A PHS Grant (MH51393) supported this work. Some of the Aplysia we use are provided by the National Resource for Aplysia of the University of Miami under Grant RR10294 from the National Center for Research Resources, NIH.

1. Preparation
<ol>
 <li>Anesthetize the animal by injecting 75-100 ml isotonic magnesium chloride solution. The Aplysia we use for imaging are generally 150-200 grams and are obtained from Marinus Scientific. </li>
 <li>Pin the anesthetized animal to a wax covered dish. Syringe needles work well for this purpose; sterile techniques are not necessary. Using gross forceps and standard scissors make an incision in the animal's foot and expose the buccal mass. Locate the buccal ganglion. Using spring sci...

<ol>
	<li>Zucker, R. S., Regehr, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+synaptic+plasticity.">Short-term synaptic plasticity.</a> Annu. Rev. Physiol. 64, 355-405 (2002).</li><li>Eberhard, M., Erne, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+binding+to+fluorescent+calcium+indicators:+Calcium+green,+calcium+orange+and+calcium+crimson.">Calcium binding to fluorescent calcium indicators: Calcium green, calcium orange and calcium crimson.</a> Biochem. Biophysical Res. Comm. 180, 209-215 (1991).</li><li>Escobar, A. L., Velez, P., Kim, A. M., Cifuentes, F., Fill, M., Vergata, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+properties+of+DM-nitrophen+and+calcium+indicators:+rapid+transient+response+to+flash+photolysis.">Kinetic properties of DM-nitrophen and calcium indicators: rapid transient response to flash photolysis.</a> Eur. J. Physiol. 434, 615-631 (1997).</li><li>Ivanov, A. I., Calabrese, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+spike-mediated+synaptic+transmission+by+presynaptic+background+Ca2++in+leech+heart+interneurons.">Modulation of spike-mediated synaptic transmission by presynaptic background Ca2+ in leech heart interneurons.</a> J. Neurosci. 23, 1206-1218 (2003).</li><li>Rosen, S. C., Miller, M. W., Evans, C. G., Cropper, E. C., Kupfermann, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+synaptic+connections+between+peptidergic+radula+mechanoafferent+neurons+and+neurons+in+the+feeding+system+of+Aplysia.">Diverse synaptic connections between peptidergic radula mechanoafferent neurons and neurons in the feeding system of Aplysia.</a> J. Neurophysiol. 83, 1605-1620 (2000).</li><li>Ludwar, B. C. h., Evans, C. G., Jing, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+mechanisms+mediate+potentiating+effects+of+depolarization+on+synaptic+transmission.">Two distinct mechanisms mediate potentiating effects of depolarization on synaptic transmission.</a> J. Neurophysiol. 102, 1976-1983 (2009).</li><li>Evans, C. G., Ludwar, B. C. h., Askansas, J., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+holding+potential+on+the+dynamics+of+homosynaptic+facilitation.">Effect of holding potential on the dynamics of homosynaptic facilitation.</a> J. Neurosci. 31, 11039-11043 (2011).</li><li>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Handbook. , (2005).</li><li>Borovikov, D., Evans, C. G., Jing, J., Rosen, S. C., Cropper, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+proprioceptive+role+for+an+exteroceptive+mechanoafferent+neuron+in+Aplysia.">A proprioceptive role for an exteroceptive mechanoafferent neuron in Aplysia.</a> J. Neurosci. 20, 1990-2002 (2000).</li><li>Goldberg, J. H., 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=Chapter+38:+A+practical+guide:+Two-photon+calcium+imaging+of+spines+and+dendrites.">Chapter 38: A practical guide: Two-photon calcium imaging of spines and dendrites.</a> Imaging in Neuroscience and Development. , (2005).</li></ol>

We demonstrate techniques that can be used to simultaneously monitor the intracellular calcium concentration and evaluate the efficacy of synaptic transmission. These techniques are useful for determining how various forms of short-term plasticity are mediated. 
The imaging is carried out with a fluorescence microscope and CCD camera. These equipment requirements are relatively modest when compared to most functional imaging set-ups. The technique is simple and easy to learn. While imaging wit...

<table border="1">
<tbody>
 <tr>
 <td>Reagent Name</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>Calcium Orange</td>
 <td>Invitrogen</td>
 <td>C-3013</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>EGTA</td>
 <td>Sigma</td>
 <td>E-4378</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Calcium calibration buffer kit</td>
 <td>Invitrogen </td>
 <td>C-3008MP</td>
 <td>useful for testing the sensitivity and dynamic range of the 			signal</td>
 </tr>
 <tr>
 <td>Magnesium chloride hexahydrate</td>
 <td>Sigma </td>
 <td>M0250</td>
 <td>used in 0.33 M solution to anesthetize animal</td>
 </tr>
 </tbody>
</table>
Table 1. Reagents used.
<table border="1">
<tbody>
 <tr>
 <td>Equipment Name</td>
 <td>Company</td>
 <td>Comment</td>
 </tr>
 <tr>
 <td>FN-1 upright fluorescence microscope</td>
 <td>Nikon Instruments</td>
 <td>with Narishige ITS-FN1 stage</td>
 </tr>
 <tr>
 <td>NMN-21 manipulators</td>
 <td>Narishige</td>
 <td>mounted on stage with magnets</td>
 </tr>
 <tr>
 <td>CoolSNAP HQ2 CCD camera</td>
 <td>Photometrics</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NIS elements AR 
	(version 3.22)</td>
 <td>Nikon Instruments</td>
 <td>imaging software used to acquire fluorescence data</td>
 </tr>
 <tr>
 <td>10X/0.3w Plan Fluor objective</td>
 <td>Nikon Instruments</td>
 <td>this water immersion lens has a very long working distance of 			3.5 mm</td>
 </tr>
 <tr>
 <td>X-Cite 120 PC metal halide lamp</td>
 <td>EXFO</td>
 <td>used for fluorescence imaging</td>
 </tr>
 <tr>
 <td>LS-DWL 
	halogen lamp</td>
 <td>Sumica</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>ET-CY3 filter set</td>
 <td>Chroma Technology</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Power 1401 A/D converter</td>
 <td>Cambridge Electronic Design </td>
 <td>sampling was done at 3 kHz</td>
 </tr>
 <tr>
 <td>Spike II 
	(version 7.07)</td>
 <td>Cambridge Electronic Design</td>
 <td>software used to acquire electrophysiology data</td>
 </tr>
 <tr>
 <td>SEC-10 LX amplifier</td>
 <td>NPI electronics</td>
 <td>used with a 10X headstage</td>
 </tr>
 <tr>
 <td>Model 410 amplifier</td>
 <td>Brownlee precision</td>
 <td>used to amplify and filter the signal</td>
 </tr>
 <tr>
 <td>WS-4</td>
 <td>minus k Technology</td>
 <td>vibration isolation for imaging</td>
 </tr>
 <tr>
 <td>cooling platform</td>
 <td>custom made</td>
 <td>brass plate through which ice water is pumped at a variable 			rate</td>
 </tr>
 </tbody>
</table>
Table 2. Equipment used.

monitoring changes in the intracellular calcium concentration and synaptic efficacy in the mollusc aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Watch this Scientific Journal Video about Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia at JoVE.com

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

monitoring-changes-intracellular-calcium-concentration-synaptic

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10.9K Views.  Mt. Sinai School of Medicine. We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.