Imaging Brain Slices

Brain slices are convenient preparations to study synapses, neurons, and neural circuits because, while they are easily accessed by experimental manipulations such as drug applications, intracellular recordings, and optical imaging, they preserve many of the essential functional properties of these circuits. In this chapter, we describe techniques of live brain-slice imaging used in our laboratory. We cover in detail experimental protocols and know-how acquired over the years about preparing neocortical and hippocampal slices and slice cultures, loading neurons with dyes or using biolistic transfection techniques, two-photon and second harmonic imaging, morphological reconstructions, and image processing and analysis. These techniques are used to study the functional or morphological dynamics of synaptic structures, including dendritic spines and axon terminals, and to characterize circuit connectivity and dynamics.

[1]  R. Yuste,et al.  Optical probing of neuronal circuits with calcium indicators. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Yuste,et al.  Detecting action potentials in neuronal populations with calcium imaging. , 1999, Methods.

[3]  M. G. Honig,et al.  Dil and DiO: versatile fluorescent dyes for neuronal labelling and pathway tracing , 1989, Trends in Neurosciences.

[4]  Rafael Yuste,et al.  Spine motility with synaptic contact , 2001, Nature Neuroscience.

[5]  Rafael Yuste,et al.  Quantitative morphologic classification of layer 5 neurons from mouse primary visual cortex , 2003, The Journal of comparative neurology.

[6]  Rafael Yuste,et al.  A custom-made two-photon microscope and deconvolution system , 2000, Pflügers Archiv.

[7]  George Paxinos,et al.  The Mouse Brain in Stereotaxic Coordinates , 2001 .

[8]  N. Heintz,et al.  A strategy for the analysis of gene expression during neural development. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[9]  J. Lichtman,et al.  Multicolor “DiOlistic” Labeling of the Nervous System Using Lipophilic Dye Combinations , 2000, Neuron.

[10]  F. A. Edwards,et al.  A thin slice preparation for patch clamp recordings from neurones of the mammalian central nervous system , 1989, Pflügers Archiv.

[11]  R. Yuste,et al.  Developmental regulation of spine motility in the mammalian central nervous system. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Rafael Yuste,et al.  Regulation of dendritic spine motility and stability by Rac1 and Rho kinase: evidence for two forms of spine motility , 2004, Molecular and Cellular Neuroscience.

[13]  R. Yuste,et al.  Regulation of dendritic spine morphology by the rho family of small GTPases: antagonistic roles of Rac and Rho. , 2000, Cerebral cortex.

[14]  R. Yuste,et al.  Stereotyped position of local synaptic targets in neocortex. , 2001, Science.

[15]  B. Connors,et al.  Thalamocortical responses of mouse somatosensory (barrel) cortexin vitro , 1991, Neuroscience.

[16]  David Kleinfeld,et al.  Principles, Design,and Construction of a Two-Photon Laser-Scanning Microscopefor In Vitro and In Vivo Brain Imaging , 2002 .

[17]  Christian Lohmann,et al.  Transmitter-evoked local calcium release stabilizes developing dendrites , 2002, Nature.

[18]  Rafael Yuste,et al.  Bidirectional Regulation of Hippocampal Mossy Fiber Filopodial Motility by Kainate Receptors A Two-Step Model of Synaptogenesis , 2003, Neuron.

[19]  R. Yuste,et al.  Thalamocortical Bursts Trigger Recurrent Activity in Neocortical Networks: Layer 4 as a Frequency-Dependent Gate , 2002, The Journal of Neuroscience.

[20]  Da-Ting Lin,et al.  Multi-photon laser scanning microscopy using an acoustic optical deflector. , 2002, Biophysical journal.

[21]  R. Yuste,et al.  Regulation of Spine Calcium Dynamics by Rapid Spine Motility Materials and Methods , 2022 .

[22]  Pietro Liò,et al.  Wavelets in bioinformatics and computational biology: state of art and perspectives , 2003, Bioinform..

[23]  Narayanan Kasthuri,et al.  Imaging calcium dynamics in the nervous system by means of ballistic delivery of indicators , 2002, Journal of Neuroscience Methods.

[24]  B. Sakmann,et al.  Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. , 1996, Biophysical journal.

[25]  R. Tsien.  Fluorescent probes of cell signaling. , 1989, Annual review of neuroscience.

[26]  Lawrence C. Katz,et al.  Neuronal transfection in brain slices using particle-mediated gene transfer , 1994, Neuron.

[27]  Leslie M. Loew,et al.  Second-harmonic generation of biological interfaces: probing the membrane protein bacteriorhodopsin and imaging membrane potential around GFP molecules at specific sites in neuronal cells of C. elegans , 1999 .

[28]  G. Feng,et al.  Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP , 2000, Neuron.

[29]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[30]  Rafael Yuste,et al.  Control of postsynaptic Ca2+ influx in developing neocortex by excitatory and inhibitory neurotransmitters , 1991, Neuron.

[31]  W. Denk,et al.  Dendritic spines as basic functional units of neuronal integration , 1995, Nature.

[32]  G. Paxinos.  The Rat nervous system , 1985 .

[33]  Rafael Yuste,et al.  A two-photon and second-harmonic microscope. , 2003, Methods.

[34]  Rafael Yuste,et al.  Imaging neurons : a laboratory manual , 1999 .