This work was funded by grants from the National Institutes of Health: EY015748, EY017964 (MEZ), and EY019517 (ASV). This work was also supported by the Research to Prevent Blindness Unrestricted Grant to the Ophthalmology Department and the Lions of Central New York. We would also like to thank our animal technician, Matthew Mellini, for his excellent care of the animals and for stepping in to star in this video.

Animal Care
The Xenopus laevis tadpoles used in this study were grown, raised and treated according to procedures approved by Upstate Medical University IACUC and the Guide for the Care and Use of Laboratory Animals.
1. Pre-behavior Setup: Tadpoles
<ol>
	<li>Obtain fertilized Xenopus embryos from a commercially available source or in vitro fertilize oocytes, as previously described6.</li>
	<li>Place blastula to neural plate stage embryos at 18 &#730;C in 60 mm Petri dishes containing 0.1X MMR [10 X Marc&#39;s Modified Ringer&#39;s (MMR) solution stock (10 mM MgCl2; 20 mM KCl; 20 mM CaCl2 ; 50 mM HEPES; 1 M NaCl; adjusted to pH 7.5, autoclaved and stored at RT)] with 50 &#181;g/ml gentamicin antibiotic. Change 0.1X MMR each day and remove any dead embryos.</li>
	<li>Once the embryos reach Nieuwkoop and Faber stage ~27 - 307,&#160;switch the embryos into 0.1X MMR without antibiotic to allow gut bacterial growth.</li>
	<li>Grow the tadpoles until they reach the feeding stage (stage 45) and move them into 100 mm Petri dishes filled with 0.1X MMR. Feed them nettle powder supernatant every other day for a week, changing the 0.1X MMR on the intervening day.</li>
	<li>After one week in the 100 mm Petri dish, move animals to half gallon tanks filled with frog water [0.5 g/L instant ocean and 2 mM Sodium phosphate dibasic (Na2HPO4: mw 141.96) pH 6.8]. Feed them as in step 2.4, but change water only 2-3 times per week when water is cloudy. Maintain the animals in a 12 hr light/12 dark light cycle with the lights turning on at 6 am.</li>
	<li>Place the half-gallon tank with the test subjects on a white surface like a lab bench underpad, at least 12 hr O/N, prior to testing.</li>
	<li>Test tadpoles at stages 45 to 50 as described in step 3 below.</li>
</ol>
2. Pre-behavior Setup: Equipment
<ol>
	<li>Set aside an area of the lab for the behavior assay. It should be in a quiet, low-traffic area with standard fluorescent lighting.</li>
	<li>Prepare the testing tanks for the animals. 
	The nested, half-gallon testing tanks are composed of two parts: an inner tank that will hold the water and tadpoles; and an outer tank with the visual stimulus.
	<ol>
		<li>For the inner tank, make a small mark on the outside corners with a sharpie, 5 cm from the bottom; this is the water line (Figure 1A, green dotted line).</li>
		<li>Also, for the inner tank, fill the divets in the tank (at the corners and the center) with an inert compound, like Sylgard elastomer. NOTE: The tadpoles will linger in these areas if they are not filled in.</li>
		<li>For the outer tank, cover exactly one half of the outside of one tank with black electrical tape and the other half with the white tissue paper Figure 1B.</li>
	</ol>
	</li>
	<li>Place the tanks on the inoculating turntable.</li>
	<li>Setup the webcam/tripod so that it is above the testing tanks. Adjust the camera to allow visualization of the testing area, as shown in&#160;Figure 1C.</li>
	<li>Connect the webcam to a computer with QuickTime Player software installed5.&#160;Turn on the camera. In QuickTime Player, under File, select &#8216;New Movie Recording.&#8217; The behavior assay setup will be visible on the computer right away.</li>
	<li>Drape a lightweight cotton cloth over the entire setup to reduce external cues that could affect tadpole behavior, as well as, reduce reflected light on the surface of the water.</li>
