Name: drosophila passive avoidance behavior as a new paradigm to study associative aversive learning
Uploaded: 2021-10-15T15:00:00
Description: Watch this Scientific Journal Video about Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning at JoVE.com

This study was supported in parts by NIH R15ES029673 (AKM).

1. Preparation of passive avoidance apparatus
<ol>
	<li>Drill a 4 mm hole perpendicular to the wall surface of the 14 mL polypropylene culture tube and 8 mm away from the tube bottom. 
	NOTE: Use an electric drill and 5/32 drill bit for best results.</li>
	<li>Using a steel utility knife, cut off the upper part of the 14 mL polypropylene culture tube to create a 45 mm long tube bottom fragment. The bottom fragment serves as the lower compartment.</li>
	<li>Cut off the tip of 1,000 &#181;L blue pip...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+courtship+conditioning+as+a+measure+of+learning+and+memory.">Drosophila courtship conditioning as a measure of learning and memory.</a> Journal of Visualized Experiments. (124), e55808 (2017).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> Journal of Visualized Experiments. (49), e2504 (2011).</li><li>Bozler, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+systems+level+approach+to+temporal+expression+dynamics+in+Drosophila+reveals+clusters+of+long+term+memory+genes.">A systems level approach to temporal expression dynamics in Drosophila reveals clusters of long term memory genes.</a> Plos Genetics. 13 (10), 1007054 (2017).</li><li>Atucha, E., Roozendaal, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+avoidance+discrimination+task+to+investigate+accuracy+of+memory.">The inhibitory avoidance discrimination task to investigate accuracy of memory.</a> Frontiers in Behavioral Neuroscience. 9, 60 (2015).</li><li>Thiels, E., Hoffman, E. K., Gorin, M. 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+reliable+behavioral+assay+for+the+assessment+of+sustained+photophobia+in+mice.">A reliable behavioral assay for the assessment of sustained photophobia in mice.</a> Current Eye Research. 33 (5), 483-491 (2008).</li><li>Detrait, E. R., Hanon, E., Dardenne, B., Lamberty, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+avoidance+test+optimized+for+discovery+of+cognitive+enhancers.">The inhibitory avoidance test optimized for discovery of cognitive enhancers.</a> Behavior Research Methods. 41 (3), 805-811 (2009).</li><li>Piper, M. D. W., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+ageing.">Drosophila as a model for ageing.</a> Biochimica et Biophysica Acta - Molecular Basis of Disease. 1864 (9), 2707-2717 (2018).</li><li>Chalmers, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicomponent+screen+for+feeding+behaviour+and+nutritional+status+in+Drosophila+to+interrogate+mammalian+appetite-related+genes.">A multicomponent screen for feeding behaviour and nutritional status in Drosophila to interrogate mammalian appetite-related genes.</a> Molecular Metabolism. 43, 101127 (2021).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Experimental Gerontology. 40 (5), 386-395 (2005).</li><li>Yang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+homemade+tools+to+handle+fruit+flies-Drosophila+melanogaster.">Simple homemade tools to handle fruit flies-Drosophila melanogaster.</a> Journal of Visualized Experiments. (149), e59613 (2019).</li><li>Barradale, F., Sinha, K., Lebestky, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+Drosophila+grooming+behavior.">Quantification of Drosophila grooming behavior.</a> Journal of Visualized Experiments. (125), e55231 (2017).</li><li>Denmark, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+chronic+social+defeat+stress+on+mouse+self-grooming+behavior+and+its+patterning.">The effects of chronic social defeat stress on mouse self-grooming behavior and its patterning.</a> Behavioural Brain Research. 208 (2), 553-559 (2010).</li><li>Kalueff, A. V., 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=Neurobiology+of+rodent+self-grooming+and+its+value+for+translational+neuroscience.">Neurobiology of rodent self-grooming and its value for translational neuroscience.</a> Nature Reviews: Neuroscience. 17 (1), 45-59 (2016).</li><li>Motulsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Intuitive biostatistics: A nonmathematical guide to statistical thinking. Fourth edition. , (2018).</li><li>Qiao, B., Li, C., Allen, V. W., Shirasu-Hiza, M., Syed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+analysis+of+long-term+grooming+behavior+in+Drosophila+using+a+k-nearest+neighbors+classifier.">Automated analysis of long-term grooming behavior in Drosophila using a k-nearest neighbors classifier.</a> Elife. 7, 34497 (2018).</li><li>Mu, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+limbic+circuitry+involved+in+emotional+stress-induced+grooming.">A limbic circuitry involved in emotional stress-induced grooming.</a> Nature Communications. 11 (1), 2261 (2020).</li><li>Song, C., Berridge, K. C., Kalueff, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stressing'+rodent+self-grooming+for+neuroscience+research.">Stressing' rodent self-grooming for neuroscience research.</a> Nature Reviews: Neuroscience. 17 (9), 591 (2016).