Name: Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes (Video) | JoVE Journal
Uploaded: 2011-03-23T13:45:00.000+00:00
Duration: 9 min 36 s
Description: Learn optical imaging of neurons using voltage-sensitive dyes in the crab stomatogastric ganglion. This method enables recording of membrane potential changes and synaptic interactions without electrode damage.

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5 minutes

We acknowledge support by the School of Computing Science and Faculty of Science, Agriculture and Engineering of Newcastle University, by the Ulm University, and by the SciMedia Ltd (Tokyo, Japan).

1. Preparation of the Crab Stomatogastric Nervous System
Adult Cancer pagurus L. were obtained from local sources (Newcastle University, Dove Marine Laboratories) and kept in filtered, aerated seawater (10 - 12°C). Animals were anesthetized by packing them in ice for 20 - 40 min before dissection. We used isolated stomatogastric nervous systems (STNS)1. The STNS was pinned down in a silicone elastomer-lined (ELASTOSIL RT-601, Wacker, Munich, Germany) Petri dish and continuously superfused (7 - 12 mL/min) with chilled saline (10-13°C). Physiological saline consisted of (mMol*l-1): NaCl, 440; MgCl2, 26; CaCl2, 13; KCl, 11; trisma base, 10; maleic acid, 5. Saline was kept at a constant temperature of 11 - 13°C and at pH 7.4 - 7.6.
The detailed steps of the STNS dissection and preparation, including the desheathing of the stomatoastric ganglion (STG), are presented in the JoVE article by Guttierez and Grashow1. All experiments were carried out in accordance with the European Communities Council Directive of 24th November 1986 (86/609/EEC). Figure 1A shows a schematic diagram of the STNS and Figure 1B shows a typical desheathed STG having its neurons arranged as a flat semicircle in the posterior part of the ganglion.
The Petri dish with the STNS is fixed to the operating platform of the imaging microscope (BW51 WI; Olympus, Tokyo, Japan) with modelling clay. Both, the microscope stage and the microscope are mounted on a vibration isolated table (Scientifica, Uckfield, UK) to prevent movement artefacts during the optical recording. The motor patterns generated in the stomatogastric ganglion (STG) are monitored using extracellular recordings2-4. This is done through the following steps:
<ol>
<li>A petroleum jelly-based cylindrical compartment is built around a section of the main motor nerve (lvn) to electrically isolate the nerve from the bath (this is done on the dissection stage before the Petri dish is placed on the operating platform of the imaging microscope).</li>
<li>One of two stainless steel electrode wires is placed in this compartment, the other one in the bath as a reference electrode.</li>
<li>The differential signal is recorded, filtered and amplified with an AC differential amplifier (Univ. Kaiserslautern, Germany).</li>
<li>The motor activity of the ganglion is monitored using an oscilloscope (DL708E; Yokogawa, Tokyo, Japan) and is additionally recorded using a data acquisition board (CED Power, 1401, Cambridge Electronic Design, Cambridge, UK) and the Spike 2 v6.10 software (Cambridge Electronic Design, Cambridge, UK). Files are analyzed using Spike 2 v6.10 (Cambridge Electronic Design, Cambridge, UK).</li>
</ol>
2. Preparation of the Dye Solution
We used the fluorescent voltage-sensitive dye Di-8-ANEPPQ (Cambridge Bioscience)5, which has a double positive charge that facilitates the loading of the dye through microelectrodes by using positive current pulses. The dye solution should be protected from light as much as possible. The dye solution is prepared as follows:
<ol>
<li>5 mg of dye is mixed with 1 mL of F-127 Pluronic acid in DMSO (Invitrogen) solution.</li>
<li>The plastic vial containing the dissolved dye is centrifuged for 20 minutes at 12,000 rotation/minute in a Sigma Microcentrifuge 1-14 (Sigma, Osterode, Germany) in order to separate larger dye particles that may clog the microelectrode.</li>
