Name: desensitization and recovery of crayfish photoreceptors upon delivery of a light stimulus
Uploaded: 2019-11-09T12:00:00
Description: Watch this Scientific Journal Video about Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus at JoVE.com

This work was supported by DGAPA-UNAM IN224616-RN224616 grant. The authors want to thank Mrs. Josefina Bolado, Head of the Scientific Paper Translation Department, from Divisi&#243;n de Investigaci&#243;n at Facultad de Medicina, UNAM, for editing the English-language version of this manuscript.

NOTE: The experiments comply with the Laws of Animal Protection of Mexico.
1. Experimental Setup
<ol>
	<li>General connections
	<ol>
		<li>Connect the amplifier to a suitable computer through an analog-to-digital converter and use an oscilloscope to monitor the experiment (Figure 1).</li>
		<li>Connect the photostimulator to the A/D converted.</li>
	</ol>
	</li>
	<li>Recording chamber
	<ol>
		<li>Place the recording chamber on top of an anti-vibration table and ...

<ol>
	<li>Barriga-Montoya, C., G&#243;mez-Lagunas, F., Fuentes-Pardo, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pigment+dispersing+hormone+on+the+electrical+activity+of+crayfish+visual+photoreceptors+during+the+24-h+cycle.">Effect of pigment dispersing hormone on the electrical activity of crayfish visual photoreceptors during the 24-h cycle.</a> Comp. Biochem. Physiol. A Comp. Physiol. 157 (4), 338-345 (2010).</li><li>Barriga-Montoya, C., de la O-Mart&#237;nez, A., Fuentes-Pardo, B., G&#243;mez-Lagunas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Desensitization+and+recovery+of+crayfish+photoreceptors.+Dependency+on+circadian+time,+and+pigment-dispersing+hormone.">Desensitization and recovery of crayfish photoreceptors. Dependency on circadian time, and pigment-dispersing hormone.</a> Comp. Biochem. Physiol. A Comp. Physiol. 203, 297-303 (2017).</li><li>Wickenden, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+electrophysiological+techniques.">Overview of electrophysiological techniques.</a> Curr. protoc. pharmacol. 11, 1-17 (2014).</li><li>Brette, R., Destexhe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+recording.">Intracellular recording.</a> Handbook of Neural Activity Measurement. , 44-91 (2012).</li><li>Cummins, D., Goldsmith, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+identification+of+the+violet+receptor+in+the+crayfish+eye.">Cellular identification of the violet receptor in the crayfish eye.</a> J. Comp. Physiol. 142 (2), 199-202 (1981).</li><li>Eguchi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhabdom+structure+and+receptor+potentials+in+single+crayfish+retinular+cells.">Rhabdom structure and receptor potentials in single crayfish retinular cells.</a> J. Cell and Comp. Physiol. 66, 411-430 (1965).</li><li>Nosaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+study+of+color+encoding+in+the+compound+eye+of+crayfish,+Procambarus+clarkii.">Electrophysiological study of color encoding in the compound eye of crayfish, Procambarus clarkii.</a> Z. vergl. Physiologie. 64 (3), 318-323 (1969).</li><li>Miller, C. S., Glantz, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+adaptation+modulates+a+potassium+conductance+in+retinular+cells+of+the+crayfish.">Visual adaptation modulates a potassium conductance in retinular cells of the crayfish.</a> Vis Neurosci. 17 (3), 353-368 (2000).</li><li>Hamill, O. P., Marty, A., Neher, E., Sakmann, B., Sigworth, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+patch-clamp+techniques+for+high-resolution+current+recording+from+cells+and+cell-free+membrane+patches.">Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.