	<li>Ensure that the luminance under the cloth measures between 35-50 cd/m2.</li>
</ol>
3. Behavior Assay
<ol>
	<li>Fill the inner testing tank to the 5 cm water mark made in step 1.2.1 with frog water.</li>
	<li>Using a small net, gently move the animal into the inner testing tank.</li>
	<li>Open QuickTime software on computer and, using the outer tank as a guide, make sure that the camera is able to visualize and record the testing area.</li>
	<li>Write the animal name, date and time on a piece of paper and record with the camera.</li>
	<li>Place the test tank into the outer tank with the black side of the tank to the right Figure 2.</li>
	<li>Start recording the movie immediately after arranging the tank and set timer for 2 min.</li>
	<li>When the timer beeps, remove the inner test tank, rotate the outer tank 180&#176;&#160;and place the inner test tank inside the outer tank. Start the timer. Note: the black side should now be on the other side.</li>
	<li>Repeat step 3.7, eight more times for a total of ten trials. Use Figure 2 as a guide, checking off each trial.</li>
	<li>Repeat the behavior assay on two separate days.</li>
</ol>
4. Retinal Axotomy
<ol>
	<li>Place animal in 0.02% tricaine until they are unresponsive to a tail pinch with a #3 forceps.</li>
	<li>Melt 1% agarose in 0.1X MMR and then add to a 60 mm Petri dish. Once cooled, make a small rectangular divot in the agarose and add the tadpole to the divot with a little 0.02% tricaine, so that the animal is partially submerged in the liquid.</li>
	<li>Pierce the skin at a 45 degree angle behind the dorsal region of the eye with a 25 G needle, while bracing the animal against forceps on the opposite side.</li>
	<li>Taking care not to snip the vein, which lies next to the optic nerve, carefully reach into the hole with #5 forceps, snip the optic nerve and flip it out of the way. If there is severe bleeding due to accidently snipping the artery, then quickly place the animal in 2% tricaine to euthanize. CAUTION: 2% tricaine solution can cause numbness in humans. This solution should be handled with gloves.</li>
	<li>To recover the animal after surgery, place animal in 100 mm Petri dish with 0.7X MMR and 50 &#181;g/ml gentamicin for 20 min. Next, transfer tadpole to recovery tank with frog water. Let animal rest O/N.</li>
	<li>The next morning, test the visual function as described above (steps 3.1-3.9).</li>
</ol>
5. Analyze Results
<ol>
	<li>After the trials are finished, measure the amount of time the tadpole stays on the black side of the tank.
	<ol>
		<li>Manually view the videos using software that displays the time and allows frequent pausing. Look at the video display time. Write down the start of the two-min&#160;trial, which is when the inner tank successfully nests inside the outer tank and is squared in view of the camera.</li>
		<li>Use the position of the tadpole&#8217;s eyes to define which side of the tank the animals are swimming on. Define crossing over from one side of the tank to the other only when both eyes have crossed the black/white line.</li>
	</ol>
	</li>
	<li>Write down the beginning and end of each interval (in sec) that the animal spends on the black side. Pause and rewind the video to ensure accuracy. Total the sec&#160;within each trial.</li>
	<li>Input these numbers into the attached Behavior Worksheet. The worksheet will calculate the ratio of time that the animal spent on the white side by subtracting the time spent on the black side of the tank from the total 120 sec&#160;trial and divide by the total time in sec&#160;[(120 &#8211; total seconds spent on the black side)/120 sec]. The worksheet averages this ratio over all 10 trials.</li>
	<li>Use a student&#8217;s T-test, paired, two-tailed distribution to determine if the trials between days are significant (P &#8804; 0.05).</li>
</ol>

