Technical Reference #1676

1676.

Serotonin Stimulates Mitochondrial Transport in Hippocampal Neurons Sigeng Chen;a Geoffrey C. Owens;a Kathryn L. Crossin;a;b and David B. Edelmana;, The Neuroscience Insitute, Molecular and Cellular Neuroscience, 36(1676), (2007)
Link To Paper

Abstract:
Axonal transport of mitochondria is critical for proper neuronal function. However little is known about the extracellular signals that regulate this process. In the present study we show that the neuromodulator serotonin (5-HT) greatly enhances mitochondrial movement in the axons of rat hippocampal neurons in vitro. Administration of a 5-HT1A receptor antagonist inhibited mitochondrial movement whereas addition of fluoxetine a selective serotonin reuptake inhibitor promoted mitochondrial movement. 5-HTreceptors are known to activate the Akt/Protein kinase B pathway. Consistent with this directional mitochondrial movement was almost completely blocked by a specific Akt inhibitor. Moreover an inhibitor of glycogen synthase kinase-3β (GSK3β) a kinase whose activity is blocked by Akt-mediated phosphorylation promoted mitochondrial movement. These findings show that 5-HT1A receptor activation stimulates mitochondrial movement in hippocampal neurons by inhibiting GSK3β activity via Akt. Our findings suggest that 5-HT may mediate the redistribution of energy sources within responsive neurons a possibility that has significant implications for understanding the global biological effects of this important neuromodulator.

Keywords:
Serotonin; 5-HT; Akt; GSK3Beta; Mitochondria; Axonal transport

Materials & Methods:
Primary cell culture Primary cultures of rat hippocampal neurons were prepared according to previously described methods with some modifications (Mistry et al. 2002). Briefly rat hippocampal cells were collected from E18 embryos and plated on poly-D-lysine- and laminin-coated 35 mm glass-bottom dishes (MatTek Ashland MA) in DMEM containing high glucose (25 mM) supplemented with B27 L-Asparagine L-proline and Vitamin B-12. This medium had been previously conditioned for 24 h on monolayers of cortical glia. Plating densities were 2800 cells/cm2 and 1200 cell/cm2 in conventional 35-mm dishes (for protein assays) and 35 mm glass-bottom dishes (for imaging studies) respectively. Cells were harvested at 14 DIV for protein assays or used at 17–21 DIV in time-lapse imaging studies. On the fourth day after initial plating and every 4 days thereafter Ara-C was added to control growth of glial populations. 5-HT 8-OH-DPAT WAY100635 and fluoxetine were purchased from Sigma-Aldrich (St. Louis MO); GSK3β inhibitor VII and Akt inhibitor VIII were obtained from Calbiochem (La Jolla CA).

Microscopic Technique
Fluorescence Microscopy

Cell Type(s)
Hippocampal