</a> Pflugers Arch. 391 (2), 85-100 (1981).</li><li>Lishko, P., Clapham, D. E., Navarro, B., Kirichok, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sperm+patch-clamp.">Sperm patch-clamp.</a> Methods Enzymol. 525, 59-79 (2013).</li><li>Finkel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axoclamp-2A+microelectrode+clamp+theory+and+operation.">Axoclamp-2A microelectrode clamp theory and operation.</a> Axon Instruments, Inc. , (1990).</li><li>Sakmann, 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> Single-channel recording. , (2013).</li><li>Van Harreveld, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+physiological+solution+for+freshwater+crustacea.">A physiological solution for freshwater crustacea.</a> Proc. Soc. Exp. Biol. Med. 34 (4), 428-432 (1936).</li><li>Oesterle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> pipette cookbook. , 97 (2008).</li><li>Fuentes-Pardo, B., Ramos-Carvajal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phase+response+curve+of+electroretinographic+circadian+rhythm+of+crayfish.">The phase response curve of electroretinographic circadian rhythm of crayfish.</a> Comp. Biochem. Physiol. A Comp. Physiol. 74 (3), 711-714 (1983).</li><li>Pace-Schott, E. F., Hobson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neurobiology+of+sleep:+genetics,+cellular+physiology+and+subcortical+networks.">The neurobiology of sleep: genetics, cellular physiology and subcortical networks.</a> Nat. Rev. Neurosci. 3 (8), 591-605 (2002).</li><li>Pittendrigh, C. S., Aschoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+the+entrainment+of+a+circadian+rhythm+by+light+cycles.">On the mechanism of the entrainment of a circadian rhythm by light cycles.</a> Circadian Clocks. , 277-297 (1965).</li><li>Pittendrigh, C. S., Aschoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+systems:+entrainment.">Circadian systems: entrainment.</a> Handbook Behavioral Neurobiology Biological Rhythms. , 94-124 (1981).</li><li>Vitaterna, M. H., Takahashi, J. S., Turek, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+circadian+rhythms.">Overview of circadian rhythms.</a> Alcohol Res. Health. 25 (2), 85-93 (2001).</li><li>Hille, 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> Ion channels of excitable membranes. 507, (2001).</li><li>Dircksen, H., Strauss, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+clocks+in+crustaceans:+identified+neuronal+and+cellular+systems.">Circadian clocks in crustaceans: identified neuronal and cellular systems.</a> Front Biosci. 15, 1040-1074 (2010).</li><li>Terakita, A., Hariyama, T., Tsukahara, Y., Katsukura, Y., Tashiro, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+of+GTP-binding+protein+Gq+with+photoactivated+rhodopsin+in+the+photoreceptor+membranes+of+crayfish.">Interaction of GTP-binding protein Gq with photoactivated rhodopsin in the photoreceptor membranes of crayfish.</a> FEBS Lett. 330, 197-200 (1993).</li><li>Terakita, A., Takahama, H., Hariyama, T., Suzuki, T., Tsukahara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+regulated+localization+of+the+beta-subunit+of+Gq-type+G-protein+in+the+crayfish+photoreceptors.">Light regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors.</a> J Comp Physiol A. 183 (4), 411-417 (1998).</li><li>Terakita, A., Takahama, H., Tamotsu, S., Suzuki, T., Hariyama, T., Tsukahara, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-modulated+subcellular+localization+of+the+alpha-subunit+of+GTP-binding+protein+Gq+in+crayfish+photoreceptors.">Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors.</a> Vis Neurosci. 13 (3), 539-547 (1996).</li></ol>