<ol>
	<li>Maurer, C. M., Huang, Y. Y., Neuhauss, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+zebrafish+oculomotor+behavior+to+model+human+disorders.">Application of zebrafish oculomotor behavior to model human disorders.</a> Rev Neurosci. 22 (1), 5-16 (2011).</li><li>Solessio, E., Scheraga, D., Engbretson, G. A., Knox, B. E., Barlow, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+modulation+of+temporal+properties+of+the+rod+pathway+in+larval+Xenopus.">Circadian modulation of temporal properties of the rod pathway in larval Xenopus.</a> J Neurophysiol. 92 (5), 2672-2684 (2004).</li><li>Moriya, T., Kito, K., Miyashita, Y., Asami, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+for+background+color+of+the+Xenopus+laevis+tadpole.">Preference for background color of the Xenopus laevis tadpole.</a> J Exp Zool. 276 (5), 335-344 (1996).</li><li>Viczian, A. S., Solessio, E. C., Lyou, Y., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+functional+eyes+from+pluripotent+cells.">Generation of functional eyes from pluripotent cells.</a> PLoS Biol. 7 (8), (2009).</li><li>Choi, R. Y., 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=Cone+degeneration+following+rod+ablation+in+a+reversible+model+of+retinal+degeneration.">Cone degeneration following rod ablation in a reversible model of retinal degeneration.</a> Invest Ophthalmol Vis Sci. 52 (1), 364-373 (2011).</li><li>Viczian, A. S., Zuber, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+determination+using+the+animal+cap+transplant+(ACT)+assay+in+Xenopus+laevis.">Tissue determination using the animal cap transplant (ACT) assay in Xenopus laevis.</a> J Vis Exp. 16 (39), (2010).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Blackiston, D., Shomrat, T., Nicolas, C. L., Granata, C., Levin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+second-generation+device+for+automated+training+and+quantitative+behavior+analyses+of+molecularly-tractable+model+organisms.">A second-generation device for automated training and quantitative behavior analyses of molecularly-tractable model organisms.</a> PLoS One. 5 (12), (2010).</li><li>Rosemberg, D. B., 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=Differences+in+spatio-temporal+behavior+of+zebrafish+in+the+open+tank+paradigm+after+a+short-period+confinement+into+dark+and+bright+environments.">Differences in spatio-temporal behavior of zebrafish in the open tank paradigm after a short-period confinement into dark and bright environments.</a> PLoS One. 6 (5), (2011).</li><li>Dong, W., 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=Visual+Avoidance+in+Xenopus+Tadpoles+is+Correlated+With+the+Maturation+of+Visual+Responses+in+the+Optic+Tectum.">Visual Avoidance in Xenopus Tadpoles is Correlated With the Maturation of Visual Responses in the Optic Tectum.</a> J Neurophysiol. 101 (2), 803-815 (2009).</li><li>Lan, L., 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=Noggin+Elicits+Retinal+Fate+In+Xenopus+Animal+Cap+Embryonic+Stem+Cells.">Noggin Elicits Retinal Fate In Xenopus Animal Cap Embryonic Stem Cells.</a> Stem Cells. 27 (9), 2146-2152 (2009).</li><li>De Robertis, E. M., Kuroda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dorsal-ventral+patterning+and+neural+induction+in+Xenopus+embryos.">Dorsal-ventral patterning and neural induction in Xenopus embryos.</a> Annu Rev Cell Dev Biol. 20, 285-308 (2004).</li></ol>

The authors have nothing to disclose.

We report here a simple vision-guided behavioral assay that can be easily performed in under a week by minimally trained lab personnel. While other assays require specialized equipment and expertise in animal behavior, this assay allows a quick test to determine visual function. Another behavioral assay, the Visual Avoidance assay, has been developed to determine how the tectum contributes to visual perception in Xenopus10. This assay measures spatial tuning and contrast sensitivity in response to a m...

