Optical recording of electrical activity in intact neuronal networks with random access second-harmonic generation microscopy.

One of the main challenges in understanding the central nervous system is to measure the network dynamics of neuronal assemblies, while preserving the computational role of individual neurons. However, this is not possible with current techniques. In this work, we combined the advantages of second-harmonic generation (SHG) with a random access (RA) excitation scheme to realize a new microscope (RASH) capable of optically recording fast membrane potential events occurring in a wide-field of view. The RASH microscope, in combination with bulk loading of tissue with FM4-64 dye, was used to simultaneously record electrical activity from clusters of Purkinje cells in acute cerebellar slices. Complex spikes, both synchronous and asynchronous, were optically recorded simultaneously across a given population of neurons. Spontaneous electrical activity was also monitored simultaneously in pairs of neurons, where action potentials were recorded without averaging across trials. These results show the strength of this technique in describing the temporal dynamics of neuronal assemblies, opening promising perspectives in understanding the computations of neuronal networks.

[1]  P. Saggau,et al.  Random-access Multiphoton (ramp) Microscopy Fast Functional Imaging of Single Neurons Using , 2005 .

[2]  T. Shiga,et al.  Deformation of polyelectrolyte gels under the influence of electric field , 1990 .

[3]  R. Segev,et al.  How silent is the brain: is there a “dark matter” problem in neuroscience? , 2006, Journal of Comparative Physiology A.

[4]  Y. Liu,et al.  Highly Photoluminescent CdTe/Poly(N‐isopropylacrylamide) Temperature‐Sensitive Gels , 2005 .

[5]  Toyoichi Tanaka,et al.  Spectrum of light scattered from a viscoelastic gel , 1973 .

[6]  Srdjan D Antic,et al.  Action Potentials in Basal and Oblique Dendrites of Rat Neocortical Pyramidal Neurons , 2003, The Journal of physiology.

[7]  D. Kleinfeld,et al.  Noninvasive detection of changes in membrane potential in cultured neurons by light scattering. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[8]  W. Webb,et al.  Nonlinear magic: multiphoton microscopy in the biosciences , 2003, Nature Biotechnology.

[9]  Daniel A Dombeck,et al.  Optical Recording of Action Potentials with Second-Harmonic Generation Microscopy , 2004, The Journal of Neuroscience.

[10]  Toyoichi Tanaka,et al.  Collapse of Gels in an Electric Field , 1982, Science.

[11]  Amiram Grinvald,et al.  VSDI: a new era in functional imaging of cortical dynamics , 2004, Nature Reviews Neuroscience.

[12]  R Y Tsien,et al.  Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. , 1997, Chemistry & biology.

[13]  D. Marr.  A theory of cerebellar cortex , 1969, The Journal of physiology.

[14]  R. Hamlen,et al.  Electrolytically Activated Contractile Polymer , 1965, Nature.

[15]  J. Léger,et al.  Ultrafast random-access scanning in two-photon microscopy using acousto-optic deflectors , 2006, Journal of Neuroscience Methods.

[16]  Y. Lam,et al.  Imaging membrane potential with voltage-sensitive dyes. , 2000, The Biological bulletin.

[17]  T. Z. Teisseyre,et al.  Nonlinear optical potentiometric dyes optimized for imaging with 1064-nm light. , 2007, Journal of biomedical optics.

[18]  Rafael Yuste,et al.  Imaging membrane potential in dendritic spines. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. Aertsen,et al.  Two-dimensional monitoring of spiking networks in acute brain slices , 2001, Experimental Brain Research.

[20]  Eric Betzig,et al.  High-speed, low-photodamage nonlinear imaging using passive pulse splitters , 2008, Nature Methods.

[21]  P. Saggau,et al.  High-speed, random-access fluorescence microscopy: I. High-resolution optical recording with voltage-sensitive dyes and ion indicators. , 1997, Biophysical journal.

[22]  W. Denk,et al.  Deep tissue two-photon microscopy , 2005, Nature Methods.

[23]  L M Loew,et al.  Probing membrane potential with nonlinear optics. , 1993, Biophysical journal.

[24]  Jongseong Kim,et al.  Hydrogel microparticles as dynamically tunable microlenses. , 2004, Journal of the American Chemical Society.

