We are grateful to our collaborators Knut Holthoff, Arthur Konnerth and Marco Canepari who participated in the initial development of this technique as well as to Leslie M. Loew for kindly providing dyes. Supported by NSF grant IOS-0817969, NIH grants NS068407 and M136043 and by Kavli Institute for Neuroscience at Yale University.

1. Equipment Setup
Step 1.1. Imaging setup
The key to recording voltage sensitive dye signals is appropriate setup design. We use an upright microscope (BX51WI Olympus or Zeiss AxioExaminer) equipped with three cameras. The setup is designed for illuminating individual neurons in brain slices by excitation light in epi-fluorescence, wide-field microscopy mode using either Nikon 60X/1.0 NA or Zeiss 63X/1.0 NA water dipping objectives. Our microscopes are bolted to a vibra...

<ol>
	<li>Bischofberger, J., Engel, D., Li, L., Geiger, J. R., Jonas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch-clamp+recording+from+mossy+fiber+terminals+in+hippocampal+slices.">Patch-clamp recording from mossy fiber terminals in hippocampal slices.</a> Nature Protocols. 1, 2075-2081 (2006).</li><li>Cohen, L. B., Salzberg, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurement+of+membrane+potential.">Optical measurement of membrane potential.</a> Ann. Rev. Neurosci. 1, 171-182 (1978).</li><li>Cohen, L. B., Canepari, M., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Historical+overview+and+general+methods+of+membrane+potential+imaging.">Historical overview and general methods of membrane potential imaging.</a> Membrane Potential Imaging in the Nervous System. , (2010).</li><li>Canepari, M., Djurisic, M., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+signals+from+rat+hippocampal+CA1+pyramidal+neurons+during+coincident+pre-+and+post-synaptic+activity:+a+combined+voltage-+and+calcium+imaging+study.">Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre- and post-synaptic activity: a combined voltage- and calcium imaging study.</a> J. Physiol. 580, 463-484 (2007).</li><li>Canepari, M., Popovic, M., Vogt, K., Holthoff, K., Konnerth, A., Salzberg, B. M., Grinvald, A., Antic, S. D., Zecevic, D., Canepari, M., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+submillisecond+membrane+potential+changes+from+individual+regions+of+single+axons,+dendrites+and+spines.">Imaging submillisecond membrane potential changes from individual regions of single axons, dendrites and spines.</a> Membrane Potential Imaging in the Nervous System. , (2010).</li><li>Djurisic, M., Antic, S., Chen, W. R., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voltage+imaging+from+dendrites+of+mitral+cells:+EPSP+attenuation+and+spike+trigger+zones.">Voltage imaging from dendrites of mitral cells: EPSP attenuation and spike trigger zones.</a> J. Neurosci. 24, 6703-6714 (2004).</li><li>Holthoff, K., Zecevic, D., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+time-course+of+action+potentials+in+spines+and+remote+dendrites+of+mouse+visual+cortical+neurons.">Rapid time-course of action potentials in spines and remote dendrites of mouse visual cortical neurons.</a> J. Physiol. 588, 1085-1096 (2010).</li><li>Kole, M. H. P., Letzkus, J. J., Stuart, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+initial+segment+Kv1+channels+control+axonal+action+potential+waveform+and+synaptic+efficacy.">Axon initial segment Kv1 channels control axonal action potential waveform and synaptic efficacy.</a> Neuron. 55, 633-647 (2007).</li><li>Kuhn, B., Fromherz, P., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+of+Stark-shift+voltage-sensing+dyes+by+one-+or+two-photon+excitation+near+the+red+spectral+edge.">High sensitivity of Stark-shift voltage-sensing dyes by one- or two-photon excitation near the red spectral edge.</a> Biophysical J. 87, 631-639 (2004).</li><li>Loew, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+characterization+of+electrochromic+membrane+probes.">Design and characterization of electrochromic membrane probes.</a> J. Biochem. Biophys. Method. 6, 243-260 (1982).</li><li>Loew, L., Canepari, M., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+use+of+organic+voltage+sensitive+dyes.">Design and use of organic voltage sensitive dyes.</a> Membrane Potential Imaging in the Nervous System. , (2010).</li><li>Palmer, L. M., Stuart, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site+of+action+potential+initiation+in+layer+5+pyramidal+neurons.">Site of action potential initiation in layer 5 pyramidal neurons.</a> J. Neurosci. 26, 1854-1863 (2006).</li><li>Popovic, M. A., Foust, A. J., McCormick, D. A., Zecevic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+spatio-temporal+characteristics+of+action+potential+initiation+in+layer+5+pyramidal+neurons:+a+voltage+imaging+study.">The spatio-temporal characteristics of action potential initiation in layer 5 pyramidal neurons: a voltage imaging study.</a> J. Physiol. 589, 4167-4187 (2011).</li><li>Ross, W. N., Salzberg, B. M., Cohen, L. B., Grinvald, A., Davila, H. V., Waggoner, A. S., Wang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+absorption,+fluorescence,+dichroism,+and+birefringence+in+stained+giant+axons:+optical+measurement+of+membrane+potential.">Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: optical measurement of membrane potential.</a> J. Membr. Biol. 33, 141-183 (1977).</li><li>Shu, Y., Duque, A., Yu, Y., Haider, B., McCormick, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+action+potential+initiation+in+neocortical+pyramidal+cells:+evidence+from+whole+cell+axon+recordings.">Properties of action potential initiation in neocortical pyramidal cells: evidence from whole cell axon recordings.</a> J Neurophysiol. 97, 746-760 (2007).</li><li>Wu, J. -. Y., Cohen, L. B., Mason, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+multisite+optical+measurement+of+membrane+potential.">Fast multisite optical measurement of membrane potential.</a> A practical guide to technology for quantitative real-time analysis. , 389-404 (1993).</li><li>Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dendritic Spines. , (2010).</li></ol>