<li>The supernatant (top 200 μL) of the dye solution is separated using a pipettor and stored in a separate plastic vial. Both vials are wrapped in aluminium foil to prevent exposure to light.</li>
<li>The dye solution is frozen at -20 °C.</li>
<li>Before use the dye solution is melted by placing the closed and wrapped vial in warm (25 °C) water for 10-15 minutes.</li>
</ol>
3. Dye Loading by Intracellular Injection using Sharp Microelectrodes
We used sharp microelectrodes to load the neurons with the dye. To check the dye loading we use the MiCAM02 imaging system (SciMedia, Tokyo, Japan)6. The imaging system has two CCD cameras: the HR camera has a larger sensor chip (6.4 mm x 4.8 mm) providing better spatial resolution with the best temporal resolution being 1.4 ms, the HS camera has a smaller (2.9 mm x 2.1 mm) but faster sensor chip allowing faster imaging having 0.7 ms as its best temporal resolution. The steps of the dye loading are as follows:
<ol>
<li>Microelectrodes are pulled with a P-97 Flaming - Brown (Sutter Instruments, Novato, CA, USA) electrode puller using 10 cm long, 1 mm outer diameter glass tubes with filament (GB100TF 8P, Science Products, Hofheim, Germany). The electrodes are pulled to have a resistance in the range of 25 - 40 MOhm in 3M KCl (for appropriate pulling parameter settings follow the pipette cookbook provided by the maker of the electrode puller).</li>
<li>The frozen dye solution is molten and a very small amount of it is sucked up into a microfil needle (MicroFil - 34G, World Precision Instruments, Sarasota, FL, USA) - the sucked up dye fills around 2/3rd of the microfil needle.</li>
<li>The dye solution in the microfil needle is injected into the tip of a microelectrode. The microelectrode is placed in a dark box for 15 minutes.</li>
<li>The microelectrode is backfilled with 3M KCl solution. The electrode is then placed back into a dark electrode storage box for another 10-20 minutes to allow the dye solution to reach the fine tip of the electrode by the suction of the capillary force acting in the microelectrode.</li>
<li>The ready microelectrode is placed into an electrode holder and fixed onto the headstage of the intracellular amplifier (IA-251A, Warner Instruments Corporation, Hamden, CT, USA).</li>
<li>The intracellular amplifier is connected to the data acquisition board that provides a pulse signal to drive current pulses into the microelectrode.</li>
<li>The microelectrode is placed gently into the membrane of an STG neuron using a microelectrode manipulator (PatchStar, Scientifica Ltd, Uckfield, UK). Since the working distance of the 10x objective (UMPLFL10XW, NA 0.30, WD 3.3mm; Olympus Corporation, Tokyo, Japan) of the imaging microscope is very small, the microelectrode should be positioned in an oblique position (around 30°) and the movement of the microelectrode into the right position requires several steps of re-focusing of the microscope objective and moving the microelectrode. To start the microelectrode is moved in a position such that its tip is above the STG. The microscope objective is lowered until the electrode tip appears in focus. Then it is lowered further but not as much to touch the microelectrode. The microelectrode is lowered until it re-appears in the focus of the objective. These steps are repeated until the neurons appear clearly in the focus plane of the objective. Then the microelectrode is lowered until it touches and gently pierces the membrane of the selected STG neuron. This is monitored using the oscilloscope.</li>
<li>The intracellular electrode is used to record the activity of the selected STG neuron in order to identify the neuron given its activity profile and the relationship between its activity and the STG activity recorded from the extracellular electrode2,3,7.</li>
<li>The intracellular amplifier is switched into current injection mode and is used for 30 minutes to inject 10 nA positive current steps into the microelectrode that last for 1 second and are separated by 1 second gaps with no current injected into the microelectrode. The injected current drives the positively charged dye molecules into the recorded neuron. If equipment is available multiple neurons may be injected simultaneously.</li>