</li><li>Wang, C., Chan, J. S., Ren, L., Yan, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity+reduces+cognitive+and+motor+functions+across+the+lifespan.">Obesity reduces cognitive and motor functions across the lifespan.</a> Neural Plasticity. 2016, 2473081 (2016).</li><li>Lewis, A. R., Singh, S., Youssef, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cafeteria-diet+induced+obesity+results+in+impaired+cognitive+functioning+in+a+rodent+model.">Cafeteria-diet induced obesity results in impaired cognitive functioning in a rodent model.</a> Heliyon. 5 (3), 01412 (2019).</li><li>Yohn, S. E., Galbraith, J., Calipari, E. S., Conn, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shared+behavioral+and+neurocircuitry+disruptions+in+drug+addiction,+obesity,+and+binge+eating+disorder:+Focus+on+Group+I+mGluRs+in+the+mesolimbic+dopamine+pathway.">Shared behavioral and neurocircuitry disruptions in drug addiction, obesity, and binge eating disorder: Focus on Group I mGluRs in the mesolimbic dopamine pathway.</a> ACS Chemical Neuroscience. 10 (5), 2125-2143 (2019).</li><li>Lopez-Taboada, I., Gonzalez-Pardo, H., Conejo, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Diet:+Implications+for+brain+function+and+behavior.">Western Diet: Implications for brain function and behavior.</a> Frontiers in Psychololgy. 11, 564413 (2020).</li><li>Murashov, A. K., 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=Preference+and+detrimental+effects+of+high+fat,+sugar,+and+salt+diet+in+wild-caught+Drosophila+simulans+are+reversed+by+flight+exercise.">Preference and detrimental effects of high fat, sugar, and salt diet in wild-caught Drosophila simulans are reversed by flight exercise.</a> FASEB Bioadvances. 3 (1), 49-64 (2021).</li><li>Krypotos, A. M., Effting, M., Kindt, M., Beckers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Avoidance+learning:+A+review+of+theoretical+models+and+recent+developments.">Avoidance learning: A review of theoretical models and recent developments.</a> Frontiers in Behavioral Neuroscience. 9, 189 (2015).</li><li>Binder, M. D., Hirokawa, N., Windhorst, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Neuroscience. , 3093 (2009).</li><li>Mery, F., Belay, A. T., So, A. K., Sokolowski, M. B., Kawecki, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+polymorphism+affecting+learning+and+memory+in+Drosophila.">Natural polymorphism affecting learning and memory in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (32), 13051-13055 (2007).</li><li>Tan, Y., Yu, D., Pletting, J., Davis, R. 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Avoidance of threatening stimuli is a crucial characteristic of adaptive behavior in various species from C. elegance to human32. Avoidance learning procedures which typically entail the escaping of an aversive event, are commonly used behavioral tasks to investigate learning and memory processes in laboratory rodents13 since the 1970&#39;s32. In active avoidance procedures, an indifferent stimulus or conditioned signal (CS) is followed by a...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.
In the Representative Results, the legend for Figure 5 was updated from:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable increase in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
to:
Figure 5: Comparison of passive avoidance and grooming behavior in D. simulans males and females. (A)&#160;Average latency (s) per trial. The graph shows no statistically significant differences between males and females in the latencies. (B) An average number of received shocks per trial. The graph shows no statistically significant differences between males and females in the number of received shocks. (C) The total duration of grooming bouts in trials 1-3. While there were no statistically significant differences between males and females, the female flies showed a considerable decrease in grooming behavior during trials 2 and 3 compared to trial 1. Abbreviations: *- P&#60;0.05. One-way ANOVA with Tukey&#39;s multiple comparisons test.
In the Discussion, the third paragraph&#160;was updated from:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly higher in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.
to:
The assay worked equally well in D. melanogaster and D. simulans male and female flies, demonstrating that the paradigm could be adapted to different D. species. The changes in fly behavior characterized by increased latencies and decreased number of shocks were statistically significant in the second trial and would strengthen with subsequent trials. Interestingly, if na&#239;ve flies were habituated to the apparatus without electric shock, they would enter the upper compartment a little faster on the second and the third trials. However, the decrease in latencies was not statistically significant (data not shown). No statistically significant differences were observed between sexes, although female flies had somewhat longer latencies and received slightly more shocks. This difference could be due to a combination of factors, including females&#39; failure to associate the shock with the upper compartment, a stronger geotaxis, or possibly because females are slightly larger and slower than males. The total duration of grooming bouts was significantly lower&#160;in the second and third trials in female flies, which draws a parallel between D. and rodent anxiety-like behaviors26.

