Name: electrophysiological method for whole-cell voltage clamp recordings from drosophila photoreceptors
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Description: Watch this Scientific Journal Video about Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors at JoVE.com

The experimental part of this research was supported by grants from the US-Israel Bi National Science Foundation (to B.M. and I.L.), the Israel Science Foundation (ISF), the Deutsch-Israelische Projektkooperation (DIP) (to B.M.), and the Biotechnology and Biological Sciences Research Council (BBSRC Grant numbers: BB/M007006/1 and BB/D007585/1) to R.C.H.

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H., Niemeyer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+protein+encoded+by+the+InaD+gene+regulates+recovery+of+visual+transduction+in+Drosophila.">A novel protein encoded by the InaD gene regulates recovery of visual transduction in Drosophila.</a> Neuron. 14 (1), 201-210 (1995).</li><li>Tsunoda, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multivalent+PDZ-domain+protein+assembles+signalling+complexes+in+a+G-protein-coupled+cascade.">A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.</a> Nature. 388 (6639), 243-249 (1997).</li><li>Tsacopoulos, M., Veuthey, A. L., Saravelos, S. G., Perrottet, P., Tsoupras, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cells+transform+glucose+to+alanine,+which+fuels+the+neurons+in+the+honeybee+retina.">Glial cells transform glucose to alanine, which fuels the neurons in the honeybee retina.</a> J. Neurosci. 14 (3 Pt 1), 1339-1351 (1994).</li><li>Hardie, R. C., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+activation+of+light-sensitive+channels+in+Drosophila+photoreceptors.">Spontaneous activation of light-sensitive channels in Drosophila photoreceptors.</a> J. Gen. Physiol. 103, 389-407 (1994).</li><li>Agam, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+stress+reversibly+activates+the+Drosophila+light-sensitive+channels+TRP+and+TRPL+in+vivo.">Metabolic stress reversibly activates the Drosophila light-sensitive channels TRP and TRPL in vivo.</a> J Neurosci. 20 (15), 5748-5755 (2000).</li><li>Agam, K., Frechter, S., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+Drosophila+TRP+and+TRPL+channels+requires+both+Ca2++and+protein+dephosphorylation.">Activation of the Drosophila TRP and TRPL channels requires both Ca2+ and protein dephosphorylation.</a> Cell Calcium. 35 (2), 87-105 (2004).</li><li>Delgado, R., Mu&#241;oz, Y., Pe&#241;a-Cort&#233;s, H., Giavalisco, P., Bacigalupo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diacylglycerol+activates+the+light-dependent+channel+TRP+in+the+photosensitive+microvilli+of+Drosophila+melanogaster+photoreceptors.">Diacylglycerol activates the light-dependent channel TRP in the photosensitive microvilli of Drosophila melanogaster photoreceptors.</a> J Neurosci. 34 (19), 6679-6686 (2014).</li><li>Parnas, M., Katz, B., Minke, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+channel+block+by+Ca2++underlies+the+voltage+dependence+of+Drosophila+TRPL+channel.">Open channel block by Ca2+ underlies the voltage dependence of Drosophila TRPL channel.</a> J. 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The authors have nothing to disclose.