<li>Ten minutes into the injection period and after the end of the injection the dye spread within the neuron is checked. To do this we use the MiCAM02 imaging system (SciMedia Ltd, Tokyo, Japan) with the 10x objective and illumination by the ultra-low ripple halogen light source (HL-151; Moritex, Tokyo, Japan) through the filter cube MSWG2 (with 480-550 nm excitation filter; Olympus Corporation, Tokyo, Japan). The imaging system is set to use the HR camera with 192 x 128 pixel resolution, 40 ms imaging time, the 'hbin' mode, and 10 frames per imaging session. If the dye filling is successful the imaging should show a dyed patch in the neuron around the location of the microelectrode, and this dyed patch should grow between the images taken after 10 minutes and after 30 minutes. If the spread of the dye is insufficient after 30 min, more injection time is granted. Alternatively, a new dye electrode may be used.</li>
<li>The electrode is pulled out of the neuron and the neuron is left to relax for at least 20-30 minutes. Within this time, the next neuron may be injected with dye.</li>
</ol>
4. Imaging of Dyed Neurons
The activity of the single neurons can be imaged after the dye filling procedure. Alternatively two or more neurons may be filled to image the simultaneous activity of several STG neurons. In principle many and possibly all STG neurons can be filled with dye. The imaging of the neurons is done with the MiCAM 02 imaging system using the HR camera. The procedure of the imaging is as follows:
<ol>
<li>The microscope objective is changed and a high light transmission efficiency 20x objective (XLUMPLFL20XW/0.95, NA 0.95, WD 2.0mm; Olympus Corporation, Tokyo, Japan) is used for the imaging data collection. This objective collects much more light that the 10x objective and also gives higher magnification providing more detailed image of the neurons. Being more light efficient means that small amount of dye loading is already visible with this objective and also that we can use lower light intensity for the imaging reducing the potential photo damage that may be caused to the neurons by the dye.</li>
<li>The microscope's operating stage is positioned such that the neurons that are meant to be imaged are in the field of view of the objective and the objective is focused onto them.</li>
<li>The extracellular recording data is also fed into the MiCAM 02 imaging system through its analogue input channel. This allows the relatively easy relating of optical signals and corresponding motor patterns or extracellularly recorded spikes in the analysis phase.</li>
<li>The MiCAM 02 system is set to use 96 x 64 pixel resolution with 2.2 ms imaging time or 48 x 32 pixel resolution with 1.5 ms imaging time. In both cases the 'hbin' setting may be used if the dyeing of the neurons is not very strong.</li>
<li>The light level is set as low as possible (i.e. the dye in the neurons should generate still sufficient fluorescence for the imaging) to avoid photo modulation and photo damage of the recorded neurons.</li>
<li>The room lighting and all remaining light sources are turned off. In addition, a black curtain may be closed around the recording rig to prevent light exposure from external sources.</li>
<li>The imaging sessions are set to be between 4 - 16 seconds long.</li>
<li>The spontaneous activity of the dyed neurons is recorded over several imaging sessions.</li>
</ol>
5. Analysis of the Optical Imaging Data
To analyse the imaging data we used the BVAna software (version 10.08; SciMedia Ltd, Tokyo, Japan) associated with the MiCAM 02 imaging system. The software supports the visualisation of the imaging data and provides a range of analysis tools as well. The steps of the data analysis and interpretation are as follows:
<ol>
<li>An appropriate circular region of interest is selected on each recorded neurons. Typically we use 2 - 3 pixel radius regions of interest for investigating neurons at the 48 x 32 pixel resolution, and 6 - 8 pixel radius regions of interest for investigating neurons at 96 x 64 resolution.</li>