An erratum was issued for:&#160;<a href="https://www.jove.com/t/63163">Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning</a>. The Representative Results and Discussion&#160;sections were&#160;updated.

Erratum: Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

Learning and memory is an evolutionarily ancient adaptation mechanism to the environment, conserved from Drosophila (D.) to human1. The fruit fly is a robust model organism to study fundamental principles of learning and memory as it offers a wide range of powerful genetic tools to dissect intrinsic molecular mechanisms2. The pioneering genetic screening studies, which identified rutabaga3, amnesiac4, and dunce5 genes critical for learning and memory2, took advantage of olfactory conditioning as the fruit flies rely on their keen sense of smell to find food, potential mates, and to avoid predators6.
Olfactory conditioning has become a popular paradigm to study the mechanism of learning and memory, thanks to the introduction of olfactory T-maze by Tully and Quinn7,8. Subsequently, other methods to measure various types of learning and memory have been proposed, including visual conditioning9, courtship conditioning10, aversive phototaxis suppression assay11, and wasp-exposure conditioning12. However, most of these assays have a complex setup that must be custom-built at a university workshop or purchased through a vendor. The paradigm described here is based on a simple behavioral assay to study aversive associative learning in flies that can be easily assembled with a few available supplies.
The described paradigm is equivalent to passive (or inhibitory) avoidance behavior in laboratory mice and rats in which animals learn to avoid a compartment where they have previously received electric foot shock13. In murids, the procedure is based on their innate avoidance of bright light and preference for darker areas14. On the first trial, the animal is placed into the bright compartment, from where the animal quickly exits, stepping into a dark compartment, where an electric foot shock is delivered. Usually, a single trial is sufficient to form a solid long-term memory, resulting in significantly increased latency 24 h later. The latency is then used as an index of the ability of the animal to remember the association between the aversive stimulus and the specific environment15.
This work describes an analogous procedure using D. as a model system which offers several advantages over rodent models including cost-effectiveness, larger sample size, the absence of regulatory oversight, and access to powerful genetic tools16,17. The procedure is based on negative geotaxis behavior, which manifests in flies&#39; urge to climb up when they are placed on a vertical surface18. The setup consists of two vertical chambers. On the first trial, a fruit fly is placed into a lower compartment. From there, it usually exits within 3-15 s, stepping into the upper compartment where it receives an electric shock. During a 1 min trial, some flies may occasionally re-enter the upper compartment, which results in an additional electric shock. During the testing phase, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first day indicating that flies formed aversive associative memory about the upper compartment. The latency, number of shocks, and the duration and frequency of grooming bouts are then used to analyze the animal behavior and the ability to form and remember the association between the aversive stimulus and the specific environment. The representative results reveal that exposure to the Western diet (WD) significantly impairs passive avoidance behavior in male flies, suggesting that the WD profoundly impacts the fly&#39;s behavior and cognition. Conversely, flight exercise alleviated the negative effect of the WD, improving passive avoidance behavior.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bloomington Formulation diet</td>