The crayfish has proven to be an excellent model due to its ability to survive under non-natural conditions. There is easy access to in vivo and in vitro electrophysiological analyses. In addition, crustaceans are a favorable group for neurobiological research in the field of comparative chronobiology21.
In this paper, the study of desensitization and recovery of the light-activated transduction-current of crayfish photoreceptor cells is shown using th...

In order to be perceived as a visual stimulus, light reaching the eyes must be transduced into an electrical signal. Hence, in all visual organisms, light triggers a transduction ion-current, which in turn produces a change in the membrane potential of photoreceptor cells, the so-called receptor potential. Due to this, the light sensitivity of the eye primarily depends on the state of the light activated conductance, which can be either available to be activated or desensitized.
In crayfish photoreceptors, light triggers a slow, transient, ionic current1. Upon illumination, the transduction current arises after a lag or latency before reaching its maximum; thereafter it decays, as the transduction channels fall into a desensitized state in which they are unresponsive to further light stimulus2. That is, light, in addition to activating the transduction current responsible of vision, also induces a transient decrement of the sensitivity of photoreceptor cells. Desensitization may represent a general protective mechanism against overexposure to an adequate stimulus. The eye&#39;s sensitivity to light is recovered as the transduction conductance recovers from desensitization.
Intracellular recording is a useful technique for measuring electrical activity of excitable cells3,4,5,6,7,8. Although intracellular recording has become less frequent with the advent of the patch-clamp technique9, it is still a convenient approach when cells are either difficult to isolate, or present a geometry that makes the formation of the patch-clamping giga-seals difficult (i.e., seals or tight-contacts between the patch electrode and membranes with electrical resistance of the order of 109ohms). Examples of the latter are sperm cells10 and the photoreceptor cells herein studied. In our experience, Procambarus clarkii photoreceptors are difficult to isolate and keep in primary culture; additionally, they are thin rods that make giga-seal formation difficult to achieve. In intracellular recordings, a sharp electrode is advanced into a cell that is kept in place by the surrounding tissue. The electrode is chopped by the high-speed switching circuitry of the amplifier, so current is sampled between voltage pulses. This mode is known as discontinuous single-electrode voltage clamp (dSEVC mode)11. The high resistance (small opening) of the electrode hinders the diffusional exchange between the cell and the pipette solutions, yielding a minimal disturbance of the intracellular milieu3. A potential drawback of this technique is that electrode insertion may produce a non-selective leak current; therefore, care must be taken to avoid recording from cells where the size of the leak current may interfere with the intended measurements4,12.
Herein, we use isolated crayfish eyestalks to assess desensitization and recovery of the light-activated ion conductance by performing intracellular electrical recordings of photoreceptor cells under voltage clamp conditions.

<table border="0" cellpadding="0" cellspacing="0" style="width:644px;" width="644">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Axoclamp2A&#160;</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td style="width:180px;">Amplifier</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Digidata 1200 Interface</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td>Digitizer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Oscilloscope TDS430A</td>
			<td style="width:129px;">Tektronix</td>
			<td style="width:119px;"></td>
			<td>Analogic Oscilloscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Photostimulator PS33 Plus</td>
			<td style="width:129px;">Grass</td>
			<td style="width:119px;"></td>
			<td>Lamp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puller PC-100</td>
			<td style="width:129px;">Narishige</td>
			<td style="width:119px;"></td>
			<td>Micropipette Puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puller P-97</td>
			<td style="width:129px;">Sutter Instruments</td>
			<td style="width:119px;"></td>
			<td>Micropipette Puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glass Capillary Tube Kimax-51</td>
			<td style="width:129px;">Kimble Products</td>
			<td style="width:119px;">34502</td>
			<td>0.8, 1.10, 100 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HS-2 Headstage</td>
			<td style="width:129px;">Axon Instruments Inc</td>
			<td style="width:119px;"></td>
			<td>Headstage</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator MX-4</td>
			<td>Narishige</td>
			<td style="width:119px;"></td>
			<td>Mechanical Micromanipulator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stereoscopic Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">pClamp</td>
			<td>Axon Instruments Inc</td>
			<td></td>
			<td style="width:180px;">Data acquisition software for digidata 1200 interface</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Clampfit</td>
			<td>Axon Instruments Inc</td>
			<td></td>
			<td style="width:180px;">Analysis software linked to pClamp</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Origin</td>
			<td>OriginLab Corp.</td>
			<td></td>
			<td style="width:180px;">Data analysis and graphing software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">S7653</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">P-9333</td>
			<td>Minimum 99%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnesium Sulfate</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">M7506</td>
			<td>Minimum 99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Calcium Chloride</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">C5080</td>
			<td>Minimum 99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hepes</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">H7523</td>
			<td>&#62;99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">S8045</td>
			<td>98.00%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hypochlorite solution</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:119px;">425044</td>
			<td style="width:180px;">Available chlorine, 10-15%&#160;</td>
		</tr>
	</tbody>
</table>

desensitization and recovery of crayfish photoreceptors upon delivery of a light stimulus

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse.
To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

First, a representative receptor potential of crayfish photoreceptor cells is obtained (Figure 4). Afterwards, a test light-flash was applied to trigger the light transduction current (Figure 5). The cationic transduction current1 activates after a lag, reaching a maximal and thereafter slowly drops into an absorbing desensitized state from which it slowly recovers.
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Watch this Scientific Journal Video about Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus at JoVE.com

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

A protocol for the study of desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time is presented.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor ...