<li>The temporal smoothing of the data may be necessary, especially if the dye generates relatively low amount of fluorescence (low background value in the data indicates this; it may be due to the low light level that is used or low level dye loading, for example in a neurite of the recorded neuron). Typically in such cases we used temporal smoothing with 3 - 5 time units.</li>
<li>In addition to the optical recordings we also display the extracellular recording by selecting to display the analogue input of the imaging system.</li>
<li>The parameters for the visualisation of the optical data and the analogue input should be set appropriately in order to see all recordings on the computer screen.</li>
<li>Considering the optical recordings of the neurons, the extracellular recording from the lvn, and the classification of the neuron based on the earlier intracellular recording, it is possible to determine the identity of the neuron and to relate its activity to the neural activities recorded from the nerve.</li>
<li>It is expected that the optical recordings show clearly, and without the need of averaging over multiple similar events, the neural activities corresponding to extracellularly recorded spikes and also subthreshold events such as inhibitory post-synaptic potentials caused by the activity of other neurons.</li>
<li>The data export features of the BVAna software can be used to export the optical recordings calculated for the selected regions of interest and also the analogue input that contains the recording from the lvn. The exported data can be processed and analyzed further using other data analysis software as well (e.g. in the simplest case Microsoft Excel (Microsoft, Redmond, WA, US) may be used for additional visualisation and data processing).</li>
</ol>
6. Representative Results
Figure 2 shows the simultaneous recording of two neurons (LP, lateral pyloric neuron; PD, pyloric dilator neuron) in the pyloric central pattern generator of a crab STG together with the recording from the lvn. The optical recordings of the LP and PD neurons identified on the basis of their intracellular recordings show that indeed these match well with the extracellular recordings corresponding to LP and PD neurons. There is a consistent 12 ms delay between the optical and lvn recordings of spike peaks (see inset of Figure 2) due to the axonal transmission delay between the ganglion and the extracellular recording site. The presented data also shows that the neural activity in the soma that corresponds to spikes of the recorded neurons is clearly detectable.
<img src="/files/ftp_upload/2567/2567fig1.jpg" alt="Crustacean stomatogastric ganglion anatomy; neural diagram and microscope image." data-altupdatedat="1788235205000" data-alt="Figure 1"> 
Figure 1. A) Schematic diagram of the stomatogastric nervous system (STNS). CoG, commissural ganglion; OG, esophageal ganglion; STG, stomatogastric ganglion; stn, stomatogastric nerve; lvn, lateral ventricular nerve. B) The crab stomatogastric ganglion (STG) - the image shows the semicircular posterior arrangement of STG neurons.
<img src="/files/ftp_upload/2567/2567fig2.jpg" alt="Neural activity analysis; electrophysiology graph, neuron firing pattern; experimental setup." data-altupdatedat="1788235205000" data-alt="Figure 2"> 
Figure 2. Simultaneous single-sweep recording of a PD and an LP neuron together with the recording of the lvn. The data shows that that the optical and nerve recordings match very well and that we can identify the neural activities corresponding to spikes recorded form the nerve. The inset shows an overlay of several sweeps of LP action potentials - both optically and electrically recorded - and their average demonstrating the match between optically and electrically recorded activities. Due to the fact that our optical recording method does not filter out the low frequency components of the data we are also able to record slow membrane potential changes characteristic for PD and LP neurons14.

