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New Biological Frontiers Illuminated by Molecular Sensors and Actuators
Poster Abstracts
31-POS
Board 31
Investigating Cellular Activity in Dissociated Neuronal Cultures Using Novel pH and PKA
Activity Biosensors.
Thorvald F. Andreassen, Sofie E. Pedersen,
Kenneth Madsen
.
University of Copenhagen, Copenhagen, Denmark.
A fundamental ambition within the field of neuroscience is to understand and describe neuronal
signaling at the single neuron level as well as within complex neuronal circuits. Consequently,
new insights into neuronal activity are a requirement in order to uncover the processes governing
brain physiology. Traditionally the investigation of neuronal function on the single cell level has
been limited to electrophysiological measurements providing great temporal resolution but poor
spatial information and flexibility. However, the advents of novel biosensors have provided the
tools to circumvent these caveats by allowing us to probe the function of neurons using less
invasive techniques with high spatial resolution and increasing flexibility. Here we describe our
work in applying biosensors in different neuronal culture systems, firstly to investigate
hippocampal acidification using pHluorin as pH sensor and secondly to monitor PKA activity in
striatal neurons.
Our group has recently described the use of the pH sensitive pHluorin-GFP variant (1) as a tool
to monitor intracellular acidification in hippocampal neurons (2). We found that chemical LTD
protocols decrease the intracellular pH in dissociated hippocampal neurons through a currently
unknown calcium dependent mechanism. We have now implemented the use of simultaneous
measurement of calcium and pH through the use of pHluorin and the calcium sensor R-GECO
(3).
We also describe the use of the PKA activity sensor AKAR (4) to investigate discrete dopamine
mediated changes in postsynaptic striatal neuron PKA activity. The sensitivity of this assay
allows us to monitor changes of PKA activity in response physiological concentrations of agonist
providing a unique method to investigate striatal dopamine physiology.