			<td>Nutri-Fly</td>
			<td>&#160;66-112</td>
			<td>Available from Genesee Scientific Inc., San Diego, CA</td>
		</tr>
		<tr>
			<td>1000 &#181;L Blue tip</td>
			<td>Fisher</td>
			<td>NC9546243</td>
			<td></td>
		</tr>
		<tr>
			<td>17 x 100 mm 14 mL polypropylene culture tube</td>
			<td>VWR</td>
			<td>&#160;60818-689</td>
			<td></td>
		</tr>
		<tr>
			<td>Aduino-based Automatic Kontrol Module</td>
			<td>In-house</td>
			<td>AKM-007</td>
			<td>This unit is optional. Complete description, schematics, wiring diagram and a code are provided at the ECU Digital Market - https://digitalmarket.ecu.edu/akmmodule</td>
		</tr>
		<tr>
			<td>Dual-Display 2-Channel&#160; Digital Clock/Timer</td>
			<td>Digi-Sense</td>
			<td>AO-94440-10</td>
			<td>https://www.amazon.com/Cole-Parmer-AO-94440-10-Dual-Display-2-Channel-Jumbo-Digit/dp/B00PR0809G/ref=sr_1_5?dchild=1&#38;keywords=Dual-Display+timer+jumbo&#38;qid=1627660660&#38;sr= 
			8-5#customerReviews</td>
		</tr>
		<tr>
			<td>Electronic Finger Counter</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>https://www.amazon.com/gp/product/B01M8IRK6F/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;psc=1</td>
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			<td>Fisherbrand Sparkleen 1 Detergent</td>
			<td>Fisher Scientific</td>
			<td>04-320-4</td>
			<td></td>
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		<tr>
			<td>Fly mouth aspirator</td>
			<td>In-house</td>
			<td></td>
			<td>Prepared as described in reference 19.</td>
		</tr>
		<tr>
			<td>Grass S88 stimulator</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Could be replaced with any stimulator which can provide described parameters</td>
		</tr>
		<tr>
			<td>Kim-wipes</td>
			<td>Fisher Scientific</td>
			<td>06-666</td>
			<td>Kimberly-Clark Professional 34120</td>
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		<tr>
			<td>Metal block for fly immobilization</td>
			<td>In-house</td>
			<td></td>
			<td>4 x 13 x 23.5cm aluminum block</td>
		</tr>
		<tr>
			<td>Nutiva USDA Certified Organic, non-GMO, Red Palm Oil</td>
			<td>Nutiva</td>
			<td>N/A</td>
			<td>https://www.amazon.com/Nutiva-Certified-Cold-Filtered-Unrefined-Ecuadorian/dp/B00JJ1E83G/ref=sxts_rp_s1_0?cv_ct_cx=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&#38;dchild=1&#38;keywords=Nutiva+USDA+Certified+Organic%2C+non-GMO%2C+Red+Palm+Oil&#38;pd_rd_i=B00JJ1E83G&#38;pd_ 
			rd_r=f35e9d2f-afe4-44b6-afc2-1c9cd705be18&#38;pd_rd_w= 
			R3Zb4&#38;pd_rd_wg=eUv1m&#38;pf_rd_ 
			p=c6bde456-f877-4246-800f-44405f638777&#38;pf 
			_rd_r=M94N11RC7NH333EMJ66Y 
			&#38;psc=1&#38;qid=1627661533&#38;sr=1-1-f0029781-b79b-4b60-9cb0-eeda4dea34d6</td>
		</tr>
		<tr>
			<td>Shock tube</td>
			<td>CelExplorer</td>
			<td>TMA-201</td>
			<td>https://www.celexplorer.com/product_detail.asp?id=217&#38;MainType=110&#38;SubType=8</td>
		</tr>
		<tr>
			<td>Stopwatch</td>
			<td>Accusplit</td>
			<td>A601XLN</td>
			<td>https://www.amazon.com/gp/product/B0007ZGZYI/ref=ppx_yo_dt_b_search_asin_title?ie=UTF8&#38;psc=1</td>
		</tr>
		<tr>
			<td>Transparent vinyl tubing (3/4&#8221; OD, 5/8&#8221; ID)</td>
			<td>Lowes</td>
			<td></td>
			<td>Avaiable from Lowes</td>
		</tr>
	</tbody>
</table>

