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.

<ol>
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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.

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

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, ...

electrophysiological-method-for-whole-cell-voltage-clamp-recordings

Research

JoVE Journal

Neuroscience

15.0K Views.  Hebrew University.  Whole-cell opnames van Drosophila melanogaster fotoreceptoren maken het mogelijk om spontaan donkere stoten, kwantumstoten, macroscopische reacties op licht en spanningsrelaties onder verschillende omstandigheden te meten. In combinatie met D. melanogaster genetische manipulatie instrumenten, deze methode maakt het mogelijk om de alomtegenwoordige inositol-lipide signalering weg en zijn doel, het TRP kanaal te onderzoeken. 

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

Whole Cell Recordings from Brain of Adult Drosophila

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by describing the dissecting solution and capture of the adult females used in our studies. The procedure for removing the whole brain intact, including both optic lobes, is illustrated. Dissection of the overlying trachea is also shown.  The isolated brain is not only small but needs special care in handling at this stage to prevent damage to the neurons, many of which are close to the outer surface of the tissue. We show how a special holder we developed is used to stabilize the brain in the recording chamber. A standard electrophysiology set up is used for recording from single neurons or pairs of neurons. A fluorescent image, viewed through the recording microscope, from a GAL4 line driving GFP expression (GH146) illustrates how projection neurons (PNs) are identified in the live brain. A high power Nomarski image shows a view of a single neuron that is being targeted for whole cell recording. When the brain is successfully removed without damage, the majority of the neurons are spontaneously active, firing action potentials and/or exhibiting spontaneous synaptic input. This in situ preparation, in which whole cell recording of identified neurons in the whole brain can be combined with genetic and pharmacological manipulations, is a useful model for exploring cellular physiology and plasticity in the adult CNS.

This video demonstrates the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons using standard whole cell technology. It includes images of GFP labeled cells and neurons viewed during recording.

In this video, we demonstrate the procedure for isolating whole brains from adult Drosophila in preparation for recording from single neurons. We begin by ...

Biology

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" (or "startle") response during the adult stage is mediated by the multi-component neuronal circuit called the Giant Fiber System (GFS). The comparative large size of the neurons, their distinctive morphology and simple connectivity make the GFS an attractive model system for studying neuronal circuitry. The GFS pathway is composed of two bilaterally symmetrical Giant Fiber (GF) interneurons whose axons descend from the brain along the midline into the thoracic ganglion via the cervical connective. In the mesothoracic neuromere (T2) of the ventral ganglia the GFs form electro-chemical synapses with 1) the large medial dendrite of the ipsilateral motorneuron (TTMn) which drives the tergotrochanteral muscle (TTM), the main extensor for the mesothoracic femur/leg, and 2) the contralateral peripherally synapsing interneuron (PSI) which in turn forms chemical (cholinergic) synapses with the motorneurons (DLMns) of the dorsal longitudinal muscles (DLMs), the wing depressors. The neuronal pathway(s) to the dorsovental muscles (DVMs), the wing elevators, has not yet been worked out (the DLMs and DVMs are known jointly as indirect flight muscles - they are not attached directly to the wings, but rather move the wings indirectly by distorting the nearby thoracic cuticle) (King and Wyman, 1980; Allen et al., 2006). The di-synaptic activation of the DLMs (via PSI) causes a small but important delay in the timing of the contraction of these muscles relative to the monosynaptic activation of TTM (~0.5 ms) allowing the TTMs to first extend the femur and propel the fly off the ground. The TTMs simultaneously stretch-activate the DLMs which in turn mutually stretch-activate the DVMs for the duration of the flight. The GF pathway can be activated either indirectly by applying a sensory (e.g."air-puff" or "lights-off") stimulus, or directly by a supra-threshold electrical stimulus to the brain (described here). In both cases, an action potential reaches the TTMs and DLMs solely via the GFs, PSIs, and TTM/DLM motoneurons, although the TTMns and DLMns do have other, as yet unidentified, sensory inputs. Measuring "latency response" (the time between the stimulation and muscle depolarization) and the "following to high frequency stimulation" (the number of successful responses to a certain number of high frequency stimuli) provides a way to reproducibly and quantitatively assess the functional status of the GFS components, including both central synapses (GF-TTMn, GF-PSI, PSI-DLMn) and the chemical (glutamatergic) neuromuscular junctions (TTMn-TTM and DLMn-DLM). It has been used to identify genes involved in central synapse formation and to assess CNS function.

Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster

The Giant Fiber System is a simple neuronal circuit of adult Drosophila melanogaster containing the largest neurons in the fly. We describe the protocol for monitoring synaptic transmission through this pathway by recording post synaptic potentials in dorsal longitudinal (DLM) and tergotrochanteral (TTM) muscles following direct stimulation of the Giant Fiber interneurons.

When startled adult D. melanogaster react by jumping into the air and flying away. In many invertebrate species, including D. melanogaster, the "escape" ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch clamp analyses of the voltage-gated channels in sensory hair cells isolated from a variety of species have been described previously1-4 but this video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...

Electrophysiological Recording of Voltage Responses of Drosophila Retinal Photoreceptors to Light Stimuli

Source: Juusola, M., et al. <a href="https://app.jove.com/v/54142">Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo.</a> J. Vis. Exp. (2016).
This video outlines the protocol for electrophysiological recording of photoreceptor activity in the eye of an immobilized Drosophila, enabling precise measurement of voltage changes in response to light stimuli.

1. Recording from R1-R6 Photoreceptors 

	Always be grounded when operating the microelectrode amplifier (for example by touching the metal surface of the ...

JoVE Encyclopedia of Experiments

Neurophysiology

Electrophysiology Techniques

Investigating Prolonged Depolarizing Afterpotential (PDA) in Drosophila Photoreceptors

Source: Gutorov, R., et al. <a href="https://app.jove.com/v/63514">Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.</a> J. Vis. Exp. (2022).
This video demonstrates an experimental protocol to investigate the prolonged depolarizing afterpotential (PDA) in white-eyed Drosophila. The fly&#39;s eye is exposed to intense blue light pulses to convert the photopigment rhodopsin to metarhodopsin, initiating a signaling cascade that opens positive ion channels and causes membrane depolarization. Because blue light prevents the reconversion of metarhodopsin to rhodopsin, the continuous influx of positive ions results in sustained depolarization, known as the prolonged depolarizing afterpotential (PDA).

1. Measuring the prolonged depolarizing afterpotential (PDA) using the electroretinogram

	Suitable rearing conditions for Drosophila melanogaster ...

Stimulation and Modulation Techniques

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Source:&#160; Katz, B., et al., <a href="https://app.jove.com/v/55627">Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.</a> J. Vis. Exp. (2017)
The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila&#39;s ommatidium by establishing a whole-cell configuration. It highlights the activation of transient receptor potential channels by light pulses, resulting in measurable quantum bumps.

NOTE: During all of the following steps, use only dim red light illumination and ensure that the ommatidia&#39;s exposure to light is minimal (i.e., work ...

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

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