Name: patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices
Uploaded: 2012-01-19T13:00:00
Description: Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

We thank Dr. Fred Rieke for his kind explanation of the agar-embedded retinal slice preparation when we started using the protocol in our lab. We also thank Lori Vaskalis for the illustration of schematic overview and Drs. Veeramuthu Balakrishnan and Soyoun Cho for helpful comments on the text and video. This work was supported by an NEI-NIH RO1 grant, and was also partially supported by a Korea Research Foundation Grant funded by the Korean Government [KRF-2008-357-E00032].

1. External and internal Solutions
<ol>
 <li>Prepare slicing solution (low calcium) from 10x stock solution and add MgCl2, CaCl2, and D-glucose daily. The final 1x solution consists of (in mM): 119 NaCl, 2.5 KCl, 3.2 MgCl2, 0.25 CaCl2, 12 D-glucose, 0.2 L-ascorbic acid, 12 HEPES. Set the pH to 7.4 (with NaOH), and adjust osmolarity to 260 mOsm (using H2O and 10x stock solution).</li>
 <li>Weigh out 3% low gelling-temperature agar (Agarose type VII-A, A0701, Sigma; ...

<ol>
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A critical and difficult step in our protocol is the transfer of the piece of retina into the agar solution (protocol 3.4). It is necessary to carefully remove the vitreous humor and residual slice solution from the retinal piece and transfer it without distortion or bending. In order to accomplish this, we use a small spatula (21-401-25B, Fisherbrand) with a tip bent to form a 90&deg; angle, along with angled tip forceps (11251-35, Fine Science Tools), to position the retinal piece throughout the transf...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Low gelling-temperature agar</td>
 <td>Sigma</td>
 <td>A0701</td>
 <td>Agarose type VII-A</td>
 </tr>
 <tr>
 <td>Patch pipette</td>
 <td>World Precision Instruments</td>
 <td>1B150F-4</td>
 <td>Thick-walled (1.5 mm outer diameter) borosilicate glass</td>
 </tr>
 <tr>
 <td>Vertical puller</td>
 <td>Narishige</td>
 <td>PP830</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dental wax</td>
 <td>Cavex</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Spring scissors</td>
 <td>Fine Science Tools</td>
 <td>15003-08</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>45&deg; angled fine tip forceps</td>
 <td>Fine Science Tools</td>
 <td>11251-35</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Razor blade</td>
 <td>Personna</td>
 <td>&nbsp;</td>
 <td>Double-edged, cleaned with 70% ethanol and H2O</td>
 </tr>
 <tr>
 <td>Cylindrical tube</td>
 <td>Fisherbrand</td>
 <td>03-338-1B</td>
 <td>Polyethylene sample vials 2.5 ml</td>
 </tr>
 <tr>
 <td>Hyaluronidase</td>
 <td>Sigma</td>
 <td>H6254</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vibratome slicer</td>
 <td>Leica</td>
 <td>VT1000S or VT1200S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Upright microscope</td>
 <td>Olympus</td>
 <td>BX51WI</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>60x water-immersion objective</td>
 <td>Olympus</td>
 <td>LUMPlanFl</td>
 <td>NA 0.90</td>
 </tr>
 <tr>
 <td>CCD camera</td>
 <td>Sony</td>
 <td>XC-75</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Camera controller</td>
 <td>Hamamatsu</td>
 <td>C2400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Monitor</td>
 <td>Sony</td>
 <td>&nbsp;</td>
 <td>13&#x201D; black and white monitor</td>
 </tr>
 <tr>
 <td>Syringe filter</td>
 <td>Nalgene</td>
 <td>&nbsp;</td>
 <td>0.2 &mu;m</td>
 </tr>
 <tr>
 <td>Micromanipulator</td>
 <td>Sutter Instrument</td>
 <td>MPC-200</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Lock-in amplifier</td>
 <td>HEKA</td>
 <td>&nbsp;</td>
 <td>EPC-9/10 amplifiers have software emulation</td>
 </tr>
 <tr>
 <td>Spinning disk laser confocal microscope</td>
 <td>Yokogawa</td>
 <td>CSU-X1</td>
 <td>Live cell imaging after patch clamp whole cell recording</td>
 </tr>
 <tr>
 <td>Slidebook software</td>
 <td>Intelligent Imaging Instruments (3i)</td>
 <td>&nbsp;</td>
 <td>Imaging data acquisition and analysis</td>
 </tr>
 <tr>
 <td>Paraformaldehyde</td>
 <td>Sigma</td>
 <td>P6148</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Phosphate buffer solution</td>
 <td>GIBCO</td>
 <td>70013</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Superfrost slide</td>
 <td>Fisher Scientific</td>
 <td>&nbsp;</td>
 <td>Slide glass</td>
 </tr>
 <tr>
 <td>Anti-fading agents</td>
 <td>Biomeda corp.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Confocal laser-scanning microscope</td>
 <td>Carl Zeiss</td>
 <td>LSM 710</td>
 <td>Imaging of fixed tissue</td>
 </tr>
 <tr>
 <td>Spatula</td>
 <td>Fisherbrand</td>
 <td>21-401-25B</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Manuel vertical slicer</td>
 <td>Narishige</td>
 <td>ST-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Oregon Green 488 BAPTA-1</td>
 <td>Invitrogen</td>
 <td>O-6806</td>
 <td>Ca2+ sensitive fluorescent dye</td>
 </tr>
 <tr>
 <td>Alexa Fluor 555 Hydrazide</td>
 <td>Invitrogen</td>
 <td>A-20501MP</td>
 <td>Fluorescent dye</td>
 </tr>
 </tbody>
</table>

patch-clamp capacitance measurements and ca2+ imaging at single nerve terminals in retinal slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Watch this Scientific Journal Video about Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices at JoVE.com

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

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