drosophila passive avoidance behavior as a new paradigm to study associative aversive learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to passive avoidance behavior in laboratory rodents in which animals learn to avoid a compartment where they have previously received an electric shock. The assay takes advantage of negative geotaxis in flies, which manifests as an urge to climb up when they are placed on a vertical surface. The setup consists of vertically oriented upper and lower compartments. On the first trial, a fly is placed into a lower compartment from where it usually exits&#160;within 3-15 s, and steps into the upper compartment where it receives an electric shock. During the second trial, 24 h later, the latency is significantly increased. At the same time, the number of shocks is decreased compared to the first trial, indicating that flies formed long-term memory about the upper compartment. The recordings of latencies and number of shocks could be performed with a tally counter and a stopwatch or with an Arduino-based simple device. To illustrate how the assay can be used, the passive avoidance behavior of D. melanogaster and D. simulans male and female were characterized here. Comparison of latencies and number of shocks revealed that both D. melanogaster and D. simulans flies efficiently learned the passive avoidance behavior. No statistical differences were observed between male and female flies. However, males were a little faster while entering the upper compartment on the first trial, while females received a slightly higher number of shocks in every retention trial. The Western diet (WD) significantly impaired learning and memory in male flies while flight exercise counterbalanced this effect. Taken together, the passive avoidance behavior in flies offers a simple and reproducible assay that could be used for studying basic mechanisms of learning and memory.

The passive avoidance was studied in D. melanogaster (Canton-S) and D. simulans. The experiments compared the latencies and number of received shocks between consecutive trials. Initially, the experiments were performed with 3-4 day old male D. melanogaster flies. Flies were maintained on the standard Bloomington Formulation diet in a climate-controlled environment at 24 &#176;C under a 12 h light-dark cycle, 70% humidity, and controlled population density. The density was controlled b...

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Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This work describes a simple behavioral paradigm that allows the analysis of aversive associative learning in adult fruit flies. The method is based on suppressing the innate negative geotaxis behavior due to the association formed between a specific environmental context and an electric shock.

Drosophila Passive Avoidance Behavior as a New Paradigm to Study Associative Aversive Learning

This protocol describes a new paradigm for analyzing aversive associative learning in adult flies (Drosophila melanogaster). The paradigm is analogous to ...

drosophila-passive-avoidance-behavior-as-new-paradigm-to-study

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The Crossmodal Congruency Task as a Means to Obtain an Objective Behavioral Measure in the Rubber Hand Illusion Paradigm

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden hand is touched. If the viewed and felt touches are given at the same time then this is sufficient to induce the compelling experience that the rubber hand is one's own hand. The RHI can be used to investigate exactly how the brain constructs distinct body representations for one's own body. Such representations are crucial for successful interactions with the external world. To obtain a subjective measure of the RHI, researchers typically ask participants to rate statements such as "I felt as if the rubber hand were my hand". Here we demonstrate how the crossmodal congruency task can be used to obtain an objective behavioral measure within this paradigm.
The variant of the crossmodal congruency task we employ involves the presentation of tactile targets and visual distractors. Targets and distractors are spatially congruent (i.e. same finger) on some trials and incongruent (i.e. different finger) on others. The difference in performance between incongruent and congruent trials - the crossmodal congruency effect (CCE) - indexes multisensory interactions. Importantly, the CCE is modulated both by viewing a hand as well as the synchrony of viewed and felt touch which are both crucial factors for the RHI.
The use of the crossmodal congruency task within the RHI paradigm has several advantages. It is a simple behavioral measure which can be repeated many times and which can be obtained during the illusion while participants view the artificial hand. Furthermore, this measure is not susceptible to observer and experimenter biases. The combination of the RHI paradigm with the crossmodal congruency task allows in particular for the investigation of multisensory processes which are critical for modulations of body representations as in the RHI.