The application of whole-cell recordings to D. melanogaster photoreceptors allowed for the discovery and the functional elucidation of novel signaling proteins, such as TRP channels27,28,29 and INAD30,31,32 scaffold protein. Ever since the initial introduction of this technique, it enabled the resolution of long-term basic questions regarding the ionic mechanism and voltage dependence of the light response. This occurred because of the conferred ability to accurately control the membrane voltage and extracellular and intracellular ionic composition19,28.
A major obstacle of the patch clamping technique in D. melanogaster has been the fragility of the isolated ommatidia preparation. Detailed studies have revealed that the integrity of the phototransduction machinery critically depends on the continuous supply of ATP, especially during light exposure, which leads to a large consumption of ATP. Unfortunately, the mechanical striping of the pigment (i.e.&#160;glia) cells, which is required to reach the photoreceptor membrane with the patch pipette, eliminates the main source of metabolites necessary for ATP production33. Application of exogenous ATP into the recording pipette only partially fulfills the requirement for large quantities of ATP. A short supply of ATP leads to spontaneous activation of the TRP channels and to the dissociation of the phototransduction machinery from the light-activated channels, causing a large increase in cellular Ca2+ and the abolishment of the normal response to light34,35. This sequence of events is not due to damage of the photoreceptors by the dissection procedure, but rather to the cellular depletion of ATP. To prevent this sequence of events from occurring and to maintain normal light responses, the photoreceptors should not be exposed to intense lights, which consume large quantities of ATP. Also, NAD must be included in the recording pipette, presumably to facilitate ATP production in the mitochondria18,36. For measurements of spontaneous and quantum bumps, the above difficulty is minimal because only dim lights are used. In practice, a stable whole-cell recording can be maintained for ~20-25 min, although there is a tendency for response kinetics to slow down over this period. A single preparation of dissociated ommatidia may remain viable for up to 2 h.
An additional shortcoming of the isolated ommatidia preparation is the inaccessibility of the microvilli, which translates to the inaccessibility of the TRP and TRPL channels to the recording pipette, preventing single-channel recordings. Using a method they developed, Bacigalupo and colleagues succeeded at directly recording single-channel activity from the rhabdomere37. However, this channel activity differs from that of TRPL channels heterologously expressed in tissue culture cells38 and from TRP channel activity derived from shot noise analysis obtained from isolated ommatidia34. Presumably, the dissection procedure greatly damaged the photoreceptor cells when using this method.

Extensive genetic studies of the fruit fly, Drosophila melanogaster (D. melanogaster), initiated more than 100 years ago, have established the D. melanogaster fly as an extremely useful experimental model for the genetic dissection of complex biological processes. The methodology described below combines the accumulated power of D. melanogaster molecular genetics with the high signal-to-noise ratio of whole-cell patch clamp recordings. This combination allows for the study of D. melanogaster phototransduction as a model of inositol-lipid signaling and TRP channel regulation and activation, both in the native environment and at the highest resolution of single molecules.
Application of the whole-cell recording method to D. melanogaster photoreceptors has revolutionized the study of invertebrate phototransduction. This method was developed by Hardie1 and independently by Ranganathan and colleagues2 ~26 years ago and was designed to exploit the extensive genetic manipulation tools of D. melanogaster and use them to uncover mechanisms of phototransduction and inositol-lipid signaling. At first, this technique suffered from a rapid reduction in light sensitivity and a low yield of ommatidia during the dissection process, which prevented detailed quantitative studies. Later, the addition of ATP and NAD to the patch pipette dramatically increased the suitability of the preparation for prolonged quantitative recordings. Thereafter, extensive characterization of the signal-transduction mechanism at the molecular level was realized.
Currently, D. melanogaster phototransduction is one of the few systems in which phosphoinositide signaling and TRP channels can be studied ex vivo at single-molecule resolution. This makes D. melanogaster phototransduction and the methodology developed to study this mechanism a highly sensitive model system. This protocol describes how to dissect the D. melanogaster retina and mechanically strip the isolated ommatidia from the surrounding pigment (glia) cells. This enables the formation of a giga-seal and a whole-cell patch clamp on the photoreceptor cell bodies. Fortunately, most signaling proteins are confined to the rhabdomere and do not diffuse. In addition, there is an immobile Ca2+ buffer called calphotin, located between the signaling compartment and the cell body3,4, and a high expression level of the Na+/Ca2+ exchanger (CalX) in the microvilli5. Together, the protein confinement to the rhabdomere, the calphotin buffer, and the high expression of the CalX allow for relatively prolonged (i.e.&#160;up to ~20 min) whole-cell recordings, without the loss of essential components of the phototransduction process and while maintaining high sensitivity to light. The following protocol describes how to obtain isolated ommatidia and perform whole-cell recordings that appear to preserve the native properties of the phototransduction cascade. Whole-cell patch clamp experiments on dissociated cockroach (Periplaneta americana)6 and cricket (Gryllus bimaculatus)7 ommatidia were performed similarly to that described for D. melanogaster. In addition, patch clamp experiments on dissociated photoreceptors of the file clam, (Lima scabra) and scallop (Pecten irradians) were performed in a slightly different manner from that conducted on D. melanogaster, allowing both whole-cell8 and single-channel measurements9. Here, the major achievements obtained in D. melanogaster using this technique are described. The Discussion includes the description of some pitfalls and limitations of this technique.

