Quantitative measurement of serotonin synthesis and sequestration in individual live neuronal cells

Synthesis and subsequent sequestration into vesicles are essential steps that precede neurotransmitter exocytosis, but neither the total neurotransmitter content nor the fraction sequestered into vesicles have been measured in individual live neurons. We use multiphoton microscopy to directly observe intracellular and intravesicular serotonin in the serotonergic neuronal cell line RN46A. We focus on how the relationship between synthesis and sequestration changes as synthesis is up-regulated by differentiation or down-regulated by chemical inhibition. Temperature-induced differentiation causes an increase of about 60% in the total serotonin content of individual cells, which goes up to about 10 fmol. However, the number of vesicles per cell increases by a factor of four and the proportion of serotonin sequestered inside the vesicles increases by a factor of five. When serotonin synthesis is inhibited in differentiated cells and the serotonin content goes down to the level present in undifferentiated cells, the sequestered proportion still remains at this high level. The total neurotransmitter content of a cell is, thus, an unreliable indicator of the sequestered amount.