We demonstrate how an objective measure can be employed in the widely employed rubber hand illusion paradigm. This measure is obtained by modifying the well-established crossmodal congruency task. This task allows the investigation of multisensory processes which are critical for modulations of body representations as in the rubber hand illusion.

The rubber hand illusion (RHI) is a popular experimental paradigm. Participants view touch on an artificial rubber hand while the participants' own hidden ...

A Cognitive Paradigm to Investigate Interference in Working Memory by Distractions and Interruptions

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that one attempts to ignore, or by interrupting information that demands attention as part of another (secondary) task goal. Both forms of external interference have been shown to detrimentally impact the ability to maintain information in working memory (WM). Emerging evidence suggests that these different types of external interference exert different effects on behavior and may be mediated by distinct neural mechanisms. Better characterizing the distinct neuro-behavioral impact of irrelevant distractions versus attended interruptions is essential for advancing an understanding of top-down attention, resolution of external interference, and how these abilities become degraded in healthy aging and in neuropsychiatric conditions. This manuscript describes a novel cognitive paradigm developed the Gazzaley lab that has now been modified into several distinct versions used to elucidate behavioral and neural correlates of interference, by to-be-ignored distractors versus to-be-attended interruptors. Details are provided on variants of this paradigm for investigating interference in visual and auditory modalities, at multiple levels of stimulus complexity, and with experimental timing optimized for electroencephalography (EEG) or functional magnetic resonance imaging (fMRI) studies. In addition, data from younger and older adult participants obtained using this paradigm is reviewed and discussed in the context of its relationship with the broader literatures on external interference and age-related neuro-behavioral changes in resolving interference in working memory.

A novel cognitive paradigm is developed to elucidate behavioral and neural correlates of interference by to-be-ignored distractors versus interference by to-be-attended interruptors during a working memory task. In this manuscript, several variants of this paradigm are detailed, and data obtained with this paradigm in younger/older adult participants is reviewed.

Goal-directed behavior is often impaired by interference from the external environment, either in the form of distraction by irrelevant information that ...

How to Study Placebo Responses in Motion Sickness with a Rotation Chair Paradigm in Healthy Participants

Placebo responses occur in every medical intervention when patients or participants expect to receive an effective treatment to relieve symptoms. However, underlying mechanisms of placebo responses are not fully understood. It has repeatedly been shown that placebo responses are associated with changes in neural activity but for many conditions it is unclear whether they also affect the target organ, such as the stomach in motion sickness. Therefore, we present a methodology for the multivariate assessment of placebo responses by subjective, behavioral and objective measures in motion sickness with a rotation chair paradigm. The physiological correlate of motion sickness is a shift in gastric myoelectrical activity towards tachygastria that can be recorded with electrogastrography. The presented study applied the so-called balanced placebo design (BPD) to investigate the effects of ginger compared to placebo and the effects of expectations by verbal information. However, the study revealed no significant main or interactional effects of ginger (as a drug) or information on outcome measures but showed interactions when sex of participants and experimenters are taken into considerations. We discuss limitations of the presented study and report modifications that were used in subsequent studies demonstrating placebo responses when rotation speed was lowered. In general, future placebo studies have to identify the appropriate target organ for the studied placebo responses and to apply the specific methods to assess the physiological correlates.

An experimental rotation chair paradigm is used to investigate whether a placebo intervention on motion sickness affects subjective outcome measures only or also affects behavioral and objective measures in a balanced placebo design.

Placebo responses occur in every medical intervention when patients or participants expect to receive an effective treatment to relieve symptoms. However, ...

