Multi-Page PapersVolume 10, Spring 2016

Optical Characterization of Potential Dampening in Dendritic Spine Electrophysiology

Samuel Kim1,

Masayuki Sakamoto2,

Rafael Yuste3

1 Columbia College, Columbia University,
2 Department of Biological Sciences, Columbia University,
3 Department of Biological Sciences, Kavli Institute for Brain Science, Columbia University

Abstract

This study aims to quantitatively characterize the electrophysiology of the dendritic spine as compared to that of its adjacent dendritic shaft, by imaging artificially induced back-propagating action potentials using a variety of different genetically encoded voltage indicators. We performed whole cell patch clamp and current injection recordings with simultaneous voltage imaging of neonatal mouse hippocampal neurons, which were transfected to express ‘ArcLight’ or one of two variants of ‘Archaerhodopsin 3 (Arch)’ known as ‘QuasAr1’ and ‘QuasAr2’. With ArcLight, we coupled electrophysiological current injection recordings with fluorescence imaging and compared the individual peak fluorescence change () value of each spine with the peak  value of its adjacent dendritic shaft in response to induced back-propagating action potentials. The results from ArcLight do not indicate a statistically significant dampening in membrane potential from the dendrite shaft across an adjacent spine, but do suggest a difference in membrane potential fluctuations between long pulse (100msec) and short pulse (20msec) current injections. With Arch, we quantified the  values from current injection recordings and voltage imaging sessions of neuronal soma for ‘QuasAr1’ and ‘QuasAr2’. Preliminary current injection and soma imaging results initially exhibited negligible signal-to-noise ratios of  in response to induced action potentials, possibly due to technical maladjustments. A subsequent, more general characterization of Arch using voltage clamp and voltage step manipulations with QuasAr1 at different laser intensities indicates that QuasAr1 shows large changes in fluorescence at much weaker laser intensities (~10 mW) than was used for aforementioned current injection voltage imaging (~110 mW). We intend to further optimize and apply QuasAr1 and QuasAr2 to spine imaging, and subsequently investigate the difference in peak  values for different current injection pulse durations as suggested by ArcLight imaging data.