<ol><li>Gutierrez, G. J., Grashow, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+borealis+stomatogastric+nervous+system+dissection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Cancer borealis stomatogastric nervous system dissection.</a> J Vis Exp. , (2009).</li>
<li>Bartos, M., Nusbaum, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intercircuit+control+of+motor+pattern+modulation+by+presynaptic+inhibition%5BTitle%5D)+AND+%22J.+Neurosci%22%5BJournal%5D)">Intercircuit control of motor pattern modulation by presynaptic inhibition.</a> J. Neurosci. 17, 2247-2256 (1997).</li>
<li>Blitz, D. M., Nusbaum, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Motor+pattern+selection+via+inhibition+of+parallel+pathways%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Motor pattern selection via inhibition of parallel pathways.</a> J Neurosci. 17, 4965-4975 (1997).</li>
<li>Stein, W., Smarandache, C. R., Nickmann, M., Hedrich, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+consequences+of+activity-dependent+synaptic+enhancement+at+a+crustacean+neuromuscular+junction%5BTitle%5D)+AND+%22J+Exp+Biol%22%5BJournal%5D)">Functional consequences of activity-dependent synaptic enhancement at a crustacean neuromuscular junction.</a> J Exp Biol. 209, 1285-12300 (2006).</li>
<li>Loew, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potentiometric+dyes%3A+Imaging+electrical+activity+of+cell+membranes%5BTitle%5D)+AND+%22Pure+%26+Appl+Chem%22%5BJournal%5D)">Potentiometric dyes: Imaging electrical activity of cell membranes.</a> Pure & Appl Chem. 68, 1405-1409 (1996).</li>
<li>Tominaga, T., Tominaga, Y., Yamada, H., Matsumoto, G., Ichikawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantification+of+optical+signals+with+electrophysiological+signals+in+neural+activities+of+Di-4-ANEPPS+stained+rat+hippocampal+slices%5BTitle%5D)+AND+%22J+Neurosci+Meth%22%5BJournal%5D)">Quantification of optical signals with electrophysiological signals in neural activities of Di-4-ANEPPS stained rat hippocampal slices.</a> J Neurosci Meth. 102, 11-23 (2000).</li>
<li>Heinzel, H. G., Weimann, J. M., Marder, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+behavioral+repertoire+of+the+gastric+mill+in+the+crab%2C+Cancer+pagurus%3A+an+in+situ+endoscopic+and+electrophysiological+examination%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">The behavioral repertoire of the gastric mill in the crab, Cancer pagurus: an in situ endoscopic and electrophysiological examination.</a> J Neurosci. 13, 1793-1803 (1993).</li>
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<li>Briggman, K. L., Kristan, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+dedicated+and+multifunctional+neural+circuits+generating+distinct+behaviors%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Imaging dedicated and multifunctional neural circuits generating distinct behaviors.</a> J Neurosci. 26, 10925-10933 (2006).</li>
<li>Nusbaum, M. P., Beenhakker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+small-systems+approach+to+motor+pattern+generation%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A small-systems approach to motor pattern generation.</a> Nature. 417, 343-350 (2002).</li>
<li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs%5BTitle%5D)+AND+%22Ann+Rev+Physiol%22%5BJournal%5D)">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Ann Rev Physiol. 69, 291-316 (2007).</li>
<li>Grillner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+pattern+generation%3A+the+cellular+and+computational+logic+of+networks+in+motion%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Biological pattern generation: the cellular and computational logic of networks in motion.</a> Neuron. 52, 751-766 (2006).</li>
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<li>Stein, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+stomatogastric+rhythms%5BTitle%5D)+AND+%22J+Comp+Physiol+A%22%5BJournal%5D)">Modulation of stomatogastric rhythms.</a> J Comp Physiol A. 195, 989-1009 (2009).</li>
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No conflicts of interest declared.