Combined Shuttle-Box Training with Electrophysiological Cortex Recording and Stimulation as a Tool to Study Perception and Learning

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the necessary equipment is now available by several commercial companies. This protocol provides a detailed description of a two-way shuttle-box avoidance learning paradigm in rodents (here Mongolian gerbils; Meriones unguiculatus) in combination with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical electrophysiological in vivo recordings. The detailed protocol is applicable to study multiple aspects of learning behavior and perception in different rodent species.
Site-specific ICMS of auditory cortical circuits as conditioned stimuli here is used as a tool to test the perceptual relevance of specific afferent, efferent and intracortical connections. Distinct activation patterns can be evoked by using different stimulation electrode arrays for local, layer-dependent ICMS or distant ICMS sites. Utilizing behavioral signal detection analysis it can be determined which stimulation strategy is most effective for eliciting a behaviorally detectable and salient signal. Further, parallel multichannel-recordings using different electrode designs (surface electrodes, depth electrodes, etc.) allow for investigating neuronal observables over the time course of such learning processes. It will be discussed how changes of the behavioral design can increase the cognitive complexity (e.g. detection, discrimination, reversal learning).

Shuttle-box avoidance learning is well-established in behavioral neuroscience. This protocol describes how shuttle-box learning in rodents can be combined with site-specific electrical intracortical microstimulation (ICMS) and simultaneous chronical in vivo recordings as a tool to study multiple aspects of learning and perception.

Shuttle-box avoidance learning is a well-established method in behavioral neuroscience and experimental setups were traditionally custom-made; the ...

Vagus Nerve Stimulation as a Tool to Induce Plasticity in Pathways Relevant for Extinction Learning

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been maintaining the behavior. There is close correspondence between fear and human anxiety, and therefore studies of extinction learning might provide insight into the biological nature of anxiety-related disorders such as post-traumatic stress disorder, and they might help to develop strategies to treat them. Preclinical research aims to aid extinction learning and to induce targeted plasticity in extinction circuits to consolidate the newly formed memory. Vagus nerve stimulation (VNS) is a powerful approach that provides tight temporal and circuit-specific release of neurotransmitters, resulting in modulation of neuronal networks engaged in an ongoing task. VNS enhances memory consolidation in both rats and humans, and pairing VNS with exposure to conditioned cues enhances the consolidation of extinction learning in rats. Here, we provide a detailed protocol for the preparation of custom-made parts and the surgical procedures required for VNS in rats. Using this protocol we show how VNS can facilitate the extinction of conditioned fear responses in an auditory fear conditioning task. In addition, we provide evidence that VNS modulates synaptic plasticity in the pathway between the infralimbic (IL) medial prefrontal cortex and the basolateral complex of the amygdala (BLA), which is involved in the expression and modulation of extinction memory.

Vagus nerve stimulation (VNS) has emerged as a tool to induce targeted synaptic plasticity in the forebrain to modify a range of behaviors. This protocol describes how to implement VNS to facilitate the consolidation of fear extinction memory.

Extinction describes the process of attenuating behavioral responses to neutral stimuli when they no longer provide the reinforcement that has been ...

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the appearance and behavior of visual stimuli, compared to manipulating live animals. Here, we present the FishSim Animation Toolchain, a software framework developed to provide researchers with an easy-to-use method for implementing 3D computer animations in behavioral experiments with fish. The toolchain offers templates to create virtual 3D stimuli of five different fish species. Stimuli are customizable in both appearance and size, based on photographs taken of live fish. Multiple stimuli can be animated by recording swimming paths in a virtual environment using a video game controller. To increase standardization of the simulated behavior, the prerecorded swimming path may be replayed with different stimuli. Multiple animations can later be organized into playlists and presented on monitors during experiments with live fish.
In a case study with sailfin mollies (Poecilia latipinna), we provide a protocol on how to conduct a mate-choice copying experiment with FishSim. We utilized this method to create and animate virtual males and virtual model females, and then presented these to live focal females in a binary choice experiment. Our results demonstrate that computer animation may be used to simulate virtual fish in a mate-choice copying experiment to investigate the role of female gravid spots as an indication of quality for a model female in mate-choice copying.
Applying this method is not limited to mate-choice copying experiments but can be used in various experimental designs. Still, its usability depends on the visual capabilities of the study species and first needs validation. Overall, computer animations offer a high degree of control and standardization in experiments and bear the potential to &#39;reduce&#39; and &#39;replace&#39; live stimulus animals as well as to &#39;refine&#39; experimental procedures.