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			<td align="left" height="21" style="height:21px;width:292px;">10 ml syringe</td>
			<td style="width:255px;"></td>
			<td style="width:309px;"></td>
			<td style="width:508px;"></td>
		</tr>
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			<td align="left" height="21" style="height:21px;">5 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">1 ml syringe with elongated tip</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td align="left" height="21" style="height:21px;">Petri dish</td>
			<td></td>
			<td></td>
			<td align="left">60 mm</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;">Silicone dissection dish/block</td>
			<td align="left">Dow Corning</td>
			<td align="left">Sylguard 184 Silicone Elastomer Kit</td>
			<td align="left">Throughly mix both components, pour into mould and cure for 2 hours at 60&#7506;C</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Syringe filters</td>
			<td align="left">Millex</td>
			<td></td>
			<td align="left">22 &#181;m PVDF filter&#160;</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;">Capillaries (for omatidia separation)</td>
			<td></td>
			<td></td>
			<td align="left">Glass, 1.2 x 0.68 mm (~7.5 cm each)</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;">Polyethylene Tubing</td>
			<td align="left">Becton Dickinson</td>
			<td></td>
			<td align="left">1.57 x 1.14 mm (35 cm)</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;">&#160;2 small beakers</td>
			<td></td>
			<td></td>
			<td align="left">50 ml or less</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">2 paraffin film sheets</td>
			<td align="left">Parafilm M</td>
			<td></td>
			<td align="left">~5 x 5 cm</td>
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		<tr height="21">
			<td align="left" height="21" style="height:21px;">Bath chamber</td>
			<td align="left">home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Cover slips</td>
			<td></td>
			<td></td>
			<td align="left">22 x 22 mm No. 0</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Paraplast Plus</td>
			<td align="left">Sigma</td>
			<td align="left">Paraffin &#8211; polyisobutylene mixture</td>
			<td align="left">To glue the coverslip onto the bottom of the bath chamber</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Ground</td>
			<td align="left">Warner Instruments&#160;</td>
			<td align="left">64-1288</td>
			<td align="left">Hybrid Assembly Ag-AgCl Wire Assembly</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Headless micro dissection needle</td>
			<td></td>
			<td></td>
			<td align="left">Entomology, 12 mm</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Micro dissecting needle holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Vise</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">2 fine tweezers + 1 rough tweezers</td>
			<td></td>
			<td align="left">Dumont #5, Biology&#160;</td>
			<td align="left">0.05 x 0.02 mm, length 110 mm, Inox</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Stereoscopic zoom Microscope&#160;</td>
			<td align="left">Nikon</td>
			<td align="left">SMZ-2B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Cold light source</td>
			<td align="left">Schott</td>
			<td align="left">KL1500 LCD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Filter (Color) for cold light source</td>
			<td align="left">Schott</td>
			<td align="left">RG620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Delicate wipers</td>
			<td align="left">Kimtech</td>
			<td></td>
			<td align="left">Kimwipes</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Electrode holder</td>
			<td align="left">Warner Instruments</td>
			<td align="left">QSW-T10P</td>
			<td align="left">Q series Holders compatible with Axon amplifiers, straight body style</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Silver Wire</td>
			<td align="left">Warner Instruments</td>
			<td></td>
			<td align="left">0.25 mm diameter, needs to be chloridized</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Micromanipulator</td>
			<td align="left">Sutter Instruments</td>
			<td align="left">MP 85</td>
			<td align="left">Huxley-Wall Style Micromanipulator</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Faraday cage</td>
			<td align="left">home made</td>
			<td></td>
			<td align="left">Electromagnetic noise shielding and black front curtain</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Anti-vibration Table</td>
			<td align="left">Newport</td>
			<td align="left">VW-3036-OPT-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Osilloscope</td>
