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Parasol retinal ganglion cells are more sensitive to luminance contrast and respond more transiently at all levels of adaptation than midget ganglion cells. This may be due, in part, to differences between bipolar cells that provide their input, and the goal of these experiments was to study these differences. Midget bipolar cells are known to be presynaptic to midget ganglion cells. To identify the bipolar cells presynaptic to parasol cells, these ganglion cells were intracellularly injected with Neurobiotin, cone bipolar cells were immunolabeled, and the double‐labeled material was analyzed. In the electron microscope, we found that DB3 diffuse bipolar cells labeled by using antiserum to calbindin D‐28k were presynaptic to OFF parasol cells. In the confocal microscope, DB3 bipolars costratified with OFF parasol cell dendrites and made significantly more appositions with them than expected due to chance. Flat midget bipolar cells were labeled with antiserum to recoverin. Although they made a few appositions with parasol cells, the number was no greater than would be expected when two sets of processes have overlapping distributions in the inner plexiform layer. DB2 diffuse bipolar cells were labeled with antibodies to excitatory amino acid transporter 2, and they also made appositions with OFF parasol cells. These results suggest that DB2 bipolar cells are also presynaptic to OFF parasol ganglion cells, but midget bipolar cells are not. We estimate that midperipheral OFF parasol cells receive ≈500 synapses from 50 DB3 bipolar cells that, in turn, receive input from 250 cones. J. Comp. Neurol. 416:6–18, 2000. © 2000 Wiley‐Liss, Inc.

Results of this study show that all three melatonin receptor subtypes are expressed in retinal and extraretinal ocular tissues of the chick eye. The finding that administration of melatonin alters the growth of several ocular tissues in both control and form-deprived eyes suggests that melatonin, acting through specific melatonin receptors in ocular tissues, plays a role in ocular growth and development. This conclusion suggests that the action of melatonin, combined with expression of melatonin receptors, is involved in the regulation of the diurnal rhythm of ocular growth.

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The serine/threonine protein kinase B (PKB)/Akt is a key mediator of signal transduction processes downstream of phosphoinositide 3-kinase (PI-3 kinase). PI 3-kinase is the key enzyme catalyzing the transfer of phosphate from ATP to the D-3 position of the inositol ring of membrane-localized phosphoinositides, thereby generating 3¢-phosphorylated phosphoinositides, such as phosphotidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P 3 , PIP3] and phosphotidylinositol 3,4-bisphosphate [PtdIns (3,4) P 2 ], upon growth factor stimulation. The production of PIP3 and PI(3, 4)P 2 recruits Akt from the cytoplasm to the plasma membrane through the Pleckstrin homology (PH) domain of Akt Meier et al. 1997;Wijkander et al. 1997;Zhang and Vik 1997;Sable et al. 1998;Currie et al. 1999). The relocalization of Akt to the plasma membrane brings Akt in proximity to regulatory kinases, such as 3-phosphoinositide-dependent protein kinase 1 (PDK1), which phosphorylate and activate Akt. Akt has also been observed to translocate to the nucleus after its activation in some cells (Testa and Bellacosa 1997;Borgatti et al. 2000;Carvalho et al. 2000;Bijur and Jope 2003).The PKB/Akt family consists of three members, PKBa/ Akt1, PKBb/Akt2, and PKBc/Akt3, which are products of three separate genes located on distinct chromosomes. The Abbreviations used: GFP, green fluorescent protein; GST, glutathione S-transferase; IR, insulin receptor; MMR, multimedia router; PDK1, phosphoinositide-dependent protein kinase 1; PH, pleckstrin homology; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; ROS, rod outer segments; SDM, site-directed mutagenesis. AbstractAkt is a phospholipid-binding protein and the downstream effector of the phosphoinositide 3-kinase (PI3K) pathway. Akt has three isoforms: Akt1, Akt2, and Akt3. All of these isoforms are expressed in rod photoreceptor cells, but the individual functions of each isoform are not known. In this study, we found that light induces the activation of Akt1. The membrane binding of Akt1 to rod outer segments (ROS) is insulin receptor (IR)/PI3K-dependent as demonstrated by reduced binding of Akt1 to ROS membranes of photoreceptor-specific IR knockout mice. Membrane binding of Akt1 is mediated through its Pleckstrin homology (PH) domain. To determine whether binding of the PH domain of Akt1 to photoreceptor membranes is regulated by light, various green fluorescent protein (GFP)/ Akt1-PH domain fusion proteins were expressed in rod photoreceptors of transgenic Xenopus laevis under the control of the Xenopus opsin promoter. The R25C mutant PH domain of Akt1, which does not bind phosphoinositides, failed to associate with plasma membranes in a light-dependent manner. This study suggests that light-dependent generation of phosphoinositides regulates the activation and membrane binding of Akt1 in vivo. Our results also suggest that actin cytoskeletal organization may be regulated through light-dependent generation of phosphoinositides.

Previous work has demonstrated that melatonin inhibits growth of cultured human uveal melanoma cells. The goal of this study was to determine the expression of mRNA encoding the melatonin receptor subtypes and the effect of specific melatonin receptor agonists on cell growth of uveal melanoma cells and melanocytes. RNA expression of the human melatonin Mel1a and Mel1b receptor subtypes was determined by reverse transcription‐polymerase chain reaction (RT‐PCR) amplification of RNA isolated from two melanoma cell lines and from one cell line of normal melanocytes. PCR‐amplified cDNA encoding the Mel1b melatonin receptor subtype, but not the Mel1a subtype, was detected in reverse‐transcribed RNA obtained from both normal uveal melanocytes and melanoma cell lines. Uveal melanoma cells and melanocytes were cultured for 24 hr, then melatonin or one of its membrane receptor agonists, 6‐chloromelatonin (Mel1a–1b) or S‐20098 (Mel1b) or its putative nuclear agonist, CGP‐52608 (Mel2), was added to the medium. After 5 days, the cells were detached, counted, and compared to untreated controls. Melatonin and its membrane receptor agonists (Mel1a–1b and Mel1b ), but not its putative nuclear receptor agonist (Mel2), inhibited the growth of uveal melanoma cells, but not normal melanocytes, at very low concentrations. In uveal melanoma cells, the expression of RNA encoding the Mel1b receptor suggests that the growth inhibiting effect of melatonin on uveal melanoma cells is related to activation of the melatonin Mel1b membrane receptor. Furthermore, the expression of RNA encoding melatonin receptors in normal uveal melanocytes suggests that melatonin may play a role in the function of these cells.

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