase conjugated to goat anti-rabbit IgG for 1 hr at room temperature followed by extensive washing. Immunoreactive material was visualized by the addition of nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (13).Immunohistochemistry. Animals at various ages from embryonic day (E)-22 to PND-14 (where PND-0 is the day of birth) were deeply anesthetized with pentobarbital (100 mg/ kg) and sacrificed by cardiac perfusion with saline followed by 4% paraformaldehyde. The brains were removed, postfixed, and immersed in 20% sucrose. Bra...

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With the abundance of chemicals in the environment that could potentially cause neurodevelopmental deficits, there is a need for rapid testing and chemical screening assays. This study evaluated the developmental toxicity and behavioral effects of 61 chemicals in zebrafish (Danio rerio) larvae using a behavioral Light/Dark assay. Larvae (n = 16–24 per concentration) were exposed to each chemical (0.0001–120 μM) during development and locomotor activity was assessed. Approximately half of the chemicals (n = 30) did not show any gross developmental toxicity (i.e., mortality, dysmorphology or non-hatching) at the highest concentration tested. Twelve of the 31 chemicals that did elicit developmental toxicity were toxic at the highest concentration only, and thirteen chemicals were developmentally toxic at concentrations of 10 µM or lower. Eleven chemicals caused behavioral effects; four chemicals (6-aminonicotinamide, cyclophosphamide, paraquat, phenobarbital) altered behavior in the absence of developmental toxicity. In addition to screening a library of chemicals for developmental neurotoxicity, we also compared our findings with previously published results for those chemicals. Our comparison revealed a general lack of standardized reporting of experimental details, and it also helped identify some chemicals that appear to be consistent positives and negatives across multiple laboratories.