Xenopus laevis have been used as a model organism to study eye formation. The eyes develop quickly, growing to maturity in less than a week for testing genes or pathways that have an effect on visual development and function. To test visual function, the optokinetic and optomotor response has been used in zebrafish and Xenopus tadpoles, respectively1,2&#160;. Because the eyes of Xenopus tadpoles are relatively smaller than zebrafish, this assay requires the use of specialized equipment and trained personnel to detect the subtle tail-flip and eye-movement behavior in Xenopus. A more robust behavior in Xenopus is the preference for swimming in a tank with a white background, which is described herein3. When placing a tadpole in a half black/half-white tank, the pre-metamorphic tadpole quickly swims to the white side of the tank. We previously used this assay to determine if pluripotent cell-derived eyes were functional4.&#160;Here, we report a detailed version of this assay, which can be used to test the visual function of premetamorphic Xenopus tadpoles.
This assay is simpler than the optomotor response assay, since it only requires a mounted digital camcorder, digital camera software and standard equipment found in most Xenopus laboratories. In addition, the recorded response requires no special training to tally results. Our representative results show that the same group of tadpoles, having undergone double retinal axotomy, swim randomly around the tank. We have also included the behavior assay results from a representative tadpole, showing how one eye can be tested for visual response. A worksheet has been included so that numbers acquired during the assay can be inserted and analyzed. This worksheet can be used to determine whether the tadpoles tested have a visual response.

<table border="0" cellpadding="0" cellspacing="0" width="654">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1/2-gal. Flex-Tank with Cover</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">SB19271M</td>
			<td style="width:225px;">size: 5-3/8&#34; x 7&#34; x 3-3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black electrical tape</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">White tissue paper</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Large Inoculating Turntable</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">50809-022</td>
			<td>size: Dia 114.3 x&#160; H 76.2 mm (4 1/2&#160; x&#160; 3&#34;)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Durasorb Underpad</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">82004-836</td>
			<td>size: 43.2 x 60.1 cm (17 x 24&#34;)</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Kim wipe</td>
			<td style="width:95px;">Krackeler Scientific, Inc.</td>
			<td style="width:119px;">1945-34155-CS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Standard Tripod</td>
			<td style="width:95px;"></td>
			<td style="width:119px;">various</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">iSight camera or webcam</td>
			<td style="width:95px;">Apple</td>
			<td style="width:119px;">M8817LL/A</td>
			<td>Good for larger tadpoles but small ones are difficult to see</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Portable computer</td>
			<td style="width:95px;">Apple/PC</td>
			<td style="width:119px;">various</td>
			<td>We used a 13&#34; MacBook, 2 GHz Intel Core 2 Duo running MacOSX Lion 10.7.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MiniDV Handycam Camcorder</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCR-HC42</td>
			<td>Connected by firewire to the computer with a 6-conductor and 4-conductor alpha FireWire 400&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Handycam Station</td>
			<td style="width:95px;">SONY</td>
			<td style="width:119px;">DCRA-C121</td>
			<td>This can be used for connecting firewire to camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">QuickTime Player software</td>
			<td style="width:95px;">Quicktime</td>
			<td style="width:119px;">Version 10.1</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">26 Gauge Needle (5/8&#34; length)</td>
			<td style="width:95px;">VWR</td>
			<td style="width:119px;">BD305115</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Dumont #5 Forceps Biology</td>
			<td style="width:95px;">Fine Science Tools</td>
			<td style="width:119px;">11295-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Disposables</td>
			<td style="width:95px;"></td>
			<td style="width:119px;"></td>
			<td style="width:225px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Gentamicin Sulfate [50 mg/ml]</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">17-528Z</td>
			<td>Stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sylgard 184 Silicone Elastomer</td>
			<td style="width:95px;">Fisher Scientific</td>
			<td style="width:119px;">NC9644388</td>
			<td></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Instant Ocean</td>
			<td style="width:95px;">Doctors Foster and Smith</td>
			<td style="width:119px;">CD-116528</td>
			<td>Stock solution = 100 g/L stored at room temperature</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Sodium phosphate dibasic</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">S0876</td>
			<td>Stock solution = 0.4 M stored at room temperature</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Tricaine (Ethyl-3-aminobenzoate methanesulfonate salt)</td>
			<td style="width:95px;">Sigma Aldrich</td>
			<td style="width:119px;">A5040</td>
			<td>Stock solution = 10% stored at -20&#730;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">In vitro fertilized embryos</td>
			<td style="width:95px;">eNasco</td>
			<td style="width:119px;">LM00490MX</td>
			<td>100 embryos/unit</td>
		</tr>
	</tbody>
</table>

a simple behavioral assay for testing visual function in xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