The overall goal of this procedure is to study desensitization and sensitivity recovery of crayfish photoreceptors as a function of circadian time, using intercellular electrical recordings of photoreceptor cells in isolated eye stocks, employing the discontinuous single electrode switched voltage clamp configuration. This method can help answer key questions in the neurobiology field, such as the circadian time dependency of so many electro-physiological properties of photoreceptors in inactivation and recovery. The main advantage of this technique, is that it allows the measurement of photoreceptors'electrical activity.In spite of the difficulty to isolate cells and without disturbing the intracellular milieu. Demonstrating this procedure will be Doctors Carolina Barriga-Montoya and Araceli de la O-Maritnez, coworkers of our laboratory. To prepare the intracellular electrode, pull the glass capillary tube with a micropipette puller to obtain a thin tip with a small opening.Next, fill the pulled capillary glass with 2.7-molar potassium chloride solution by capillarity and then, fill half of the pipette with a fine injection needle. If necessary, tap the electrode pipette to eliminate air bubbles. After that, place the electrode on its holder.Connect the holder to amplifier head stage with the amplifier. Following that, position the electrode holder head stage with a stable 3-D micromanipulator. Lower the electrode until bath solution covers its tip.Select the bridge mode of the amplifier and measure the electrode resistance. Make sure that the resistance is about 50 megaohms. Null the offset current and compensate capacitive transients using the holding position button in the section voltage clamp of the amplifier and the capacitance neutralization button in the Microelectrode 1 section of the amplifier.To construct the super fusion system, pour the bath solution into a suitable receptacle and connect it to an irrigation tubing set, thus connecting the recording chamber. Use a gravity-driven superfusion system. Regulate the flow rate to 0.5 milliliters per second.Connect the chamber to a suction device. Regulate the solution suction system with a vacuum pump in such a way that the total volume of recording chamber does not vary. To isolate the eye stock of an adult crayfish, detach it from the base with a pair of fine scissors.Make an opening of one square millimeter in the dorsal cornea using a razor blade to access the retina. Next, place the eye stock in the center of the recording chamber with the opening to the retina facing up. Keep the eye stock in darkness for 20 minutes.Place the microelectrode parallel to the eye stocks longitudinal axis in such a way that the microelectrode is centered to access the retina. Use a stereoscopic microscope to place the devices in the correct configuration. Next, monitor the voltage difference between the reference and recording electrodes by selecting the bridge mode of the amplifier.Lower the electrode into the bath and place it right over the retina. Remove the microscope and position the photostimulator lamp parallel to the eye stocks longitudinal axis. Then, slowly lower the microelectrode until a sudden voltage drop is detected.Subsequently, deliver a test light flash to record the photoreceptor's receptor potential. To obtain appropriate recordings of light-elicited currents, it is critical to make a good impairment of a healthy photoreceptor cell. In this procedure, clamp the voltage at the measured resting membrane potential of the cell by selecting holding amplitude in the data acquisition software.Then, select the DSEVC mode of the amplifier. In the mode section of the amplifier, select the SEVC button and switch the lever to the Discont. SEVC position.Set the switching rate to 500 to 1, 000 hertz, as determined by the speed of electrode. After that, deliver a light flash and observe the evoked ion flux. Then, deliver a pair of light pulses and apply the second flash after a desired time interval.Digitize the currents at 10 kilohertz sampling with the data acquisition software and save the data for offline analysis. Shown here is a two-pulse protocol. Light stimuli were applied at zero milliseconds and 700 milliseconds.This figure shows the recovery from desensitization. Here is latency recovery, here is peak current recovery, and here is the desensitization time constant tau recovery. Experiments were performed at zero hours circadian time.This figure shows the recovery from desensitization as a function of circadian time. This is iP recovery, this is L recovery, and this is tau recovery, and this graph shows the weighted time constants. Biphasic processes are marked with an arrow.Once mastered, this technique can be done in two hours, if it's done properly. While attempting this procedure, it is important to remember to aim for a low noise and low leakage current, photoreceptor impairment. Following this procedure, other methods, like pharmacological assays, can be done in order to answer additional questions, like the involvement of particular channels, receptors, or signaling pathways.After its development, this technique paved the way for researchers in the field of neurobiology to explore the circadian time dependence of electrophysiological properties of photoreceptor cells. After watching this video, you should have a good understanding of how to study desensitization and recovery of light-activated transduction current by using the intracellular recording technique.

