Host: The Japanese Pharmacological Society, The Japanese Society of Clinical Pharmacology
Name : WCP2018 (18th World Congress of Basic and Clinical Pharmacology)
Location : Kyoto
Date : July 01, 2018 - July 06, 2018
Background
Cyclic adenosine monophosphate (cAMP) is the well-known second messenger in various cell types and subcellular regions. Presynaptic terminal is the one of the specialized compartments in neuronal cell. cAMP signaling might be different between somata and presynaptic terminals. For example, the presynaptic [cAMP]i is predicted determinant for hippocampal mossy fiber LTP (Long-Term Potentiation) as the major cellular basis for learning and memory. In spite of the large amount of information about the organization and function of mossy fiber synapses, there is no evidence about when and where cAMP is generated then degraded. So, the temporal and spatial analyses of [cAMP]i during mossy fiber LTP induction are needed for understanding the presynaptic LTP mechanisms. In addition, the other presynaptic terminals also have cAMP dependency in its transmitter release. Therefore, we have tried to elucidate the cAMP dynamics in presynaptic terminals.
Methods
For seeing [cAMP]i dynamics in presynaptic terminals specifically, we constructed newly developed genetically encoded green fluorescence indicator fused to Synapsin I, which is abundant protein in presynaptic terminal (g-CC-Syn). Hippocampal slices dissected from newborn Wistar rats (P5～P7) were maintained for several weeks in culture. Primary hippocampal neurons were prepared from ICR mice(E16～18). For long-term and high-level g-CC-Syn expression, we used lentivirus or AAV mediated expression.
Results
We report here the g-CC-Syn works well in dissociated culture and slice culture of hippocampus. The g-CC expressed evenly in axons and dendrites in hippocampal dissociated culture, while the g-CC-Syn targeted bouton-like structure (Fig.1a). The g-CC-Syn could response to [cAMP]i elevation induced by Forskolin+Ibudilast application (Fig.1b). The g-CC-Syn was co-localized with the immunostaining of Synaptophysin or Rab3a, both of which were presynapse specific molecules. These results indicate that g-CC-Syn express in presynapse.
In g-CC-Syn expressed cultured hippocampal neuron, we detected presynaptic [cAMP]i elevation under the LTP stimulation or adenylate cyclase activation.
Conclusions
We succeeded in presynapse targeting of genetically encoded green fluorescence cAMP indicator and detecting presynaptic [cAMP]i elevation induced by Forskolin application and electrical stimulation.