Previous reports have shown that premetamorphic Xenopus tadpoles prefer to swim on the white side of a black/white tank and called this assay, the Background Color Preference Assay3 We have changed this assay in order to test the visual function of either eye of tadpoles in less than a week. In this way, their eyes can be collected for histological examination.
We show here how the response is due to visual cues. In Movie 1, the tadpoles in the left and rig...

Watch this Scientific Journal Video about A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis at JoVE.com

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

a-simple-behavioral-assay-for-testing-visual-function-xenopus

Research

JoVE Journal

Neuroscience

9.8K 次观看.  Center for Vision Research, SUNY Eye Institute, Upstate Medical University.  非洲爪蟾蝌蚪喜欢上一个黑/白罐的白边游泳。此行为是由他们的愿景指导。基于这种情况，我们提出了一个简单的实验来测试蝌蚪的视觉功能。 

视频: A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

The Vermicelli and Capellini Handling Tests: Simple quantitative measures of dexterous forepaw function in rats and mice

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended upon this work to develop a simple quantitative measure of forepaw dexterity that is sensitive to lateralized impairments and age-dependent changes. Rodents learn skillful forepaw and digit movements to manage thin pasta pieces, which they eagerly consume. We have previously described methods for quantifying vermicelli handling in rats and showed that the measures are very sensitive to forelimb impairments resulting from unilateral ischemic lesions, middle cerebral artery occlusions and unilateral striatal dopamine depletion [Allred, R.P., Adkins, D.L., Woodlee, M.T., Husbands, L.C., Maldonado M.A., Kane, J.R., Schallert, T. &amp; Jones, T.A. The Vermicelli Handling Test: a simple quantitative measure of dexterous forepaw function in rats. J. Neurosci. Methods 170, 229-244 (2008)]. Here we present a more detailed protocol for this test in rats and compare it with a newly developed version for mice, the Capellini Handling Test. Rats and mice are videotaped while handling short lengths of uncooked vermicelli or capellini pasta, respectively, with a camera positioned to optimize the view of paw movements. Slow motion video playback allows for the identification of forepaw adjustments, defined as any distinct removal and replacement of the paw, or of any number of digits, on the pasta piece after eating commences. Forepaw adjustments per piece are averaged over trials per each testing session. Repeated testing permits sensitive quantitative analysis of changes in forepaw dexterity over time. Protocols for pre-testing habituation and handling practice, as well as procedures for characterizing atypical handling patterns, are described. Because rats and mice perform the pasta handling tests slightly differently, species-specific differences in administration and scoring of these tests are highlighted. All animal use was in accordance with protocols approved by the University of Texas at Austin Animal Care and Use Committee.

The Vermicelli and Capellini Handling Tests of forepaw dexterity take advantage of the natural inclination of rodents to manipulate food items using skillful forepaw and digit movements. Animals are videotaped while handling short strands of uncooked dry pasta. Slow motion video playback allows for the quantification of forepaw adjustments.

