ABSTRACT
We evaluated the signalling framework of immortalized cells from the hypothalamic suprachiasmatic nucleus (SCN) of the mouse. We selected a vasoactive intestinal peptide (VIP)-positive sub-clone of immortalized mouse SCN-cells stably expressing a cAMP-regulated-element (CRE)-luciferase construct named SCNCRE. We characterized these cells in terms of their status as neuronal cells, as well as for important components of the cAMP-dependent signal transduction pathway and compared them to SCN ex vivo. SCNCRE cells were treated with agents that modulate different intracellular signalling pathways to investigate their potency and timing for transcriptional CRE-dependent signalling. Several activating pathways modulate SCN neuronal signalling via the cAMP-regulated-element (CRE: TGACGCTA) and phosphorylation of transcription factors such as cAMP-regulated-element-binding protein (CREB). CRE-luciferase activity induced by different cAMP-signalling pathway-modulating agents displayed a variety of substance-specific dose and time-dependent profiles and interactions relevant to the regulation of SCN physiology. Moreover, the induction of the protein kinase C (PKC) pathway by phorbol ester application modulates the CRE-dependent signalling pathway as well. In conclusion, the cAMP/PKA- and the PKC-regulated pathways individually and in combination modulate the final CRE-dependent transcriptional output.
Subject(s)
Suprachiasmatic Nucleus Neurons , Vasoactive Intestinal Peptide , Mice , Animals , Vasoactive Intestinal Peptide/genetics , Vasoactive Intestinal Peptide/pharmacology , Vasoactive Intestinal Peptide/metabolism , Cyclic AMP Response Element-Binding Protein/metabolism , Suprachiasmatic Nucleus Neurons/metabolism , Suprachiasmatic Nucleus/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Luciferases/metabolism , Phorbol EstersABSTRACT
Macaque retinae were immunostained with monoclonal antibodies directed against the protein synaptotagmin-2 (Syt2). Syt2 was localized in a population of small-field amacrine cells, whose cell bodies formed a regular mosaic within the inner nuclear layer, indicating they represent a single amacrine cell type. The labeled amacrine cells had a bistratified appearance with a dense dendritic plexus in the OFF-layer and only a few lobular processes extending into the ON-layer of the inner plexiform layer, similar to A8 amacrine cells described in cat and human retina. Syt2-labeled cells were immunoreactive for glycine but lacked immunoreactivity for γ-aminobutyric acid (GABA), suggesting they use glycine as their neurotransmitter. The density of these cells increases from â¼200/mm(2) in peripheral retina to â¼1,400/mm(2) in central retina. Their bipolar cell input was studied by immunolabeling experiments using various bipolar cell markers combined with CtBP2, a marker of presynaptic ribbons. Our data show that Syt2-labeled amacrine cells receive input from both OFF and ON cone bipolar cells, as well as from rod bipolar cells. The OFF input is dominated by the diffuse bipolar cell DB1 (44%) and the OFF midget bipolar cell (38%). Here we describe a population of bistratified small-field amacrine cells closely resembling A8 amacrine cells and their cone-dominated bipolar cell input. J. Comp. Neurol. 521:709-724, 2013. © 2012 Wiley Periodicals, Inc.