			<td align="left">GW</td>
			<td align="left">GOS-622G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Perfusion system</td>
			<td align="left">Warner Instruments</td>
			<td align="left">VC-8P</td>
			<td align="left">Pinch valve control system</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Perfusion valve controller</td>
			<td align="left">Scientific instruments</td>
			<td align="left">BPS-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td align="left" height="20" style="height:20px;">Suction system</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Amplifier</td>
			<td align="left">Molecular Device</td>
			<td align="left">Axopatch-1D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Head-stage</td>
			<td align="left">Molecular Device</td>
			<td align="left">CV - 4</td>
			<td align="left">Gain: x 1/100</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">A/D converter</td>
			<td align="left">Molecular Device</td>
			<td align="left">Digidata 1440A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Clampex</td>
			<td align="left">Molecular Device</td>
			<td dir="LTR">10</td>
			<td align="left">software</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">pCLAMP</td>
			<td align="left">Molecular Device</td>
			<td>10</td>
			<td align="left">software</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Light source (Xenon Arc lamp)</td>
			<td align="left">Sutter Instruments</td>
			<td align="left">Lambda LS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Light detector</td>
			<td align="left">home made</td>
			<td></td>
			<td align="left">phototransistor</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Filter wheel and shutter controller</td>
			<td align="left">Sutter Instruments</td>
			<td align="left">Lambda 10-2 with a Uniblitz shutter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Filters (Natural density filter)</td>
			<td align="left">Chroma</td>
			<td align="left">6,5,4,3,2,1,0.5,0.3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Filter (Color)</td>
			<td align="left">Schott</td>
			<td align="left">OG590, Edge filter</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Xenon Flash Lamp system</td>
			<td align="left">Dr. Rapp OptoElecftronic</td>
			<td align="left">JML-C2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Light guide</td>
			<td></td>
			<td></td>
			<td align="left">Quartz&#160;</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Pulse generator</td>
			<td align="left">AMPI</td>
			<td align="left">Master 8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Microscope</td>
			<td align="left">Olympus</td>
			<td align="left">IX71, Inverted</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Red illumination filter (Microscope)</td>
			<td></td>
			<td></td>
			<td align="left">RG630 / RG645 &#248;45mm</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Microscope objective</td>
			<td align="left">Olympus</td>
			<td align="left">X60/0.9 UplanFL N air or X60/1.25 UplanFI oil</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">CCD Camera</td>
			<td align="left">Andor</td>
			<td align="left">iXon DU885K</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">NIS Element&#160;</td>
			<td align="left">Nikon</td>
			<td align="left">AR</td>
			<td align="left">software</td>
		</tr>
		<tr height="24">
			<td align="left" height="24" style="height:24px;">Ca+2 indicator</td>
			<td align="left">Invitrogen</td>
			<td align="left">Calcium green 5N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Excitation &#38; emission filters and dichroic mirror</td>
			<td align="left">Chroma</td>
			<td align="left">19002 - AT - GFP/FITC Longpass set</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Vertical pipette puller</td>
			<td align="left">Narishige&#160;</td>
			<td align="left">Model PP83</td>
			<td align="left">Use either vertical or horizontal puller, as preferred.</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Horizontal pipette puller</td>
			<td align="left">Sutter Instrument&#160;</td>
			<td align="left">Model P-1000 Flaming/Brown Micropipette Puller</td>
			<td></td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Filament</td>
			<td align="left">Sutter Instrument</td>
			<td></td>
			<td align="left">3 mm trough or square box</td>
		</tr>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Capillaries</td>
			<td align="left">Harvard Apparatus</td>
			<td align="left">borosilicate glass capillaries</td>
			<td align="left">1 x 0.58 mm</td>
		</tr>
	</tbody>
</table>

