The induction of Per1 expression by the combined treatment with glutamate, 5-hydroxytriptamine and dopamine initiates a ripple effect on Bmal1 and Cry1 mRNA expression via the ERK signaling pathway in cultured rat spinal astrocytes.

Clock genes contribute to the regulation of spinal cord astrocytic function. Although it was previously found that noradrenaline has a pivotal role in the regulation of clock genes expression in cultured rat spinal astrocytes, it is still unknown whether other neurotransmitters might affect clock gene expression. Thus, the effect of spinal neurotransmitters glutamate (Glu), 5-hydroxytriptamine (5-HT) and dopamine (DA) on clock genes expression was examined in cultured rat spinal astrocytes. Simultaneous treatment with Glu (100 μM), 5-HT (10 μM) and DA (10 μM) led to a transient induction of Per1 expression, and a delayed increase of Bmal1 expression and a decrease of Cry1 expression. By contrast, treatment with either Glu, 5-HT or DA alone increased only Per1 expression. The increase in Per1 mRNA by simultaneous treatment with Glu, 5-HT and DA was dependent on extracellular signal-regulated kinase (ERK) activation, since pretreatment with ERK inhibitor U0126 (3 μM) blocked Per1 expression. Second messengers p38 and c-jun N-terminal kinase were not involved in the neurotransmitter effect on Per1 expression, since pretreatment with SB202190 (3 μM) and SP600125 (10 μM), a p38 inhibitor and c-jun N-terminal kinase inhibitor, respectively, had no effect. Blockade of ERK signaling also prevented changes in Bmal1 and Cry1 mRNA expression induced by co-treatment with Glu, 5-HT and DA. In addition to modulating clock gene expression, co-treatment with Glu, 5-HT and DA significantly increased ERK1/2 phosphorylation. Per1 expression itself modulates the expression of other clock gene as knockdown of Per1 expression by using short interference RNA significantly blocked the increase of Bmal1 mRNA expression and the decrease of Cry1 mRNA expression. Thus, neurotransmitters Glu, 5-HT and DA regulate spinal astrocytic clock genes mRNA expression through the ERK pathway and Per1 is a key clock gene that likely modulates the oscillation of clock genes thereby regulating astrocytic function.