Using the FishSim Animation Toolchain to Investigate Fish Behavior: A Case Study on Mate-Choice Copying In Sailfin Mollies

Using the novel FishSim Animation Toolchain, we present a protocol for non-invasive visual manipulation of public information in the context of mate-choice copying in sailfin mollies. FishSim Animation Toolchain provides an easy-to-use framework for the design, animation and presentation of computer-animated fish stimuli for behavioral experiments with live test fish.

Over the last decade, employing computer animations for animal behavior research has increased due to its ability to non-invasively manipulate the ...

Using the Visual World Paradigm to Study Sentence Comprehension in Mandarin-Speaking Children with Autism

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is currently a paucity of research exploring how preschool children with autism understand sentences using different types of cues. The mechanisms underlying sentence comprehension remains largely unclear. The present study presents a protocol to examine the sentence comprehension abilities of preschool children with autism. More specifically, a visual world paradigm of eye-tracking is used to explore the moment-to-moment sentence comprehension in the children. The paradigm has multiple advantages. First, it is sensitive to the time course of sentence comprehension and thus can provide rich information about how sentence comprehension unfolds over time. Second, it requires minimal task and communication demands, so it is ideal for testing children with autism. To further minimize the computational burden of children, the present study measures eye movements that arise as automatic responses to linguistic input rather than measuring eye movements that accompany conscious responses to spoken instructions.

We present a protocol to examine the use of morphological cues during real-time sentence comprehension by children with autism.

Sentence comprehension relies on the ability to rapidly integrate different types of linguistic and non-linguistic information. However, there is ...

A Methodology for Capturing Joint Visual Attention Using Mobile Eye-Trackers

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency sensors, such as eye-trackers, electrodermal activity wristbands, EEG bands, and motion sensors provide observations at the millisecond level. This level of precision allows researchers to collect large datasets on social interactions. In this paper, I discuss how multiple eye-trackers can capture a fundamental construct in social interactions, joint visual attention (JVA). JVA has been studied by developmental psychologists to understand how children acquire language, learning scientists to understand how small groups of learners work together, and social scientists to understand interactions in small teams. This paper describes a methodology for capturing JVA in colocated settings using mobile eye-trackers. It presents some empirical results and discusses implications of capturing microobservations to understand social interactions.

Using multimodal sensors is a promising way to understand the role of social interactions in educational settings. This paper describes a methodology for capturing joint visual attention from colocated dyads using mobile eye-trackers.

With the advent of new technological advances, it is possible to study social interactions at a microlevel with unprecedented accuracy. High frequency ...

Experimental Paradigm for Measuring the Effect of Induced Emotion on Grammar Learning

Previous studies have found that emotion has significant influence on the learning of foreign language vocabulary and textual comprehension. However, little attention has been given to the effect of induced emotion on grammar learning. This research examined the influence of positive induced emotion on the learning of Japanese grammatical rules among a group of learners with Chinese as their native language, using a semi-artificial language (i.e. Chipanese), which combines the grammatical rules of Japanese and the vocabulary of Chinese. Music was used to invoke positive emotional conditions in participants. Participants were required to learn Chipanese sentences in a training session through practice and then a grammaticality judgment task was administered to measure learning outcomes. We found that participants in positive emotional states performed less accurately and efficiently than those in the control group. The findings suggest that the protocol is effective in identifying the effect of positive induced emotion on grammar learning. The implications of this experimental paradigm for investigating foreign language learning are discussed.

Here, we present a protocol to measure the effect of positive induced emotion on grammar learning in foreign language learners using a semi-artificial language that integrates the grammatical rules of a foreign language with the lexicon of the learners&#39; native language.

Previous studies have found that emotion has significant influence on the learning of foreign language vocabulary and textual comprehension. However, ...

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders.

Fear- and anxiety-related behaviors significantly contribute to an organism&#8217;s survival. However, exaggerated defensive responses to perceived threat ...