electrophysiological method for whole-cell voltage clamp recordings from drosophila photoreceptors

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions. This technique allows control over the intracellular and extracellular media; the membrane voltage; and the fast application of pharmacological compounds, such as a variety of ionic or pH indicators, to the intra- and extracellular media. With an exceptionally high signal-to-noise ratio, this method enables the measurement of dark spontaneous and light-induced unitary currents (i.e.&#160;spontaneous and quantum bumps) and macroscopic Light-induced Currents (LIC) from single D. melanogaster photoreceptors. This protocol outlines, in great detail, all the key steps necessary to perform this technique, which includes both electrophysiological and optical recordings. The fly retina dissection procedure for the attainment of intact and viable ex vivo isolated ommatidia in the bath chamber is described. The equipment needed to perform whole-cell and fluorescence imaging measurements are also detailed. Finally, the pitfalls in using this delicate preparation during extended experiments are explained.

The described method has enabled the accurate recording of the fundamental unitary currents that generate spontaneous and light-evoked quantum bumps, which sum to produce the macroscopic response to light, under defined conditions. It also allowed the comparison between wildtype and mutant flies that have defects in critical signaling molecules (Figures 3 and 5)14,15,16,17,18. In addition, the ability to measure reversal potential under bi-ionic conditions revealed fundamental biophysical properties of the TRP and TRP-like (TRPL) channels18,19. It also enabled the measurement of the effects of amino acid substitutions in the pore region of TRP that modified its Ca2+ permeability20.
The light response obtained by patch clamp whole-cell recordings depends linearly on light intensity for at least 4 orders of magnitude. This could not be resolved by using ERG and intracellular recording methods. Accordingly, a series of responses to brief flashes of increasing intensity and a plot of the intensity response function revealed a strict linearity of the flash response with increasing light intensity. The strict linearity holds up to at least several hundred pA, but it is debatable whether thereafter it is linearity or clamp control that breaks down (Figure 6). These results suggest that the macroscopic responses to light are a linear summation of the unitary responses to light (i.e.&#160;quantum bumps).
It has been well established using voltage recordings that dim light stimulation induces discrete voltage fluctuations (i.e.&#160;quantum bumps) in most invertebrate species. The D. melanogaster quantum bumps result from the concerted opening of ~15 TRP channels and ~2 TRPL channels at the peak of the bump18. Each bump is generated by the absorption of a single photon, while the macroscopic response to more intense lights is the summation of these elementary responses14,21. The bumps vary significantly in latency, time course, and amplitude, even when the stimulus conditions are identical. Bump generation is a stochastic process described by Poisson statistics, whereby each effectively absorbed photon elicits only one bump. The single-photon-single-bump relationship requires that each step in the cascade includes not only an efficient &#34;turn-on&#34; mechanism, but also an equally effective &#34;turn-off&#34; mechanism. The functional advantage is the production of a very sensitive photon counter with a fast transient response very well suited to both the sensitivity and the temporal resolution required by the visual system. The requirement for an efficient turn-off mechanism is revealed when either the active photopigment (i.e.&#160;metarhodopsin, M) or its target, the Gq&#945;, fails to inactivate and leads to the continuous production of bumps long after light is turned off (Figure 3)15,22,23,24 .
