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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C., Franze, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photomechanical+responses+in+Drosophila+photoreceptors.">Photomechanical responses in Drosophila photoreceptors.</a> Science. 338 (6104), 260-263 (2012).</li><li>Huang, J., 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=Activation+of+TRP+channels+by+protons+and+phosphoinositide+depletion+in+Drosophila+photoreceptors.">Activation of TRP channels by protons and phosphoinositide depletion in Drosophila photoreceptors.</a> Curr. Biol. 20 (3), 189-197 (2010).</li><li>Minke, B., Wu, C. F., Pak, W. 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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+light-activated+conductance+is+composed+of+the+two+channels+TRP+and+TRPL.">The Drosophila light-activated conductance is composed of the two channels TRP and TRPL.</a> Cell. 85 (5), 651-659 (1996).</li><li>Huber, A., 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=The+transient+receptor+potential+protein+(Trp),+a+putative+store-+operated+Ca+2++channel+essential+for+phosphoinositide-mediated+photoreception,+forms+a+signaling+complex+with+NorpA,+InaC+and+InaD.">The transient receptor potential protein (Trp), a putative store- operated Ca 2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.</a> EMBO J. 15 (24), 7036-7045 (1996).</li><li>Shieh, B. 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. 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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 border="0" cellpadding="0" cellspacing="0" width="1363">
	<tbody>
		<tr height="21">
			<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>
		<tr height="21">
			<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>
		<tr height="21">
			<td align="left" height="21" style="height:21px;">Petri dish</td>
			<td></td>
			<td></td>
			<td align="left">60 mm</td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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.

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, ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

14.9K Views.  Hebrew University. 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.

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

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" ...

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  ...

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

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

Whole Cell Patch Clamp for Investigating the Mechanisms of Infrared Neural Stimulation

It has been demonstrated in recent years that pulsed, infrared laser light can be used to elicit electrical responses in neural tissue, independent of any further modification of the target tissue. Infrared neural stimulation has been reported in a variety of peripheral and sensory neural tissue in vivo, with particular interest shown in stimulation of neurons in the auditory nerve. However, while INS has been shown to work in these settings, the mechanism (or mechanisms) by which infrared light causes neural excitation is currently not well understood. The protocol presented here describes a whole cell patch clamp method designed to facilitate the investigation of infrared neural stimulation in cultured primary auditory neurons. By thoroughly characterizing the response of these cells to infrared laser illumination in vitro under controlled conditions, it may be possible to gain an improved understanding of the fundamental physical and biochemical processes underlying infrared neural stimulation.

Infrared nerve stimulation has been proposed as an alternative to electrical stimulation in a range of nerve types, including those associated with the auditory system. This protocol describes a patch clamp method for studying the mechanism of infrared nerve stimulation in a culture of primary auditory neurons.

It has been  demonstrated in recent years that pulsed, infrared laser light can be used to  elicit electrical responses in neural tissue, independent of ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the researcher to measure gustatory responses directly and quantitatively, reflecting the sensory input that the insect nervous system receives from taste stimuli in its environment. This protocol outlines all key steps in performing this technique. The critical steps in assembling an electrophysiology rig, such as selection of necessary equipment and a suitable environment for recording, are delineated. We also describe how to prepare for recording by making appropriate reference and recording electrodes, and tastant solutions. We describe in detail the method used for preparing the insect by insertion of a glass reference electrode into the fly in order to immobilize the proboscis. We show traces of the electrical impulses fired by taste neurons in response to a sugar and a bitter compound. Aspects of the protocol are technically challenging and we include an extensive description of some common technical challenges that may be encountered, such as lack of signal or excessive noise in the system, and potential solutions. The technique has limitations, such as the inability to deliver temporally complex stimuli, observe background firing immediately prior to stimulus delivery, or use water-insoluble taste compounds conveniently. Despite these limitations, this technique (including minor variations referenced in the protocol) is a standard, broadly accepted procedure for recording Drosophila neuronal responses to taste compounds.

Electrophysiological Recording From Drosophila Labellar Taste Sensilla

This protocol describes extracellular recording of the action potential responses fired by labellar taste neurons in Drosophila.

The peripheral taste response of insects can be powerfully investigated with electrophysiological techniques. The method described here allows the ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

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.