The voltage sensitive dye imaging8,9 of STG neurons in combination with traditional electrophysiology methods (intra- and extracellular electrode recordings) allows an improved understanding of how this small, well-known and still complex neural system works. The STG is a prototype of central pattern generator (CPG) neuronal networks (it includes the pyloric and gastric mill rhythm networks)10,,11, thus a better understanding of the emergent functional properties of the STG will also help to understand how in general CPG networks generate their network level functionality. CPG networks play a critical role in motor control12 and also in higher cognitive functions13, consequently the better understanding of their emergent properties may have a major impact in many areas of neuroscience.

<table><tbody><tr><td>MiCAM02</td><td>SciMedia</td><td></td><td>Imaging system</td></tr><tr><td>BVAna v.10.08</td><td>SciMedia</td><td></td><td>Data analysis software for the MiCAM 02 imaging system</td></tr><tr><td>HL-151</td><td>Moritex, Tokyo, Japan</td><td></td><td>Ultra-low ripple halogen light source</td></tr><tr><td>BX51 WI</td><td>Olympus Corporation</td><td></td><td>Upright microscope for epifluorescence imaging</td></tr><tr><td>20x imaging objective </td><td>Olympus Corporation</td><td>XLUMPLFL20XW/0.95</td><td>NA 0.95, WD 2.0mm</td></tr><tr><td>10x imaging objective </td><td>Olympus Corporation</td><td>UMPLFL10XW</td><td>NA 0.30, WD 3.3mm</td></tr><tr><td>Filter cube with 480-550 nm excitation filter and 590nm emission filter </td><td>Olympus Corporation</td><td>MSWG2</td><td></td></tr><tr><td>Antivibration table</td><td>Scientifica Ltd</td><td>63-534</td><td></td></tr><tr><td>PatchStar electrode manipulator</td><td>Scientifica Ltd</td><td>PS-7000C</td><td>Micro-electrode manipulator</td></tr><tr><td>CED Power 1401</td><td>Cambridge Electronic Design</td><td></td><td>Data acquisition board</td></tr><tr><td>DL708E Oscilloscope</td><td>Yokogawa</td><td></td><td>Intracellular amplifier</td></tr><tr><td>AC differential amplifier</td><td>University of Kaiserslautern, Germany</td><td></td><td>Extracellular amplifier</td></tr><tr><td>P-97 Flaming &amp;#8211; Brown type electrode puller</td><td>Sutter Instrument Co.</td><td></td><td>Electrode puller</td></tr><tr><td>Sigma Microcentrifuge 1-14</td><td>Sigma-Aldrich</td><td></td><td>Centrifuge</td></tr><tr><td>Genex Pipettors Mline</td><td>Scientific Laboratory Supplies LTD</td><td>PIP7774</td><td>PIP7774</td></tr><tr><td>Glass micropipette with filament</td><td>Science Products</td><td>GB100TF 8P</td><td>Glass for micr&amp;#8211;lectrodes</td></tr><tr><td>MicroFil &amp;#8211; 34G</td><td>World Precision Instruments, Inc.</td><td></td><td>Microfil needle</td></tr><tr><td>Spike 2 v6.10</td><td>Cambridge Electronic Design</td><td></td><td>Electrophysiology software</td></tr><tr><td>Di-8-ANEPPQ</td><td>Cambridge BioScience</td><td>BT61014</td><td>Fluorescent voltage sensitive dye</td></tr><tr><td>F-127 Pluronic Acid, DMSO</td><td>Invitrogen</td><td>P-3000MP</td><td>Solvent</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td>Crab saline component</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt; . 2H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>C3881</td><td>Crab saline component</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td>Crab saline component, electrolyte solution</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt; . 6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Aldrich</td><td>M9272</td><td>Crab saline component</td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td>Crab saline component</td></tr><tr><td>Maleic acid</td><td>Sigma-Aldrich</td><td>M0375</td><td>Crab saline component</td></tr><tr><td>Elastosil RT-601</td><td>Wacker, Munich, Germany</td><td></td><td>Lining of Petri dishes</td></tr></tbody></table>