The bump represents the cooperative activity of the TRP/TRPL channels in a microvillus. As such, any hypothesis of channel activation should also explain cooperative channel activation. Recently, Hardie and colleagues have demonstrated that light evokes rapid contractions of the photoreceptors, suggesting that the light-sensitive channels (TRP/TRPL) may be mechanically gated25. This mechanical activation, together with the observed protons released by PLC-mediated PIP2 hydrolysis, promote the opening of the TRP/TRPL channels and explain the cooperative nature of bump production26. Currently, D. melanogaster photoreceptors are one of the few systems in which phosphoinositide signaling and TRP channels can be studied in vivo, thus making D. melanogaster phototransduction and the methodology developed to study this mechanism a highly valuable model system.
<img alt="Figure 3" src="/files/ftp_upload/55627/55627fig3.jpg" /> 
Figure 3: The inaCP209 and inaDP215 Mutants Reveal Slow Response Termination of the Macroscopic Response to Light and of the Single Quantum Bumps. (A)The isolated ommatidium preparation with a patch pipette filled with fluorescent Lucifer Yellow CH dye (excitation: 430 nm; emission: 540 nm) is presented during a whole-cell recording. Note that the fluorescent dye diffused and labeled a single photoreceptor cell body and that the photoreceptor cell bodies are detached from their elongated axons but still maintain viability. This preparation is suitable for simultaneous whole-cell recordings and imaging experiments. (B-D) Upper panels: Whole-cell voltage clamped quantum bump responses to continuous dim light (open bar) in WT, inaCP209, and inaDP215 mutant flies. A slow termination of the bumps is observed in inaCP209 and inaDP215 mutants relative to WT flies. The inset below displays the magnified shape of single bumps. Bottom panels: Normalized whole-cell recorded macroscopic responses to a 500-ms light pulse (1.5 x 105 photons per s) of the above wildtype and mutant flies. (E-G) Upper panels: Whole-cell voltage clamped quantum bump responses to a brief (1 ms), dim light eliciting single-photon responses in wildtype, arr23, and ninaCP235 mutant flies. Note the train of bumps observed in arr23 and ninaCP235 mutant flies in response to a single photon absorption. Bottom panels: Whole-cell voltage clamped normalized responses to a 500&#160;ms light pulse (1.5 x&#160;104 photons/s) in the corresponding mutants. Note the slow termination of the macroscopic responses observed in arr23 and ninaCP235 mutant flies relative to WT. <a href="http://cloudflare.jove.com/files/ftp_upload/55627/55627fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55627/55627fig4.jpg" /> 
Figure 4: Cellular Ca2+&#160;Dynamics Following Signal-induced Ca2+&#160;Influx is Affected by Calphotin.&#160; A time series of photoreceptor images of wildtype and Cpn1% flies showing the fluorescence of the Ca2+ indicator during light stimulation. Raw intensity images are plotted using false-color coding (bar = 10 &#181;m; arrowheads indicate the pipette). Figure reprinted with permission from Weiss et al.4. <a href="http://cloudflare.jove.com/files/ftp_upload/55627/55627fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55627/55627fig5.jpg" /> 
Figure 5: The Electrophysiological Properties of WT, trp, and trpl Mutants. (A) Whole-cell voltage clamp recordings of quantum bumps in response to continuous dim light (open bar) in WT, trpl302, and trpP343 null mutant flies. Highly reduced amplitudes of trpP343 bumps are observed. Inset: Magnified single quantum bumps of wildtype and trpP343 null mutant flies are shown. (B) Whole-cell voltage clamp recordings in response to a 3&#160;s light pulse of wildtype and the corresponding mutants. The transient steady-state response of the trpP343 mutant is observed. Inset: Magnified light responses of WT and trpP343mutant are shown. (C) A family of superimposed light-induced currents of the above fly strains, elicited in response to a 20&#160;ms light pulse, at voltage steps of 3 mV, measured around the reversal potential (Erev). (D) A histogram plotting the mean Erev of wildtype and the various mutants. The error bars are the S.E.M. The reversal potential (Erev) of WT is between the positive Erev of trpl302, which expresses only TRP, and the Erev of the trpP343 null mutant, which expresses only TRPL. <a href="http://cloudflare.jove.com/files/ftp_upload/55627/55627fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55627/55627fig6.jpg" /> 
Figure 6: The Flash Response is Strictly Linear with Increasing Light Intensity. 