[25]  J Mertz,et al.  Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy. , 2000, Optics letters.

[26]  O. Güven,et al.  Equilibrium swelling behavior of pH‐ and temperature‐sensitive poly(N‐vinyl 2‐pyrrolidone‐g‐citric acid) polyelectrolyte hydrogels , 2000 .

[27]  Leonardo Sacconi,et al.  Optical recording of fast neuronal membrane potential transients in acute mammalian brain slices by second-harmonic generation microscopy. , 2005, Journal of neurophysiology.

[28]  Toshio Kurauchi,et al.  Bending of poly(vinyl alcohol)-poly(sodium acrylate) composite hydrogel in electric fields , 1992 .

[29]  Robert Pelton,et al.  Preparation of aqueous latices with N-isopropylacrylamide , 1986 .

[30]  P. Saggau,et al.  Fast three-dimensional laser scanning scheme using acousto-optic deflectors. , 2005, Journal of biomedical optics.

[31]  J. Eccles.  The cerebellum as a computer: patterns in space and time. , 1973, The Journal of physiology.

[32]  Thomas Knöpfel,et al.  Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins. , 2003, Methods.

[33]  K. Kubota,et al.  Solution Properties of Poly(N-isopropylacrylamide) in Water , 1990 .

[34]  Egidio D'Angelo,et al.  The Spatial Organization of Long-Term Synaptic Plasticity at the Input Stage of Cerebellum , 2007, The Journal of Neuroscience.

[35]  R. Llinás,et al.  Electrophysiological properties of in vitro Purkinje cell dendrites in mammalian cerebellar slices. , 1980, The Journal of physiology.

[36]  Rafael Yuste,et al.  Two-photon photostimulation and imaging of neural circuits , 2007, Nature Methods.

[37]  Professor Dr. John C. Eccles,et al.  The Cerebellum as a Neuronal Machine , 1967, Springer Berlin Heidelberg.

[38]  V. Conticello,et al.  In-Lens Cryo-High Resolution Scanning Electron Microscopy: Methodologies for Molecular Imaging of Self-Assembled Organic Hydrogels , 2003, Microscopy and Microanalysis.

[39]  Leslie M Loew,et al.  Second-harmonic imaging microscopy for visualizing biomolecular arrays in cells, tissues and organisms , 2003, Nature Biotechnology.

[40]  Masayuki Yamato,et al.  Thermally responsive polymer-grafted surfaces facilitate patterned cell seeding and co-culture. , 2002, Biomaterials.

[41]  Jun Gao,et al.  Cross-Linker-Free N-Isopropylacrylamide Gel Nanospheres , 2003 .

[42]  Electric-Field-Induced Phase Transition in Polymer Gels , 1985 .

[43]  Jianzhong Wu,et al.  Phase behavior of thermally responsive microgel colloids. , 2003, Physical review letters.

[44]  D. Linden,et al.  Differential Maturation of Climbing Fiber Innervation in Cerebellar Vermis , 2004, The Journal of Neuroscience.

[45]  M. Zrínyi,et al.  Intelligent polymer gels controlled by magnetic fields , 2000 .

[46]  Ehud Y Isacoff,et al.  A Genetically Encoded Optical Probe of Membrane Voltage , 1997, Neuron.

[47]  R. Llinás,et al.  Electrophysiological properties of in vitro Purkinje cell somata in mammalian cerebellar slices. , 1980, The Journal of physiology.

[48]  Preparation of stimuli-responsive water absorbent , 2002 .

[49]  Arjun G Yodh,et al.  Two-photon excitation of potentiometric probes enables optical recording of action potentials from mammalian nerve terminals in situ. , 2008, Journal of neurophysiology.

[50]  Teruo Okano,et al.  Pulsatile drug release control using hydrogels. , 2002, Advanced drug delivery reviews.

[51]  John D. Mackenzie,et al.  Room temperature optical gain in sol‐gel derived CdS quantum dots , 1996 .

[52]  L Sacconi,et al.  Overcoming photodamage in second-harmonic generation microscopy: real-time optical recording of neuronal action potentials. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[53]  K. Deisseroth,et al.  Millisecond-timescale, genetically targeted optical control of neural activity , 2005, Nature Neuroscience.