Mechanisms of raphe neuron differentiation: Control by protein kinase C and cyclic adenosine monophosphate-dependent protein kinase and regulation of tryptophan hydroxylase expression

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)



First Committee Member

Scott R. Whittemore, Committee Chair


The specific mechanisms that regulate the differentiation of brain stem raphe neurons are not well understood. Using the glutamatergic raphe-derived neuronal cell line, RN33B, the effects of altering cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) on raphe neuron differentiation were examined. PKA activity increased 2.25-fold with differentiation and pharmacological activation of PKA potentiated their glutamatergic phenotype. Activation of PKC inhibited neuronal differentiation, as manifested in reduced neuronal specific proteins, by inducing proliferation. Inhibition of either PKC or PKA did not significantly alter neuronal differentiation. That both PKA and PKC can regulate, but are not necessary for, RN33B cell differentiation, suggests the existence of parallel pathways regulating raphe neuron differentiation.Neurotransmitter biosynthesis in serotonergic raphe neurons is dependent upon activation of tryptophan hydroxylase (TPH). The mechanisms that underlie increased TPH activity during ontogeny are unknown. Using the serotonergic raphe-derived neuronal cell line, RN46A, we tested the hypothesis that BDNF and high potassium increase TPH expression by activating the TPH gene promoter. We determined that the previously documented increase in TPH immunoreactivity by BDNF and high potassium occurs through the actions of high potassium, which acts at a minimum, by inducing TPH promoter activity resulting from increased osmolarity. High potassium significantly increased both the activity of a 3.1 kb TPH promoter sequence in 3 stably transfected RN46A cell lines and TPH immunoreactivity in untransfected RN46A cells. Equiosmolar replacement of high potassium with choline or N-methylglucamine increased TPH promoter activation to equivalent levels. These results suggest that BDNF may not play a role in the early serotonergic development of raphe neurons.The developmental regulation of TPH mRNA expression and enzyme activity in the rat raphe and its target fields was studied using quantitative in situ hybridization and a radioenzymatic assay, respectively, to test the hypothesis that developmental changes in TPH enzyme activities correlate with corresponding changes in steady-state TPH mRNA levels. Developmental changes in TPH mRNA levels were correlated with TPH enzyme activities in the raphe which contain the serotonergic soma, but not in the cortex, hippocampus, or spinal cord, which contain the serotonergic terminals. These data indicate that during development, somal TPH enzyme activity is controlled, at least in part, by regulation of TPH mRNA levels. Following transport of TPH protein out of the cell bodies and into the synaptic terminals, TPH activity may then be regulated post-translationally. This would permit TPH enzyme activity to be matched to the immediate and local needs of those synapses.Comparison of TPH mRNA levels per cell and per brain section revealed a potential serotonergic neuron loss between P22 and P61 in both the dorsal raphe nucleus and nucleus raphe obscuris. Decreased TPH enzyme activities in the postnatal raphe and spinal cord are consistent with this interpretation. This loss may reflect serotonergic neuron death or a switch to another neurotransmitter phenotype.


Biology, Neuroscience

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