A series of current responses to brief flashes of increasing light intensity and a plot of the dependence of the peak amplitude of the light response on the increasing intensity of brief light flashes. This relationship reveals a strict linearity between the flash response and increasing light intensity. This strict linearity holds up to at least several hundred pA, with light intensity spanning over 4 orders of magnitude, while it is debatable whether it is linearity or the clamp control that breaks down thereafter. <a href="http://cloudflare.jove.com/files/ftp_upload/55627/55627fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pH</td>
			<td>7.15 (adjust with NaOH)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>120</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>5</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>4</td>
		</tr>
		<tr>
			<td>TES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Proline</td>
			<td>25</td>
		</tr>
		<tr>
			<td>Alanine</td>
			<td>5</td>
		</tr>
		<tr>
			<td></td>
			<td>Store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td colspan="2">Note: This solution is nominally Ca2+ free but has no Ca2+ buffers added and hence will have approximately 5 - 10 &#181;M trace Ca2+. Extracellular solution (ES) = ES-0Ca2+ with 1.5 mM CaCl2, made by adding CaCl2 from a 0.5 or 1 M stock solution to ES-0Ca2+.</td>
		</tr>
	</tbody>
</table>
Table 1: Ca+2-free Extracellular Solution (ES). Chemical description and the specific quantities required to produce Ca+2-free ES.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>1.5 g</td>
		</tr>
		<tr>
			<td colspan="2">Divide into 150 &#181;L aliquots in 1.5 mL vials and store at -20 &#176;C.</td>
		</tr>
		<tr>
			<td>Trituration solution (TS)</td>
			<td>Fill 1 vial of 150 mL of the stock solution with 1,350 mL ES or ES-0Ca2+, to match the solution used during the dissection.</td>
		</tr>
	</tbody>
</table>
Table 2: Fetal Bovine Serum (FBS) + Sucrose - Stock Solution. Chemical description and the specific quantities required to producing fetal bovine serum (FBS) + sucrose - stock solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pH</td>
			<td>7.15 (adjust with KOH)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td>Potassium gluconate (Kglu)</td>
			<td>140</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>TES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>ATP magnesium salt (MgATP)</td>
			<td>4</td>
		</tr>
		<tr>
			<td>GTP sodium salt (Na2GTP)</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide hydrate (NAD)</td>
			<td>1</td>
		</tr>
		<tr>
			<td></td>
			<td>Store at -20 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 3: Intracellular Solution (IS1). Chemical description and the specific quantities required to produce&#160;IS1, which is mostly used for intensity response and quantum bump measurements.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>pH</td>
			<td>7.15 (adjust with CsOH)</td>
		</tr>
		<tr>
			<td>Reagent</td>
			<td>Concentration (mM)</td>
		</tr>
		<tr>
			<td>CsCl</td>
			<td>120</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>2</td>
		</tr>
		<tr>
			<td>TES</td>
			<td>10</td>
		</tr>
		<tr>
			<td>ATP magnesium salt (MgATP)</td>
			<td>4</td>
		</tr>
		<tr>
			<td>GTP sodium salt&#160; (Na2GTP)</td>
			<td>0.4</td>
		</tr>
		<tr>
			<td>&#946;-Nicotinamide adenine dinucleotide hydrate (NAD)</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Tetra-ethyl-ammonium chloride (TAE)</td>
			<td>15</td>
		</tr>
		<tr>
			<td></td>
			<td>Store at -20 &#176;C.</td>
		</tr>
	</tbody>
</table>
Table 4: Intracellular Solution (IS2). Chemical description and the specific quantities required to produce Intracellular Solution&#160;IS2, which is mostly used for reversal potential measurements of the light-induced current.

Watch this Scientific Journal Video about Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors at JoVE.com

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

Whole-cell recordings from Drosophila melanogaster photoreceptors enable the measurement of spontaneous dark bumps, quantum bumps, macroscopic responses to light, and current-voltage relationships under various conditions. In combination with D. melanogaster genetic manipulation tools, this method enables the study of the ubiquitous inositol-lipid signaling pathway and its target, the TRP channel.

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Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors

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Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

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