Previous characterizations of rodent eating behavior have revealed that they use coordinated forepaw movements to manipulate food pieces. We have extended ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance imaging. However, use of such methods in animal models requires anesthesia and hence both alters the brain state and prevents behavioral measures. An alternative method is the use of in vivo microdialysis to take continuous measurement of brain extracellular fluid concentrations of glucose, lactate, and related metabolites in awake, unrestrained animals. This technique is especially useful when combined with tasks designed to rely on specific brain regions and/or acute pharmacological manipulation; for example, hippocampal measurements during a spatial working memory task (spontaneous alternation) show a dip in extracellular glucose and rise in lactate that are suggestive of enhanced glycolysis1-3,4-5, and intrahippocampal insulin administration both improves memory and increases hippocampal glycolysis6. Substances such as insulin can be delivered to the hippocampus via the same microdialysis probe used to measure metabolites. The use of spontaneous alternation as a measure of hippocampal function is designed to avoid any confound from stressful motivators (e.g. footshock), restraint, or rewards (e.g. food), all of which can alter both task performance and metabolism; this task also provides a measure of motor activity that permits control for nonspecific effects of treatment. Combined, these methods permit direct measurement of the neurochemical and metabolic variables regulating behavior.

Hippocampal Insulin Microinjection and In vivo Microdialysis During Spatial Memory Testing

Modulation of hippocampally-dependent spatial working memory by direct intrahippocampal microinjection, accompanied and followed by in vivo microdialysis for metabolites in conscious, behaving animals.

Glucose metabolism is a useful marker for local neural activity, forming the basis of methods such as 2-deoxyglucose and functional magnetic resonance ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense organs provide an excellent model system for the study of mechanosensation. This protocol describes a simple touch behavior in fruit flies which can be used to identify mutations that interfere with mechanosensation. The tactile stimulation of a macrochaete bristle on the thorax of flies elicits a grooming reflex from either the first or third leg. Mutations that interfere with mechanotransduction (such as NOMPC), or with other aspects of the reflex arc, can inhibit the grooming response. A traditional screen of adult behaviors would have missed mutants that have essential roles during development. Instead, this protocol combines the touch screen with mosaic analysis with a repressible cell marker (MARCM) to allow for only limited regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP). By testing MARCM clones for abnormal behavioral responses, it is possible to screen a collection of lethal p-element mutations to search for new genes involved in mechanosensation that would have been missed by more traditional methods.

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. However, there is piling evidence that olfaction is not solely limited to chemical sensitivity, but also includes temperature sensitivity. Premetamorphic Xenopus laevis are translucent animals, with protruding nasal cavities deprived of the cribriform plate separating the nose and the olfactory bulb. These characteristics make them well suited for studying olfaction, and particularly thermosensitivity. The present article describes the complete procedure for measuring temperature responses in the olfactory bulb of X. laevis larvae. Firstly, the electroporation of olfactory receptor neurons (ORNs) is performed with spectrally distinct dyes loaded into the nasal cavities in order to stain their axon terminals in the bulbar neuropil. The differential staining between left and right receptor neurons serves to identify the &#947;-glomerulus as the only structure innervated by contralateral presynaptic afferents. Secondly, the electroporation is combined with focal bolus loading in the olfactory bulb in order to stain mitral cells and their dendrites. The 3D brain volume is then scanned under line-illumination microscopy for the acquisition of fast calcium imaging data while small temperature drops are induced at the olfactory epithelium. Lastly, the post-acquisition analysis allows the morphological reconstruction of the thermosensitive network comprising the &#947;-glomerulus and its innervating mitral cells, based on specific temperature-induced Ca2+ traces. Using chemical odorants as stimuli in addition to temperature jumps enables the comparison between thermosensitive and chemosensitive networks in the olfactory bulb.

Recording Temperature-induced Neuronal Activity through Monitoring Calcium Changes in the Olfactory Bulb of Xenopus laevis

Here we describe a protocol for measuring and analyzing temperature responses in the olfactory bulb of Xenopus laevis. Olfactory receptor neurons and mitral cells are differentially stained, after which calcium changes are recorded, reflecting a sensitivity of some neural networks in the bulb to temperature drops induced at the nose.

The olfactory system, specialized in the detection, integration and processing of chemical molecules is likely the most thoroughly studied sensory system. ...

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 ...