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Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

Gene Delivery to Postnatal Rat Brain by Non-ventricular Plasmid Injection and Electroporation

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals are not viable in those cases where the modified gene is expressed or deleted in the whole organism. Moreover, a variety of compensatory mechanisms often make it difficult to interpret the results. The compensatory effects can be alleviated by either timing the gene expression or limiting the amount of transfected cells.
The method of postnatal non-ventricular microinjection and in vivo electroporation allows targeted delivery of genes, siRNA or dye molecules directly to a small region of interest in the newborn rodent brain. In contrast to conventional ventricular injection technique, this method allows transfection of non-migratory cell types. Animals transfected by means of the method described here can be used, for example, for two-photon in vivo imaging or in electrophysiological experiments on acute brain slices.

This protocol describes a non-viral method of delivery of genetic constructs to a certain area of living rodent brain. The method consists of plasmid preparation, micropipette fabrication, neonatal rat pup surgery, microinjection of the construct, and in vivo electroporation.

Creation of transgenic animals is a standard approach in studying functions of a gene of interest in vivo. However, many knockout or transgenic animals ...

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings with a force transducer to monitor the strength of contraction. In addition, we demonstrate how to visually monitor peristaltic activity, which can be used as a bioassay for various peptides, biogenic amines and neurotransmitters. This preparation is amenable to student laboratories in physiology and for demonstrating pharmacological concepts to students. This preparation has been in use for over 100 years, and it still offers much as a model for investigating the generation and regulation of peristaltic rhythms and for describing the mechanisms underlying their modulation. The pharmacological assays and receptor sub-typing that were started over 50 years ago on the hindgut still contribute to research today. This robust preparation is well suited to training students in physiology and pharmacology.

In this report we demonstrate techniques that can be used to investigate the biology of the crayfish hindgut. We show how to dissect a crayfish abdomen and study the associated anatomy, physiology and modulation of activity. The peristaltic activity and strength of contractions are measured using a force transducer.

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Behavioral Determination of Stimulus Pair Discrimination of Auditory Acoustic and Electrical Stimuli Using a Classical Conditioning and Heart-rate Approach

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural auditory prostheses, such as auditory brainstem implants1-3 and auditory midbrain implants4,5. While acute experiments can give initial insight to the effectiveness of the implant, testing the chronically implanted and awake animals provides the advantage of examining the psychophysical properties of the sensations induced using implanted devices6,7.
Several techniques such as reward-based operant conditioning6-8, conditioned avoidance9-11, or classical fear conditioning12 have been used to provide behavioral confirmation of detection of a relevant stimulus attribute. Selection of a technique involves balancing aspects including time efficiency (often poor in reward-based approaches), the ability to test a plurality of stimulus attributes simultaneously (limited in conditioned avoidance), and measure reliability of repeated stimuli (a potential constraint when physiological measures are employed).
Here, a classical fear conditioning behavioral method is presented which may be used to simultaneously test both detection of a stimulus, and discrimination between two stimuli. Heart-rate is used as a measure of fear response, which reduces or eliminates the requirement for time-consuming video coding for freeze behaviour or other such measures (although such measures could be included to provide convergent evidence). Animals were conditioned using these techniques in three 2-hour conditioning sessions, each providing 48 stimulus trials. Subsequent 48-trial testing sessions were then used to test for detection of each stimulus in presented pairs, and test discrimination between the member stimuli of each pair. 
This behavioral method is presented in the context of its utilisation in auditory prosthetic research. The implantation of electrocardiogram telemetry devices is shown. Subsequent implantation of brain electrodes into the Cochlear Nucleus, guided by the monitoring of neural responses to acoustic stimuli, and the fixation of the electrode into place for chronic use is likewise shown.

The application of a classical fear conditioning behavioral paradigm for auditory prosthetic research in rats is described. This paradigm provides a mechanism for identifying both detection of, and discrimination between, distinct acoustic and electrical stimuli using heart-rate as an outcome measure.

Acute animal preparations have been used in research prospectively investigating electrode designs and stimulation techniques for integration into neural ...