This article describes a voltage-sensitive dye recording method for monitoring electrical activity of individual neurons with sub-micrometer and sub-millisecond spatiotemporal resolution. Laser excitation at near-optimal wavelength (regarding signal size) improved the sensitivity of recording by a factor of ~50 over previous approaches. The current sensitivity enables monitoring electrical signals from all parts of individual neurons, including dendrites, axons, axon collaterals and axon terminals as well as individual d...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the component</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Setup components</td>
 </tr>
 <tr>
 <td>Upright Microscope</td>
 <td>Olympus Inc.</td>
 <td>BX51WI</td>
 <td>With three camera ports</td>
 </tr>
 <tr>
 <td>Motorized Movable Stage</td>
 <td>Siskiyou</td>
 <td>MXOPi.2</td>
 <td></td>
 </tr>
 <tr>
 <td>Epi-fluorescence Condenser for Olympus BX51</td>
 <td>TILL Photonics</td>
 <td>0000-560-11659</td>
 <td></td>
 </tr>
 <tr>
 <td>Upright Microscope</td>
 <td>Carl Zeiss, LLC </td>
 <td>AxioExaminer D1</td>
 <td>With three camera ports</td>
 </tr>
 <tr>
 <td>Motorized Top Plate</td>
 <td>Scientifica Limited</td>
 <td>MMBP</td>
 <td></td>
 </tr>
 <tr>
 <td>Epi-fluorescence Condenser for Zeiss AxioExaminer</td>
 <td>TILL Photonics</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Data Acquisition Camera</td>
 <td>RedShirtImaging LLC</td>
 <td>NeuroCCD-SM</td>
 <td>High speed, low read noise</td>
 </tr>
 <tr>
 <td>CCD for IR-DIC</td>
 <td>Dage-MTI</td>
 <td>IR-1000</td>
 <td></td>
 </tr>
 <tr>
 <td>Spinning-Disc Confocal Scanner</td>
 <td>Yokogawa</td>
 <td>CSU-10</td>
 <td></td>
 </tr>
 <tr>
 <td>High Spatial Resolution CCD on Confocal Scanner</td>
 <td>PCO AG</td>
 <td>PixelFly</td>
 <td>1392x1024 pixels </td>
 </tr>
 <tr>
 <td>DPSS CW Laser (532 Nm)</td>
 <td>CNI Optoelectronics Tech. Co., Ltd</td>
 <td>MLL-III-532 400mW</td>
 <td>Excitation light source</td>
 </tr>
 <tr>
 <td>Multi-Mode Fiber Launcher</td>
 <td>Siskiyou</td>
 <td>SM-CFT</td>
 <td></td>
 </tr>
 <tr>
 <td>Light Guide </td>
 <td>TILL Photonics</td>
 <td>0000-515-11524</td>
 <td></td>
 </tr>
 <tr>
 <td>Shutter</td>
 <td>Vincent Associates</td>
 <td>LS6</td>
 <td></td>
 </tr>
 <tr>
 <td>Vibration Isolation Table</td>
 <td>Minus k Technology</td>
 <td>MK26</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Specific reagents</td>
 </tr>
 <tr>
 <td>Di-2-ANEPEQ (JPW 1114)</td>
 <td>Life Technologies</td>
 <td>D-6923</td>
 <td>Voltage sensitive dye</td>
 </tr>
 <tr>
 <td>Crym-EGFP Mouse Line</td>
 <td>GENSAT (MMRRC)</td>
 <td>STOCK Tg(Crym-EGFP)GF82Gsat/Mmcd</td>
 <td>Sparsely expressing EGFP in Layer 5 cortical neurons</td>
 </tr>
 </tbody>
</table>

voltage-sensitive dye recording from axons, dendrites and dendritic spines of individual neurons in brain slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

Successful confocal microscopy should allow clear identification of intact neuronal processes which are close to the surface of the slice and located in one plane of focus. The selection of nerve cells which are appropriate for voltage imaging prior to voltage-sensitive dye loading is critical. An example of confocal images of L5 pyramidal neurons expressing EGFP in a cortical slice (Crym transgenic mouse line) is shown in Figure 2. Axons of individual neurons are clearly visible. Cells with long intact ...

Watch this Scientific Journal Video about Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices at JoVE.com

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

voltage-sensitive-dye-recording-from-axons-dendrites-dendritic-spines

Research

JoVE Journal

Neuroscience

16.6K visualizações.  Yale University School of Medicine. Uma técnica de imagem para monitoramento de mudanças potenciais de membrana com sub-micrômetro espacial e sub-milissegundo resolução temporal é descrito. A técnica, com base na excitação do laser de corantes sensíveis à voltagem, permite a medição de sinais em axónios e colaterais, ramos terminais axonais e dendríticas individuais espinhas dendriticas.