optical imaging of neurons in the crab stomatogastric ganglion with voltage-sensitive dyes

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

The aim of the following experiment is to use voltage sensitive dyes to optically record membrane potential changes and synaptic interactions between neurons in the central pattern generators of the crustacean St Somatic asterisk ganglion. This is achieved by loading identified neurons with the DD eight and at PQ by ion retic injection using sharp micro electrodes and simultaneous extracellular recording of motor nerve activity as a second step, the fluorescence of the voltage sensitive dye is excited by green light, which allows the imaging of fluorescence changes that correspond to the changes in the cell membrane potential. Next, the image data is analyzed by selecting a region of interest in the neuron's membrane and comparing the optical signal to the recorded extracellular bike activity in order to identify action potentials and synaptic potentials.In the image neurons results are obtained that show the optical recording of synaptically interacting pattern generating neurons can supplement traditional recording techniques and open new opportunities for understanding how central pattern generator neural networks function. The main advantage of this technique over existing methods, like extra and intercellular recordings, is that after dive filling, optical recordings of member potential changes and synaptic interactions can be achieved without the steric restrictions imposed by electrode placement and without further damage to the neurons during long-term recordings. Though this method can provide additional insight into pattern generation in the crustacean stomach gastric ganglion.It can also be applied to other systems such as locomotion and respiration in vertebrates. Generally, individuals new to this method will struggle because D selection injection and preparation are difficult, and the parameters for the analysis of the image data have to be set adequately to detect membrane potential changes. Begin the preparations by making the fluorescent dye solution in a plastic vial mix five milligrams of the fluorescent voltage sensitive dye D eight and FPQ with one milliliter of F1 27 onic acid in DMSO solution.Next, centrifuge the dissolved dye for 20 minutes at 12, 000 rotations per minute. In order to separate larger D particles that may clog the micro electrode following centrifugation user prepared to aspirate the top 200 microliters of the supinate, expel the supinate into a separate plastic vial. Wrap the vial in aluminum foil to prevent exposure to light.Store the D solution in a minus 20 degrees Celsius freezer on the day of the experiment. Defrost the dye solution by placing the wrapped vial in 25 degrees Celsius water for 10 to 15 minutes. To prepare for the recordings, start by isolating the STS somatic gastric nervous system of a crab and de sheathing the somatic gastric ganglion.Apply petroleum jelly around a section of the lateral ventricular nerve or LVN to create an electrically isolated compartment. Use modeling clay to attach the Petri dish to the operating platform of the imaging microscope. Place a stainless steel electrode wire in the petroleum jelly compartment to record from the LVN place, another stainless steel electrode in the bath as a reference electrode.When the D solution is defrosted, draw up enough of the D solution to fill approximately two thirds of a microfill needle. Inject the D solution from the microfill needle into the tip of a glass micro electrode. Cover the dye filled electrode with a box and wait 15 minutes.Then backfill the micro electrode with a three molar potassium chloride solution. Place the micro electrode back into a dark electrode storage box for 10 to 20 minutes to allow capillary action to pull the dice solution into the fine tip of the electrode. After 10 to 20 minutes, place the micro electrode into an electrode holder and attach it to the head stage of the intracellular amplifier.The intracellular amplifier is connected to the data acquisition board that provides a pulse signal to drive current pulses into the micro electrode. Next, adjust the micro electrode to an oblique angle and position its tip above the somatic gastric ganglion. Lower the microscope objective until the electrode tip is in focus.Then lower the objective, being careful to stop before touching the micro electrode. Then lower the micro electrode until it reappears. In the focus of the objective monitoring progress on the oscilloscope, gently pierce the membrane of the selected somatic gastric ganglia neuron.At this point, compare intracellular and extracellular recorded action potentials. In order to identify the impaled neuron, begin dye injection by switching the intracellular amplifier into current injection mode and injecting 10 nano positive current steps into the micro electrode. The current steps should last for one second, separated by one second gaps of no current.Continue this current injection for 30 minutes. After 10 minutes of dye injection, use the MYCA zero two imaging system to check the dye spread within the neuron. Repeat this, check after 30 minutes.If the dye filling is successful, the dyed patch around the micro electrode should grow between the 10 minute and 30 minute images. Pull the electrode out of the neuron and allow the neuron to relax for at least 20 to 30 minutes to allow the dye to spread further in the cell. During this time, another neuron can be injected with dye.Switch to the highlight transmission efficiency 20 times objective on the MYAM zero two imaging system. Reposition the microscope's operating stage as needed, and focus on the D field neuron. Feed the extracellular recording data into the imaging system through its analog input channel.Turn off the room lighting and all remaining light sources. In addition, close a black curtain around the recording rig to prevent light exposure from external sources. Set the imaging system to the appropriate pixel resolution and imaging time shown in the chart.If the dye in the neurons is not very strong, use the H bin setting. Set the microscopes light level as low as possible to avoid photo modulation and photo damage of the recorded neurons. Set the imaging sessions to between four to 16 seconds long.Then record the spontaneous activity of the DY neurons over several imaging sessions. To begin analysis of the optical responses using the BV A NA imaging software, select an appropriate circular region on each recorded neuron if temporal smoothing of the data is necessary. For example, if the D generates a relatively low amount of fluorescence, use the software to apply temporal smoothing with three to five time units.The two dye injected neurons in the upper left of this figure are identified as a PD neuron and an LP neuron on the basis of their intracellular recordings. The voltage traces to their right show the spontaneous optical responses from each neuron. The extracellular recording of the LVN is shown at the bottom of the figure.The LP and PD neurons are part of the pyloric central pattern generator of a crab somatic gastric ganglion and a mutually inhibitory. Their optical recordings can be matched well to the extracellular recordings of the LVN. The inset at the bottom shows an overlay of several sweeps of LP action potentials and their average demonstrating the match between optically and electrically recorded activities.Once mastered, this technique can be done in one and a half hours if it is performed properly. Following this procedure, other methods like immunostaining can be performed in order to answer additional questions such as which type of synapsis was involved in eliciting the observed membrane potential changes. After watching this video, you should have a good understanding of how to record optically, multiple identified and ethal interacting neurons in the stomach gastric ganglia.

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Research

JoVE Journal

Neuroscience

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Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

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