Extinction Training During the Reconsolidation Window Prevents Recovery of Fear

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents the return of fear. Pavlovian fear-conditioning paradigms are commonly employed to create a controlled, novel fear association in the laboratory. After pairing an innocuous stimulus (conditioned stimulus, CS) with an aversive outcome (unconditioned stimulus, US) we can elicit a fear response (conditioned response, or CR) by presenting just the stimulus alone1,2 . Once fear is acquired, it can be diminished using extinction training, whereby the conditioned stimulus is repeatedly presented without the aversive outcome until fear is no longer expressed3. This inhibitory learning creates a new, safe representation for the CS, which competes for expression with the original fear memory4. Although extinction is effective at inhibiting fear, it is not permanent. Fear can spontaneously recover with the passage of time. Exposure to stress or returning to the context of initial learning can also cause fear to resurface3,4.
Our protocol addresses the transient nature of extinction by targeting the reconsolidation window to modify emotional memory in a more permanent manner. Ample evidence suggests that reactivating a consolidated memory returns it to a labile state, during which the memory is again susceptible to interference5-9. This window of opportunity appears to open shortly after reactivation and close approximately 6hrs later5,11,16, although this may vary depending on the strength and age of the memory15. By allowing new information to incorporate into the original memory trace, this memory may be updated as it reconsolidates10,11. Studies involving non-human animals have successfully blocked the expression of fear memory by introducing pharmacological manipulations within the reconsolidation window, however, most agents used are either toxic to humans or show equivocal effects when used in human studies12-14. Our protocol addresses these challenges by offering an effective, yet non-invasive, behavioral manipulation that is safe for humans.
By prompting fear memory retrieval prior to extinction, we essentially trigger the reconsolidation process, allowing new safety information (i.e., extinction) to be incorporated while the fear memory is still susceptible to interference. A recent study employing this behavioral manipulation in rats has successfully blocked fear memory using these temporal parameters11. Additional studies in humans have demonstrated that introducing new information after the retrieval of previously consolidated motor16, episodic17, or declarative18 memories leads to interference with the original memory trace14. We outline below a novel protocol used to block fear recovery in humans.

Conditioned fear can be diminished through an inhibitory process called extinction, but can resurface under conditions such as the passage of time or exposure to stress. Our protocol presents a novel way of preventing fear recovery by introducing extinction during the reconsolidation window (the re-storage phase of a reactivated memory).

Fear is maladaptive when it persists long after circumstances have become safe. It is therefore crucial to develop an approach that persistently prevents ...

The Swimmeret System of Crayfish: A Practical Guide for the Dissection of the Nerve Cord and Extracellular Recordings of the Motor Pattern

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of abdominal ganglia (A1 to A6). This chain of ganglia includes the part of the central nervous system (CNS) that drives coordinated locomotion of the pleopods (swimmerets): the swimmeret system. It is known for over five decades that in crayfish each swimmeret is driven by its own independent pattern generating kernel that generates rhythmic alternating activity 1-3. The motor neurons innervating the musculature of each swimmeret comprise two anatomically and functionally distinct populations 4. One is responsible for the retraction (power stroke, PS) of the swimmeret. The other drives the protraction (return stroke, RS) of the swimmeret. Motor neurons of the swimmeret system are able to produce spontaneously a fictive motor pattern, which is identical to the pattern recorded in vivo 1.
The aim of this report is to introduce an interesting and convenient model system for studying rhythm generating networks and coordination of independent microcircuits for students&#8217; practical laboratory courses. The protocol provided includes step-by-step instructions for the dissection of the crayfish&#8217;s abdominal nerve cord, pinning of the isolated chain of ganglia, desheathing the ganglia and recording the swimmerets fictive motor pattern extracellularly from the isolated nervous system.
Additionally, we can monitor the activity of swimmeret neurons recorded intracellularly from dendrites. Here we also describe briefly these techniques and provide some examples. Furthermore, the morphology of swimmeret neurons can be assessed using various staining techniques. Here we provide examples of intracellular (by iontophoresis) dye filled neurons and backfills of pools of swimmeret motor neurons. In our lab we use this preparation to study basic functions of fictive locomotion, the effect of sensory feedback on the activity of the CNS, and coordination between microcircuits on a cellular level.

Here we describe the dissection of the crayfish abdominal nerve cord. We also demonstrate an electrophysiological technique to record fictive locomotion from swimmeret motor neurons.

Here we demonstrate the dissection of the crayfish abdominal nerve cord. The preparation comprises the last two thoracic ganglia (T4, T5) and the chain of ...

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.