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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 rapidly and turn off the dissecting light source and chamber red illumination used for viewing the ommatidia when performing tasks that do not demand viewing the ommatidium). In addition, perform all of the following steps according to standard electrophysiological protocol.
<ol>
	<li>Under an inverted microscope (40X objective), carefully inspect all of the ommatidia in the bath and choose a suitable ommatidium for the experiment.&#8203;
	<ol>
		<li>Ensure that the outer membrane of the ommatidium is smooth and intact, that the long axis is approximately at a right angle relative to the electrode approach direction (as seen in Figure 1A), and that the distal section of the ommatidium is not surrounded by any excess tissue. Place the chosen ommatidium at the optical axis of the objective lens (in the center of the field of vision) to ensure uniform illumination.</li>
	</ol>
	</li>
	<li>Fill a patch pipette with intracellular solution (IS1 or IS2). 
	NOTE: To measure the intensity response and bump analysis, use IS1. To measure the reversal potential of the light-sensitive channels, use IS2.</li>
	<li>Mount the patch pipette onto the electrode holder.</li>
	<li>Blow into the pipette by mouth through the tube connected to the electrode holder, causing it to fill with positive pressure. Close the tube valve to maintain the pressure.</li>
	<li>Using the micromanipulator, insert the electrode into the bath chamber.</li>
	<li>Guide the electrode close to the distal section of the ommatidium, such that there is no contact between the electrode and the ommatidium, until a small dimple (due to the positive pressure in the patch pipette) can be observed in the ommatidium.</li>
	<li>Open the recording software (see the Table of Materials). Open the &#34;membrane test module&#34; to apply continuous square voltage pulses of 2 mV at a rate of 100 Hz.</li>
	<li>Set the junction potential to &#34;zero,&#34; by adjusting the appropriate knob in the patch clamp amplifier to set the base of the square pulse to &#34;zero&#34; current 
	NOTE: The electrophysiological setup includes a head stage (i.e. first-stage amplification) connected to an amplifier (i.e. second-stage amplification). The amplified analog signal is converted to a digital signal using the A/D converter, which is controlled by software installed on a PC computer.</li>
	<li>Release the positive pressure in the pipette by opening the valve of the tube connected to the electrode holder. Gently create negative pressure in the pipette by sucking out of the tube, leading to the association of the pipette to the cell membrane. Close the tube valve to maintain the pressure.</li>
	<li>Ensure that the electrode resistance viewed on the computer screen is elevated to 100 - 150 M&#8486;. Release the negative pressure in the pipette by manually opening the valve of the tube connected to the electrode holder.</li>
	<li>Ensure that the electrode resistance is elevated to at least 1-2 G&#8486;. 
	NOTE: At this point, a seal has been formed between the electrode and the photoreceptor.</li>
	<li>Offset the capacitive currents of the pipette by adjusting the appropriate knob in the patch clamp amplifier.</li>
	<li>Create rapid, short, and forceful bouts of negative pressure in the electrode, sucking by mouth out of the tube connected to the electrode holder to &#34;break&#34; into the photoreceptor membrane and create a whole-cell configuration. Alternatively, use the &#34;Zap button&#34; to apply short, rectangular electrical pulses, starting with a duration of &#34;0.1 ms,&#34; or apply a combination of both methods. 
	NOTE: The generation of the whole-cell configuration is revealed by a sudden increase in pipette capacitance (typically ~60 pF for a wildtype R1-6 photoreceptor; a capacitance of only ~20 pF indicates a recording from an R7 photoreceptor; a capacitance above ~90 pF indicates a recording from two photoreceptors).</li>
	<li>Set the holding potential of the photoreceptor to the required voltage (usually -70 mV) manually using the appropriate knob in the patch clamp amplifier. 
	NOTE: This step can be performed after a seal has been obtained and before the whole-cell configuration has been achieved.</li>
	<li>Offset the capacitive currents and series resistance (a measured series resistance value greater than 25 M&#8486; indicates that the electrode pipette has been clogged) and, if required (i.e. for larger currents), apply series resistance compensation using the appropriate knobs in the patch clamp amplifier.</li>
	<li>Close the black front curtain of the Faraday cage to obtain maximal darkness and electrical isolation.</li>
	<li>Begin the recording process using the software and administer light stimuli and/or pharmacological substances according to the desired experimental procedure.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Polyethylene Tubing</td>
			<td>Becton Dickinson</td>
			<td></td>
			<td>1.57 x 1.14 mm (35 cm)</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 &#8211; polyisobutylene mixture</td>
			<td>To glue the coverslip onto the bottom of the bath chamber</td>
		</tr>
		<tr>
			<td>Ground</td>
			<td>Warner Instruments</td>
			<td>64-1288</td>
			<td>Hybrid Assembly Ag-AgCl Wire Assembly</td>
		</tr>
		<tr>
			<td>Stereoscopic zoom Microscope</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>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>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</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 &#248;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</td>
			<td>Nikon</td>
			<td>AR</td>
			<td>software</td>
		</tr>
		<tr>
			<td>Calcium indicator</td>
			<td>Invitrogen</td>
			<td>Calcium green 5N</td>
			<td></td>
		</tr>
		<tr>
			<td>Excitation &#38; 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</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</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>
	</tbody>
</table>

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.

<img alt="Figure 1" src="/files/ftp_upload/22840/22840_Figure1.jpg" /> 
Figure 1: 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 an...

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

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 ...

Take a Drosophila ommatidium in a recording chamber perfused with an extracellular solution containing calcium ions.
The ommatidium comprises a photoreceptor cell array, which processes visual information.
Assemble a patch pipette comprising an intracellular solution and an electrode connected to an amplifier.
Apply positive pressure to the pipette and advance it perpendicular to the ommatidium&#39;s long axis.
The tip forms a dimple on the photoreceptor membrane upon contact.
Calibrate the amplifier parameters for accurate measurements.
Apply negative pressure to form a high-resistance seal.
Then, apply negative pressure pulses to establish a whole-cell configuration.
Set the patched photoreceptor&#39;s holding potential to a constant negative value, stabilizing the cell.
Ensure electrical isolation and optimal darkness for accurate measurements.
Apply brief light pulses, triggering signaling pathways, opening the photoreceptor&#39;s transient receptor potential channels.
The resulting cation influx causes a transient current signal called a quantum bump, which is recorded.&#160;

whole-cell-voltage-clamp-recordings-from-drosophila-photoreceptors

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37 Views. Source:  Katz, B., et al., Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors. J. Vis. Exp. (2017) The video demonstrates recording electrical activity in the photoreceptor neurons of Drosophila'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. 

Video: Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors - Concept

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

JoVE Journal

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

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 ...

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Transcript

Whole-Cell Voltage Clamp Recordings from Drosophila Photoreceptors

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors